CN115725784A

CN115725784A - Kit and method for detecting pathogens related to respiratory tract infection

Info

Publication number: CN115725784A
Application number: CN202210963755.6A
Authority: CN
Inventors: 苏莹; 雷湘华; 郭永超; 王艳平; 袁利花; 蔡锦刚
Original assignee: Shenzhen Uni Medica Technology Co ltd
Current assignee: Shenzhen Uni Medica Technology Co ltd
Priority date: 2022-05-31
Filing date: 2022-08-11
Publication date: 2023-03-03
Also published as: CN117604136A; CN115948607A; CN115948607B; CN117568499A

Abstract

The present disclosure describes a kit and a method for detecting pathogens related to respiratory tract infection, the kit comprises a primer set for detecting target regions of genes of a first pathogen and a second pathogen, the primer set comprises a first forward primer and a first reverse primer, the first forward primer comprises a first sequencing primer and an upstream primer matched with the 5' end of the target region; the first reverse primer comprises a second sequencing primer and a downstream primer matched with the 3' end of the target region; wherein the first pathogen and the second pathogen are pathogens associated with respiratory tract infections, and the first pathogen comprises the second pathogen; the first pathogen comprises genus coronavirus α, genus coronavirus β, genus coronavirus δ, genus coronavirus γ, influenza a virus, influenza b virus, adenovirus and nontuberculous mycobacteria. According to the kit and the method, the detection comprehensiveness of pathogens related to respiratory tract infection is high, the detection rate is high, and the authenticity is good.

Description

Kit and method for detecting pathogens related to respiratory tract infection

Technical Field

The invention relates to the field of gene detection, in particular to a kit and a method for detecting pathogens related to respiratory tract infection.

Background

According to the statistics of the world health organization, about 260 million people worldwide die from respiratory tract infection, and the incidence rate is up to 0.05-0.12%. Most of the acute respiratory infections are caused by infection of nasopharynx, lung, bronchus and other parts of a human body with viruses, bacteria, fungi, mycoplasma and chlamydia, wherein the viruses account for about 70 percent, and the detected pathogens account for more alphavirus, respiratory syncytial virus, rhinovirus, human metapneumovirus, respiratory adenovirus and the like. About 20% of the bacteria cause the bacteria, and streptococcus pneumoniae, streptococcus pyogenes, staphylococcus aureus and the like are mainly detected. The prior respiratory tract infection detection methods generally comprise a pathogen isolation culture method, a smear microscopy method, a PCR method and an enzyme-linked immunosorbent assay method. The methods for co-detecting more than one pathogen include a multiplex PCR method, a metagenome technology, a DNA microarray, a liquid chip technology and the like.

Among them, multiplex PCR is widely used for pathogen detection with advantages of low cost, applicability to simultaneous detection of multiple pathogens, and the like.

However, the existing detection kit for respiratory tract infection pathogens covers respiratory tract infection-related pathogens not completely, and most of the respiratory tract infection-related pathogens can only be localized to the genus or species of the pathogens, so that the detection has limitations.

Disclosure of Invention

The present disclosure has been made in view of the above-mentioned problems, and an object of the present disclosure is to provide a kit and a method for detecting pathogens associated with respiratory tract infection, which can detect pathogens more comprehensively, and can improve the detection rate of detection for some pathogens including detection sites from species to species or from species to subspecies, and can perform multiple tests to improve the authenticity of detection results.

To this end, the present disclosure provides in a first aspect a kit for detecting a pathogen associated with a respiratory infection, the kit comprising a primer set for detecting a target region of a gene of a first pathogen and a gene of a second pathogen, the primer set comprising a first forward primer and a first reverse primer, the first forward primer comprising a first sequencing primer and an upstream primer matched to the 5' end of the target region; the first reverse primer comprises a second sequencing primer and a downstream primer matched with the 3' end of the target region; wherein the first pathogen and the second pathogen are pathogens associated with respiratory tract infections, and the first pathogen comprises the second pathogen; the first pathogen comprises coronavirus genus alpha, coronavirus genus beta, coronavirus genus delta, coronavirus genus gamma, influenza A virus, influenza B virus, adenovirus, nontuberculous mycobacterium and Haemophilus influenzae; the forward primer comprises a universal forward primer designed based on a target region of a gene of the first pathogen or a species forward primer designed based on a target region of a gene of the second pathogen; the downstream primer includes a universal downstream primer designed based on a target region of a gene of the first pathogen or a species downstream primer designed based on a target region of a gene of the second pathogen.

In the kit disclosed by the disclosure, a primer group capable of detecting a first pathogen and a second pathogen is included, wherein the second pathogen belongs to the first pathogen, and in this case, the detection results can be mutually verified between the primer group of the first pathogen and the primer group of the second pathogen, so that the authenticity of detection is improved. For example, if a sample is positive for a second pathogen, the detection result of the first pathogen corresponding to the second pathogen is also positive, and if the second pathogen is positive but the detection result of the corresponding first pathogen is negative, the result needs to be further verified (the verification method may be, for example, other methodologies are used to detect the pathogen in the sample), so that the authenticity of the detection result can be improved. In addition, if the detection result of the second pathogen in the sample is negative but the detection result of the corresponding first pathogen is positive, it indicates that the genes of pathogens other than the second pathogen are present in the sample, and thus the detection rate can be improved. In addition, the first pathogen comprises coronavirus alpha, coronavirus beta, coronavirus delta, coronavirus gamma, influenza A virus, influenza B virus, adenovirus, nontuberculous mycobacterium and haemophilus influenzae, and has the advantages of comprehensive coverage on pathogens related to respiratory infection and high detection comprehensiveness. In conclusion, the kit provided by the disclosure has the advantages of high detection comprehensiveness, high detectable rate and good authenticity.

In a kit contemplated by the present disclosure, optionally, the second pathogen comprises coronavirus 229E, coronavirus NL63, coronavirus OC43, coronavirus HKU1, middle east respiratory syndrome coronavirus, SARS virus, novel coronavirus, influenza a H1N1 virus, influenza a H3N2 virus, influenza a H5N1 virus, influenza a H7N9 virus, influenza B virus Victoria line, influenza B virus Yamagata line, adenovirus group B, adenovirus group C, adenovirus group E, mycobacterium abscessus, mycobacterium intracellulare, mycobacterium avium, mycobacterium shallowlii, mycobacterium terrae, mycobacterium tortoise, mycobacterium kansasii, and haemophilus influenzae type B. Thereby, pathogens associated with respiratory infections can be more fully covered.

In kits contemplated by the present disclosure, optionally, a primer set is included for detecting a target region of a gene of a third pathogen comprising parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, respiratory syncytial virus type a, respiratory syncytial virus type B, rhinovirus, human metapneumovirus, bocavirus, rubella virus, measles virus, nipah virus, mumps virus, varicella zoster virus, mycobacterium tuberculosis complex, staphylococcus aureus, klebsiella pneumoniae, streptococcus pneumoniae, legionella pneumophila, haemophila parainfluenza, nocardia, neisseria meningitidis, yersinia pneumocystis, mycoplasma pneumoniae, and chlamydia pneumoniae; the upstream primer comprises a universal upstream primer designed based on a target region of a gene of the third pathogen or a species upstream primer designed based on a target region of a gene of the third pathogen; the downstream primer comprises a universal downstream primer designed based on a target region of a gene of the third pathogen or a species downstream primer designed based on a target region of a gene of the third pathogen. Thereby, the pathogens related to respiratory tract infection can be covered more comprehensively.

In a kit to which the present disclosure relates, optionally, the primer set comprises a second reverse primer, a second forward primer and a third reverse primer, the second reverse primer comprises the second sequencing primer, a first barcode and a first sequencing adaptor, the first barcode is configured to identify different samples; the second forward primer comprises a second sequencing adaptor and the first sequencing primer; the third reverse primer comprises the first sequencing adapter.

In the kit related to the present disclosure, optionally, the first forward primer is the first sequencing primer and the sequence matching the 5 'end of the target region in sequence from the 5' end to the 3 'end, the first reverse primer is the second sequencing primer and the sequence matching the 3' end of the target region in sequence from the 5 'end to the 3' end, the second reverse primer is the first sequencing adaptor, the first barcode and the second sequencing primer in sequence from the 5 'end to the 3' end, the second forward primer is the second sequencing adaptor and the first sequencing primer in sequence from the 5 'end to the 3' end, and the third reverse primer is the first sequencing adaptor.

In the kit related to the present disclosure, optionally, the first sequencing primer and the second sequencing primer are sequencing primers of an illumina sequencing platform, the first sequencing linker is a P7 linker of the illumina sequencing platform, the second sequencing linker is a P5 linker of the illumina sequencing platform, and the first barcode is a random base sequence of 6 to 12bp. Thus, the library of interest can be adapted to the illumina sequencing platform.

In the kit related to the present disclosure, optionally, each of the pathogens has 3 target regions, and the upstream primer and the downstream primer include an upstream primer and a downstream primer corresponding to at least 1 target region of the 3 target regions of each pathogen.

In the kit related to the present disclosure, optionally, the forward primer and the backward primer are forward primers and backward primers corresponding to 3 target regions of each pathogen. Thus, 3 target regions are detected for each pathogen gene, enabling an increase in detection accuracy.

A second aspect of the present disclosure provides a method for detecting pathogens associated with respiratory tract infections for non-diagnostic purposes, comprising: preparing a nucleic acid sample to be detected; performing PCR amplification on the nucleic acid sample to be detected by using the kit provided by the first aspect of the disclosure to obtain a target library; sequencing analysis is carried out on the target library to obtain sequencing data; and obtaining a detection result based on the sequencing data. Thus, pathogens associated with respiratory infections can be detected.

In the method related to the present disclosure, optionally, performing a first round of PCR amplification on the nucleic acid sample to be detected by using the first forward primer, the first reverse primer and the second reverse primer to obtain a first round of PCR amplification product; and carrying out second round PCR amplification on the first round PCR amplification product by using a second forward primer and a third reverse primer to obtain the target library.

According to the present disclosure, a kit and a method for detecting pathogens associated with respiratory tract infection can be provided, the detected pathogens are more comprehensive, and the detection rate of detection can be improved for part of pathogens including detection sites from subordinate to species or from species to subspecies, and multiple tests can be performed to improve the authenticity of the detection result.

Drawings

Fig. 1 shows a schematic diagram of a primer set, a kit, a method and an application for detecting pathogens associated with respiratory tract infection according to an example of the present disclosure.

Fig. 2 shows a schematic of the extraction of multiple target regions from a gene of a pathogen in accordance with examples of the present disclosure.

Fig. 3 shows a schematic diagram of a process for PCR amplification of a target region by a primer set according to an example of the present disclosure.

Fig. 4 shows a schematic diagram of a kit to which examples of the present disclosure relate.

Fig. 5 shows a flow diagram of a method of detecting pathogens associated with respiratory tract infections in accordance with examples of the present disclosure.

Fig. 6 shows a flow diagram of PCR amplification according to an example of the present disclosure.

Fig. 7 shows a flow diagram of PCR amplification and purification according to an example of the present disclosure.

Fig. 8 shows a schematic diagram of a scenario of PCR amplification according to an example of the present disclosure.

Detailed Description

Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same components are denoted by the same reference numerals, and redundant description thereof is omitted. The drawings are schematic and the ratio of the dimensions of the components and the shapes of the components may be different from the actual ones.

It is noted that, as used herein, the terms "comprises," "comprising," or any other variation thereof, such that a process, method, system, article, or apparatus that comprises or has a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include or have other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

In addition, the subtitles and the like referred to in the following description of the present invention are not intended to limit the content or the scope of the present invention, and serve only as a cue for reading. Such a subtitle should neither be understood as a content for segmenting an article, nor should the content under the subtitle be limited to only the scope of the subtitle.

The present disclosure relates to a primer set for detecting a pathogen associated with respiratory infection (hereinafter sometimes simply referred to as "primer set"), an application of a primer set for preparing a reagent for detecting a respiratory infection (hereinafter sometimes simply referred to as "application"), a kit for detecting a pathogen associated with respiratory infection (hereinafter sometimes simply referred to as "kit"), and a method for detecting a pathogen associated with respiratory infection (hereinafter sometimes simply referred to as "method" or "detection method"). The primer group, the kit, the application and the method for detecting the pathogens related to the respiratory tract infection have the advantages that the detected pathogens are more comprehensive, the effect of multiple verification is achieved for part of the pathogens including detection sites from subordinate to species or from species to subspecies, and the detection rate and the detection authenticity are improved. Has the effects of high detection comprehensiveness and high detectable rate.

Pathogens are generic terms for agents that cause or transmit disease, including viruses, bacteria, fungi, and parasites. By detecting the existence condition of pathogen genes in the detection sample, the auxiliary diagnosis can be carried out on diseases, and the subsequent symptomatic treatment and the like are facilitated.

The primer group, the kit, the application and the method can detect the target region of each pathogen gene, and the target region can be a conserved region selected from the pathogen genes. In other words, the target region may be selected from genes or gene fragments specific for the pathogen, and the pathogen can be identified by detecting the target region.

The primer group, the kit, the application and the method can be used for detecting pathogens related to respiratory tract infection. In other words, the test subject is a pathogen associated with respiratory tract infection. A pathogen associated with a respiratory infection may refer to one or more pathogens capable of causing a respiratory infection.

In some examples, in the primer sets, kits, uses and methods related to the present disclosure, pathogens associated with respiratory infection may include viral-based pathogens, bacterial-based pathogens and other microorganisms. In other words, the test subjects include viral pathogens, bacterial pathogens, and other microorganisms associated with respiratory infections.

The primer group, the kit, the application and the method can detect common respiratory tract infection related pathogens, wherein part of pathogens are detected from species (subspecies) to species (subspecies or serotypes). Thereby, the detection rate and the authenticity can be increased.

The primer set, the kit, the method and the application for detecting pathogens related to respiratory tract infection according to the present disclosure are described below with reference to the accompanying drawings.

Fig. 1 shows a schematic scenario of primer sets, kits, methods and applications for detecting pathogens associated with respiratory infections according to examples of the present disclosure.

In the present embodiment, as shown in FIG. 1, a nucleic acid sample 20 to be tested can be obtained from a test object such as a human body in general. The number of the nucleic acid samples 20 to be detected may be multiple, and for example, the number includes a nucleic acid sample 21 to be detected, a nucleic acid sample 22 to be detected, a nucleic acid sample 23 to be detected, and the like. Subsequently, the nucleic acid sample to be tested may be subjected to PCR amplification using, for example, the PCR instrument 400. Then, the sequencing is performed by the sequencer 500 to obtain sequence information of each nucleic acid sample to be tested (see fig. 1).

In some examples, the primer set according to the present embodiment can be used for PCR amplification of a nucleic acid sample to be detected.

In some examples, a primer set may refer to a primer set that detects a target region of a gene of a pathogen.

In some examples, the species of pathogen may be multiple. In other words, the primer set according to the present embodiment can detect a plurality of pathogens. In this embodiment, the primer set can simultaneously detect pathogens associated with respiratory infections.

In some examples, the pathogens contemplated by the present embodiments may include a first pathogen and a second pathogen. In other words, the primer set according to the present embodiment can detect a first pathogen and a second pathogen.

In some examples, the first pathogen may include a second pathogen. In other words, the second pathogen is subordinate to the first pathogen.

In some examples, the first pathogen may include genus coronavirus α, genus coronavirus β, genus coronavirus δ, genus coronavirus γ, influenza a virus, influenza b virus, adenovirus, nontuberculous mycobacterium and haemophilus influenzae.

In some examples, the second pathogen may include coronavirus 229E, coronavirus NL63, coronavirus OC43, coronavirus HKU1, middle east respiratory syndrome coronavirus, SARS virus, novel coronavirus, influenza a H1N1 virus, influenza a H3N2 virus, influenza a H5N1 virus, influenza a H7N9 virus, influenza B virus Victoria line, influenza B virus Yamagata line, adenovirus group B, adenovirus group C, adenovirus group E, mycobacterium abscessus, mycobacterium intracellularis, mycobacterium avium, mycobacterium flavum, mycobacterium terrae, mycobacterium cheloni, mycobacterium kansasii, and haemophilus influenzae type B.

In some examples, the target region may be a conserved region in a gene selected from a pathogen. In other words, the target region may be selected from a gene or gene fragment specific to the pathogen, and the pathogen can be recognized by detecting the target region.

In some examples, the sequences of the genes of the pathogen are known or published and can be obtained by public database queries or downloads, such as the National Center for Biotechnology Information (NCBI) DNA sequence database (GenBank), and software (e.g., clone Manager) can be used to find conserved sequence regions of the genes of the pathogen. In some examples, specific primers may be designed by selecting target regions from conserved sequence regions of genes of pathogens. In some examples, designing a specific primer allows for an increase in degenerate bases in the primer, thereby enabling increased primer coverage of conserved regions. Thus, a primer set capable of performing PCR amplification of a target region of a pathogen gene can be designed.

In some examples, one or more target regions may be selected from the genes of each pathogen. For example, 1, 2, 3, 5 or 10 target regions are selected from the genes of a pathogen. In some examples, from the standpoint of primer design difficulty and cost, 2 to 5 target regions can be selected from the gene sequence of each pathogen, and primer sets can be designed for the 2 to 5 target regions, respectively. For example, 2, 3, 4 or 5 target regions may be selected from the gene sequence of each pathogen. Preferably, 3 target regions can be selected from the gene sequence of each pathogen.

In some examples, as shown in fig. 2, 3 target regions, namely target region 100, target region 200, and target region 300, may be extracted from the gene sequence of gene 10 of the pathogen.

In some examples, the multiple target regions selected from the gene sequences of each pathogen may not overlap with each other. In other examples, the multiple target regions selected from the gene sequences of each pathogen may also only partially overlap with each other. That is, when multiple target regions are extracted from a gene of a pathogen, the multiple target regions do not completely overlap with each other. This enables selection of a plurality of different target regions from the gene sequence of each pathogen.

In some examples, one primer set may be designed for each target region. In some examples, each primer set may include a plurality of primers. In some examples, each primer set can include a first forward primer and a first reverse primer, respectively. In some examples, each primer set may further include a second reverse primer, a second forward primer, and a third reverse primer (described in detail later). For each target region, the first forward primer and the first reverse primer in the designed primer set are typically different (specific), while the second reverse primer, the second forward primer and the third reverse primer are common.

In some examples, the first forward primer may include a first sequencing primer and an upstream primer that matches the 5' end of the target region. In some examples, the first forward primer may consist of a first sequencing primer and an upstream primer that matches the 5' end of the target region. Specifically, the first forward primer may be, from its 5' end to its 3' end, a first sequencing primer and an upstream primer that matches the 5' end of the target region in that order.

In some examples, the first reverse primer may include a second sequencing primer and a downstream primer that matches the 3' end of the target region. In some examples, the first reverse primer may consist of the second sequencing primer and a downstream primer that matches the 3' end of the target region. Specifically, the first reverse primer may be, from its 5' end to its 3' end, a second sequencing primer and a downstream primer that matches the 3' end of the target region in that order.

In some examples, an upstream primer designed based on a target region of a gene of a first pathogen may be referred to as a universal upstream primer. An upstream primer designed based on a target region of a gene of a second pathogen may be referred to as a species upstream primer.

In some examples, a downstream primer designed based on a target region of a gene of a first pathogen may be referred to as a universal downstream primer. A downstream primer designed based on a target region of a gene of a second pathogen may be referred to as a species downstream primer.

In this case, the first forward primer containing the universal upstream primer, the first reverse primer containing the universal downstream primer, the first forward primer containing the species upstream primer and the first reverse primer containing the species downstream primer are combined to detect the first pathogen and the second pathogen, so that the effect of multiple verification can be achieved, and the detection rate and the authenticity of detection can be improved.

Of course, the primer set according to the present embodiment is not limited thereto, and in some examples, the primer set may also detect a third pathogen. Therefore, the detection of pathogens related to respiratory tract infection can be more comprehensive.

In some examples, the third pathogen may include parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, respiratory syncytial virus type a, respiratory syncytial virus type B, rhinovirus, human metapneumovirus, bocavirus, rubella virus, measles virus, nipah virus, mumps virus, varicella zoster virus, mycobacterium tuberculosis complex, staphylococcus aureus, klebsiella pneumoniae, streptococcus pneumoniae, legionella pneumophila, haemophilus parainfluenza, nocardia, neisseria meningitidis, pneumocystis yeri, mycoplasma pneumoniae, and chlamydia pneumoniae.

In some examples, an upstream primer (which may be referred to as a universal upstream primer or a species upstream primer) may be designed based on the 5' end of the target region of the gene of the third pathogen. In some examples, a downstream primer (which may be referred to as a universal downstream primer or a species downstream primer) may be designed based on the 3' end of the target region of the gene of the third pathogen. Thus, the first forward primer and the first reverse primer are capable of capturing a target region of a gene of a third pathogen.

In some examples, in the present embodiment, each pathogen has 3 target regions, and the upstream primer and the downstream primer in the primer set of the present embodiment may include the upstream primer and the downstream primer corresponding to at least 1 target region of the 3 target regions of each pathogen. For example, as shown in table 1 below, for coronavirus 229E, the upstream and downstream primers can be the upstream and downstream primers of at least one of the target regions 1-3. Preferably, the primer set of the present embodiment may be an upstream primer and a downstream primer corresponding to 3 target regions of each pathogen. In other words, each pathogen detects 3 target regions. In this case, the detection accuracy can be improved.

In some examples, pathogen and primer sequence information contemplated by the present embodiments may be as shown in tables 1 through 15 below:

table 1:

table 2:

table 3:

table 4:

table 5:

table 6:

table 7:

table 8:

table 9:

table 10:

table 11:

table 12:

table 13:

table 14:

table 15:

in some examples, each primer set can further include a second reverse primer, a second forward primer, and a third reverse primer.

In some examples, when the primer set is used for detecting a nucleic acid sample to be detected, a first forward primer, a first reverse primer and a second reverse primer may be used to perform a first round of PCR amplification on the nucleic acid sample to be detected and obtain a first round of PCR amplification product, and then a second forward primer and a third reverse primer may be used to perform a second round of PCR amplification on the first round of PCR amplification product to obtain a target library.

In some examples, the second reverse primer may include a second sequencing primer, a first barcode, and a first sequencing adapter. In some examples, the second reverse primer may consist of the second sequencing primer, the first barcode, and the first sequencing adapter. Specifically, the second reverse primer may be, in order from its 5 'end to its 3' end, a first sequencing adaptor, a first barcode, and a second sequencing primer.

In some examples, the first barcode in the second reverse primer may be configured to identify different nucleic acid samples to be tested. That is, the first barcodes used for the same sample of nucleic acid to be tested have the same sequence, and different first barcodes are used for different samples of nucleic acid to be tested. In some examples, different first barcodes are used for at least the nucleic acid samples to be detected in the same batch, and in this case, the influence of aerosol pollution and the like on the detection results of different nucleic acid samples to be detected can be reduced by adding the first barcodes to the nucleic acid samples to be detected in the first PCR amplification process.

In some examples, the first barcode may be a random sequence. For example, a first barcode may consist of several bases, with different base orderings representing different first barcodes. In some examples, the first barcode may be a random sequence with a base number of 6 to 12. For example, the first barcode may be a random sequence with a number of bases of 6, 7, 8, 9, 10, 11, or 12.

In some examples, the second forward primer may include a second sequencing adaptor and a first sequencing primer. Specifically, the second forward primer may be, in order from its 5 'end to its 3' end, the second sequencing adaptor and the first sequencing primer.

In some examples, the second forward primer may also include a second barcode. Specifically, the second forward primer may be, in order from its 5 'end to its 3' end, a second sequencing adaptor, a second barcode, and a first sequencing primer. The second barcode may be configured to identify different batches of samples. That is, the sequence of the second barcode used for the nucleic acid sample to be tested in the same lot is the same, and the sequence of the second barcode used for the nucleic acid sample to be tested in different lots is different. Therefore, the influence of aerosol pollution and the like on the detection results of different batches of samples can be reduced.

In some examples, the second barcode may be a random sequence. For example, the second barcode may consist of several bases, with different base orderings representing different second barcodes. In some examples, the first barcode may be a random sequence with a base number of 6 to 12. For example, the first barcode may be a random sequence with a number of bases of 6, 7, 8, 9, 10, 11, or 12.

In some examples, the second barcode may preferably be different from the first barcode for the sake of more convenient sequencing data analysis. Specifically, the number of bases of the second barcode may be different from that of the first barcode, or the base sequence of the second barcode may be different from that of the first barcode.

In some examples, the third reverse primer may include a first sequencing adaptor. In some examples, the third reverse primer can be a first sequencing adaptor.

In some examples, in the first round of PCR amplification, the first forward primer can be used as a forward primer for the target region, and the first reverse primer and the second reverse primer can be used as reverse primers for the target region, and the target region is captured and amplified by PCR to obtain a first round of PCR amplification product.

In some examples, since different first barcodes can be added to different nucleic acid samples to be detected through the second reverse primer during the first round of PCR amplification, after the first round of PCR amplification is completed, first round PCR amplification products of different samples can be mixed, and then the second round PCR amplification is performed uniformly after the mixing.

In some examples, during the second round of PCR amplification, the second forward primer can be used as a forward primer for the first round PCR amplification product, and the third reverse primer can be used as a reverse primer for the first round PCR amplification product, and the first round PCR amplification product is subjected to the second round PCR amplification to obtain the target library.

In some examples, as can be seen from the composition of the first forward primer, the first reverse primer, the second forward primer and the third reverse primer, in the actual primer design, the first forward primer and the first reverse primer are designed mainly for each target region separately, while the second reverse primer, the second forward primer and the third reverse primer may be universal for each target region, i.e. the second reverse primer, the second forward primer and the third reverse primer do not need to be designed separately for different target regions.

In some examples, as with the target region 100, a first forward primer and a first reverse primer that specifically bind to the target region 200 upstream and downstream may also be designed and obtained for the target region 200; the second reverse primer, the second forward primer and the third reverse primer are universal primers. Similarly, for the target region 300, a first forward primer and a first reverse primer that specifically bind to the target region 300 upstream and downstream may also be designed; the second reverse primer, the second forward primer and the third reverse primer are universal primers.

In the case of multiple pathogens, likewise, a first forward primer and a first reverse primer that specifically bind to the upstream and downstream of each target region are designed and obtained for each target region of the gene of each pathogen. And the second reverse primer, the second forward primer and the third reverse primer for each target region of each pathogen are universal primers.

As shown in fig. 3, in some examples, in a primer set contemplated by examples of the present disclosure, the first sequencing primer and the second sequencing primer may be sequencing primers of an illumina sequencing platform, the first sequencing linker may be sequencing linker P7 of the illumina sequencing platform, and the second sequencing linker may be sequencing linker P5 of the illumina sequencing platform. Thus, a library of interest obtained by amplifying a target region with a primer set can be sequenced by the illumina sequencing platform to obtain sequencing data.

In some examples, the forward primer of the first forward primer can complementarily pair with the 5 'end of the target region 100 and the reverse primer of the first reverse primer can complementarily pair with the 3' end of the target region 100. Thus, the first forward primer, the first reverse primer and the second reverse primer can be used for performing a first round of PCR amplification on the target region 100 to obtain a first round of PCR amplification product 101. The first round PCR amplification product 101 may be, from its 5 'to 3' end, a first sequencing primer, the target region 100, a second sequencing primer, a first barcode, and a sequencing adaptor P7 in that order.

In some examples, the second forward primer and the third reverse primer can be used to perform a second round of PCR amplification on the first round PCR amplification product 101 to obtain the target library 102. The library of interest 102 may be, from its 5 'to 3' end, a sequencing adaptor P5, a first sequencing primer, the target region 100, a second sequencing primer, a first barcode, and a sequencing adaptor P7, in that order. Thus, the resulting library of interest 102 can be used directly for sequencing by the illumina sequencing platform.

In other examples, the first sequencing primer, the second sequencing primer, the first sequencing adapter and the second sequencing adapter may also be universal sequencing primers and universal sequencing adapters of other sequencing platforms, and may be selected according to actual situations.

Hereinafter, a kit for detecting pathogens associated with respiratory tract infections (hereinafter, simply referred to as "kit") according to the present disclosure will be described in detail with reference to fig. 4. Fig. 4 shows a schematic view of a kit 1 according to an example of the present disclosure.

In this embodiment, the kit 1 may include the primer set described above. In some examples, kit 1 may include reagent vial 810 containing a first forward primer, reagent vial 820 containing a first reverse primer, reagent vial 830 containing a second reverse primer, reagent vial 840 containing a second forward primer, and reagent vial 850 containing a third reverse primer. The first forward primer, the first reverse primer, the second forward primer, the third reverse primer, the pathogen gene, and other substances are described in detail in the description of the primer set, and are not repeated herein.

In some examples, kit 1 may further include a collection of artificial plasmids. The artificial plasmid collection may include a plurality of artificial plasmids capable of binding to the first forward primer and the first reverse primer. Among them, various artificial plasmids can be designed based on the sequences of target regions of different pathogens but have differences from the sequences of the target regions.

When multiple pathogen genes are detected, the multiple PCR primers Chi Zhongbao contain primers for specific amplification of hundreds of pathogenic microorganisms, but in actual samples to be detected, only a small number of types of microorganisms exist or even no microorganisms exist, under the condition, only a certain pair of primers of hundreds of primers actually can be amplified, and the rest of a large number of primers can form a large amount of dimers or non-specific amplification due to no consumption of a target DNA template, so that the quality of an amplified library is poor. In this case, the artificial plasmid set can consume the first forward primer and the first reverse primer in the system by binding to the first forward primer and the first reverse primer, thereby reducing the formation of primer dimers and improving the library quality.

In some examples, the kit 1 may further include at least one of a positive quality control, a negative quality control, a reverse transcription reagent, a nucleic acid extraction reagent, a banking reagent (including PCR buffer, DNA polymerase, dNTPs, etc.), a quantification reagent, a purification reagent, a sequencing reagent. Here, the positive quality control product, the negative quality control product, the reverse transcription reagent, the nucleic acid extraction reagent, the library construction reagent (including PCR buffer solution, DNA polymerase, dNTPs and the like), the quantification reagent, the purification reagent and the sequencing reagent can be made by oneself or sold on the market.

In some examples, a positive quality control can be a sample of a target region that includes a gene of a pathogen and a negative quality control can be a sample of a target region that does not include a gene of a pathogen. Thus, true positive and false positive samples can be provided for each target region of a pathogen gene, for establishing a ROC curve to obtain a detection threshold for each target region, or for performing a positive control experiment or a negative control experiment.

In some examples, the kit 1 may further include instructions that can describe how to use the kit of the present disclosure to detect pathogens associated with respiratory infections, and instructions that can also describe how to interpret the results of the detection.

According to the kit related by the disclosure, the detected pathogens are more comprehensive, and for part of pathogens including detection sites from subordinate to species or from species to subspecies, the effect of multiple verification is achieved, and the detection rate and the authenticity of detection are improved.

In addition, the disclosure also relates to an application of the primer group in preparing a reagent related to respiratory tract infection detection (hereinafter, referred to as "application").

In the application related to the present disclosure, the primer set refers to the above primer set.

According to the application provided by the disclosure, the detected pathogens are more comprehensive, and for part of pathogens including detection sites from subordinate to species or from species to subspecies, the effect of multiple verification is achieved, and the detection rate and the authenticity of detection are improved.

In addition, the disclosure relates to a method for detecting pathogens associated with respiratory infections (some are referred to as "methods" or "detection methods")

Fig. 5 shows a flow diagram of a method of detecting pathogens associated with respiratory tract infections in accordance with examples of the present disclosure. Fig. 6 shows a flow diagram of PCR amplification according to an example of the present disclosure. Figure 7 shows a flow diagram of PCR amplification and purification according to an example of the present disclosure. Fig. 8 shows a schematic diagram of a scenario of PCR amplification according to an example of the present disclosure. Fig. 7 shows a flow diagram of PCR amplification and purification according to an example of the present disclosure. Fig. 8 shows a schematic diagram of a scenario of PCR amplification according to an example of the present disclosure.

In this embodiment, as shown in fig. 5, the method for detecting pathogens associated with respiratory infections may comprise the steps of: preparing a sample of nucleic acid to be tested (step S10); carrying out PCR amplification on a nucleic acid sample to be detected by using a primer group or the kit 1 to obtain a target library (step S20); sequencing the target library to obtain sequencing data (step S30); the detection result is obtained based on the sequencing data (step S40).

In some examples, a method of detecting a pathogen associated with a respiratory infection may include preparing a nucleic acid sample to be tested (step S10).

In some examples, in step S10, a test nucleic acid sample can be obtained from a subject. For example, a sample of a nucleic acid to be tested can be obtained by collecting a sample of tissue, body fluid, etc. that may contain a pathogen to be tested from a subject. In some examples, the sample of a pathogen associated with a respiratory infection may be obtained from at least one of a pharyngeal swab, a nasal swab, sputum, alveolar lavage fluid, a bronchofiberscope antipollution brush, peripheral blood, and serosal cavity effusion of the subject.

In some examples, a nucleic acid extraction kit can be used to extract and obtain a test nucleic acid sample. Wherein, different nucleic acid extraction kits can be adopted for extraction according to different sample types. For example, a DNA extraction kit or an RNA extraction kit may be used, or a DNA/RNA co-extraction kit may be used for extraction. In some examples, for a sample containing cells that are difficult to break, ultrasonic breaking may be performed in advance, and then nucleic acid extraction may be performed.

In some examples, after the nucleic acid extraction is completed, the concentration can be measured by using a fluorescence quantitative kit and a fluorescence quantitative instrument, and the concentration of the nucleic acid in each nucleic acid sample is made uniform as much as possible. In some examples, the extracted test nucleic acid sample can be stored at-20 ℃ to-80 ℃.

In some examples, the nucleic acid sample to be tested may include at least one of a DNA sample and an RNA sample, and if the nucleic acid sample to be tested includes an RNA sample, after obtaining the nucleic acid sample to be tested, a step of reverse transcription of the nucleic acid sample to be tested is further included. Thus, a nucleic acid sample to be tested containing an RNA sample can be detected. For example, if the nucleic acid sample to be tested is an RNA sample such as a novel coronavirus gene, the nucleic acid sample to be tested needs to be subjected to reverse transcription and then to be subjected to reverse transcription into a DNA sample.

In some examples, as described above, the method for detecting pathogens associated with respiratory infection may include performing PCR amplification on a test nucleic acid sample using a primer set or a kit to obtain a target library (step S20). The primer set referred to herein refers to the primer set described above for detecting pathogens associated with respiratory infections. The kit referred to herein is a kit for detecting pathogens associated with respiratory infections as described above.

As described above, each primer set may include a plurality of primers. In some examples, each primer set can include a first forward primer, a first reverse primer, a second forward primer, and a third reverse primer, respectively. The first forward primer may be, from its 5' end to its 3' end, in turn, a first sequencing primer and a sequence matching the 5' end of the target region. The first reverse primer may be, in order from its 5' end to its 3' end, a second sequencing primer and a sequence matching the 3' end of the target region. The second reverse primer may be, in order from its 5 'end to its 3' end, a first sequencing adaptor, a first barcode, and a second sequencing primer. The first barcode in the second reverse primer can be configured to identify a different nucleic acid sample to be tested. The second forward primer may be, in order from its 5 'end to its 3' end, a second sequencing adaptor, a second barcode, and a first sequencing primer. The third reverse primer may be a first sequencing adapter. In this case, the primer set can perform PCR amplification on the nucleic acid sample to be detected and obtain the target library.

In some examples, referring to fig. 6, step S20 may include the steps of: performing a first round of PCR amplification on a nucleic acid sample to be detected by using a first forward primer, a first reverse primer and a second reverse primer to obtain a first round of PCR amplification product (step S201); and performing second PCR amplification on the first PCR amplification product by using the second forward primer and the third reverse primer to obtain a second PCR amplification product (step S202).

In some examples, a step of magnetic bead purification of the amplification product is further included after each round of PCR amplification. In other words, the method further comprises a step of magnetic bead purification of the first PCR amplification product after the first PCR amplification, and a step of magnetic bead purification of the second PCR amplification product after the second PCR amplification. Referring to fig. 7, step S20 may include the steps of: performing a first round of PCR amplification on a nucleic acid sample to be detected by using a first forward primer, a first reverse primer and a second reverse primer to obtain a first round of PCR amplification product (step S21); performing magnetic bead purification on the first round PCR amplification product (step S23); performing a second PCR amplification on the first PCR amplification product by using a second forward primer and a third reverse primer to obtain a second PCR amplification product (step S25); and (5) performing magnetic bead purification on the second PCR amplification product to obtain a target library (step S27).

In some examples, a first round PCR amplification product obtained by performing a first round PCR amplification on a test nucleic acid sample using a first forward primer, a first reverse primer and a second reverse primer is stored in a test tube and sealed, and then the first round PCR amplification product in the test tube is subjected to a magnetic bead purification step (step S23). In this case, the nucleic acid can be purified by magnetic bead purification, and a nucleic acid fragment of a desired length can be retained, whereby a purified PCR amplification product can be obtained. In addition, in the purification process of step S23, the test tube storing the first round PCR amplification product needs to be uncapped, but due to the high content of the amplified nucleic acid, the uncapping is easy to generate aerosol, and the aerosol diffuses into the laboratory environment, so that aerosol pollution is generated. In this case, since different nucleic acid samples to be detected are labeled with the first barcode carried by the second reverse primer after the first round of PCR amplification is performed on the nucleic acid samples to be detected, even if aerosol is generated, the influence of aerosol contamination and the like on the detection results between different samples can be reduced.

In some examples, the mixed amplification product is PCR amplified using the second forward primer and the third reverse primer, and then a magnetic bead purification step (step S27) is performed to obtain the target library. In this case, the nucleic acid can be purified by magnetic bead purification, the nucleic acid can be separated from other components such as proteins, and a nucleic acid fragment having a desired length can be retained, whereby a purified target library can be obtained. Similarly, in the purification process, a cap opening operation is usually performed, and due to the high content of amplified nucleic acid, aerosol is easily generated and diffuses into the laboratory environment, so that aerosol pollution is generated. In this case, since different nucleic acid samples to be detected are labeled with the first barcode carried by the second reverse primer and different batches of samples are labeled with the second barcode carried by the second forward primer, the influence of aerosol contamination and the like on the detection results of samples between different batches can also be reduced.

In some examples, during steps S23, S25 and S27, operations of adding an experimental reagent to the sample, collecting or transferring the purified sample, and the like may be included. During the above operation, there may be problems of sample mixing, sample splashing, reagent contamination, etc. caused by human error, which may cause contamination between samples. In this case, even if the above-mentioned contamination problem exists, since each nucleic acid sample to be tested is "attached" with a different first barcode after being amplified using the second reverse primer (after step S21), the influence of the above-mentioned contamination problem on the detection result can be reduced.

In some examples, in step S23, the first round PCR amplification products of each nucleic acid sample to be detected may be mixed to obtain a mixed amplification product, and then the mixed amplification product is uniformly subjected to magnetic bead purification. In other examples, in step S23, the first round PCR amplification products of each nucleic acid sample to be detected may be subjected to magnetic bead purification, and then the purified first round PCR amplification products are mixed and then subjected to the second round PCR amplification in a unified manner. Therefore, reagent and labor cost can be saved.

Referring to fig. 8, a first round of PCR amplification may be performed on a nucleic acid sample to be detected (e.g., a nucleic acid sample to be detected 21, a nucleic acid sample to be detected 22, and a nucleic acid sample to be detected 23) stored in different test tubes by using a first forward primer, a first reverse primer, and a second reverse primer (where a second reverse primer with a different first barcode is used for different nucleic acid samples to be detected), so as to obtain first round PCR amplification products (including a first round PCR amplification product 31, a first round PCR amplification product 32, and a first round PCR amplification product 33, respectively). Then, the first round amplification products (including the first round PCR amplification product 31, the first round PCR amplification product 32, and the first round PCR amplification product 33) of each nucleic acid sample to be detected are mixed to obtain a mixed amplification product 34, and then the second round PCR amplification is performed on the mixed amplification product 34 by using the second forward primer and the third reverse primer to obtain a target product 35. In this case, by performing magnetic bead purification and/or second PCR amplification on the mixed amplification products in a unified manner, it is possible to reduce the cost of reagents and labor compared to performing magnetic bead purification and second PCR amplification on the first PCR amplification product of each nucleic acid sample to be tested.

In some examples, in step S20, step S201, or step S21, the test nucleic acid sample may be subjected to multiplex PCR amplification using the first forward primer, the first reverse primer, and the second reverse primer to obtain PCR amplification products.

In some examples, in step S201 or step S21, a plurality of annealing temperatures may be selected when performing a first round of PCR amplification on a test nucleic acid sample using a first forward primer, a first reverse primer, and a second reverse primer. For example, 3 temperatures can be selected from 57 ℃ to 62 ℃ as the annealing temperature, for example, 57 ℃, 60 ℃ and 62 ℃ as the annealing temperature. This facilitates binding of each primer to the template, thereby increasing the coverage of the target library.

In some examples, an artificial plasmid set may also be added in step S20, step S201, or step S21, i.e., during the first round of PCR amplification. In this case, in the first round of PCR amplification, the artificial plasmid set can consume the first forward primer and the first reverse primer in the system by binding to the first forward primer and the first reverse primer, and thus, the formation of primer dimers can be reduced; the second reverse primer in the first round PCR amplification system can be combined with the artificial plasmid assembly through the first reverse primer, so that the artificial plasmid assembly can also consume the second reverse primer, and the formation of primer dimer is reduced. That is, the formation of primer dimers during the first round of PCR amplification can be reduced by adding artificial plasmid pools. It will be appreciated that due to the sequence differences between the artificial plasmid and the target region, the target region products can be distinguished from the artificial plasmid products when the sequence alignment is performed after sequencing.

In some examples, the number of cycles used in the first round of PCR amplification or the second round of PCR amplification may be selected according to the requirements of detection sensitivity and the like. Preferably, in the present embodiment, the number of cycles per PCR amplification may be 10 to 40 cycles. Thus, it can help meet the sensitivity requirements for pathogen detection.

In some examples, as described above, the detection method may include performing sequencing analysis on the library of interest, obtaining sequencing data (step S30). Here, the target library is obtained by PCR amplification of a nucleic acid sample to be tested using a primer set or a kit in step S20.

In some examples, in step S30, the library of interest may be sequenced by the illumina sequencing platform to obtain sequencing data. Of course, in other examples, the primer set may be composed of sequencing primers and sequencing adaptors of other sequencing platforms, and thus other sequencing platforms may also be used to sequence the library of interest.

In some examples, the sequencing data may include the sequence of the target library (i.e., the sequence of each DNA fragment). In other words, the sequencing data may include the sequence of each of the reads. In some examples, the sequencing data can specifically include a sequence of the target region, a sequence of the first barcode, and/or a sequence of the second barcode.

In some examples, as described above, the detection method may include obtaining a detection result based on the sequencing data (step S40).

In some examples, whether each of the reads is a sequence of a target region may be determined based on the sequence of the read, that is, whether each target region is detected may be determined. In some examples, which sample the detected reads belong to may be identified based on the sequence of the first barcode, and which sample of which lot the detected reads belong to may also be identified based on the sequence of the second barcode, thereby obtaining the lot and sample information to which the reads belong. This makes it possible to determine the detection of each target region in each sample nucleic acid to be tested based on the sequencing data.

In some examples, further, a detection threshold for each target region may be obtained based on the positive quality control material and the negative quality control material of each target region, and whether each target region is detected may be determined based on the detection threshold. In some examples, further, a receiver operating characteristic curve (ROC curve) may be obtained by using the positive quality control material and the negative quality control material, and a detection threshold value of each target region is established by the ROC curve, and then the detection condition of each target region in the nucleic acid sample to be detected is determined according to the detection threshold value.

In some examples, step S40 may include determining whether the nucleic acid sample to be tested contains a pathogen based on the detection of the target region.

In some examples, for each test nucleic acid sample, if the ratio of the number of detected target regions to the number of target regions selected in designing the primer set is greater than a predetermined ratio, it is determined that the test nucleic acid sample contains a pathogen.

In some examples, for each test nucleic acid sample, if the number of detected target regions compared to the number of target regions selected when designing the primer set is not greater than a preset ratio, the test nucleic acid sample is determined to be free of a pathogen. It will be appreciated that if a test sample contains a pathogen, then ideally every target region of the pathogen in the sample should be detected, so that if only a small fraction (not more than a predetermined percentage) of the target regions in the sample are detected, the sample may be contaminated with other samples. In addition, if a sample to be tested does not contain a pathogen, then theoretically every target region of the pathogen in the sample should not be detected, so if a target region in the sample is detected (but not more than a predetermined percentage), the sample may be contaminated by other samples. Thus, the influence of contamination on the detection result can be further eliminated based on the relationship between the ratio of the number of detected target regions to the number of target regions selected at the time of designing the primer set and the preset ratio.

In this embodiment, 3 target regions are selected for each pathogen gene, and the 3 target regions are detected. For example, if 2 target regions are detected in a pathogen gene according to sequencing data, the ratio of the number of detected target regions to the total number of target regions is 2/3. Then, comparing the ratio (2/3) with a preset ratio, if the ratio is greater than the preset ratio, determining that the nucleic acid sample to be detected contains the pathogen, and under a normal condition, the detection result of the nucleic acid sample to be detected can be called as the pathogen positive; if the ratio is not greater than the predetermined ratio, the sample is determined to not contain the pathogen, and the detection result of the sample can be said to be negative.

In some examples, further, the preset proportion may be 50% to 80%. For example, if the predetermined ratio is 50%, if 3 target regions are selected for a gene of a certain pathogen when designing primers, it is necessary to see whether at least 2 (2 or 3) target regions are detected when determining whether a nucleic acid sample to be tested contains the pathogen. That is, if more than half of the target region is detected, the sample is determined to contain the pathogen (i.e., the detection result is positive).

The primer set, the kit, the application and the method for detecting pathogens associated with respiratory tract infection provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

[ examples ]

1. Primer design

In the examples, primers were designed for all pathogens in tables 1 to 15 above. The nucleic acid sequences of the pathogens were downloaded by NCBI (www.ncbi.nlm.nih.gov) and the conserved sequence regions of the pathogens were searched using the software (Clone Manager). 3 regions from the conserved sequence region of each pathogen were selected as target regions, and specific forward and reverse complementary sequences were designed for each target region. The specific sequence information is shown in SEQ ID NO. 1-SEQ ID NO.348 of tables 1 to 15 above.

In an embodiment, the first forward primer of each region of each pathogen is, from its 5 'end to 3' end, the first sequencing primer and the corresponding upstream primer in tables 1 to 15, respectively, and the first reverse primer of each region of each pathogen is, from its 3 'end to 5' end, the corresponding downstream primer and the second sequencing primer in tables 1 to 15, respectively.

In embodiments, the second reverse primer, the second forward primer, and the third reverse primer of each region of each pathogen are universal primers. The second reverse primer is respectively a second sequencing primer, a first barcode and a sequencing joint P7 from the 3 'end to the 5' end, the second forward primer is respectively a sequencing joint P5, a second barcode and a first sequencing primer from the 5 'end to the 3' end, and the third reverse primer is a sequencing joint P7.

In an embodiment, the first barcode is a random sequence of base number 8, the same first barcode being used for the same sample. The second barcode is a random sequence with a base number of 8.

In embodiments, sequencing linker P5 and sequencing linker P7 are universal linker sequences for the illumina sequencing platform; the first sequencing primer and the second sequencing primer are universal sequencing primers of an illumina sequencing platform, and the specific sequences are as follows:

sequencing linker P5: AATGATACGGCGACCACCGAGATCTACAC (SEQ ID: 349);

sequencing linker P7: CAAGCAGAAGACGGCATACGAGAT (SEQ ID: no. 350);

first sequencing primer: ACACTCTTTCCCTACACGACGCTCTTCCGAT (SEQ ID: no. 351);

a second sequencing primer: GTGACTGGAGTTCAGACGTGTGCTCTT (SEQ ID: NO. 352).

2. Sample extraction

7 oropharyngeal swab samples were collected and extracted separately. Specifically, a DNA/RNA co-extraction kit was used, and the procedures were performed with reference to the kit instructions. And respectively storing the 7 parts of nucleic acid samples to be detected which are respectively extracted in test tubes at the temperature of minus 20 ℃. The DNA/RNA co-extraction kit is a commercially available kit.

7 nucleic acid samples to be tested were verified using a siemer respiratory microfluidics chip, and the specific sample information is shown in table 16 below:

table 16 sample information:

3. library construction

3.1 Reverse transcription

The extracted nucleic acids are reverse transcribed separately with random primers. Specifically, 2. Mu.l of random primers (25 ng/. Mu.l) and 6. Mu.l of the extracted nucleic acids were added to the octant tube, and after 5min (min) of denaturation by incubation at 65 ℃, the mixture was left on ice for 2min. Next, 10. Mu.l of 2 XTT Mix and 2. Mu.l of HiScript II Enzyme Mix were added. Then, carrying out reverse transcription program under the reaction condition of 25 ℃ for 5min;50 ℃ for 45min;85 ℃ for 2min. Finally, the resulting reverse transcription product (cDNA/DNA template) was stored at 4 ℃. In the examples, the reagents and equipment used were all commercially available products unless otherwise specified.

3.2 First round PCR amplification

The first round of PCR reaction system mainly adds the first sequencing primer, the second sequencing primer, the first barcode and the sequencing joint P7 to each sample, and also adds an artificial plasmid set to reduce primer dimers.

PCR capture of all target regions of the samples was performed in 2 aliquots, using the reverse transcription product cDNA/DNA as template, to formulate 2 separate PCR reaction systems per sample.

Specifically, the PCR amplification buffer (Amplicon PCR buffer) was thawed at room temperature, and after thawing, shaking and centrifugation were performed. The amplification enzyme mixture (Amplicon enzyme Mix) was centrifuged. For each pathogen gene in tables 1 to 15, the first forward primers of the multiple target regions of each pathogen gene are divided into two first forward primer mixing pools, namely a first forward primer mixing pool 1 and a first forward primer mixing pool 2, according to the target region. For each pathogen gene in tables 1 to 15, the first reverse primers of the multiple target regions of each pathogen gene are divided into two first reverse primer mixing pools, a first reverse primer mixing pool 1 and a first reverse primer mixing pool 2, according to the target region. In this example, the first forward primer mix pool 1 and the first reverse primer mix pool 1 are used for PCR capture and amplification of a part of 3 target regions of each pathogen gene; the first forward primer mix pool 2 and the first reverse primer mix pool 2 are used for PCR capture and amplification of the remaining target region of the 3 target regions of each pathogen gene. In addition, a second reverse primer and a cDNA/DNA template were prepared. The prepared reagent is placed on an ice box for standby.

Next, the first round PCR reaction system 1 and the first round PCR reaction system 2 for each sample were prepared according to the PCR reaction systems shown in table 17 and table 18 below, respectively. Different second reverse primers are added to different samples, and the same second reverse primer is added to two PCR reaction systems of the same sample. Preparing premixed reaction liquid according to the number of samples, subpackaging the premixed reaction liquid into 0.2ml of PCR tubes, and then adding a second reverse primer and a cDNA/DNA template.

Table 17 first round PCR reaction system 1:

table 18 first round PCR reaction system 2:

then, the PCR tube was placed in a PCR instrument and operated according to the first round of PCR reaction procedure shown in table 19 below to obtain the first round PCR amplification product.

Table 19 first round PCR reaction procedure:

3.3 first round PCR amplification product purification

In the examples, after the first round of PCR amplification was completed, the reaction solutions of the above first round PCR reaction system 1 and first round PCR reaction system 2 were mixed to obtain a mixed solution of 50 μ l volume. The mixed solution was then purified using 35. Mu.l volume of 0.7 XXP beads, rinsed with 80% ethanol and after the beads were air dried, eluted with 53. Mu.l of eluent (1 XTE buffer). The above steps were repeated once, and 20. Mu.l of eluent (1 XTE buffer) was used for elution to obtain the first round of PCR amplification product after purification.

3.4 second round PCR amplification

The second round of PCR amplification mainly adds the sequencing linker P5 and the second barcode, and enriches the first round PCR amplification products.

Specifically, the second round of PCR reaction solution was prepared according to the reaction system shown in Table 20 below.

Table 20 second round PCR reaction system:

then, the PCR tube was placed in a PCR instrument and run according to the reaction procedure shown in table 21 below to obtain a second round of PCR amplification products.

Table 21 second round PCR reaction procedure:

3.5 second round PCR amplification product purification

Mu.l of the second PCR amplification product was taken, 30. Mu.l of 1 XTE buffer was added, and then purified once using 35. Mu.l of 0.7 XP magnetic beads, followed by rinsing with 80% ethanol. Then, 20. Mu.l of eluent (1 XTE buffer) was used for elution to obtain the purified second round PCR amplification product, i.e., the target library.

4. Library quantification

The purified second round PCR amplification product (target library) was accurately quantified with reference to the Qubit fluorometer 4.0 specification. The requirement of quality control on the library construction is required to be not lower than 2 ng/mul, otherwise, the library construction fails. Library concentrations are shown in table 22 below:

table 22 library concentrations:

5. on-machine sequencing, sequencing data analysis

The target library was subjected to on-machine sequencing using PE150 of the illumina sequencing platform, steps performed strictly according to the supplier requirements. The data from the sequencing was filtered for low quality sequences and linker sequences. Then, the comparison software BWA is used for comparing the detected numbers of the pathogens to a reference pathogen database, and the detected numbers of the pathogens are judged by analyzing the sequencing depths (reads) and the detection threshold values of different amplicons, wherein more than 50 percent of target areas in the same pathogen are detected and are regarded as the pathogen is detected, otherwise, the target areas are regarded as undetected.

6. The result of the detection

The results are shown in table 23 below:

table 23 test results:

among the above results, the detection results of the sample 2, the sample 3, the sample 4 and the sample 5 are double positive, which indicates that the detection of the nucleic acid sample to be detected by using the primer set or the kit according to the present embodiment has a multiple verification effect and can improve the authenticity of the detection.

While the present disclosure has been described in detail in connection with the drawings and the embodiments, it should be understood that the above description is not intended to limit the present disclosure in any way. Those skilled in the art can make modifications and variations to the present disclosure as needed without departing from the true spirit and scope of the disclosure, which fall within the scope of the disclosure.

Claims

1. A kit for detecting pathogens associated with respiratory tract infections, the kit comprising a primer set for detecting target regions of genes of a first pathogen and genes of a second pathogen, wherein the primer set comprises a first forward primer and a first reverse primer, and the first forward primer comprises a first sequencing primer and an upstream primer matched with the 5' end of the target region; the first reverse primer comprises a second sequencing primer and a downstream primer matched with the 3' end of the target region;

wherein the first pathogen and the second pathogen are pathogens associated with respiratory tract infections, and the first pathogen comprises the second pathogen; the first pathogen comprises genus coronavirus α, genus coronavirus β, genus coronavirus δ, genus coronavirus γ, influenza a virus, influenza b virus, adenovirus, nontuberculous mycobacterium and haemophilus influenzae;

the upstream primer comprises a universal upstream primer designed based on a target region of a gene of the first pathogen or a species upstream primer designed based on a target region of a gene of the second pathogen;

the downstream primer includes a universal downstream primer designed based on a target region of a gene of the first pathogen or a species downstream primer designed based on a target region of a gene of the second pathogen.

2. The kit of claim 1, wherein the second pathogen comprises coronavirus 229E, coronavirus NL63, coronavirus OC43, coronavirus HKU1, middle east respiratory syndrome coronavirus, SARS virus, novel coronavirus, influenza a H1N1 virus, influenza a H3N2 virus, influenza a H5N1 virus, influenza a H7N9 virus, influenza B virus Victoria line, influenza B virus Yamagata line, adenovirus group B, adenovirus group C, adenovirus group E, mycobacterium abscessus, mycobacterium intracellulare, mycobacterium avium, mycobacterium shallowlii, mycobacterium terrae, mycobacterium cheloni, mycobacterium kansasii, and haemophilus influenzae type B.

3. The kit of claim 1, comprising a primer set for detecting a target region of a gene of a third pathogen comprising parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, respiratory syncytial virus type a, respiratory syncytial virus type B, rhinovirus, human metapneumovirus, bocavirus, rubella virus, measles virus, nipah virus, mumps virus, varicella zoster virus, mycobacterium tuberculosis complex, staphylococcus aureus, klebsiella pneumoniae, streptococcus pneumoniae, legionella pneumophila, haemophilus parainfluenza, nocardia, neisseria meningitidis, yersinia pneumocystis, mycoplasma pneumoniae, and chlamydia pneumoniae;

the upstream primer comprises a universal upstream primer designed based on a target region of a gene of the third pathogen or a species upstream primer designed based on a target region of a gene of the third pathogen;

the downstream primer includes a universal downstream primer designed based on a target region of a gene of the third pathogen or a species downstream primer designed based on a target region of a gene of the third pathogen.

4. The kit of claim 1, wherein the primer set comprises a second reverse primer comprising the second sequencing primer, a first barcode configured to identify a different sample, a second forward primer, and a third reverse primer; the second forward primer comprises a second sequencing adaptor and the first sequencing primer; the third reverse primer comprises the first sequencing adapter.

5. The kit of claim 4, wherein the first forward primer is the first sequencing primer and the sequence matching the 5 'end of the target region in sequence from the 5' end to the 3 'end, the first reverse primer is the second sequencing primer and the sequence matching the 3' end of the target region in sequence from the 5 'end to the 3' end, the second reverse primer is the first sequencing adaptor, the first barcode and the second sequencing primer in sequence from the 5 'end to the 3' end, the second forward primer is the second sequencing adaptor and the first sequencing primer in sequence from the 5 'end to the 3' end, and the third reverse primer is the first sequencing adaptor.

6. The kit of claim 4 or 5, wherein the first sequencing primer and the second sequencing primer are sequencing primers of an illumina sequencing platform, the first sequencing linker is a P7 linker of the illumina sequencing platform, the second sequencing linker is a P5 linker of the illumina sequencing platform, and the first barcode is a random base sequence of 6 to 12bp.

7. The kit of claim 1, wherein each of the pathogens has 3 target regions, and the forward and reverse primers comprise forward and reverse primers corresponding to at least 1 of the 3 target regions of each pathogen.

8. The kit of claim 7, wherein the forward primer and the reverse primer are corresponding to 3 target regions of each pathogen.

9. A method for detecting pathogens associated with respiratory infections for non-diagnostic purposes, comprising:

preparing a nucleic acid sample to be detected;

performing PCR amplification on the nucleic acid sample to be detected by using the kit according to any one of claims 1 to 8 to obtain a target library;

sequencing analysis is carried out on the target library to obtain sequencing data; and

obtaining a detection result based on the sequencing data.

10. The method according to claim 9, wherein the first forward primer, the first reverse primer and the second reverse primer are used for performing a first round of PCR amplification on the nucleic acid sample to be detected to obtain a first round of PCR amplification product; and carrying out second round PCR amplification on the first round PCR amplification product by using a second forward primer and a third reverse primer to obtain the target library.