CN110714097A

CN110714097A - Method for simultaneously detecting A, B, C three groups of rotaviruses

Info

Publication number: CN110714097A
Application number: CN201911112786.5A
Authority: CN
Inventors: 靖相密; 张通; 刘奕; 邓雪萍
Original assignee: Shandong Kaye Biological Technology Co Ltd
Current assignee: Shandong Kaye Biological Technology Co Ltd
Priority date: 2019-11-14
Filing date: 2019-11-14
Publication date: 2020-01-21

Abstract

The invention discloses a method for simultaneously detecting A, B, C three groups of rotaviruses, belonging to the field of biological detection. The method of the invention designs a pair of specific primers and a specific probe with different fluorescent labels aiming at target genes of A group of rotavirus, B group of rotavirus and C group of rotavirus respectively, and designs a pair of universal primers at the same time, and the specific primers with universal primer labels are adopted for enrichment amplification during PCR amplification; then, the universal primer label is adopted for exponential amplification, and the fluorescence signal of the exponential amplification product is collected for detection. The method can simultaneously detect the group A rotavirus, the group B rotavirus and the group C rotavirus, has strong specificity, high sensitivity and short detection time, and can be used for the early rapid diagnosis of rotavirus infection and the epidemiological research of rotavirus.

Description

Method for simultaneously detecting A, B, C three groups of rotaviruses

Technical Field

The invention relates to the field of biological detection, in particular to a method for simultaneously detecting A, B, C three groups of rotaviruses.

Background

Rotavirus (RV) is a major pathogen causing infantile diarrhea and animal viral diarrhea in autumn and winter, belongs to the genus rotavirus of reoviridae, and is a double-stranded RNA virus. Rotavirus is an important cause of severe diarrhea of infants, children and adults, is also an important cause of diarrhea of various young, adult mammals and birds, is widely popularized in the world and causes great loss every year. It is currently divided into 8 groups, numbered A, B, C, D, E, F, G and H in English. A, B, C three groups of rotavirus are pathogenic to human, group A rotavirus is the most common and most harmful and is one of the main pathogens causing infantile diarrhea, group B rotavirus (also called adult diarrhea rotavirus) mainly infects young and middle-aged people and can cause epidemic outbreak of gastroenteritis of young and middle-aged people, group C rotavirus is found individually to infect human and teenagers are the main infection objects.

Rotavirus is diagnosed definitely mainly by a laboratory, and the traditional diagnosis method mainly comprises morphology and immunology, and the two methods have the disadvantages of complicated operation and long identification period. In recent years, with the rapid development of molecular biology technology, PCR technology has also been widely applied to the detection of rotavirus. The PCR technology has high sensitivity and good specificity, and is suitable for rapid detection and identification, but the common PCR technology can only detect one target gene at a time, and once the detection direction is wrong, time and reagent waste is caused. The multiplex PCR technology can detect a plurality of target genes in one reaction system, detect a plurality of pathogens at one time, improve the detection efficiency and make up for the defects of the common PCR. However, it is difficult to amplify multiple target sequences simultaneously using multiple pairs of primers in the conventional multiplex PCR technique, because each different target sequence has its own optimal amplification conditions, it is difficult to unify them, and even if unified, each target sequence has different amplification efficiency, so that low concentration/titer pathogens are not amplified and not detected, and results such as low detection sensitivity and false negative occur.

Therefore, a real high-efficiency rotavirus multiplex PCR detection method is urgently needed to be established for the rapid and sensitive identification and detection of rotavirus.

Disclosure of Invention

In order to make up for the deficiencies of the prior art, the invention provides a method for simultaneously detecting A, B, C three groups of rotaviruses.

The technical scheme of the invention is as follows:

a method for detecting A, B, C three groups of rotaviruses at the same time, a pair of specific primers and a specific probe with different fluorescent labels are respectively designed aiming at target genes of A group of rotaviruses, B group of rotaviruses and C group of rotaviruses, a pair of universal primers are simultaneously designed, and the specific primers with universal primer labels are firstly adopted for enrichment amplification during PCR amplification; then, performing exponential amplification by adopting a universal primer label, and collecting a fluorescent signal of an exponential amplification product for detection;

wherein, the specific primers and the specific probe sequence with fluorescent labels of the group A rotavirus are as follows:

an upstream primer: 5'-ACTTTATTGGCGAATGTTAC-3'

A downstream primer: 5'-GCTTCTGATAGAGGCTACTG-3'

And (3) probe: 5 '-VIC-CTATACCAGTTGGACCAGTATTTCCACC-BHQ 1-3';

the specific primers and the specific probe sequence with the fluorescent label of the B group rotavirus are as follows:

an upstream primer: 5'-ATAGGTATGACTGGTGGAAAT-3'

A downstream primer: 5'-ATGTGGGAATGTAGACGATGT-3'

And (3) probe: 5 '-FAM-CGATGTTAATTATTCCACAAGTGC-BHQ 3-3';

the specific primers and the specific probe sequence with the fluorescent label of the group C rotavirus are as follows:

an upstream primer: 5'-AATTTGGAAGCGTCATGGGTTTA-3'

A downstream primer: 5'-AAGCGTTGCGTATTGTTTGGT-3'

And (3) probe: 5 '-CY 5-ACAACAAGTAATACTTGTCAAATTGCAGC-BHQ 2-3';

the universal primers of the group A rotavirus, the group B rotavirus and the group C rotavirus are all as follows:

an upstream primer: 5'-Gatccaatcgcgtacttgc-3'

A downstream primer: 5'-Tgccatgcataaccgcagt-3' are provided.

The enrichment amplification in the invention is to use the extracted rotavirus RNA as a template and apply the specific primer with the universal primer label to be specifically combined with the extracted RNA template to obtain a PCR amplification product with the universal primer label at the 5' end. The exponential amplification is to take PCR amplification products with universal primer labels at the 5 ' end generated by enrichment amplification as templates, and apply the universal primers to carry out amplification to generate PCR products with the universal primer labels at the 5 ' end and the 3 ' end. Enrichment amplification produces enough amplification product to serve as the basis for exponential amplification.

In the method, three groups of specific primers and universal primers are determined by repeated design, experiment, comparison and optimization. In addition to the specific primers and the universal primers of the present invention, the specific primers of group A rotavirus, group B rotavirus and group C rotavirus may have other sequences, and the universal primers of group A rotavirus, group B rotavirus and group C rotavirus may have other sequences. However, the combination of different specific primers and universal primers has a great influence on the detection result (such as accuracy, sensitivity, etc.). The method has high detection sensitivity and accuracy.

Preferably, the enrichment amplification is:

the following were added to the PCR tube: 5 uL of 5 × RT-PCR Buffer, 0.4mM of dNTP Mix final concentration, 0.1uM of specific primer with universal primer label, 0.2 uL of RNase inhibitor and Mg²⁺The final concentration is 2.5mM, the mixture of reverse transcriptase and Taq enzyme is 2unit, the sample is 3 mu L, DEPC water is added to make up the volume to 25 mu L;

amplification was performed under the following amplification conditions:

reverse transcription is carried out for 20min at 50 ℃; activating the hot start enzyme at 95 ℃ for 15 min; denaturation at 94 ℃ for 15sec, annealing at 55 ℃ for 50sec, and extension at 72 ℃ for 30sec, for 10 cycles; denaturation at 94 ℃ for 15sec, annealing at 65 ℃ for 50sec, and extension at 72 ℃ for 30sec, for 15 cycles; storing at 4 ℃.

Preferably, the exponential amplification comprises the following specific steps:

the following were added to the PCR tube: 5 XPCR Buffer 5.0 uL, dNTP Mix final concentration 0.3mM, general upstream primer final concentration 0.1uM, general downstream primer final concentration 0.3 uM, each probe final concentration 0.1uM, Mg²⁺Final concentration 2.5mM, Taq enzyme 1unit, PCR enrichment amplification product 5 uL, adding DEPC water to make up volume to 25 uL;

amplification was performed under the following amplification conditions:

activating the hot start enzyme at 95 ℃ for 15 min; denaturation at 94 ℃ for 15sec and annealing at 58 ℃ for 35sec, 40 cycles were performed.

Preferably, the group A rotavirus probe is labeled with a VIC fluorescent group at 5 'and a BHQ1 fluorescent quenching group at 3'.

Preferably, the group B rotavirus probe is labeled with a FAM fluorescent group at the 5 'end and a BHQ3 fluorescent quenching group at the 3' end.

Preferably, the group C rotavirus probe is labeled with a 5 'fluorescent group CY5 and 3' fluorescent quenching group BHQ 2.

Preferably, the universal primer has no homology with any known sequence deposited in GenBank. So that the universal primer is not complementary to all target gene sequences and does not produce specific binding.

As a preferred scheme, the fluorescence signal of the exponential amplification product is collected, and the detection is carried out by applying a multiplex real-time fluorescence quantitative PCR technology platform.

Further, in the method for simultaneously detecting A, B, C three groups of rotaviruses, the detection result interpretation standard is as follows: when the CT value is less than 35, the index is positive, the CT value is more than 38 or underended, the index is negative, and the CT value is more than 35 and less than 38, which is a detection gray level interval, and needs to be detected again.

The method for simultaneously detecting A, B, C three groups of rotavirus has the detection sensitivity of 1 multiplied by 10 on A group of rotavirus, B group of rotavirus and C group of rotavirus³copies/ml。

The invention has the beneficial effects that:

the method overcomes the defects of low detection sensitivity, false negative and other results caused by the fact that pathogens with low concentration/titer are not amplified and are not detected when a plurality of target sequences are simultaneously amplified by a plurality of pairs of primers in the multiple PCR technology in the prior art through the optimal specific primer and the optimal universal primer; the method has the detection accuracy up to 100 percent, and the detection sensitivity of the method to A group rotavirus, B group rotavirus and C group rotavirus reaches 1 multiplied by 10³copies/ml。

The method can simultaneously detect the group A rotavirus, the group B rotavirus and the group C rotavirus, has strong specificity, high sensitivity and short detection time, and can be used for the early rapid diagnosis of rotavirus infection and the epidemiological research of rotavirus.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a graph showing the sensitivity amplification of group A rotavirus of the VIC channel in example 2;

FIG. 2 is a graph of the amplification sensitivity of the FAM channel group B rotavirus of example 2;

FIG. 3 is a graph of the CY5 channel group C rotavirus sensitivity amplification curve in example 2;

FIG. 4 is a graph of the amplification curve of sample 1 in example 3;

FIG. 5 is a graph of the amplification curve of sample 2 in example 3;

FIG. 6 is a graph of the amplification curve of sample 3 in example 3;

FIG. 7 is a graph of the amplification curve of sample 4 in example 3;

FIG. 8 is a graph of the amplification curve of sample 5 in example 3;

FIG. 9 is a graph of the amplification curve of sample 6 in example 3;

FIG. 10 is a graph of the amplification curve of sample 7 in example 3;

FIG. 11 is a graph of the amplification curve of sample 8 in example 3;

FIG. 12 is a graph of the amplification curve of sample 9 in example 3;

FIG. 13 is a graph of the amplification curve of sample 10 in example 3.

Detailed Description

The present invention is further illustrated by the following specific examples in conjunction with the attached drawings, but these examples are only illustrative and not limiting the scope of the invention.

Example 1 primer Probe design and Synthesis

A pair of specific primers, a specific probe with different fluorescent labels and a pair of universal primers are respectively designed aiming at the group A rotavirus, the group B rotavirus and the group C rotavirus.

TABLE 1 list of detected pathogens

Serial number	Viral name	English abbreviation	Target genes
				1	Group A rotavirus	RV-A	VP6
2	Group B rotavirus	RV-B	VP6
				3	Group C rotavirus	RV-C	VP6

A plurality of gene sequences covering RV-A, RV-B, RV-C viruses at home and abroad are downloaded from an NCBI gene library. Homology comparisons were performed using the BioEditor software to determine conserved regions of the above viral genomes. The Primer premier5.0 software is used for designing highly specific primers and probes in a conserved area, and the sequences of the primers and the probes are verified by Blast, so that the primers and the probes have better specificity. Marking a VIC fluorescent group at the 5 'of the RV-A probe, and marking a BHQ1 fluorescent quenching group at the 3' of the RV-A probe; FAM fluorescent group is marked on the 5 'of RV-B probe, and BHQ3 fluorescent quenching group is marked on the 3' of RV-B probe. The 5 'of the RV-C probe is marked with a CY5 fluorescent group, and the 3' of the RV-C probe is marked with a BHQ2 fluorescent quenching group. Meanwhile, a pair of universal primers are designed, and have no homology with any known sequence recorded in GenBank.

The designed primer probe sequences are shown in Table 2, and the synthesis of primers and probes was performed according to the sequences shown in Table 2.

Example 2 evaluation of sensitivity of multiplex PCR detection method for Rotavirus

1. Preparing a sample to be detected:

the construction method of the RV-A plasmid comprises the following steps: a specific primer (namely the RV-A specific primer in the invention) is used for amplifying nucleic acid of a group A rotavirus sample by a PCR method to obtain a group A rotavirus target gene fragment, the group A rotavirus target gene fragment is cloned to a pMD18-T vector through TA after purification, a recombinant vector with a correct sequencing result is transformed into DH5 alpha competent cells through sequencing identification, and the RV-A plasmid is obtained through amplification.

The construction method of the RV-B plasmid comprises the following steps: amplifying nucleic acid of a B group rotavirus sample by using a specific primer (namely the RV-B specific primer in the invention) by adopting a PCR method to obtain a B group rotavirus target gene fragment, cloning the B group rotavirus target gene fragment to a pMD18-T vector by TA after purification, transforming a recombinant vector with a correct sequencing result into DH5 alpha competent cells through sequencing identification, and amplifying to obtain the RV-B plasmid.

The construction method of the RV-C plasmid comprises the following steps: the method comprises the steps of amplifying nucleic acid of a C group rotavirus sample by using a specific primer (namely the RV-C specific primer in the invention) by adopting a PCR method to obtain a C group rotavirus target gene fragment, cloning the C group rotavirus target gene fragment to a pMD18-T vector by TA after purification, transforming a recombinant vector with a correct sequencing result into DH5 alpha competent cells through sequencing identification, and amplifying to obtain the RV-C plasmid.

Mixing the above three plasmids, and calibrating the concentration until the concentration of each plasmid in the mixture is 1 × 10⁶copies/ml, according to 1: 10. 1: 100. 1: 1000, and finally obtaining positive control samples S1, S2, S3 and S4 to be detected, wherein the concentrations are respectively 1 multiplied by 10⁶copies/ml、1×10⁵copies/ml、1×10⁴copies/ml、1×10³copies/ml。

2. PCR amplification reaction

The present embodiment is performed as follows:

the sensitivity detection is performed with 4 concentration gradients, corresponding to the above samples S1, S2, S3, and S4, and adding a negative control (DEPC-ddH)₂O)。

1) Enrichment amplification

The following were added to the PCR tube: 5 uL of 5 × RT-PCR Buffer, 0.4mM of dNTP Mix final concentration, 0.1uM of specific primer with universal primer label, 0.2 uL of RNase inhibitor and Mg²⁺The final concentration is 2.5mM, the mixture of reverse transcriptase and Taq enzyme is 2unit, 3 mu L of sample to be detected is added with DEPC water to complement the volume to 25 mu L;

amplifying according to the following amplification conditions to obtain PCR enrichment amplification products:

2) Exponential amplification

amplifying according to the following amplification conditions, and activating the enzyme at 95 ℃ for 15 min; denaturation at 94 ℃ for 15sec and annealing at 58 ℃ for 35sec, (fluorescence signal collected at the end of this period), were performed for 40 cycles.

The detection channels are set as follows: collecting a fluorescent signal by an RV-A-VIC channel; collecting a fluorescent signal by an RV-B-FAM channel; the RV-C-CY 5 channel collects fluorescence signals.

3. Analysis of results

The fluorescence amplification curves of this experiment are shown in FIGS. 1 to 3, respectively.

The CT values for each sample are shown in table 3 below:

TABLE 3 results of sensitivity detection

As can be seen from fig. 1 to 3 and table 3: the positive control samples S1-S4 to be detected with different concentrations are all positive in the fluorescence detection process, which shows that the detection sensitivity of the kit for detecting A group rotavirus, B group rotavirus and C group rotavirus can reach 1 multiplied by 10³copies/ml。

Example 3 evaluation of detection Effect of multiplex PCR detection method for Rotavirus

1. Sample processing

2 cases of excrement samples of patients confirmed to be positive by RV-A, RV-B, RV-C in clinical detection and 2 cases of healthy human serum samples are selected, and 8 cases of samples are counted for RNA extraction. RNA can be extracted using a commercial Kit, such as the QIAGEN viral RNA Mini Extraction Kit (CAT: 52904), and the Extraction procedure is well known to those skilled in the art and will not be described herein.

2. PCR amplification reaction

And selecting 8 extracted virus RNA samples, adding one negative control/positive control reaction, and carrying out 10-tube PCR reaction. Respectively adding RNA samples of the viruses to be detected into No. 1-8 reaction tubes, and adding 1 multiplied by 10 into No. 9 reaction tubes⁶The negative control, DEPC-ddH2O, was added to a copies/ml plasmid positive control, reaction tube No. 10.

The present embodiment is performed as follows:

1) enrichment amplification

amplification was performed according to the following amplification conditions to obtain PCR amplification product 1:

2) Exponential amplification

3. Analysis of results

The amplification curves for each sample are shown in FIGS. 4-13.

The results of the measurements for each sample are shown in table 4 below:

TABLE 4 sample test results

As shown in the table, the test results were: the samples No. 1 and No. 2 are single positive RV-A, the samples No. 3 and No. 4 are single positive RV-B, the samples No. 5 and No. 6 are single positive RV-C, the samples No. 7 and No. 8 are healthy people, all indexes are negative, the sample No. 9 is a positive reference substance, and the three detection indexes are positive, so that the PCR amplification reaction is proved to be normal, and the fluorescence detection system is proved to be normal; no. 10 is negative control, and the three detection indexes are negative, so that the PCR amplification reaction is proved to be pollution-free. All the samples with positive detection results are verified by sequencing through a Sanger method without errors, and the accuracy rate of the positive results is 100%. The detection method has good specificity and accuracy, and can be used for identifying and detecting A, B, C group rotavirus.

SEQUENCE LISTING

<110> Shandong Kai Jing Biotechnology Co., Ltd

<120> a method for simultaneously detecting A, B, C three groups of rotaviruses

<130>2019

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<170>PatentIn version 3.5

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gcttctgata gaggctactg 20

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ataggtatga ctggtggaaa t 21

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Claims

1. A method for simultaneously detecting A, B, C three groups of rotaviruses is characterized in that: designing a pair of specific primers and a specific probe with different fluorescent labels aiming at target genes of A group of rotavirus, B group of rotavirus and C group of rotavirus respectively, and designing a pair of universal primers at the same time, wherein the specific primers with universal primer labels are adopted for enrichment amplification during PCR amplification; then, performing exponential amplification by adopting a universal primer label, and collecting a fluorescent signal of an exponential amplification product for detection;

an upstream primer: 5'-ACTTTATTGGCGAATGTTAC-3'

A downstream primer: 5'-GCTTCTGATAGAGGCTACTG-3'

And (3) probe: 5 '-VIC-CTATACCAGTTGGACCAGTATTTCCACC-BHQ 1-3';

an upstream primer: 5'-ATAGGTATGACTGGTGGAAAT-3'

A downstream primer: 5'-ATGTGGGAATGTAGACGATGT-3'

And (3) probe: 5 '-FAM-CGATGTTAATTATTCCACAAGTGC-BHQ 3-3';

an upstream primer: 5'-AATTTGGAAGCGTCATGGGTTTA-3'

A downstream primer: 5'-AAGCGTTGCGTATTGTTTGGT-3'

And (3) probe: 5 '-CY 5-ACAACAAGTAATACTTGTCAAATTGCAGC-BHQ 2-3';

an upstream primer: 5'-Gatccaatcgcgtacttgc-3'

A downstream primer: 5'-Tgccatgcataaccgcagt-3' are provided.

2. The method of claim 1 wherein three groups of rotavirus are simultaneously detected A, B, C,

the enrichment amplification is as follows:

the following were added to the PCR tube: 5 xRT-PCR Buffer 5 muL, dNTP Mix final concentration 0.4mM, specific primer final concentration 0.1 muM with universal primer label, RNase inhibitor 0.2 muL, Mg²⁺The final concentration is 2.5mM, the mixture of reverse transcriptase and Taq enzyme is 2unit, the sample is 3 muL, and DEPC water is added to complement the volume to 25 muL;

amplification was performed under the following amplification conditions:

3. The method of claim 1 wherein three groups of rotavirus are simultaneously detected A, B, C,

the exponential amplification comprises the following specific steps:

the following were added to the PCR tube: 5 XPCR Buffer 5.0 muL, dNTP Mix final concentration 0.3mM, universal upstream primer final concentration 0.1 muM, universal downstream primer final concentration 0.3 muM, each probe final concentration 0.1uM, Mg²⁺The final concentration is 2.5mM, 1unit of Taq enzyme, 5 muL of PCR enrichment amplification product is added with DEPC water to complement the volume to 25 muL;

amplification was performed under the following amplification conditions:

4. A method for simultaneously detecting A, B, C three groups of rotaviruses as claimed in any one of claims 1-3, wherein: the group A rotavirus probe is labeled with a VIC fluorescent group at 5 'and a BHQ1 fluorescent quenching group at 3'.

5. A method for simultaneously detecting A, B, C three groups of rotaviruses as claimed in any one of claims 1-3, wherein: the group B rotavirus probe is marked with FAM fluorescent group at 5 'and BHQ3 fluorescent quenching group at 3'.

6. A method for simultaneously detecting A, B, C three groups of rotaviruses as claimed in any one of claims 1-3, wherein: group C rotavirus probes are 5 'labeled CY5 fluorophore and 3' labeled BHQ2 fluorescence quencher.

7. The method of claim 1 for simultaneously detecting A, B, C three groups of rotaviruses, wherein: the universal primers do not share homology with any of the sequences deposited in the known GenBank.

8. The method of claim 1 for simultaneously detecting A, B, C three groups of rotaviruses, wherein: and (3) collecting the fluorescence signal of the exponential amplification product, and detecting by using a multiplex real-time fluorescence quantitative PCR technology platform.

9. The method of claim 8, wherein the interpretation criteria for the results of the simultaneous detection of A, B, C three groups of rotaviruses is: when the CT value is less than 35, the index is positive, the CT value is more than 38 or underended, the index is negative, and the CT value is more than 35 and less than 38, which is a detection gray level interval, and needs to be detected again.

10. The method of claim 8, wherein the three groups of rotaviruses are simultaneously detected A, B, C, wherein: the detection sensitivity of the kit to group A rotavirus, group B rotavirus and group C rotavirus is 1 multiplied by 10³copies/ml。