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CN111647670A - Nephrotic syndrome-related enterobacteria Faecaliallea and application thereof - Google Patents

Nephrotic syndrome-related enterobacteria Faecaliallea and application thereof Download PDF

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CN111647670A
CN111647670A CN201910161867.8A CN201910161867A CN111647670A CN 111647670 A CN111647670 A CN 111647670A CN 201910161867 A CN201910161867 A CN 201910161867A CN 111647670 A CN111647670 A CN 111647670A
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nephrotic syndrome
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abundance
primer
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张森
王琰
张东明
王伟达
李昭君
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Abstract

The invention discloses application of intestinal flora in nephrotic syndrome diagnosis, which is characterized in that 16SrRNA sequencing is carried out on a sample of a nephrotic syndrome subject, so that the significant difference of Faecalialtalea abundance in a patient is found for the first time, and the early accurate diagnosis of nephrotic syndrome can be realized by detecting the Faecaltalea abundance.

Description

Nephrotic syndrome-related enterobacteria Faecaliallea and application thereof
Technical Field
The invention belongs to the field of biomedicine, and relates to application of intestinal flora in nephrotic syndrome diagnosis.
Background
Nephrotic Syndrome (NS) is currently the most common one of kidney diseases, and patients with nephrotic syndrome are mainly manifested by clinical symptoms of hypoproteinemia, profuse proteinuria, hyperlipidemia and edema, wherein the profuse proteinuria and hypoproteinemia are essential conditions for nephrotic syndrome diagnosis. Nephrotic syndrome includes both primary and secondary conditions, depending on the cause of the disease.
The progression and prognosis of nephrotic syndrome are related to a number of factors, including underlying disease, complications, and renal pathology. Infection, thromboembolism, acute kidney injury, endocrine and metabolic abnormalities, and the like are common complications of NS. The thromboembolic event is the most serious complication except infection, the patient has a high coagulation state generally, the incidence rate of the thromboembolic event is as high as 2-50%, the incidence rate and the mortality rate are very high, and the treatment and the prognosis of the thromboembolic event are seriously influenced.
There are many factors that can affect the occurrence of NS thromboembolic events, such as age, length of disease course, serum albumin level, blood coagulation function, type of pathology, severity of disease, etc. that are reported, but there is no consistent conclusion. Studies have shown that the pathological type of membranous nephropathy is an independent risk factor for thromboembolic events in glomerulonephritis patients. NS-complicated thromboembolism includes venous thrombosis and arterial thrombosis, and the incidence of venous thrombosis is higher than that of arterial thrombosis clinically. Pulmonary embolism, renal vein and deep vein thrombosis are common in clinic, and blood vessels such as heart and brain are also high-incidence parts of thrombosis. Meanwhile, the thrombus has great harm to the organism, when the lower limb has thrombus and is not treated in time, the patient may have paralysis of the lower limb or even be disabled, and the thrombus of important organs such as heart, brain, kidney and the like can be threatened to life if the thrombus is not treated in time. Therefore, early diagnosis and active prevention are particularly important for NS complicated thrombosis patients.
The relationship between microbial communities and hosts is very close, and according to research, the intestinal flora imbalance is closely related to various clinical diseases. The role of the intestinal flora in the development process of nephrotic syndrome is not clear, and the important significance is provided for researching the correlation of the intestinal flora and the nephrotic syndrome, particularly the nephrotic syndrome with the pathological type of membranous nephropathy and searching for a microbial marker for early diagnosis.
Disclosure of Invention
In order to remedy the deficiencies of the prior art, the object of the present invention is to provide a means and a product for the early diagnosis of nephrotic syndrome.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an application of a reagent for detecting microorganisms in preparation of a product for diagnosing nephrotic syndrome, wherein the microorganisms are Faecalialatalea (faecium).
Further, the product determines whether or not a subject has nephrotic syndrome by measuring the abundance of the microorganism Faecaliallae (faecium) in a sample including cells, tissues, organs, body fluids (blood, lymph fluid, etc.), digestive juice, urine, feces, etc.
Further, the sample is a stool sample.
Further, the reagent includes a primer, a probe, an antisense oligonucleotide, an aptamer, or an antibody that detects specificity of faecaliallea (faecium sp).
Further, the specific primer is a primer for detecting 16S rRNA of Faecaltalalea (genus faecium).
Further, the pathological type of nephrotic syndrome is membranous glomerulonephritis.
The present invention provides a system for predicting nephrotic syndrome using microbial markers, comprising:
a nucleic acid sample separation unit for separating a nucleic acid sample of the intestinal flora from a detection object;
the sequencing unit is used for sequencing the separated intestinal flora nucleic acid sample to obtain a sequencing result;
the data processing unit is used for detecting the relative abundance of the microbial markers in the intestinal flora according to the sequencing result, and analyzing the obtained relative abundance value to obtain the critical value of the microbial markers;
and the result judging unit is used for comparing the critical value of the microbial marker obtained by the data processing unit with the set diagnostic value.
Further, the microbial marker includes faecalaleea (faecium sp.).
The invention provides a product for diagnosing nephrotic syndrome, which comprises a reagent for detecting the abundance of Faecaliallea (faecium), wherein the product comprises a kit, a chip and a nucleic acid membrane strip.
Further, the reagent includes a primer, a probe, an antisense oligonucleotide, an aptamer, or an antibody that detects specificity of faecaliallea (faecium sp).
Further, the specific primer is a primer for detecting 16S rRNA of Faecalialeae (genus faecium).
As an alternative embodiment, the present invention provides a kit for diagnosing nephrotic syndrome based on the detection of faecalialeae (leguminous bacteria) abundance. The kit comprises the following components: a DNA extraction reagent, a primer pair for specifically detecting Faecalalitaea (faecium) 16SrRNA, a reaction buffer solution, base triphosphate deoxynucleotides (dNTPs), Taq-polymerase reverse transcriptase, DNase, an RNAse inhibitor, DEPC-water, sterile water and SYBR Green fluorescent dye.
The invention provides application of Faecalialteae (faecium) in preparing a calculation model for predicting nephrotic syndrome.
In the present invention, the term "biomarker" is to be understood in a broad sense and includes any detectable biomarker capable of reflecting an abnormal state, which may include genetic markers, species markers (species/genus markers) and functional markers. The meaning of the gene marker is not limited to the existing gene that can be expressed as a biologically active protein, and includes any nucleic acid fragment, which may be DNA, RNA, modified DNA or RNA, or unmodified DNA or RNA. Genetic markers may also sometimes be referred to herein as signature fragments. In particular, the biomarkers of the invention are microbial markers.
Stool samples of nephrotic syndrome were analyzed according to embodiments of the invention. And (3) counting intestinal flora presenting difference in nephrotic syndrome based on high-throughput sequencing data, so as to determine a specific sequence related to the nephrotic syndrome.
As a preferred embodiment, the method comprises the following steps:
(1) collecting and processing samples: collecting related excrement samples, and performing DNA extraction by using the kit to obtain nucleic acid samples;
(2) library construction and sequencing: constructing and sequencing a DNA library by using high-throughput sequencing so as to obtain a nucleic acid sequence of the intestinal microorganisms contained in the fecal sample;
(3) determining the nucleic acid sequence of specific intestinal microorganisms related to the nephrotic syndrome by using a bioinformatics analysis method.
First, obtaining sequencing data of a nucleic acid sequence in a fecal sample of the individual, the sequencing data comprising a plurality of reads; assembling the reads to obtain a gene set, wherein the gene set comprises a plurality of assembling fragments, and the assembling fragments in the gene set are non-redundant sequences; determining the assembly fragments comprised by each microorganism in the marker; determining the abundance of each assembled fragment in the gene set according to the sequencing data, wherein the abundance of each assembled fragment contained in each microorganism in the marker is determined; determining the abundance of each microorganism according to the determined abundance of the assembled fragments.
In the present invention, sequencing may be selected from, but is not limited to, semiconductor sequencing technology platform such as PGM, sequencing by synthesis technology platform such as HISeq, Miseq sequence platform of Illumina, and single molecule real-time sequencing platform such as pacdio sequence platform, depending on the sequencing platform selected. The sequencing mode can select single-ended sequencing or double-ended sequencing, and the obtained off-line data is a fragment obtained by sequencing and reading, and is called a read segment.
In the present invention, the assembly referred to may be performed using known sequence assembly methods or software, for example using SOAPdenovo, velvet, etc.
According to one embodiment of the present invention, the assembled fragments in the gene set are aligned with a microbial reference sequence using the MetaGeneMark software to determine whether the assembled fragments are from a certain type of microbe based on their similarity to the microbial reference sequence. The reference sequence refers to a predetermined sequence, and may be any reference template of a biological category to which a sample to be tested belongs or which is obtained in advance, for example, if the target is a microorganism in the sample to be tested, the reference sequence may be selected from reference genomes of various microorganisms in the NCBI database and/or DAcc enteric genomes disclosed in the MetaHIT project, and further, a resource library including more reference sequences may be configured in advance, for example, a more similar sequence may be selected or determined to be assembled as the reference sequence according to factors such as the status and region of an individual from which the sample to be tested originates. According to one embodiment of the present invention, determining the assembled fragments comprised by the various microorganisms in the nephrotic syndrome marker comprises: and (3) respectively comparing the assembled fragments in the gene set with the reference sequences of various microorganisms, and determining that the assembled fragments with the similarity of more than or equal to 90 percent of the reference sequences of a microorganism are from the microorganism.
According to an embodiment of the present invention, in the step of determining the abundance of each microorganism in the nephrotic syndrome marker according to the determined abundance of the assembled fragments, the abundance of the microorganism is the median or average of the abundances of all the assembled fragments contained in the microorganism.
The term "chip", also referred to as an "array", refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays are also known as "microarrays".
Faecalitarea: the genus faecium.
A "microarray" is an ordered array of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The matrix may be a solid matrix, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid matrix, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any permutation thereof.
The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.
The probe is typically labeled directly, e.g., with an isotope, chromophore, luminophore, chromogen, or indirectly, e.g., with biotin to which the streptavidin complex can subsequently bind. Thus, the detectable label used in the assays of the invention may be a primary label (wherein the label comprises a directly detectable element or an element capable of producing a directly detectable element) or a secondary label (wherein a detectable label is associated with a primary label, e.g., as commonly used in immunolabeling). Typically, a labeled signal nucleic acid is used to detect hybridization. The complementary nucleic acid or signal nucleic acid can be labeled by any of several methods commonly used to detect the presence of hybridized polynucleotides. The most commonly used detection method is an autoradiography method using a probe or the like labeled with 3H, 125I, 35S, 14C, or 32P. Other labels include, for example, ligands that bind to the labeled antibody, fluorophores, chemiluminescent agents, enzymes, and antibodies that are specific binding pair members for the labeled ligand.
The term "kit" includes a detection effective amount of a reagent for detecting Faecalialteae selected from one or more of the following: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent. For example, a solution for suspending or immobilizing cells, a detectable label or label, a solution for facilitating hybridization of nucleic acids, a solution for lysing cells, or a solution for nucleic acid purification.
The kit of the present invention may further comprise instructions for use of the kit, wherein the instructions describe how to use the kit for detection and how to use the detection results to determine the development of a disease.
With the kit of the present invention, Faecalialteae can be detected by various methods selected from the group consisting of (including but not limited to): real-time quantitative reverse transcription PCR, biochip detection method, DNA blotting or in situ hybridization method, and immunoassay method. The detection mode can be adjusted and changed by those skilled in the art according to actual conditions and needs.
As a preferred embodiment, the kit detects the abundance of faecaliallea by real-time quantitative reverse transcription PCR; more preferably, the kit comprises a specific primer for Faecalialteae, a reaction buffer, enzymes such as deoxynucleotide triphosphate (dNTPs) and Taq-polymerase reverse transcriptase, DNase, an RNAse inhibitor, DEPC-water, sterile water and the like.
In the present invention, "subject" includes animals capable of suffering from or suffering from nephrotic syndrome, and examples of subjects include mammals, e.g., humans, non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, and in certain embodiments, the subject is a rat.
The "sample" includes cells, tissues, organs, body fluids (blood, lymph, etc.), digestive juices, expectoration, alveolar bronchial lavage, urine, feces, etc. Preferably, the sample is tissue, blood, stool. In a particular embodiment of the invention, the sample is feces.
The term "abundance difference" refers to a higher or lower level of microorganisms obtained in a patient with nephrotic syndrome compared to the normal or control in vivo microorganism levels. For the purposes of the present invention, an "abundance difference" is considered to be a phenomenon that occurs when the level of microorganisms taken from a normal or disease-suffering subject, or from each stage of a disease-suffering subject, differs by 1.5-fold or more, about 4-fold or more, about 6-fold or more, about 10-fold or more.
The invention has the advantages and beneficial effects that:
the invention discovers that the abundance level of Faecalialtalea in patients with nephrotic syndrome shows significant difference for the first time, can realize early diagnosis of nephrotic syndrome by detecting the abundance of Faecaltaleaa, and has high specificity and strong sensitivity when being diagnosed by using the microbial marker of the invention.
Drawings
Figure 1 is a graph of the abundance of Faecalialtea in nephrotic syndrome samples.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Example 1 screening of intestinal flora associated with nephrotic syndrome
1. Collection of samples
1.1 construction of Membrane-type glomerulonephritis model
A typical stable model of membranous glomerulonephritis is replicated by immunizing rats with C-BSA, using Sprague-Dawley rats as experimental animals, according to the Border method (Border WA, Ward HJ, KamileS, et al.indication of molecular neuropathology in libraries by administration of an exogenous cationic antigen.J. Clin Invest,1982, 69: 451-461).
1.1.1 preparation of antigen-cationized BSA (C-BSA)
Natural bovine serum albumin fraction V: electrophoretically pure, with an isoelectric point of 4.5, Ameresco. Carbodiimide (EDC): beijing chemical company. Anhydrous Ethylenediamine (EDA): analytically pure, Beijing Chemicals, Inc.
Adding 500mL of double distilled water into 67mL of EDA, then slowly adding 350mL of 6M hydrochloric acid, adjusting the pH value to 4.75, cooling the solution to 25 ℃ on ice, dissolving 5g of BSA in 25mL of double distilled water, then slowly adding the solution into the EDA solution, continuously stirring, adding 1.8g of EDC, reacting for 2h at constant temperature of 25 ℃, and stopping the reaction by using 30mL of acetic acid buffer solution with pH4.75 to obtain the C-BSA solution with the improved isoelectric point. Dialyzing with double distilled water at 4 deg.C for 48h (changing water every 8 hr), freeze drying to obtain C-BSA powder with isoelectric Point (PI) of above 8.4, and storing at-70 deg.C.
1.1.2 construction of Membrane-type glomerulonephritis model
The experimental animals are healthy female Sprague-Dawley rats with the weight of 160-180 g provided by Beijing Wintoli Hua. After 3 days of acclimatization, randomized into 2 groups: pathological groups 10, the nephritis model was replicated with C-BSA; the normal control group was 10, and was normally kept without any treatment.
1.5mg of C-BSA was dissolved in 0.5mL of physiological saline, and mixed with 0.5mL of Freund's incomplete adjuvant to prepare "water-in-oil", which was used for subcutaneous multipoint injection for pre-immunization, and C-BSA was injected daily after 1 week. Intraperitoneal injection is carried out for 1 week, the doses from day 1 to day 7 are 0.5mg, 1mg, 2mg and 2.5mg in sequence, and then tail vein injection is carried out for 6 weeks. The doses on days 1 to 7 of the tail vein injection were 2.5mg, 3mg, 4mg, 5mg in this order, and thereafter 5mg daily until the end of the test.
1.2 Collection of samples
Feces samples of 10 normal control rats and nephrotic syndrome rats were collected, stored in a-80 ℃ refrigerator at low temperature, and the information of the samples was recorded, which was approved and approved by the ethical committee.
2. 16S rRNA sequencing
Sequencing of sample 16S rRNA was performed by Shanghai Meiji biomedical science and technology, Inc., and the specific steps were as follows:
2.1 extraction of DNA
Bacterial DNA was extracted from the fecal specimen using the fecal genomic DNA extraction Kit OMEGA-soil DNA Kit from Omega Bio-Tek, and the procedure was as described in the specification.
2.2 DNA sample purity and concentration determination
Genomic DNA was detected by electrophoresis on a 1% agarose gel.
2.3 PCR amplification and product purification
Carrying out PCR amplification reaction by adopting TransGen AP221-02(TransStart Fastpfu DNA Polymerase), carrying out all samples according to formal experimental conditions, repeating the samples for 3 times, mixing PCR products of the same sample, carrying out electrophoresis detection by using 2% agarose gel, cutting gel by using an AxyPrepDNA gel recovery kit (AXYGEN company), recovering the PCR products, and eluting with Tris-HCl; and (5) detecting by 2% agarose electrophoresis.
2.3.1 primer design
Synthesizing specific primers with barcode according to the designated sequencing region, wherein the primer sequences are as follows:
338F：5’-ACTCCTACGGGAGGCAGCAG-3’(SEQ ID NO.1)；
806R:5’-GGACTACHVGGGTWTCTAAT-3’(SEQ ID NO.2)
2.3.2 PCR amplification
1) A20. mu.l reaction system was prepared, as shown in Table 1.
TABLE 1 PCR reaction System
Figure BDA0001984926940000081
2) Amplification of
Using ABI
Figure BDA0001984926940000082
Performing PCR amplification by a 9700 type PCR instrument, wherein the amplification procedure comprises the following steps:
95 ℃ for 3min, (95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 45s) times 27 cycles, 72 ℃ for 10 min.
2.4 fluorescent quantitation
The PCR product was treated with QuantiFluorTMThe quantitative determination was carried out by the ST blue fluorescence quantitative system (Promega Co.).
2.5 Miseq library construction
Library construction was performed using the TruSeqTM DNA Sample Prep Kit from Illumina, following the instructions.
2.6 Miseq sequencing
And (3) synthesizing a target DNA fragment to be detected by using the DNA fragment as a template through PCR, carrying out bridge PCR amplification on the cBot to generate a DNA cluster, and carrying out sequencing of 2 × 150bp on a Hiseq4000 sequencing platform.
3. Data analysis
3.1 data preprocessing
Splicing PE reads obtained by Miseq sequencing by using FLASH, trimmatic and other software according to an overlap relation, and simultaneously performing quality control and filtration on sequence quality; clustering was performed using Usearch software, the sequences were classified as many OUT's according to their similarity, statistical analysis of the biological information was performed using the RDP classifier Bayesian algorithm for OTU at 97% similarity level, and comparisons were performed using the Silva database.
3.2 intestinal flora species differential analysis
Screening different species by using LEfSe multistage species difference discriminant analysis, detecting the characteristic with significant abundance difference by using non-parametric factor Kruskal-Wallis (KW) sum-rank test, and finding a group with significant difference with abundance; linear Discriminant Analysis (LDA) was used to estimate the magnitude of the effect of abundance of each component (species) on the difference effect. Screening criteria: LDA value >2 and P value < 0.05.
4. Results
As a result, as shown in fig. 1, the abundance level of faecaliallea was significantly reduced in nephrotic syndrome samples compared to normal controls (P <0.05) and the value of LDA was about 2.82, suggesting that faecaliallea can be applied as a microbial marker in the diagnosis of nephrotic syndrome.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Sequence listing
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Claims (12)

1. Use of a reagent for the detection of a microorganism in the manufacture of a product for the diagnosis of nephrotic syndrome, characterized in that said microorganism is Faecalialtalea.
2. The use according to claim 1, wherein the product is used to determine whether a subject has nephrotic syndrome by determining the abundance of the microorganism Faecalialtalea in a sample.
3. Use according to claim 2, wherein the sample is a stool sample.
4. The use according to claim 1, wherein the agent comprises a primer, probe, antisense oligonucleotide, aptamer or antibody specific for detection of Faecalialteae.
5. The use according to claim 4, wherein the specific primer is a primer for the detection of Faecaliallea 16 SrRNA.
6. The use according to any one of claims 1 to 5, characterized in that the pathological type of nephrotic syndrome is membranous glomerulonephritis.
7. A system for predicting nephrotic syndrome using microbial markers, comprising:
a nucleic acid sample separation unit for separating a nucleic acid sample of the intestinal flora from a detection object;
the sequencing unit is used for sequencing the separated intestinal flora nucleic acid sample to obtain a sequencing result;
the data processing unit is used for detecting the relative abundance of the microbial markers in the intestinal flora according to the sequencing result, and analyzing the obtained relative abundance value to obtain the critical value of the microbial markers;
and the result judging unit is used for comparing the critical value of the microbial marker obtained by the data processing unit with the set diagnostic value.
8. The system of claim 7, wherein the microbial marker comprises Faecaliallea.
9. A product for diagnosing nephrotic syndrome, comprising a reagent for detecting the abundance of Faecalitalea in a sample.
10. The product of claim 9, wherein the reagents comprise primers, probes, antisense oligonucleotides, aptamers, or antibodies that detect specificity of Faecalialteae.
11. The product according to claim 10, wherein the specific primer is a primer that detects Faecaliallea 16S rRNA.
Use of Faecalalitaea in the preparation of a computational model for the prediction of nephrotic syndrome.
CN201910161867.8A 2019-03-04 2019-03-04 Nephrotic syndrome-related enterobacteria Faecaliallea and application thereof Pending CN111647670A (en)

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CN113025730A (en) * 2021-01-14 2021-06-25 江苏省肿瘤防治研究所（江苏省肿瘤医院） Intrahepatic flora marker related to liver cirrhosis and application thereof
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Application publication date: 20200911