CN106399303A - Host-enteric microbial interacting gene-based specific molecular marker 1-38 and detection method for pollution by excrement of pig - Google Patents

Abstract

The invention relates to the field of detection of pig feces pollutants, in particular to a pig feces pollution-specific molecular marker 1-38 based on a host-intestinal microorganism interaction gene and a detection method thereof. The present invention adopts the method of competitive hybrid gene fragment enrichment (GFE) to enrich the pig-specific metagenome, constructs the metagenome library, and classifies the library with sequence flora and function, thereby targeting and screening the host (pig)-intestinal microorganisms Interaction targets are related to specific genes, and molecular markers are designed for pig-specific interaction genes, and a monitoring method with high sensitivity, strong specificity, and efficient indication of fecal pollution sources has been established.

Description

A specific analysis of pig fecal contamination based on host-gut microbe interaction genes Submarkers 1-38 and detection methods thereof

technical field

The invention relates to the field of detection of pig feces pollutants, in particular to a specific molecular marker 1-38 for pig feces pollution based on a host-intestinal microorganism interaction gene and a detection method thereof.

Background technique

Pork products are one of the most popular meat products among domestic consumers, and their huge demand has led to the rapid development of the pig industry. According to the statistics of the Ministry of Agriculture, the number of live pigs slaughtered in my country has always ranked first in the world in recent years, accounting for about 56% and maintaining a growth trend. Due to the lack of effective management of the rapid development of the intensive livestock and poultry farming industry, the manure of pigs and other livestock and poultry and their sewage mostly enter the environment in the form of non-point sources, causing pollution and health threats. Pig feces not only contain a large number of pathogenic microorganisms (Hepatitis E virus, Salmonella, Listeria, Campylobacter spp.), which directly threaten human health, but also contain nitrogen, phosphorus Such substances, if discharged without effective treatment, will cause soil, groundwater, surface water and irrigation water pollution, and eutrophication of water bodies, which will lead to the accumulation of a large amount of harmful substances in aquatic products and affect the quality of aquatic products. Therefore, the establishment of a It is particularly urgent to develop a monitoring method with high sensitivity, strong specificity and high efficiency to indicate the source of fecal contamination.

Some studies have identified the source of contamination from the screening tags of 16S rRNA and virulence genes of host fecal-derived flora such as Frimicutes, Bifidobacteria spp, Methanobrevibacter smithii, etc. . Most of the identified porcine-specific molecular markers are based on the conserved region of 16S rRNA of dominant porcine intestinal bacteria. There are also screening of virulence genes from mitochondrial DNA and Escherichia coli in feces to determine the source of pig feces contamination. However, although the above-mentioned specific molecular markers have certain specificity and sensitivity, there are major limitations: 1) host specificity is not high enough, 2) there is cross-reactivity among different hosts, 3) there are geographical differences , 4) The accuracy of judging the source of animals is not high enough, so it will bring a high rate of misjudgment.

The intestinal microbial community and its host are co-evolved, and the strong selection and co-evolution between the host and the intestinal microbes can form host-microbe interactions that are beneficial to the host. Points are an important factor in the formation of intestinal microbial diversity and specificity. Therefore, more and more studies have screened host-microbe interaction genes as host-specific molecular markers from the intestinal microbial communities of humans, cattle, and chickens, and proved that such molecular markers have high specificity and sensitivity. However, there are no related studies on the use of host-microbe interaction genes as specific molecular markers to trace non-point source pollution of pig feces in water or food.

Contents of the invention

In order to solve the above-mentioned technical problems, one object of the present invention is to provide a specific molecular marker 1-38 based on the host-intestinal microorganism interaction gene for pig feces pollution. Another object of the present invention is to provide For the detection method of markers 1-38, the present invention designs molecular markers for pig-specific interaction genes, and establishes a monitoring method with high sensitivity, strong specificity, and efficient indication of feces pollution sources.

In order to achieve the above-mentioned first purpose, the present invention adopts the following technical solutions:

A pig feces pollution-specific molecular marker 1-38 based on host-intestinal microorganism interaction genes, which consists of the following primers and probes:

Upstream primer: GGAGGTGGTTAAGCCGATATGTT

Downstream primer: GCCCCTTTCTTGATACTTTGGA

Probe: Fam-AAACTGATTGGAGAAGAATACAGGCG-Tam.

In order to achieve the above-mentioned second purpose, the present invention adopts the following technical solutions:

A method for detecting pig feces contamination based on host-intestinal microorganism interaction genes, the method uses the primers and probes to be mixed with the sample to be tested, performs a fluorescent PCR reaction, and reads after each cycle of extension; the sample No Ct value and no amplification curve, indicating that there is no pig feces contamination in the sample; sample amplification Ct value ≤ 35, and a typical amplification curve appears, indicating that there is pig feces contamination in the sample; samples with Ct value > 35 are recommended to repeat If there is no Ct value, it is negative, otherwise it is positive.

Preferably, the concentrations of the primers and probes are 0.25 μM and 0.3 μM respectively.

Preferably, the fluorescent PCR reaction conditions are: pre-denaturation at 95°C for 30s, 40 cycles of denaturation at 95°C for 5s and annealing at 60°C for 30s.

The present invention adopts the method of competitive hybridization gene fragment enrichment (GFE) to enrich the pig-specific metagenome, constructs the metagenome library, and classifies the sequence flora and function of the library, thereby targeting and screening the host (pig)-intestinal microorganisms Interaction targets are related to specific genes, and molecular markers are designed for pig-specific interaction genes, and a monitoring method with high sensitivity, strong specificity, and efficient indication of fecal pollution sources has been established.

Description of drawings

Figure 1 is a fluorescent PCR detection map of molecular marker 1-38.

Figure 2 is a fluorescent PCR detection map of the molecular marker 3-53.

Fig. 3 is a standard curve and amplification efficiency diagram of the molecular marker 1-38 fluorescent PCR system.

Fig. 4 is a standard curve and amplification efficiency diagram of the molecular marker 3-53 fluorescent PCR system.

Fig. 5 is a diagram of the inter-batch stability test of molecular marker 1-38 fluorescent PCR.

Fig. 6 is a diagram of the inter-batch stability test of molecular marker 3-53 fluorescent PCR.

detailed description

The specific implementation manner of the present invention will be described in detail below in conjunction with the accompanying drawings.

Example 1 Enrichment screening of specific pig-intestinal microorganism interaction genes

1.1 A total of 145 feces samples from 6 species were collected, including 34 swine feces samples, 20 cow feces samples, 12 goat feces samples, 8 sheep feces samples, 7 chicken feces samples, 20 duck feces samples, Five goose feces samples and 13 human feces samples were collected. Place the sample in a sterile container, add 3mL GITC buffer (5M guanidine isothiocyanate, 100Mm EDTA (pH 8.0), sodium lauryl sarcosine) to every 200mg (wet weight) sample, and store it at low temperature. DNA extraction from feces Fast DNA Stool Kit (Qiagen, Valencia, CA) instructions. And its concentration and purity were determined on a UV spectrophotometer NanoDrop ND-2000UV (NanoDrop Technologies, Thermofisher).

1.2 Host (pig) specific genome enrichment (GFE)

Genomic DNA of all single pig source samples was mixed to form a genomic mixed library to increase the diversity of the genome (group to be enriched), and the genomic mixed library of cattle, sheep, chicken, duck, goose and other species samples was used as a control group.

1.2.1 Preparation of enriched DNA

The group to be enriched was further divided into two groups (groups to be enriched A and B).

Preparation of group A DNA: After the DNA was cleaved by sonic shock, the K9 tag was labeled with Klenow I polymerase; the DNA fragment labeled with the K9 tag was used Multiwell PCR Purification Kit (Qiagen, Valencia, CA) was purified and amplified to obtain a sufficient amount of DNA fragments with K9 random primers.

Preparation of group B DNA (biotin-labeled DNA fragments): DNA was labeled with biotin (PAB, Sigma-Aldrich, Atlanta, GA) after sonication.

1.2.2 DNA prehybridization and competitive hybridization

The DNA of the enriched group B and the control group were denatured and then pre-hybridized, and then the pre-hybridized mixture was competitively hybridized with the pig-derived DNA of group A to form DNA double strands in various combinations. Biotin-streptavidin (Streptavidin) ELISA capture assay captures biotin-labeled hybrid double-stranded DNA (that is, double-stranded DNA containing at least one single strand to be enriched) and amplifies and enriches . Ultimately, high-abundance porcine source-specific fecal genomic DNA samples will be obtained.

1.3 Construction of metagenomic library

The PCR products of each round in the above enrichment process were subjected to Multiwell l PCRPurification Kit (Qiagen, Valencia, CA) was used for purification, and the PCR purified product was cloned into a vector according to the instructions of pCR TOPO 4.0 (Invitrogen), and transformed into competent Escherichia coli DH5α. Positive clones were obtained by screening with LB solid medium containing ampicillin (50 μg/mL). After identification, 500 recombinant bacteria were randomly selected and sent to Shanghai Sangon Co., Ltd. for sequencing. DNAstar was used to assemble the sequences obtained in each round, and 382 pig-specific non-redundant sequences were screened to construct a metagenomic library.

1.4 DNA sequence analysis

In the GeneBank database, BLASTX analysis was used to predict the protein function of each non-redundant sequence according to the biological function of the similar sequence. Among them, sequences with E value ≤ 10 ^-3 and recognition rate ≥ 30% were considered similar protein sequences. The resulting DNA sequences were classified into functional genes by the clustering of neighboring classes (COG) database. The enriched non-redundant sequences were classified according to the results of BLASTX alignment in the GeneBank database (minimum E value). Finally, 60 possible pig-specific interaction targets related to information storage and processing, cell information transduction and metabolism were screened out.

1.5 Identification of pig-microbe-specific interaction genes

The primers were designed for 60 possible specific interaction targets, and the results showed that genes 1-38 and genes 3-53 were pig-specific, but could not amplify fecal DNA of other species.

Genes 1-38:

AAATACTAATGGATAAAATAGAAATAATCTCCGGCAAACTCGGAATTGAGCACCGGAAGGTAGCCAACACCGTAAAACTGCTTGAAGATGGCGCTACAGTGCCATTTATCTCGCGATACCGCAAAGAAGCAACCGGCTCACTCGATGAAGTTGCCATCATGAATATCAGCACGCTCTTGGGACAACTCGAAGAATTGGACAAGAGGCGTCGTTACATCCTCGAAAGCATTGAGGCAAGCGGTGCCTTGACTCCGGAATTGAATTCACGCATTATGGTGTGCGATGATGCAACGACACTCGAGGATATCTACCTCCCATTCAAGCCCAAACGCCGCACCAAGGCGGAAGTGGCACGAAACAACGGTCTCGAACCGTTGGCAAAAATAATTATGGCACAAAAATCGATCGATATTCATTCTCAAGCCGGTCGTTTTATTGGGGAAAATGTGCCTGACGAA。

Gene 3-53:

AAATACTAATGGATAAAATAGAAATAATCTCCGGCAAACTCGGAATTGAGCACCGGAAGGTAGCCAACACCGTAAAA CTGCTTGAAGATGGCGCTACAGTGCCATTTATCTCGCGATACCGCAAAGAAGCAACCGGCTCACTCGATGAAGTTGC CATCATGAATATCAGCACGCTCTTGGGACAACTCGAAGAATTGGACA AGAGGCGTCGTTACATCCTCGAAAGCATTGAGGCAAGCGGTGCCTTGACTCCGGAATTGAATTCACGCATTATGGTGTGCGATGATGCAACGACACTCGAGGATATCTACCTCCCATTCAAGCCCAAACGCCGCACCAAGGCGGAAGTGGCACGAAACAACGGTCTCGAACCGTTGGCAAAAATAATTATGGCACAAAAATCGATCGATATTCATTCTCAAGCCGGTCGTTTTATTGGGGAAAATGTGCCTGACGAA。

Screening and reaction conditions of embodiment 2 molecular markers

A porcine-specific DNA gene library was established by competitive hybrid gene fragment enrichment (GFE). After the library was cloned into pCR TOPO 4.0, 500 clones were randomly selected for sequencing analysis, and a total of 382 non-redundant porcine-specific sequences were obtained. BLAXTS analysis found that 60 non-redundant sequences were related to host-microbe interaction proteins such as surface proteins such as Bacteroidetes and Clostridials, membrane secreted proteins, and carbohydrate metabolism proteins. As a target for porcine-specific molecular marker screening. Molecular markers were designed for these 60 genes, and after screening, two sets of molecular markers were found to be missing from pig-derived fecal contamination (Table 1).

Table 1 Molecular marker sequence

Using positive DNA as a template, repeated experiments found that when the concentrations of molecular markers 1-38 primers and probes were 0.2 μM and 0.3 μM, and 3-53 primers and probes were respectively 0.25 μM and 0.3 μM, two sets of The detection system has high amplification efficiency and sensitivity respectively. Therefore, the 20 fluorescent PCR reaction system is as follows:

The fluorescent PCR reaction conditions were: 95°C pre-denaturation for 30s, 40 cycles of 95°C denaturation for 5s and 60°C annealing extension for 30s, and read after each cycle of extension. Figure 1 is a fluorescent PCR detection map of molecular marker 1-38, and Figure 2 is a fluorescent PCR detection map of molecular marker 3-53. The sample has no Ct value and no amplification curve, indicating that there is no pig feces contamination in the sample; the sample amplification Ct value ≤ 35, and a typical amplification curve appears, indicating that there is pig feces contamination in the sample; samples with a Ct value > 35 are recommended Redo, redo result without Ct value is negative, otherwise it is positive.

Example 3 standard curve and system repeatability

Dilute the two positive DNA standards 1-38 and molecular markers 3-53 10 times to 1.2×10 ⁸ to 1.2×10 ¹ copies/μl, and then perform fluorescent PCR reactions respectively, with 3 repetitions for each dilution, and draw the standards The curve calculates the amplification efficiency. The results showed that the two sets of molecular markers had wide amplification linear ranges and high amplification efficiencies, which were 1.07 and 0.95, respectively (Fig. 3 and Fig. 4). At the same time, the minimum detection limits of molecular markers 1-38 and 3-53 were 600 copies/reaction and 60 copies/reaction, respectively, indicating that both detection systems had good detection sensitivity.

Perform 10 repeated detections on the two positive standard DNAs with multiple dilutions by fluorescent PCR. The standard deviations of the Ct values of the molecular marker 1-38 and 3-53 reaction systems were between 0.01-0.28 and 0.03-0.76, and the coefficient of variation Both are lower than 2.17% (Table 2), indicating that the two sets of fluorescent PCR based on specific molecular markers have high reproducibility.

Table 2 Repeatability test of two molecular marker fluorescent PCR systems

Stability test between batches of embodiment 4

In order to analyze the stability of the two sets of molecular marker fluorescent PCR methods, four independent tests were carried out, and the positive standard DNA was diluted to 1.2×10 ⁸ to 1.2×10 ¹ copies/μl in each test, and each dilution was set Four repetitions were performed, and the average CT value of different dilutions and the standard deviation of four independent experiments were calculated. The results showed that the molecular markers 1-38 and 3-53 fluorescent PCR systems had good detection stability, and the standard deviations of the detection Ct values between different batches were only 0.32-1.56 and 0.07-0.51 (Figure 5 and Figure 6).

The host specificity and sensitivity of embodiment 5 actual sample detection

Pig, cow, goat, sheep, chicken, duck, goose, dog and human feces are collected, DNA is extracted, and the specificity (S) and sensitivity verification (R) of the pig-specific molecular marker and its detection system in the present invention are performed. The fluorescent PCR reaction system and conditions are the same as above. And the detection result is calculated by the formula R=a/(a+b) and S=c/(c+d), wherein a and b represent the number of positive samples and the number of false negative samples of the molecular marker for the specific host respectively, and c and d represent the number of negative samples and false positive samples detected by molecular markers against other species, respectively. The results showed that the molecular marker 1-38 fluorescent PCR detection system had 85% host specificity and 94% host sensitivity for 72 pig feces samples or farm sewage and 79 other species of feces samples; 3-53 detection system It has a host specificity of 90% and a host sensitivity of 99% (Table 3), indicating that the two sets of molecular markers can efficiently and specifically detect whether a sample contains pig feces contamination in practical applications.

Table 3 Host specificity and sensitivity of molecular markers to actual sample detection

<110> Zhejiang Institute of Inspection and Quarantine Science and Technology; Zhejiang Gongshang University

<120> A specific molecular marker 1-38 based on host-gut microbe interaction gene and its detection method

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<211>458

<212>DNA

<213> Pig - Gut Microbiome

<400>1

AAATACTAAT GGATAAAATA GAAATAATCT CCGGCAAACT CGGAATTGAG CACCGGAAGG 60

TAGCCAACAC CGTAAAACTG CTTGAAGATG GCGCTACAGT GCCATTTATC TCGCGATACC 120

GCAAAGAAGC AACCGGCTCA CTCGATGAAG TTGCCATCAT GAATATCAGC ACGCTCTTGG 180

GACAACTCGA AGAATTGGAC AAGAGGCGTC GTTACATCCT CGAAAGCATT GAGGCAAGCG 240

GTGCCTTGAC TCCGGAATTG AATTCACGCA TTATGGTGTG CGATGATGCA ACGACACTCG 300

AGGATATCTA CCTCCCATTC AAGCCCCAAAC GCCGCACCAA GGCGGAAGTG GCACGAAACA 360

ACGGTCTCGA ACCGTTGGCA AAAATAATTA TGGCACAAAA ATCGATCGAT ATTCATTCTC 420

AAGCCGGTCG TTTTATTGGG GAAAATGTGC CTGACGAA 458

<210>2

<211>458

<212>DNA

<213> Pig - Gut Microbiome

<400>2

AAATACTAAT GGATAAAATA GAAATAATCT CCGGCAAACT CGGAATTGAG CACCGGAAGG 60

TAGCCAACAC CGTAAAACTG CTTGAAGATG GCGCTACAGT GCCATTTATC TCGCGATACC 120

GCAAAGAAGC AACCGGCTCA CTCGATGAAG TTGCCATCAT GAATATCAGC ACGCTCTTGG 180

GACAACTCGA AGAATTGGAC AAGAGGCGTC GTTACATCCT CGAAAGCATT GAGGCAAGCG 240

GTGCCTTGAC TCCGGAATTG AATTCACGCA TTATGGTGTG CGATGATGCA ACGACACTCG 300

AGGATATCTA CCTCCCATTC AAGCCCCAAAC GCCGCACCAA GGCGGAAGTG GCACGAAACA 360

ACGGTCTCGA ACCGTTGGCA AAAATAATTA TGGCACAAAA ATCGATCGAT ATTCATTCTC 420

AAGCCGGTCG TTTTATTGGG GAAAATGTGC CTGACGAA 458

<210>3

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GGAGGTGGTT AAGCCGATAT GTT 23

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GCCCCTTTCT TGATACTTTG GA 22

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AAACTGATTGGAGAAGAATACAGGCG26

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<211>21

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GCGTCGTTAC ATCCTCGAAA G 21

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<211>18

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GCGTTTGGGCTTGAATGG 18

<210>8

<211>29

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TTCACGCATT ATGGTGTGCG ATGATGCAA 29

Claims (4)

1. a kind of swine excrement pollution specific molecular marker 1-38 based on host-enteric microorganism interacting genes, its feature exists It is made up of following primer and probe in this molecular labeling：

Upstream primer：GGAGGTGGTTAAGCCGATATGTT

Downstream primer：GCCCCTTTCTTGATACTTTGGA

Probe：Fam-AAACTGATTGGAGAAGAATACAGGCG-Tam.

2. a kind of based on host-enteric microorganism interacting genes swine excrement pollution detection method it is characterised in that：The method Using the primer described in claim 1 and probe and detected sample mix, carry out Fluorescence PCR, extend in each circulation Reading after end；Sample no Ct value and no amplification curve, represent no swine excrement pollution in sample；Sample amplification Ct value≤35, And typical amplification curve occurs, represent in sample, there is swine excrement pollution；Sample Ct value>35 sample suggestion is reformed, and reforms Result no Ct value person is feminine gender, otherwise for the positive.

3. detection method according to claim 2 it is characterised in that：Primer and concentration and probe concentration are respectively 0.2 μM and 0.3 μM.

4. detection method according to claim 2 is it is characterised in that Fluorescence PCR condition is：95 DEG C of denaturation 30 s, 5 s and 60 DEG C of annealing of 95 DEG C of denaturation of 40 circulations extend 30 s.