CN106381295B - It is a kind of that specific molecular marker 3-53 and its detection method are polluted based on host-enteric microorganism interacting genes swine excrement - Google Patents

It is a kind of that specific molecular marker 3-53 and its detection method are polluted based on host-enteric microorganism interacting genes swine excrement Download PDF

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CN106381295B
CN106381295B CN201610847949.4A CN201610847949A CN106381295B CN 106381295 B CN106381295 B CN 106381295B CN 201610847949 A CN201610847949 A CN 201610847949A CN 106381295 B CN106381295 B CN 106381295B
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swine excrement
pig
primer
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CN106381295A (en
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帅江冰
傅玲琳
张晓峰
曾若雪
莫虹斐
何永强
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ZHEJIANG ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
Zhejiang Gongshang University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

The present invention relates to swine excrement pollutant monitoring field more particularly to a kind of specific molecular marker 3-53 and its detection method are polluted based on host-enteric microorganism interacting genes swine excrement.The present invention is using the macro genome of competitive hybridization genetic fragment enrichment method (GFE) enrichment pig specificity, construct macro gene library, sequence flora and function classification are carried out to library, host (pig)-related specific gene of enteric microorganism interaction target spot has been screened to target, and molecular labeling is designed for pig specificity interacting genes, establish high sensitivity, high specificity and the monitoring method that can efficiently indicate fecal pollution source.

Description

It is a kind of to be divided based on host-enteric microorganism interacting genes swine excrement pollution specificity Son label 3-53 and its detection method
Technical field
The present invention relates to swine excrement pollutant monitoring fields, more particularly to one kind to be based on host-enteric microorganism interaction base The swine excrement pollution specific molecular marker 3-53 and its detection method of cause.
Background technique
For pork product as deep by one of favorite meat products of domestic consumer, huge demand leads to live pig industry It grows rapidly.It being counted according to the Ministry of Agriculture, China's live pig amount of delivering for sale accounts for always the first in the world in recent years, and accounting maintains 56% or so, And keep growing trend.Due to lacking effective management of the intensive livestock and poultry breeding industry discharge to fast development, the animal dungs such as pig Just and its sewage mostly enters environment in a manner of non-point source, causes and pollutes and induce health threat.In swine excrement excreta not only Containing a large amount of pathogenic microorganism bacterium (Hepatitis E virus, salmonella (Salmonella), Listeria (Listeria), Campylobacter jejuni (Campylobacter spp.), directly threatens human health, and contain nitrogen, the substances such as phosphorus, without having Effect handles any discharge, will cause soil, underground water, the pollution of surface water and irrigation water, and make water eutrophication, and then lead It causes aquatic products to accumulate a large amount of harmful substance, influences aquatic products quality.Therefore, a kind of high sensitivity, high specificity and energy are established Efficiently the monitoring method in instruction fecal pollution source seems especially urgent.
There is research from host's excrement source flora such as Firmicutes (Frimicutes), Bifidobacterium The 16S rRNA of (Bifidobacteria spp), Shi Shi methane phase bacillus (Methanobrevibacter smithii) etc. and Virulence gene etc. screens label to differentiate pollution source.It is excellent that current identified pig specific molecular marker is mostly based on chitling road The conserved region of gesture bacterium 16S rRNA.Also have and screen virulence gene from excrement Mitochondria DNA, escherichia coli [to judge pig Fecal pollution source.However, there are biggish although above-mentioned specific molecular marker has certain specificity and sensitivity Limitation: 1) host specificity is not high enough, 2) between different hosts there are cross reactivity, 3) there is a geographical area otherness, 4) sentence The accuracy of other animal origin is not high enough, therefore can bring higher false determination ratio.
Intestinal microflora and its host are common evolutionaries, through selecting and assisting strongly between host and enteric microorganism The function reserved area (interaction target spot) beneficial to host of host-microorganism interaction, these interaction targets can be formed with evolving Point is the key factor to form intestine microbial diversity and specificity.Therefore intestines of more and more researchs from people, ox, chicken etc. Host-microorganism interacting genes are screened in road microbiologic population as host specificity molecular labeling, and prove such molecule mark Remember specificity and sensitivity with higher.However, having not yet to see using host-microorganism interacting genes as specific molecular Label carries out the correlative study of swine excrement non-point pollution tracer in water body or food.
Summary of the invention
In order to solve the above technical problems, it is an object of the present invention to provide one kind to be based on host-enteric microorganism The molecular labeling 3-53 of the swine excrement pollution specificity of interacting genes, another object of the present invention are to provide using above-mentioned The detection method of molecular labeling 3-53, the present invention for pig specificity interacting genes design molecular labeling, establish high sensitivity, High specificity and the monitoring method that can efficiently indicate fecal pollution source.
In order to realize first above-mentioned purpose, present invention employs technical solutions below:
It is a kind of that specific molecular marker 3-53, this point are polluted based on host-enteric microorganism interacting genes swine excrement Son label is made of primer and probe below:
Upstream primer: GCGTCGTTACATCCTCGAAAG
Downstream primer: GCGTTTGGGCTTGAATGG
Probe: Fam-TTCACGCATTATGGTGTGCGATGATGCAA-Tam.
In order to realize second above-mentioned purpose, present invention employs technical solutions below:
It is a kind of based on host-enteric microorganism interacting genes swine excrement pollution detection method, this method use described in Primer and probe mixed with tested sample, carry out Fluorescence PCR, each circulation extension after read；Sample without Ct value and without amplification curve, indicates to pollute in sample without swine excrement；Value≤35 sample amplification Ct, and it is bent typical amplification occur Line indicates that there are swine excrement pollutions in sample；The sample suggestion of value > 35 sample Ct is reformed, and reforming result without Ct value person is feminine gender, It otherwise is the positive.
Preferably, the primer and probe concentration is respectively 0.25 μM and 0.3 μM.
Preferably, the Fluorescence PCR condition are as follows: 95 DEG C of initial denaturation 30s, 95 DEG C of denaturation 5s of 40 circulations with 60 DEG C of annealing extend 30s.
The present invention is constructed macro using the macro genome of competitive hybridization genetic fragment enrichment method (GFE) enrichment pig specificity Gene library carries out sequence flora and function classification to library, has screened host (pig)-enteric microorganism interaction target to target The related specific gene of point, and molecular labeling is designed for pig specificity interacting genes, establish high sensitivity, high specificity simultaneously It can efficiently indicate the monitoring method in fecal pollution source.
Detailed description of the invention
Fig. 1 is molecular labeling 1-38 fluorescent PCR test map.
Fig. 2 is molecular labeling 3-53 fluorescent PCR test map.
Fig. 3 is the standard curve and amplification efficiency figure of molecular labeling 1-38 fluorescent PCR system.
Fig. 4 is the standard curve and amplification efficiency figure of molecular labeling 3-53 fluorescent PCR system.
Fig. 5 is stability test figure between molecular labeling 1-38 fluorescent PCR batch.
Fig. 6 is stability test figure between molecular labeling 3-53 fluorescent PCR batch.
Specific embodiment
A specific embodiment of the invention is made a detailed explanation with reference to the accompanying drawing.
The enrichment isolation of the specific pig-enteric microorganism interacting genes of embodiment 1
1.1 acquire 6 species, 145 fecal specimens altogether, including 34, swine excrement sample, milk cow cow dung sample 20 parts, 12 parts of goat fecal specimens, 8 parts of sheep dung sample, 7 parts of chicken manure sample, 20 parts of duck fecal specimens, goose fecal specimens 5 parts and 13 parts of human faecal mass sample.Sample is placed in sterile chamber, 3mL GITC buffering is added in every 200mg (weight in wet base) sample Liquid (5M guanidinium isothiocyanate, 100Mm EDTA (pH 8.0), sarcosyl), cryo-conservation.The extraction of excrement sample DNA is pressedFast DNA Stool Kit (Qiagen, Valencia, CA) specification carries out.And in ultraviolet specrophotometer Its concentration and purity (NanoDrop Technologies, Thermofisher) are measured on NanoDrop ND-2000UV.
1.2 hosts (pig) specific gene group is enriched with (GFE)
All single pig source sample gene group DNA are mixed to form genome mixing library to increase the diversity of genome (group to be enriched with), as a control group with other species sample gene group mixing such as ox, sheep, chicken, duck, goose library.
1.2.1 the preparation of enrichment group DNA
Two groups will be divided into wait be enriched with group (wait be enriched with group A and B).
The preparation of A group DNA: with K9 label on Klenow I polymerase tag after DNA sound wave shock is cracked；By K9 label The DNA fragmentation of label is usedMultiwell PCR Purification Kit (Qiagen, Valencia, CA) is pure Change and expand to obtain the DNA fragmentation with K9 random primer of sufficient amount.
The preparation (biotinylated DNA fragments) of B group DNA: DNA is marked after sonication biotin (PAB, Sigma-Aldrich, Atlanta, GA).
1.2.2 DNA prehybridization and competitive hybridization
Prehybridization will be carried out after being enriched with group B and control group DNA and being denaturalized respectively, then by this prehybridization mixture and A group After pig source DNA competitive hybridization, various combined DNA double chains are formed.It is enzyme-linked through biotin-streptavidin (Streptavidin) Immuno absorbence captive test, by the heteroduplex DNA for being marked with biotin (i.e. at least containing one wait be enriched with the single-stranded double-strand of group DNA it) captures and expands enrichment.The pig source specificity excrement sample genome DNA sample of high abundance is obtained at last.
The 1.3 macro genomic libraries of building
By the PCR product warp of each round in above-mentioned enrichment processMultiwell PCR Purification Kit (Qiagen, Valencia, CA) is purified, and is purified PCR according to (Invitrogen) specification of pCR TOPO 4.0 and is produced Object is cloned into carrier, and is transferred in competent E.coli DH5 α.Positive colony, which is used, ampicillin (50 μ g/mL) The screening of LB solid medium obtains, and selects 500 recombinant bacteriums after identification at random and serves Hai Shenggong limited liability company and is sequenced. Sequence obtained by every wheel is spliced using DNAstar, filters out 382 pig source specificity non-redundant sequences, to construct macro base Because of a group library.
1.4 DNA sequence analysis
Analysis is compared using BLASTX in GeneBank database, it is non-superfluous to every according to the biological function of similar sequences Remaining sequence carries out protein function prediction, wherein value≤10 E-3, the sequence of discrimination >=30% is considered as similar protein sequence. Functional gene classification is carried out as the adjacent similar cluster of albumen (COG) database DNA sequence dna by obtained by.According in GeneBank data The result (minimum E value) that library BLASTX is compared carries out classification of flora to the non-redundant sequence of enrichment.Finishing screen selects 60 and letter Breath storage has the equiprobable pig specificity interaction target spot of correlation gene with processing, cellular informatics conduction and metabolism.
The identification of 1.5 pigs-microorganism specificity interacting genes
It is identified respectively for 60 possible specific interaction shot design primers, the results showed that wherein gene 1-38 There is pig source specificity with gene 3-53, and the faeces DNA of other species cannot be expanded.
Gene 1-38:
AAATACTAATGGATAAAATAGAAATAATCTCCGGCAAACTCGGAATTGAGCACCGGAAGGTAGCCAAC ACCGTAAAACTGCTTGAAGATGGCGCTACAGTGCCATTTATCTCGCGATACCGCAAAGAAGCAACCGGCTCACTCG ATGAAGTTGCCATCATGAATATCAGCACGCTCTTGGGACAACTCGAAGAATTGGACAAGAGGCGTCGTTACATCCT CGAAAGCATTGAGGCAAGCGGTGCCTTGACTCCGGAATTGAATTCACGCATTATGGTGTGCGATGATGCAACGACA CTCGAGGATATCTACCTCCCATTCAAGCCCAAACGCCGCACCAAGGCGGAAGTGGCACGAAACAACGGTCTCGAAC CGTTGGCAAAAATAATTATGGCACAAAAATCGATCGATATTCATTCTCAAGCCGGTCGTTTTATTGGGGAAAATGT GCCTGACGAA。
Gene 3-53:
AAATACTAATGGATAAAATAGAAATAATCTCCGGCAAACTCGGAATTGAGCACCGGAAGGTAGCCAAC ACCGTAAAACTGCTTGAAGATGGCGCTACAGTGCCATTTATCTCGCGATACCGCAAAGAAGCAACCGGCTCACTCG ATGAAGTTGCCATCATGAATATCAGCACGCTCTTGGGACAACTCGAAGAATTGGACAAGAGGCGTCGTTACATCCT CGAAAGCATTGAGGCAAGCGGTGCCTTGACTCCGGAATTGAATTCACGCATTATGGTGTGCGATGATGCAACGACA CTCGAGGATATCTACCTCCCATTCAAGCCCAAACGCCGCACCAAGGCGGAAGTGGCACGAAACAACGGTCTCGAAC CGTTGGCAAAAATAATTATGGCACAAAAATCGATCGATATTCATTCTCAAGCCGGTCGTTTTATTGGGGAAAATGT GCCTGACGAA。
The screening of 2 molecular labeling of embodiment and reaction condition
Pig specific DNA gene library is established using competitive hybridization genetic fragment enrichment (GFE) method.By library clone To pCR TOPO 4.0, randomly selects 500 clones and carry out sequencing analysis, obtain 382 nonredundancy pig source specificity sequences altogether Column.Analysis, which is compared, through BLAXTS finds wherein 60 non-redundant sequences and bacteroid group (Bacteroidetes), bacillus fusiformis group (Clostridials) the host-microorganisms interaction albumen such as surface proteins, film secretory protein and carbohydrate metabolism albumen such as It is related, it can be used as the target spot of pig specific molecular marker screening.Molecular labeling is designed for this 60 genes, is found after screening Wherein it can be used as the missing molecular labeling (table 1) of pig source fecal pollution for two sets.
1 molecule labelled series of table
Using positive DNA as template, the test of multiplicating property finds that molecular labeling 1-38 primer and probe concentration is respectively 0.2 μM and 0.3 μM, 3-53 primer and probe concentration is when being respectively 0.25 μM and 0.3 μM, two sets of detection architectures are respectively provided with higher Amplification efficiency and sensibility.Therefore, 20 Fluorescence PCR systems are as follows:
Fluorescence PCR condition are as follows: 5s and 60 DEG C of annealing of 95 DEG C of denaturation of 95 DEG C of initial denaturation 30s, 40 circulations extend 30s is read after each circulation extends.Fig. 1 is molecular labeling 1-38 fluorescent PCR test map, and Fig. 2 is molecular labeling 3- 53 fluorescent PCR test maps.Sample indicates to pollute in sample without swine excrement without Ct value and without amplification curve；Sample amplification Ct Value≤35, and there is typical amplification curve, indicate that there are swine excrement pollutions in sample；The sample suggestion weight of value > 35 sample Ct It does, otherwise it is the positive that reforming result without Ct value person, which is feminine gender,.
3 standard curve of embodiment and system repeatability
By two kinds of 10 times of doubling dilutions of positive DNA standard of 1-38 and molecular labeling 3-53 to 1.2 × 108To 1.2 × 101It copies Fluorescence PCR is carried out after shellfish/μ l respectively, 3 repetitions of each dilution draw standard curve and calculate amplification efficiency.As a result table Bright two sets of molecular labelings have the wider amplification range of linearity and high amplification efficiency, respectively 1.07 and 0.95 (Fig. 3 and Fig. 4). Meanwhile the minimum detection limit of molecular labeling 1-38 and 3-53 are respectively 600 copies/reaction and 60 copies/reaction, show two sets of inspections Survey system all has good detection sensitivity.
10 repetitions are carried out respectively by glimmering PCR to two kinds of positive criteria DNA of doubling dilution to detect, molecular labeling 1-38 and For the Ct value standard deviation of 3-53 reaction system respectively between 0.01-0.28 and 0.03-0.76, the coefficient of variation is below 2.17% (table 2) shows that two sets of fluorescent PCRs based on specific molecular marker all have very high repeatability.
2 two kinds of molecular labeling fluorescent PCR system repetitive tests of table
4 batches of stability tests of embodiment
For the stability for analyzing two sets of molecular labeling fluorescence PCR methods, 4 independent detections are carried out respectively, every time when test Positive criteria DNA ploidy ratio is diluted to 1.2 × 108To 1.2 × 101Copy/μ l, each dilution are arranged 4 repetitions, calculate not With the standard deviation of dilution mean CT-number and 4 independent experiments.The result shows that molecular labeling 1-38 and 3-53 fluorescent PCR system is equal With good detection stability, detection Ct value standard deviation is only 0.32-1.56 and 0.07-0.51 respectively between different batches (Fig. 5 and Fig. 6).
The host specificity and sensibility of 5 actual sample of embodiment detection
Pig, milk cow, goat, sheep, chicken, duck, goose, dog and human faecal mass are acquired, is extracted special to the pig in the present invention after DNA Property molecular labeling and its detection architecture carries out specific (S) and sensibility verifies (R).Fluorescence PCR system and condition are same as above. And testing result is calculated by formula R=a/ (a+b) and S=c/ (c+d), wherein a and b respectively represents molecular labeling for special Property host positive sample number and false negative sample number, c and d respectively represent the negative sample that molecular labeling is directed to the detection of other species Product number and false positive sample number.The result shows that for 72 parts of swine excrement samples or the excrement of sewage of farm and 79 parts of other species Just sample, molecular labeling 1-38 fluorescent PCR detection architecture have 85% host specificity and 94% host's sensibility；3-53 Detection architecture has 90% host specificity and 99% host's sensibility (table 3), shows that two sets of molecular labelings are actually being answered Whether contain swine excrement pollution in middle test sample that can be efficiently special.
The host specificity and sensibility that 3 molecular labeling of table detects actual sample
<110>Zhejiang Entry-Exit Inspection and Quarantine Bureau；Zhejiang Prov Industrial And Commercial University
<120>it is a kind of based on host-enteric microorganism interacting genes swine excrement pollute specific molecular marker 3-53 and Its detection method
<160>8
<210>1
<211>458
<212>DNA
<213>pig-enteric microorganism
<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 AAGCCCAAAC GCCGCACCAA GGCGGAAGTG GCACGAAACA 360
ACGGTCTCGA ACCGTTGGCA AAAATAATTA TGGCACAAAA ATCGATCGAT ATTCATTCTC 420
AAGCCGGTCG TTTTATTGGG GAAAATGTGC CTGACGAA 458
<210>2
<211>458
<212>DNA
<213>pig-enteric microorganism
<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 AAGCCCAAAC GCCGCACCAA GGCGGAAGTG GCACGAAACA 360
ACGGTCTCGA ACCGTTGGCA AAAATAATTA TGGCACAAAA ATCGATCGAT ATTCATTCTC 420
AAGCCGGTCG TTTTATTGGG GAAAATGTGC CTGACGAA 458
<210>3
<211>23
<212>DNA
<213>artificial sequence
<400>3
GGAGGTGGTT AAGCCGATAT GTT 23
<210>4
<211>22
<212>DNA
<213>artificial sequence
<400>4
GCCCCTTTCT TGATACTTTG GA 22
<210>5
<211>26
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<213>artificial sequence
<400>5
AAACTGATTG GAGAAGAATA CAGGCG 26
<210>6
<211>21
<212>DNA
<213>artificial sequence
<400>6
GCGTCGTTAC ATCCTCGAAA G 21
<210>7
<211>18
<212>DNA
<213>artificial sequence
<400>7
GCGTTTGGGC TTGAATGG 18
<210>8
<211>29
<212>DNA
<213>artificial sequence
<400>8
TTCACGCATT ATGGTGTGCG ATGATGCAA 29

Claims (4)

1. a kind of primer of detection based on host-enteric microorganism interacting genes swine excrement pollution specific molecular marker 3-53 And probe, it is characterised in that be made of primer and probe below:
Upstream primer: GCGTCGTTACATCCTCGAAAG
Downstream primer: GCGTTTGGGCTTGAATGG
Probe: Fam-TTCACGCATTATGGTGTGCGATGATGCAA-Tam.
2. a kind of detection method based on the pollution of host-enteric microorganism interacting genes swine excrement, it is characterised in that: this method It is mixed using primer and probe described in claim 1 with tested sample, carries out Fluorescence PCR, extended in each circulation After read；Sample indicates to pollute in sample without swine excrement without Ct value and without amplification curve；Value≤35 sample amplification Ct, And there is typical amplification curve, indicate that there are swine excrement pollutions in sample；The sample suggestion of value > 35 sample Ct is reformed, and is reformed As a result it is feminine gender without Ct value person, is otherwise the positive.
3. detection method according to claim 2, it is characterised in that: primer and probe concentration is respectively 0.25 μM and 0.3 μ M。
4. detection method according to claim 2, it is characterised in that Fluorescence PCR condition are as follows: 95 DEG C of initial denaturation 30s, 5s and 60 DEG C of annealing of 95 DEG C of denaturation of 40 circulations extends 30s.
CN201610847949.4A 2016-09-23 2016-09-23 It is a kind of that specific molecular marker 3-53 and its detection method are polluted based on host-enteric microorganism interacting genes swine excrement Active CN106381295B (en)

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CN105255877A (en) * 2015-11-16 2016-01-20 上海市农业科学院 SNP molecular markers used in chromosome 6 of pig for traceability and application thereof
CN105296640A (en) * 2015-11-16 2016-02-03 上海市农业科学院 SNP molecular marker used for source tracing on pig chromosome 6 and application thereof

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CN105255877A (en) * 2015-11-16 2016-01-20 上海市农业科学院 SNP molecular markers used in chromosome 6 of pig for traceability and application thereof
CN105296640A (en) * 2015-11-16 2016-02-03 上海市农业科学院 SNP molecular marker used for source tracing on pig chromosome 6 and application thereof

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