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A specific molecular marker 1-38 for porcine feces contamination based on host-gut microbe interaction genes and its detection method

A technology of molecular markers and detection methods, applied in the field of detection of pig feces pollutants, can solve the problems of high misjudgment rate, insufficient host specificity, and insufficient accuracy of animal sources, etc., and achieve the effect of strong specificity and high sensitivity

Active Publication Date: 2019-02-05
ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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
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

Method used

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  • A specific molecular marker 1-38 for porcine feces contamination based on host-gut microbe interaction genes and its detection method
  • A specific molecular marker 1-38 for porcine feces contamination based on host-gut microbe interaction genes and its detection method
  • A specific molecular marker 1-38 for porcine feces contamination based on host-gut microbe interaction genes and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Enrichment and screening of specific pig-gut microbial interaction genes

[0024] 1.1 Collect 145 manure samples from 6 species, including 34 pig manure samples, 20 cow manure samples, 12 goat manure samples, 8 sheep manure samples, 7 chicken manure samples, 20 duck manure samples, 5 samples of goose feces and 13 samples of human feces. Place the sample in a sterile container, add 3 mL of GITC buffer (5M guanidine isothiocyanate, 100Mm EDTA (pH 8.0), sodium lauryl sarcosinate) to every 200 mg (wet weight) of the sample, and store at low temperature. Fecal DNA extraction press The instructions for Fast DNA Stool Kit (Qiagen, Valencia, CA) were performed. And its concentration and purity were measured on NanoDrop ND-2000UV (NanoDrop Technologies, Thermofisher).

[0025] 1.2 Host (pig) specific genome enrichment (GFE)

[0026] The genomic DNA of all single pig samples are mixed to form a genomic mixed library to increase the diversity of the genome (the group to be ...

Embodiment 2

[0043] Example 2 Screening of molecular markers and reaction conditions

[0044] Competitive hybridization gene fragment enrichment (GFE) method was used to establish pig-specific DNA gene library. After cloning the library into pCR TOPO 4.0, 500 clones were randomly selected for sequencing analysis, and a total of 382 non-redundant pig-source specific sequences were obtained. The BLAXTS comparison analysis revealed that 60 of the non-redundant sequences were related to the Bacteroidetes, Clostriials and other surface proteins, membrane secreted proteins and carbohydrate metabolism proteins and other host-microbe interaction proteins. As a target for pig-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 feces contamination (Table 1).

[0045] Table 1 Molecular marker sequence

[0046]

[0047] Using positive DNA as a template, repeated experiments found that ...

Embodiment 3

[0050] Example 3 Standard curve and system repeatability

[0051] Dilute the two positive DNA standards of 1-38 and molecular marker 3-53 by 10 times to 1.2×10 8 To 1.2×10 1 After copying / μl, perform fluorescent PCR reaction separately, with 3 replicates for each dilution, draw a standard curve to calculate the amplification efficiency. The results show that the two sets of molecular markers have a wide linear range of amplification and high amplification efficiency, respectively 1.07 and 0.95 ( image 3 with Figure 4 ). At the same time, the minimum detection limits of molecular markers 1-38 and 3-53 are 600 copies / reaction and 60 copies / reaction, respectively, indicating that both sets of detection systems have good detection sensitivity.

[0052] The two positive standard DNAs diluted by multiples were tested in 10 replicates by fluorescence PCR. The standard deviations of the Ct values ​​of the molecular markers 1-38 and 3-53 reaction systems were between 0.01-0.28 and 0.03-0....

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Abstract

The invention relates to the field of the detection of pollutants in the excrement of a pig, and particularly relates to a host-enteric microbial interacting gene-based specific molecular marker 1-38 and a detection method for pollution by the excrement of the pig. According to the host-enteric microbial interacting gene based specific molecular marker 1-38 and the detection method for the pollution by the excrement of the pig, a specific metagenome of the pig is enriched by adopting a competitive hybrid gene fragment enrichment (GFE) method; a metagenome library is constructed; the sequential flora and function classification is carried out on the library; thus, a specific gene relevant to a host (pig)-enteric microbial interacting target point is screened in a targeting manner; the molecular marker is designed aiming at the specific interacting genet of the pig; a monitoring method which is high in sensitivity and high in specificity and can be used for efficiently indicating a pollution source of the excrement is established.

Description

Technical field [0001] The present invention relates to the detection field of pig feces pollutants, in particular to a specific molecular marker 1-38 of pig feces pollution based on host-gut microbial interaction genes and a detection method thereof. Background technique [0002] Pork products are one of the favorite meat products of domestic consumers, and its huge demand has led to the rapid development of the pig industry. According to statistics from the Ministry of Agriculture, in recent years, the number of slaughtered pigs in my country has been the largest in the world, accounting for 56% and maintaining a growth trend. Due to the lack of effective management of emissions from the rapidly developing intensive livestock and poultry breeding industry, most of the livestock and poultry manure and sewage from pigs enter the environment through non-point sources, causing pollution and health threats. Pig fecal excrement not only contains a large number of pathogenic microorg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/689C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2563/107
Inventor 帅江冰傅玲琳张晓峰莫虹斐曾若雪何永强
Owner ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE