Unlock instant, AI-driven research and patent intelligence for your innovation.

A pig-intestinal microorganism specific interaction gene 3-53 indicating pig feces contamination and its application

A technology of intestinal microorganisms and pig feces, which is 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: 2019-07-26
ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
View PDF3 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A pig-intestinal microorganism specific interaction gene 3-53 indicating pig feces contamination and its application
  • A pig-intestinal microorganism specific interaction gene 3-53 indicating pig feces contamination and its application
  • A pig-intestinal microorganism specific interaction gene 3-53 indicating pig feces contamination and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Enrichment screening of specific pig-intestinal microorganism interaction genes

[0020] 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).

[0021] 1.2 Host (pig) specific genome enrichment (GFE)

[0022] Genomic DNA of all single pig source samples was mixed to form a genomic mixed library to increase the diversit...

Embodiment 2

[0039] Screening and reaction conditions of embodiment 2 molecular markers

[0040] 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).

[0041] Table 1 Molecular marker sequence

[0042]

[0043]

[0044] Using positive DNA as a template, repeated experiments found that wh...

Embodiment 3

[0047] Example 3 standard curve and system repeatability

[0048] Dilute the two positive DNA standards 1-38 and 3-53 molecular markers 10 times to 1.2×10 8 to 1.2×10 1 After the copy / μl, the fluorescent PCR reaction was carried out separately, and each dilution was repeated three times, and the standard curve was drawn to calculate the amplification efficiency. The results showed that the two sets of molecular markers had wide amplification linear range and high amplification efficiency, which were 1.07 and 0.95 ( image 3 and Figure 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.

[0049] 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 bet...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to the field of porcine fecal pollutant detection, and particularly relates to a porcine-intestinal microorganism specific interactive gene 3-53 for indicating porcine fecal pollution and application thereof. The sequence of the porcine-intestinal microorganism specific interactive gene 3-53 for indicating porcine fecal pollution is as shown in SEQ ID NO:2. According to the invention, porcine specific metagenomes are enriched through a competitive hybridization genome fragment enrichment (GFE) method to construct a metagenome library, and sequence flora and function classification is performed on the library, thereby realizing targeted screening of the host (porcine)-intestinal microorganism interactive target spot related specific gene, designing a molecular marker for the porcine specific interactive gene, and establishing a monitoring method capable of efficiently indicating a fecal pollution source with high sensitivity and strong specificity.

Description

technical field [0001] The invention relates to the field of detection of pig feces pollutants, in particular to a pig-intestinal microorganism specific interaction gene 3-53 indicating pig feces pollution and its application. Background technique [0002] 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 micro...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12Q1/689C12N15/11
CPCC07K14/195C07K14/33C12Q1/689
Inventor 帅江冰张晓峰曾若雪莫虹斐何永强
Owner ZHEJIANG ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE