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Method for detecting cowpat and detection kit

A kit and cow dung technology, which is applied in the field of biotechnology detection, can solve the problems of methods and inadequacies in the detection of cow feces pollution, and achieve the effects of high sensitivity, strong specificity, and strong stability

Inactive Publication Date: 2018-10-19
WENZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the complex fecal pollution that may occur in water, this distinction is not enough in water pollution detection
[0005] At present, there is no report on bovine Faecalibacterium, let alone a method for detecting bovine feces pollution in water bodies against bovine Faecalibacterium

Method used

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  • Method for detecting cowpat and detection kit
  • Method for detecting cowpat and detection kit
  • Method for detecting cowpat and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Primer Design

[0056] 224 animal feces samples (chicken, duck, human, dog, pig, cow) were collected, and 984,224 bacterial 16SrDNA sequences were obtained by high-throughput sequencing. By comparing the obtained sequences to the database, the 16S rDNA gene sequences of Faecalibacterium in all feces samples were selected, and detailed comparisons were made between species to obtain the specific sequence of Faecalibacterium bovine. Therefore, it is speculated that the bovine Faecalibacterium can be used as a tracer microorganism to detect and distinguish cow feces, and this will be verified in follow-up experiments.

[0057] Two pairs of DNA primers CFY-1 and CFY-2 were designed for the specific sequence of bovine Faecalibacterium. The primer sequences are shown in Table 1, and FUP is the general primer for Faecalibacterium.

[0058] Table 1 Primer Sequence

[0059]

[0060] The above primers were synthesized, and the primer synthesis was completed by Meij...

Embodiment 2

[0061] Embodiment 2 specific detection

[0062] The fecal samples of six species of chicken, duck, human, dog, pig, and cow are tested, and the specific detection steps are as follows:

[0063] 1) Extract the sample genomic DNA:

[0064] A total of 224 feces samples from six species of chicken, duck, human, dog, pig, and cow were collected using MO-BIO DNAIsolation kit extracts genomic DNA from samples;

[0065] 2) Specificity verification:

[0066] Take the genomic DNA in step 1), mix the DNA samples of the same species, use CFY-1, CFY-2, FUP (universal primer) as primers respectively, add Taq PCR Master Mix (2×) 7.5 microliters, positive Add 0.5 microliters of primers, 0.5 microliters of reverse primers, and 1 microliter of genomic DNA; and add sterile water to 15 microliters; PCR reaction, the reaction temperature is as follows:

[0067] ①CFY-1: Pre-denaturation at 96°C / 300s, 1 cycle; 96°C / 30s, 54°C / 30s, 32 cycles.

[0068] ②CFY-2: Pre-denaturation at 96°C / 300s, 1 cyc...

Embodiment 3

[0073] Embodiment 3 minimum detection limit

[0074] 1) draw quantitative PCR standard curve

[0075] ① Amplification of the target fragment: Use CFY-1 as a primer to amplify using ordinary PCR, and the amplification system is 100 μl.

[0076] ② Purification of PCR products: Configure 1% agarose gel electrophoresis, add 100ul PCR products into a large sample well, after the electrophoresis is completed, cut the gel under UV observation to recover the electrophoresis products, and use the recovery kit to purify the PCR products.

[0077] ③ Ligation and transformation of the target fragment with the vector: connect the target fragment with pMD 19-T vector, and pour it into DH5α Escherichia coli competent cells.

[0078] ④ Screening of recombinant plasmids: Escherichia coli in step ④ of blue-white screening, select white single colony liquid medium for expansion and culture.

[0079] ⑤ Extraction of recombinant plasmids: Use a plasmid extraction kit to extract recombinant plasm...

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Abstract

The invention belongs to the field of biotechnology detection and particularly relates to a method for detecting cowpat and a detection kit. Firstly, the invention verifies that the cowpat can be detected by detecting Faecalibacterium of cows; secondly, the invention provides detection primers and the detection kit for the cowpat. The detection primers can specifically react with the Faecalibacterium of the cows to generate a band while cannot react with the Faecalibacterium of chickens, ducks, human beings, dogs and pigs; in 6 specifies, the specificity of the primers reaches 100 percent. When the primers disclosed by the invention are used for detecting the Faecalibacterium of the cows, a lowest detectable limit (LOQs) is 3273 copies / [mu]l, and the sensitivity effect is excellent; when the primers are used for detecting interspecies of a cowpat sample, positive rat reaches 95.8 percent (46 / 48), and the stability is better. When the detection primers and the detection kit are used fordetecting water body pollution, whether cowpat pollution exists in the water body or not can be quickly judged and a pollution source is located.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a method for detecting cow feces and a detection kit. Background technique [0002] Cattle are one of the most widely distributed and stocked large livestock in the world, and beef is also one of the meat varieties with the largest audience. Due to the increasing demand for beef in the international market and the continued tight beef market, the number of live cattle in the world is on the rise. The number of live cattle in 2010 was 1.012 billion, and in 2013 it has increased to 1.027 billion. The increase in the number of live cattle has brought about a serious problem of cow manure pollution, and the drinking water pollution caused by its manure has brought great harm to human health and the natural environment. [0003] Most intestinal infectious diseases can be transmitted through the water environment, but the source of pollution is difficult to determine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12Q1/6837C12N15/11
CPCC12Q1/6837C12Q1/689
Inventor 孙达张坤覃弦王媛彭仁义高艺恬嵇豪
Owner WENZHOU UNIVERSITY
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