Primers to detect cow dung contamination and detection kit

A kit and technology of feces, applied in the field of biotechnology detection, can solve problems such as shortage and detection method of cow feces pollution, and achieve the effect of strong specificity, high sensitivity and good stability

Inactive Publication Date: 2018-08-17
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, the

Method used

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  • Primers to detect cow dung contamination and detection kit
  • Primers to detect cow dung contamination and detection kit
  • Primers to detect cow dung contamination and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Primer Design

[0048] 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 with the database, select the 16SrDNA gene sequence of Faecalibacterium in all feces samples, and make a detailed comparison between species, design two pairs of DNA primers CFY-1 and CFY-2, and the universal primer FUP, primer sequence As shown in Table 1:

[0049] Table 1 Primer Sequence

[0050]

[0051] The above primers were synthesized, and the primer synthesis was completed by Meiji Biomedical Technology Co., Ltd.

Embodiment 2

[0052] Embodiment 2 specific detection

[0053] The fecal samples of six species of chicken, duck, human, dog, pig, and cow are tested, and the specific detection steps are as follows:

[0054] 1) Extract the sample genomic DNA:

[0055] A total of 224 feces samples from six species of chicken, duck, human, dog, pig, and cow were collected using MO-BIO DNA Isolation kit extracts sample genomic DNA;

[0056] 2) Specificity verification:

[0057] 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; add sterile water to 15 microliters; PCR reaction, the reaction temperature is as follows:

[0058] ①CFY-1: pre-denaturation at 96°C / 300s, 1 cycle; denaturation at 96°C / 30s, annealing at 54°C / 30s, 32 cycles.

[0059] ②CFY-2: pre-denaturation...

Embodiment 3

[0064] Embodiment 3 minimum detection limit

[0065] 1) draw quantitative PCR standard curve

[0066] ① Amplification of the target fragment: Use CFY-1 as a primer to amplify using ordinary PCR, and the amplification system is 100 μl.

[0067] 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.

[0068] 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.

[0069] ④ Screening of recombinant plasmids: Escherichia coli in step ④ of blue-white screening, select white single colony liquid medium for expansion and culture.

[0070] 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 biological technology and particularly relates to primers to detect cow dung contamination and a detection kit. The detection primers can specifically react withcattle Faecalibacterium to generate a strip; chicken, duck, human, dog and pig Faecalibacterium cannot react; in the six species, the primer specificity reaches 100%. The primers applied to the detection of cattle Faecalibacterium has lowest detection limits (LOQ)s reaching 2490 c/ mu l, and the sensitivity is excellent; same-species cow dung samples in detection reach 93.8% (45/48), and good stability is provided. The detection primers and kit can be used to detect water pollution so as to judge whether cow dung contamination is present in water, and to position a pollution source.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a primer and a detection kit for detecting cow feces pollution. 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...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 孙达张坤覃弦王媛金立波林苏娥王德仲
Owner WENZHOU UNIVERSITY
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