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Quadruple PCR detection primer group and kit for simultaneously detecting shigella, salmonella, clostridium welchii and escherichia coli

A technology for Salmonella and Clostridium welchii, applied in the field of quadruple PCR detection primer sets and kits, which can solve the problems of economic loss, reduced feed conversion rate, and few patents related to pathogenic bacteria

Pending Publication Date: 2021-04-30
青岛迪诺瓦基因科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These enteric pathogenic bacteria affect the digestion and absorption of chickens, resulting in reduced feed conversion rate and slow growth, which can cause greater economic losses
[0004] At present, there are several reports on multiple PCR-related detection methods for chicken-derived bacteria. For example, the patent CN111534619A published in 2020 is about the establishment of multiple methods for chicken-derived E. CN201910677701.1 patent for multiple bacterial detection of Bacillus, Proteus mirabilis, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, and Pseudomonas maltophilia; Escherichia coli was written in the patent CN201810317451.6 in 2018 , specific Pasteurella, Proteus mirabilis, etc., but there are not many patents related to the detection of common avian enteropathogenic bacteria

Method used

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  • Quadruple PCR detection primer group and kit for simultaneously detecting shigella, salmonella, clostridium welchii and escherichia coli
  • Quadruple PCR detection primer group and kit for simultaneously detecting shigella, salmonella, clostridium welchii and escherichia coli
  • Quadruple PCR detection primer group and kit for simultaneously detecting shigella, salmonella, clostridium welchii and escherichia coli

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Effect test

Embodiment 1

[0038] The present embodiment provides simultaneous detection of Shigella, Salmonella, Clostridium welchii, a quadruple PCR detection primer set for Escherichia coli, including four pairs of primers, respectively Shigella upstream and downstream primer pairs are respectively SEQ ID NO: 1, SEQ ID NO: 2. The upstream and downstream primers for Salmonella are SEQIDNO:3 and SEQIDNO:4 respectively; the primers for Clostridium welchii type A are SEQIDNO:5 and SEQIDNO:6 respectively; the primers for Escherichia coli are SEQIDNO:7 and SEQIDNO:8 respectively.

[0039] The primers were designed in the NCBI database according to the Shigella (ipah) gene, Salmonella (invA) gene, Clostridium welchii (plc) gene, and Escherichia coli (phoA) gene to extract the corresponding base sequences.

[0040] The primer sequence information is as follows:

[0041]

Embodiment 2

[0042] Embodiment 2 mixed template PCR amplification

[0043] Put 1 μL of templates of Shigella, Salmonella, Clostridium welchii, and Escherichia coli into the mixed system. from the result figure 1Analysis: The results and sizes of the bands of interest shown in the result plots were as expected. In order to prepare for the detection of actual clinical samples in the later stage, the specificity of the established multiplex PCR method was verified.

Embodiment 3

[0044] Example 3 Mixed Template Multiplex PCR Amplification Specificity Experiment

[0045] Utilize the primers and detection method provided by the present invention to carry out mixed template multiplex PCR amplification specificity experiment, specific design is as follows:

[0046] Lane 1: Shigella 133bp + Salmonella 423bp + Escherichia coli 697bp + Clostridium welchii 840bp;

[0047] Lane 2: Shigella 133bp;

[0048] Lane 3: 423bp;

[0049] Swimming lane 4: Escherichia coli 697bp;

[0050] Swimming lane 5: Clostridium welchii 840bp;

[0051] Lane 6: Campylobacter;

[0052] Lane 7: Vibrio parahaemolyticus;

[0053] Lane 8: Staphylococcus aureus;

[0054] Lane 9: Streptococcus;

[0055] Lane 10: Proteus;

[0056] Lane 11: Vibrio cholerae;

[0057] Lane 12: Pseudomonas aeruginosa;

[0058] Lane 13: negative control.

[0059] see results figure 2 : Only Shigella, Salmonella, Clostridium welchii, and Escherichia coli were positive for the target, Campylobacter, Vib...

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Abstract

The invention discloses a quadruple PCR detection primer group for simultaneously detecting shigella, salmonella, clostridium welchii and escherichia coli, which comprises four pairs of primers, namely a shigella upstream primer pair and a shigella downstream primer pair which are respectively SEQIDNO: 1 and SEQIDNO: 2; an upstream primer pair and a downstream primer pair of salmonella are respectively SEQ ID NO: 3 and SEQ ID NO: 4; the clostridium welchii A type primer pair is shown as SEQ ID NO: 5 and SEQ ID NO: 6 respectively; and the primer pairs of the escherichia coli are respectively SEQ ID NO: 7 and SEQ ID NO: 8. A multiple detection method is established for common enteropathogenic bacteria in chicken farms, on the basis of high detection accuracy and good specificity, the detection time is shortened, the detection efficiency is improved, and the multiple detection method is more beneficial to application in large-scale breeding production.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a quadruple PCR detection primer set and a kit for simultaneously detecting Shigella, Salmonella, Clostridium welchii and Escherichia coli. Background technique [0002] Escherichia coli, Clostridium welchii, Salmonella, and Shigella are all intestinal parasites of chickens. Chicken Escherichia coli mainly parasitizes the upper intestinal tract of chickens and can cause diarrhea in chickens after infection. And will cause the death of embryos and young chicks, sepsis, peritonitis and other complications. Clostridium welchii, also known as Clostridium perfringens, parasitizes the intestinal tract of chickens. After chickens are infected with Clostridium welchii, it can cause intestinal bleeding, ulcers and necrosis. After Salmonella infection in the chicken intestines, the chickens will have diarrhea and discharge thin feces such as white paste, causing ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12Q1/04C12N15/11C12R1/42C12R1/19C12R1/145C12R1/01
CPCC12Q1/689C12Q1/686C12Q2600/16
Inventor 李贵阳李彦段笑笑沈巍
Owner 青岛迪诺瓦基因科技有限公司
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