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Campylobacteria sixtuple PCR detection kit

A detection kit, Campylobacter technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of complex identification process, shorten detection time, reduce detection cost, specificity strong effect

Inactive Publication Date: 2016-06-22
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the identification of different strains of Campylobacter mainly depends on the characteristics of biochemical reactions and PCR technology for different strains, and the identification process is complicated and tedious.

Method used

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  • Campylobacteria sixtuple PCR detection kit
  • Campylobacteria sixtuple PCR detection kit
  • Campylobacteria sixtuple PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 Specific determination of sequence and screening of primers

[0053] Obtain the specific genes and specific nucleic acid sequences of Campylobacter genus 16s, Campylobacter jejuni, Campylobacter coli, Campylobacter fetalis, Campylobacter gullium and Campylobacter uppsala species, using BLASTn and vectorNITsuite6.0 software The DNA sequences of all specific genes were compared to determine 6 specific sequence fragments, and the NCBI database was screened to determine the DNA sequence specificity (including all sequenced strains).

[0054] 1. Screening of Campylobacter-specific DNA sequences and primers:

[0055] Usually, the common nucleic acid within the bacterial genus is mostly 16srRNA or 23srRNA sequence, which can be used as the main target gene of the specific nucleic acid fragment within the bacterial genus. By comparing the 16srRNA nucleic acid sequence of the genus Campylobacter, the specific nucleic acid for the genus Campylobacter can be obtained ...

Embodiment 2

[0071] Example 2 Establishment of Campylobacter six-fold PCR detection method

[0072] The 6 pairs of specific primers screened in Example 1 were used as six-fold PCR amplification primers, and bacterial DNA was used as a template to establish a target for Campylobacter jejuni, Campylobacter coli, Campylobacter fetalis, Campylobacter seagull and Campylobacter uppsala Six-plex PCR detection method for five species of Campylobacter.

[0073] 1. Culture medium and reagent preparation

[0074]1.1 Columbia basal medium: Weigh 13.85g of Columbia agar and place in 300mL of distilled water to mix well, then pressurize at 121°C for 15 minutes for later use. Cool to 50-54°C, add 5% defibrinated sheep blood, mix evenly, pour into 90mm plate for sterility test and set aside.

[0075] 1. 22% agarose gel: Weigh 2g agarose, add 20ml 5×TBEbuffer, add distilled water to 100ml, mix well, heat in a microwave oven until the agarose is completely dissolved, cool to 60°C, add 2μl Gel-Red dye, mix...

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Abstract

The invention provides a campylobacteria sixtuple PCR detection kit, and belongs to the technical field of bacterium PCR detection. The kit contains six pairs of primers which have nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.12. The kit can authenticate whether a to-be-detected strain is campylobacteria or not and authenticate the five frequently-seen strains of campylobacteria including campylobacter jejuni, campylobacter coli, campylobacter fitus, campylobacter loridis and campylobacter liensis. The kit has sensitivity and specificity of 100% on detecting the five strains of campylobacteria, is easy to operate, greatly reduces detection cost and operation difficulty, and has good clinical application value.

Description

technical field [0001] The invention relates to the technical field of bacteria detection, in particular to a six-fold PCR detection kit for detecting Campylobacter jejuni, Campylobacter coli, Campylobacter fetalis, Campylobacter gullifera and Campylobacter uppsala. Background technique [0002] Campylobacter is the main food-borne pathogen, and the disease caused by its infection is called campylobacteriosis, which is one of the emerging infectious diseases of human beings. At present, Campylobacter infection in European and American countries is listed as the first food-borne pathogen infection, and its infection rate is significantly higher than that of other food-borne pathogens. In addition to enteritis, infection with Campylobacter jejuni can lead to Guillain-Barré syndrome (GBS). GBS is an autoimmune cause of acute neuromuscular paralysis, with a disability rate of 7-15% and a mortality rate of 3%-10%. Currently, there is no specific drug and the disease burden is he...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143Y02A50/30
Inventor 张茂俊王敏张建中
Owner ICDC CHINA CDC
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