A multiple PCR primer used for simultaneously detecting five pathogenic bacteria and three virulence genes in marine, and a designing method thereof

A technology for virulence genes and pathogenic bacteria, which is applied in the field of multiplex PCR primers and can solve problems such as loss of bands and loss of target fragments.

Active Publication Date: 2014-09-03
ZHEJIANG WANLI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is because a single pair of primers can amplify a single template to amplify the target fragment with clear bands, but the phenomenon of band loss will occur after the amplification of various primers is mixed.
There is also a competitive r

Method used

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  • A multiple PCR primer used for simultaneously detecting five pathogenic bacteria and three virulence genes in marine, and a designing method thereof
  • A multiple PCR primer used for simultaneously detecting five pathogenic bacteria and three virulence genes in marine, and a designing method thereof
  • A multiple PCR primer used for simultaneously detecting five pathogenic bacteria and three virulence genes in marine, and a designing method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0100] Using the primer design software Oligo6.0 to design a multiplex PCR for the simultaneous detection of Enterococcus faecalis, Edwardsiella tarda, Carnobacterium thuringiensis, Vibrio alginolyticus and Enterobacter cloacae gyrB gene, Vibrio parahaemolyticus tlh gene, Enterococcus faecalis cylA gene The primer combination, the formed primer combination contains 23 primer bases, the melting temperature Tm value of the primer is 60°C, and the GC% of the primer is 60%;

[0101] Step 2, screening the primers in the primer combination, retaining primers that do not form primer dimers;

[0102] Step 3, judging the competitive status of the retained primers, comparing the GC% of the above-mentioned retained primers with the number of bases, when the base numbers of the primers are the same and the GC% of the inner primer is less than the GC% of the outer primer, or When the number of bases of the primers is not exactly the same and the base number of the inner primer is one base ...

Embodiment 2

[0123] Example 2 Multiple PCR primer design method for simultaneous detection of Enterococcus faecalis, Edwardsiella tarda, Carnobacterium thuringiensis, Vibrio alginolyticus and Enterobacter cloacae gyrB gene, Vibrio parahaemolyticus tlh gene, Enterococcus faecalis cylA gene , the method includes the following steps:

[0124] Step 1, use the primer design software Primer Premier5.0 to design and simultaneously detect Enterococcus faecalis, Edwardsiella tarda, Carnobacterium thuringiensis, Vibrio alginolyticus and Enterobacter cloacae gyrB gene, Vibrio parahaemolyticus tlh gene, Enterococcus faecalis cylA The multiple PCR primer combination of the gene, the number of primer bases contained in the formed primer combination is 22, the melting temperature Tm value of the primer is 54°C, and the GC% of the primer is 40%;

[0125] Step 2, screening the primers in the primer combination, retaining primers that do not form primer dimers;

[0126] Step 3, judging the competitive stat...

Embodiment 3

[0148] 1. Design and synthesize primers for Enterococcus faecalis, Edwardsiella tarda, Carobacillus, Bacillus thuringiensis and Vibrio alginolyticus, Enterobacter cloacae gyrB gene, Vibrio parahaemolyticus tlh gene, Enterococcus faecalis cylA gene

[0149] Referring to the corresponding gene sequences of the marine isolates and the corresponding gene sequences of the four bacteria published on GenBank, a pair of specific primers were designed by using primer express2.0. The size of the PCR amplified fragment corresponding to the primers is 708bp, the size of the PCR amplified fragment corresponding to the Bacillus thuringiensis primer is 566bp, the size of the PCR amplified fragment corresponding to the primer of Bacillus thuringiensis is 458bp, and the size of the PCR amplified fragment corresponding to the primer of Vibrio alginolyticus is 159bp, the size of the PCR amplification fragment corresponding to the primers of the Enterobacter cloacae gyrB gene is 1250bp, the size o...

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Abstract

The invention relates to a multiple PCR primer used for simultaneously detecting five pathogenic bacteria and three virulence genes in marine, and a designing method thereof. The method includes: designing a multiple PCR primer composition capable of simultaneously detecting enterococcus faecalis, edwardsiella tarda, carnobacterium, bacillus thuringiensis, vibrio alginolyticus, an enterobacter cloacae gyrB gene, a vibrio parahaemolyticus tlh gene, and an enterococcus faecalis cylA gene by utilization of a primer designing software, screening primers dominant in competitive states, subjecting the primers dominant in the competitive states to amplification and verification by utilization of a PCR method, and finally removing primers weak in the competitive states in the amplification primers so as to obtain a multiple PCR primer composition. A detection method of the multiple PCR is also disclosed. Compared with the prior art, the multiple PCR primer, the designing method and the detection method are advantageous in that: the multiple PCR primer composition simultaneously detecting the five pathogenic bacteria and the three virulence genes in marine has advantages of simple and convenient operation, rapidness, strong specificity, high sensitivity, and the like.

Description

technical field [0001] The invention belongs to the field of microbial detection, and specifically relates to a method for simultaneously detecting marine Enterococcus faecalis, Edwardsiella tarda, Carnobacterium thuringiensis, Vibrio alginolyticus, Enterobacter cloacae gyrB gene, Vibrio parahaemolyticus tlh gene, fecal enterococci Multiplex PCR primers of coccus cylA gene and its design method. Background technique [0002] Enterococcus faecalis, Edwardsiella tarda, Carobacillus, Bacillus thuringiensis and Vibrio alginolyticus are pathogenic bacteria that can be transmitted through food and water. There are certain differences in their biological characteristics and pathogenicity, but they can all cause food poisoning and digestive system diseases. [0003] Enterococcus faecalis is ubiquitous in nature and can cause many diseases such as endocarditis, cholecystitis, meningitis, urinary tract infection and wound infection. It has low nutritional requirements and can also g...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12Q1/04C12N15/11C12N15/10
CPCC12Q1/6811C12Q1/686C12Q2537/143C12Q2531/113Y02A50/30
Inventor 杨季芳管峰陈吉刚毛芝娟
Owner ZHEJIANG WANLI UNIV
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