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Multiplex PCR primer for synchronously detecting vibrio parahaemolyticus and vibrio alginolyticus, and design method thereof

A technology of Vibrio alginolyticus and Vibrio hemolyticus, which is applied in the field of multiplex PCR primers and their design, can solve problems such as shortening detection time and detection cost, and achieve the effects of convenient customs clearance speed, strong specificity and high sensitivity

Active Publication Date: 2012-01-11
INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The improved technology of multiplex PCR simplifies the detection process of food microorganisms, shortens the detection time, reduces the detection cost and improves the sensitivity of food microorganism detection; however, there are some problems in the improved technology of multiplex PCR

Method used

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  • Multiplex PCR primer for synchronously detecting vibrio parahaemolyticus and vibrio alginolyticus, and design method thereof
  • Multiplex PCR primer for synchronously detecting vibrio parahaemolyticus and vibrio alginolyticus, and design method thereof

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Embodiment 1

[0048] A method for designing multiple PCR primers for simultaneous detection of Vibrio parahaemolyticus and Vibrio alginolyticus, the method comprising the following steps:

[0049] Step 1, using the primer design software Oligo6.0 to design multiple PCR primer combinations for simultaneous detection of Vibrio parahaemolyticus and Vibrio alginolyticus, the formed primer combination contains 23 primer bases, and the melting temperature Tm value of the primers 56°C-63°C (60°C is the best), the GC% of the primer is 40%;

[0050] Step 2, screening the primers in the primer combination, retaining primers that cannot synthesize primer dimers;

[0051] Step 3, judging the competitive status of the retained primers, comparing the GC% of the above-mentioned retained primers with the base number, 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 ...

Embodiment 2

[0061] The multiple PCR primer design method for simultaneously detecting Vibrio parahaemolyticus and Vibrio alginolyticus is characterized in that the method comprises the following steps:

[0062] Step 1, using the primer design software Primer5.0 to design multiple PCR primer combinations for simultaneous detection of Vibrio parahaemolyticus and Vibrio alginolyticus, the formed primer combination contains 22 primer bases, and the melting temperature Tm value of the primers 56°C-63°C (preferably 60°C), the GC% of the primer is 60%;

[0063] Step 2, screening the primers in the primer combination, retaining primers that cannot synthesize primer dimers, performing PCR amplification on the primers used, and screening the primer combination with the best amplification effect;

[0064] Step 3, judging the competitive status of the retained primers, comparing the GC% of the above-mentioned retained primers with the base number, when the base numbers of the primers are the same and...

Embodiment 3

[0074] 1. Design and synthesize primers for Vibrio parahaemolyticus and Vibrio alginolyticus

[0075] Referring to the toxR gene sequences of Vibrio parahaemolyticus and Vibrio alginolyticus published on GenBank, a pair of specific primers were designed by using Primer2.0. The fragment size is 159 bp, synthesized by Shanghai Dingan Biotechnology Co., Ltd.;

[0076] V. para. (ToxR) F1: 5'-CGAAAGCCGTATACTCCTGATG-3'

[0077] V. para. (ToxR) R1: 5'-GAGTTGATAGCCTCGTTTTGGA-3'

[0078] V. algi. (ToxR) F1: 5'-GATCGTTTTGACCTGCGAAA-3'

[0079] V. algi.(ToxR) R1:5'-GCATTGAGCGCTACGTGAA-3';

[0080] 2. Explore the extraction method of bacterial template

[0081] According to the "Guidelines for Molecular Biology Experiments", five methods were used to extract bacterial DNA, including traditional methods (phenol-chloroform extraction method), direct bacterial cell addition method, repeated freeze-thaw method, thermal cracking method and kit extraction method. The extracted DNA templa...

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Abstract

The invention relates to multiplex PCR primer for amplifying sequences of vibrio parahaemolyticus and vibrio alginolyticus, and a design method thereof. The design method comprises the following steps: 1, adopting a primer design software to design a series of multiplex PCR primer compositions for simultaneous amplification of the sequences of the vibrio parahaemolyticus and the vibrio alginolyticus; 2, screening the multiplex PCR primer compositions, and selecting the primer composition with the best amplification effect; 3, carrying out reaction condition selection for the selected best primer composition; 4, carrying out selections of specificity, reproducibility, stability and the like for the established method. According to the present invention, the problem of long separation and detection period of the conventional bacterial during the detection process of the vibrio parahaemolyticus and the vibrio alginolyticus is solved; the vibrio parahaemolyticus and the vibrio alginolyticus are detected synchronously; advantages of rapidness, specificity, sensitivity, cost saving and the like are provided.

Description

technical field [0001] The invention relates to the field of detection of Vibrio parahaemolyticus and Vibrio alginolyticus, in particular to a multiplex PCR primer for simultaneous detection of Vibrio parahaemolyticus and Vibrio alginolyticus and a design method thereof. Background technique [0002] Both Vibrio parahaemolyticus and Vibrio alginolyticus belong to the genus Vibrio, and are pathogenic bacteria transmitted through food and water. Vibrio alginolyticus and Vibrio parahaemolyticus have similar biological characteristics and the same pathogenicity, and both can cause diarrhea and food poisoning. [0003] Vibrio parahaemolyticus was first isolated from a food poisoning patient in Japan by Fujino et al. in 1953. It is also called halophilic bacteria, because it can grow in medium containing more salt (3%-4%) , so named because they cannot reproduce in a medium without salt. Vibrio parahaemolyticus is distributed all over the world, and usually exists in bays and co...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12N15/10
CPCY02A50/30
Inventor 孔繁德徐淑菲刘阳陈信忠龚艳清杨俊萍郭树林
Owner INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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