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Multiplex PCR primer set and detecting method and kit for simultaneous detection of four pathogenic Vibrio

A technology for detecting primers and primer sets, applied in the field of microbial detection, can solve the problems of difficult PCR system and complex influencing factors, etc.

Inactive Publication Date: 2018-08-10
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Double and triple PCR detection have been reported repeatedly, but due to the complex factors affecting multiple PCR, different primers, templates, primer concentrations, template concentrations, Mg 2+ Concentration, dNTP concentration and its ratio will produce complex comprehensive effects, so the more target microorganisms are detected at the same time, the more difficult it is to establish a PCR system
According to domestic and foreign literature inquiries, there are no relevant reports on the application of multiplex PCR in the rapid detection of Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio harveyi and Vibrio mimicus

Method used

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  • Multiplex PCR primer set and detecting method and kit for simultaneous detection of four pathogenic Vibrio
  • Multiplex PCR primer set and detecting method and kit for simultaneous detection of four pathogenic Vibrio
  • Multiplex PCR primer set and detecting method and kit for simultaneous detection of four pathogenic Vibrio

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Experimental program
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Effect test

Embodiment 1

[0075] Design and screening of embodiment 1 primer (represented by Vibrio parahaemolyticus)

[0076] According to the virulence gene and housekeeping gene sequence of Vibrio parahaemolyticus published on GenBank, combined with relevant literature reports, a candidate amplified DNA fragment of Vibrio parahaemolyticus, namely the flaE gene of Vibrio parahaemolyticus was obtained , use Oligo7 and Primer Primer5.0 software to design primers, screen among the primers with high scores, adjust and modify them manually, and then perform Blast analysis on the obtained primer design scheme on the NCBI website. There is a cross-reaction in the target bacteria, and the position and sequence length of the primers need to be readjusted until a highly specific flaE gene primer sequence is obtained. Finally, two primer alternatives as shown in Table 4 were obtained.

[0077] When designing screening primers, you need to pay attention to the following points:

[0078] (1) Primers should be d...

Embodiment 2

[0095] Example 2 is used to simultaneously detect the formation of multiplex PCR detection kits of four kinds of pathogenic Vibrio

[0096] The kit consists of PCR reaction system buffer (100mM Tris-HCl buffer (pH8.3), 15mM Mg 2+ , 500mMKCl), Taq DNA polymerase (5U / μl), dNTP, primer mixture (four kinds of pathogenic Vibrio primers and IAC primer mixture), positive control template (mixed template of four kinds of pathogenic Vibrio, each 10 6 CFU / ml), double distilled water, the reaction system of the kit is 20ul, the specific configuration is as follows:

[0097] Table 5. Configuration of Multiplex PCR Detection Kit

[0098]

Embodiment 3

[0099] Embodiment 3 detects the kit multiplex PCR detection test in common agarose gel electrophoresis

[0100] Test samples: select the pathogen genome DNA of Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio harveyi and Vibrio mimicus as the template of the test, the concentration of each template is about 10 5 CFU / ml.

[0101] Kit assembly: the kit in Example 2 was used.

[0102] Kit operation: 2mL bacterial suspension, extract template DNA according to the instructions of the bacterial genome extraction kit. Put the PCR tube into the Bio-Rad C1000 PCR instrument, open the hot lid, and carry out the PCR reaction according to the following procedure: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 40 seconds, 57°C annealing for 1 minute, 72°C extension for 1.5 minutes, cycle 35 times; extend at 72°C for 10 min.

[0103] The results of the multiplex PCR detection test were as follows: figure 1 shown.

[0104] Depend on figure 1 It can be seen that the corres...

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Abstract

The invention discloses a multiplex PCR primer set and detecting method and kit for simultaneous detection of four pathogenic Vibrio. A detection primer set comprises primer pairs for detecting Vibrioparahaemolyticus, Vibrio vulnificus, Vibrio harveyi and Vibrio mimicus. The invention further provides a multiplex PCR detecting method and kit for the simultaneous detection of the four pathogenic Vibrio, by means of the primer set obtained by designing, multiplex PCR reaction is conducted on genome DNA of bacteria extracted from to-be-detected samples in the same reaction system, and whether ornot the pathogenic Vibrio is contained in the samples is determined according to the electrophoretic analysis on reaction products. The multiplex PCR primer set and detecting method and kit have theadvantages of being rapid and accurate, low in cost and high in specificity and sensitivity.

Description

technical field [0001] The invention belongs to the field of microorganism detection, and relates to a multiple PCR detection primer set, a detection method and a kit for simultaneously detecting four pathogenic Vibrio. Background technique [0002] Vibrio belongs to the genus Vibrio in the Vibrio family (Vibrionaceae), which also includes three genera including Aeromonas, Plesiomonas and Photorhabdus , their common feature is a group of Gram-negative bacilli that are straight or slightly curved, have extreme flagella, motility positive, oxidase positive, and ferment glucose. Except for Photobacterium, the other three genera are pathogenic to humans. [0003] Vibrio is one of the common dominant bacterial flora in the marine environment. In recent years, with the expansion of the scale of marine fish farming and the impact of negative factors brought about by high-density intensive farming, Vibrio diseases of fish and shellfish have occurred from time to time, serious hinde...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/63
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2600/166C12Q2537/143C12Q2545/101
Inventor 陈鲤群黄蓝青林峻蔡伟文赵依依
Owner FUZHOU UNIV
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