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Multiplex PCR primer set, detection method and kit for simultaneously detecting four kinds of pathogenic vibrio bacteria

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
After searching domestic and foreign literature, there are no relevant reports on the application of multiplex PCR in the rapid detection of Vibrio cholerae, Vibrio alginolyticus, Vibrio riverine and Vibrio eel

Method used

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  • Multiplex PCR primer set, detection method and kit for simultaneously detecting four kinds of pathogenic vibrio bacteria
  • Multiplex PCR primer set, detection method and kit for simultaneously detecting four kinds of pathogenic vibrio bacteria
  • Multiplex PCR primer set, detection method and kit for simultaneously detecting four kinds of pathogenic vibrio bacteria

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Design and screening of embodiment 1 primer (represented by Vibrio cholerae)

[0075] According to the virulence genes and housekeeping gene sequences of Vibrio cholerae published on GenBank, combined with relevant literature reports, the candidate amplified DNA fragments of Vibrio cholerae were obtained, that is, the DNA fragments of Vibrio cholerae among different biotypes or serotypes of Vibrio cholerae strains The sequence of OmpW, a highly conserved outer membrane protein gene, was designed using Oligo7 and Primer Primer5.0 software, and screening was performed among the primers with high scores. After proper manual adjustment and modification, the obtained primer design scheme was published on the NCBI website. Perform Blast analysis. If any primer pair has cross-reaction with non-target bacteria, the position and sequence length of the primers need to be readjusted until a highly specific OmpW gene primer sequence is obtained. Finally, two primer alternatives as ...

Embodiment 2

[0093] Embodiment 2 simultaneously detects the formation of the multiple PCR detection kit of four kinds of pathogenic Vibrio

[0094] 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:

[0095] Table 5 Configuration of Multiplex PCR Detection Kit

[0096]

[0097]

Embodiment 3

[0098] Embodiment 3 detects the kit multiplex PCR detection test in common agarose gel electrophoresis

[0099] Test samples: select the pathogen genome DNA of Vibrio cholerae, Vibrio alginolyticus, Vibrio riverine and Vibrio anguillarum as the template of the test, the concentration of each template is about 10 5 CFU / ml.

[0100] Kit assembly: the kit in Example 2 was used.

[0101] 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 procedures: 93°C pre-denaturation for 5 minutes; 92°C denaturation for 40 seconds, 58°C annealing for 1 minute, 72°C extension for 1.5 minutes, cycle 35 times; extend at 72°C for 7min.

[0102] The results of the multiplex PCR detection test were as follows: figure 1 shown.

[0103] Depend on figure 1 It can be seen that the corresp...

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Abstract

The invention discloses a multiplex PCR primer set, detection method and kit for simultaneously detecting four kinds of pathogenic vibrio bacteria. The detection primer set comprises primer pairs fordetecting vibrio cholerae, vibrio alginolyticus, vibrio fluvialis and vibrio anguillarum. The multiplex PCR detection method and the kit are established. The primer set obtained through design is utilized to perform a multiplex PCR reaction on genomic DNA of the bacteria extracted from a to-be-tested sample in the same reaction system, and by means of electrophoresis analysis of a reaction product, whether or not the sample contains thepathogenic vibrio bacteria is determined. The multiplex PCR primer set, detection method and kit for simultaneously detecting four kinds of pathogenic vibrio bacteria have the advantages of being high in detection speed, 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 (Vibrio) is one of the common dominant bacterial flora in the marine environment. It is short, facultatively anaerobic, and grows in alkaliphiles. Gram-negative bacteria widely distributed in bays, estuaries, brackish water, sediments, and surfaces and guts of marine organisms. [0003] According to the DNA homology, antigenicity, biochemical characteristics and pathogenicity of bacteria, the World Health Organization divides Vibrio into four categories: O1 Vibrio cholerae, non-O1 Vibrio cholerae, atypical O1 Vibrio cholerae and other vibrios. There are many kinds of Vibrio. Since Filippo Pacini first named the microorganism isolated from cholera patients as Vibrio in 1854, the increasing intensity of mariculture and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/63
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2600/166C12Q2537/143C12Q2545/101Y02A50/30
Inventor 陈鲤群黄蓝青林峻蔡伟文赵依依
Owner FUZHOU UNIV
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