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PCR detection system, kit and detection method for detecting vibrio parahaemolyticus

A hemolytic Vibrio and detection method technology, applied in the field of biochemistry, can solve the problems of time-consuming, cumbersome operation, and low specificity, and achieve the effects of avoiding sample loss, good compatibility, and high sensitivity

Active Publication Date: 2020-04-03
GUANGDONG SHUNDE IND DESIGN INST GUANGDONG SHUNDE INNOVATIVE DESIGN INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For the detection of Vibrio parahaemolyticus, the national standard method currently adopted in my country is GB 4789.7-2013 "National Food Safety Standard Food Microbiology Inspection (Vibrio parahaemolyticus inspection) 》Using conventional detection methods is still selective culture, biochemical identification, Kanagawa phenomenon and serological reaction, etc., but these detection methods are not only cumbersome to operate, time-consuming, but also have a low detection rate
With the rapid development of biological technology, a variety of biological techniques have been used for the detection of Vibrio parahaemolyticus, such as PCR technology. bacteria count) or false negative (pathogenic Vibrio parahaemolyticus missed), the specificity is not high, and it is impossible to distinguish the specific species of Vibrio parahaemolyticus

Method used

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  • PCR detection system, kit and detection method for detecting vibrio parahaemolyticus
  • PCR detection system, kit and detection method for detecting vibrio parahaemolyticus
  • PCR detection system, kit and detection method for detecting vibrio parahaemolyticus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] This example employs the primer pairs and detection probes shown in the table below.

[0046]

[0047] 1. First prepare the reaction system

[0048] Primer: tlh 900nM

[0049] tdh 900nM

[0050] ureR 900nM

[0051] Probe: tlh 0.125 μM

[0052] tdh 0.625 μM

[0053] ureR 0.25 μM

[0054] PCR Mix: 10μL

[0055] Add 2 μL of the bacterial solution of the sample to be tested, supplement ddH 2 0 to 20 μL.

[0056] 2. Generate microdroplets

[0057] The oil phase is 40 μL of the generated oil, and the water phase is the 20 μL reaction system prepared above. After mixing, 60,000 to 70,000 microdroplets are generated on a microdroplet generator. Transfer the generated micro-droplets to a 96-well plate, and use a heat sealer to seal the film for PCR amplification.

[0058] 3. PCR amplification

[0059]

[0060] Put the 96-well plate into the PCR instrument for PCR amplification.

[0061] 4. Result detection

[0062] After completing the PCR amplification, put t...

Embodiment 2

[0064] The method of this embodiment 2 is basically the same as that of embodiment 1, the only difference is that the bacterial liquid of the sample to be tested is extracted by a DNA extraction kit to obtain a DNA sample, and the ddPCR reaction is carried out with the DNA sample as a template, and the results are as follows: figure 2 shown.

[0065] Such as figure 1 and figure 2 As shown, the detection effect of the detection using the complete single cell as the template is equivalent to the detection effect of the detection using the DNA sample as the template, and 8 clear partitions can be obtained, which can realize the one-step method from sample to detection without DNA extraction. easy to use. in addition, figure 1 It can be seen that there is a three-gene overlapping partition, that is, a cluster containing three genes at the same time in the same cell, while figure 2 The partition does not exist in . This is because Vibrio parahaemolyticus has two chromosomes...

Embodiment 3

[0067] The method of this example is basically the same as that of Example 1, the only difference is the use of primer pairs and detection probes as shown in the table below. Test results such as image 3 As shown, 16 clear partitions can be obtained, and it can be seen that the effect of quadruple PCR detection using the complete cell as a template is still relatively good.

[0068]

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Abstract

The present invention relates to a PCR detection system, a kit and a detection method for detecting vibrio parahaemolyticus. The PCR detection system comprises a first primer pair and a first detection probe for detecting tlh gene, a second primer pair and a second detection probe for detecting tdh gene, and a third primer pair and a third detection probe for detecting ureR gene. The PCR detectionsystem for detecting the vibrio parahaemolyticus is based on simultaneous detection of three genes of the tlh, tdh and ureR, and can accurately distinguish specific types of the vibrio parahaemolyticus. The PCR detection system has good compatibility, very high specificity and sensitivity, and a detection limit of 15 copies / ml. At the same time, the PCR detection system can directly use cells asa template for detection, and a detection effect is equivalent to that of using DNA as a template. Therefore, a one-step method from samples to detection can be achieved without DNA extraction of thesamples.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a PCR detection system, a kit and a detection method for detecting Vibrio parahaemolyticus. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus, Vp) is a Gram-negative halophilic bacterium belonging to the genus Vibrio, which is widely found in seawater, crabs, shrimps, mackerel, sardines, cod, scallops, and oysters near the coast. It is one of the main pathogens causing seafood vibriosis in seafood and salted foods, and is also an important food-borne pathogen. Using traditional culture methods to detect virulent strains in clinical and food samples can meet the actual enumeration requirements, but because virulent strains have no obvious growth phenotype, they may not be able to be distinguished from non-virulent strains. [0003] The detection rate of Vibrio parahaemolyticus in seafood is very high, sometimes up to 100%. However, 95% of Vibrio parahaemo...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/686C12Q2563/107Y02A50/30
Inventor 古晓奎钟青萍雷舒文
Owner GUANGDONG SHUNDE IND DESIGN INST GUANGDONG SHUNDE INNOVATIVE DESIGN INST
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