RPA (Recombinase Polymerase Amplification) specific primers and kit for detecting vibrio parahaemolyticus in food and application

A specific technology for Vibrio hemolyticus, applied in the direction of DNA / RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of different temperatures and limited application range, etc., to achieve sensitive detection methods and a wide range of applications , the effect of short detection time

Active Publication Date: 2017-07-21
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional PCR-based method requires three steps of denaturation, annealing, and extension. The temperature of each ste...

Method used

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  • RPA (Recombinase Polymerase Amplification) specific primers and kit for detecting vibrio parahaemolyticus in food and application
  • RPA (Recombinase Polymerase Amplification) specific primers and kit for detecting vibrio parahaemolyticus in food and application
  • RPA (Recombinase Polymerase Amplification) specific primers and kit for detecting vibrio parahaemolyticus in food and application

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Experimental program
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Embodiment 1

[0043] The selection of embodiment 1 target gene and the design of RPA primer

[0044] The known specific genes of Vibrio parahaemolyticus were analyzed by bioinformatics, and the target gene toxR was selected for detection. Through the public BLAST software in Genbank, the sequence of the toxR gene was compared with other microorganisms, and the sequence segment with higher specificity was selected (GeneBank ID: AB029908). Use the software Primer 5.0 to design specific primers in this sequence according to the requirements of RPA primer design, and use bioedit to compare the primers with species with high homology of Vibrio parahaemolyticus, see figure 1 , 2 , from which primer pairs with low homology to other species were selected. The invention designs RPA primers of Vibrio parahaemolyticus, and the product size is 426bp. The specific sequence is as follows:

[0045] Upstream primer F14: 5'-ACTCGTATGAGAACGTGACATTGCGTATTT-3'; (SEQ ID No: 1)

[0046] Downstream primer R1...

Embodiment 2

[0047] The design and construction of embodiment 2 amplification internal standard

[0048] After amplifying the internal standard primers YXF and YXR, and PCR amplification, the reaction conditions were: 94°C for 5 min; 94°C for 30 s, 59°C for 60 s, 72°C for 30 s, 30 cycles; 72°C for 10 min. The Vibrio parahaemolyticus fragment CP011406 (271bp) with a very low degree of homology to the target gene fragment was obtained. Target detection primers F14 and R14 were connected to the 5' ends of the amplification internal standard primers YXF and YXR to obtain long primers IACF and IACR. CP011406 was amplified by long primers IACF and IACR to obtain a 331bp amplified internal standard fragment. The reaction conditions were: 94°C for 5min; 94°C for 30s, 57°C for 30s, 72°C for 60s, 30 cycles; 72°C for 10min. The designed amplified internal standard sequence was artificially synthesized and cloned into the vector Mach1-T1 (Beijing Quanshijin Co., Ltd., pEASY-T1 Cloning Kit, item numbe...

Embodiment 3

[0056] Example 3. Preliminary construction of the RPA-IAC detection method for detecting Vibrio parahaemolyticus

[0057] 1. Extraction of DNA from the sample to be tested

[0058] Take 1mL of the bacterial solution in a 1.5mL centrifuge tube, centrifuge at 12000rpm for 1min, discard the supernatant, and extract the bacteria of Vibrio parahaemolyticus according to the kit operation (Bacterial Genomic DNA Extraction Kit, Tiangen Biotechnology Co., Ltd., Cat. No.: DP302). Liquid DNA. The obtained DNA was stored at -20°C for future use.

[0059] 2. RPA-IAC amplification

[0060] Using TwistAmp Basic kit (TwistDX, TABAS03KIT, Cambridge, UK), the configuration method of RPA-IAC for detecting Vibrio parahaemolyticus is as follows:

[0061] Add the following solutions to the 0.2 mL TwistAmp reaction tube containing lyophilized enzyme powder:

[0062]

[0063] Vortex the above mixture to mix well, and centrifuge briefly to remove water beads on the wall. Add 280mM MgAc into a ...

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Abstract

The invention discloses RPA specific primers and a kit for detecting vibrio parahaemolyticus in food and application and belongs to the technical field of biology. The sequences of the RPA specific primers are shown as SEQ ID No:1 and SEQ ID No:2; the sequences of internal amplification control is shown as SEQ ID No:8. The RPA specific primers disclosed by the invention are low in time cost, i.e., a PCR (Polymerase Chain Reaction) reaction usually takes up 2 to 3 hours, but an RPA technology only needs 20 minutes to complete amplification; amplification can be realized at a normal temperature: constant-temperature reaction at 37DEG C is needed only and no special thermal cycle equipment is needed. The results are easy to judge; unlike a dispersion zone of an LAMP (Loop-Mediated Isothermal Amplification) product, the RPA only needs one pair of primers to complete amplification, and the amplified product has a strip with specific size according to design sites of the primers and also comprises the internal amplification control which can be used for indicating the false negative of the RPA reaction.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an RPA-specific primer, a kit and application for detecting Vibrio parahaemolyticus in food. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) is one of the important pathogens causing food poisoning in aquatic products, and the carrier rate in seafood is as high as 45.7%. In my country's coastal areas, food poisoning caused by Vibrio parahaemolyticus ranks first in bacterial food poisoning incidents, including Taiwan Province; in Japan, food poisoning caused by this bacteria ranks second. Therefore, Vibrio parahaemolyticus is an important indicator in food safety testing. However, the current statutory detection method for Vibrio parahaemolyticus is still a traditional culture method, which is cumbersome, time-consuming, and laborious, and often takes 5 to 6 days to complete. Moreover, the biochemical indicators for identifying Vibrio parahaemolyticus ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2600/166C12Q2521/507C12Q2521/101C12Q2545/101Y02A50/30
Inventor 吴希阳杨欢岚魏霜冼钰茵
Owner JINAN UNIVERSITY
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