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RPA (recombinase polymerase amplification) primer, probe, kit and method for detecting vibrio parahaemolyticus

A hemolytic vibrio and probe technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems that have not been applied in the detection of vibrio parahaemolyticus, achieve high sensitivity, Overcome less specific effects

Inactive Publication Date: 2019-04-26
深圳市计量质量检测研究院(国家高新技术计量站国家数字电子产品质量监督检验中心) +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, RPA technology has been applied to the rapid detection of viruses, bacteria, mycoplasma, parasites, etc., but it has not been applied to the detection of Vibrio parahaemolyticus

Method used

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  • RPA (recombinase polymerase amplification) primer, probe, kit and method for detecting vibrio parahaemolyticus
  • RPA (recombinase polymerase amplification) primer, probe, kit and method for detecting vibrio parahaemolyticus
  • RPA (recombinase polymerase amplification) primer, probe, kit and method for detecting vibrio parahaemolyticus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: the selection of target gene and RPA primer probe

[0034] Use bioinformatics knowledge and related analysis software to analyze the common target genes of Vibrio parahaemolyticus, such as tlh , toxR , gyrb , pR72H etc., through comparative analysis of these genes of Vibrio parahaemolyticus and other microorganisms, the irgb As the target gene, select its specific conserved region as the target region for primer probe design. According to RPA's design requirements for primers and probes, design specific primers and probes, and then analyze and compare the sequences of primers and probes in https: / / www.thermofisher.com for the secondary structure of multiple primers and probes Yes, blat the selected primer probe on NCBI to verify its specificity. The primers and probes in this example were synthesized by Shanghai Bioengineering Co., Ltd.

[0035] Primer of the present invention is specifically as follows:

[0036] The primers include irgb The up...

Embodiment 2

[0044] Embodiment 2: detect the RPA kit of vibrio parahaemolyticus

[0045] The present embodiment provides a kind of RPA test kit that detects Vibrio parahaemolyticus, comprises for irgb Gene primers, probes, and RPA reaction buffer. The primers may include one of the upstream primer Seq ID No.1: 5'-CGCCAAACTACATCGGAAAACATGGCCCCATC-3' and the downstream primer Seq ID No.2: 5'- CTAGAAAAAACTCCCCTTGTTCTGGGTGCGAG-3' or the upstream primer Seq ID No.1, The base sequence of a single sequence homology of 50% or more in the complementary strand sequence of the downstream primer Seq ID No.2 sequence. The probe sequence is Seq ID No. 3: 5'-CCACCCGAGAGAACTAAACAAACATCCGTGGATAGATTTTGGGTCG-3' or a base sequence with a single sequence homology of 50% or more in its complementary strand sequence. Preferably, the 31bp position of the 5' end of the probe sequence Seq ID No.3 is modified with dSpacer, and the thymines on both sides of the dSpacer molecule are replaced by fluorescent groups an...

Embodiment 3

[0046] Embodiment 3: the RPA method of vibrio parahaemolyticus

[0047] (1) Extract the genomic DNA of the sample to be tested

[0048] In this example, Vibrio parahaemolyticus CICC21617 was inoculated into 3% NaCl alkaline peptone water, placed on a shaker, cultured overnight at 37°C, 1 mL of the culture solution was added dropwise to a 1.5 mL centrifuge tube, and centrifuged at 12,000 rpm After 2 minutes, discard the supernatant, add 500 µL sterile saline, suspend and mix well, centrifuge at 12,000 rpm for 1 minute, discard the supernatant, add sterile saline repeatedly, centrifuge again, and discard the supernatant. Add 50 µL of normal saline, place in a 100°C water bath for 10-15 minutes, and centrifuge at 12,000 rpm for 1 minute after cooling down to room temperature. The supernatant is used as the genomic DNA of the sample to be tested and stored at -20°C for later use.

[0049] In other embodiments, the sample can be grown and cultured to extract DNA or directly extrac...

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Abstract

The invention provides an RPA (recombinase polymerase amplification) primer, probe, kit and method for detecting vibrio parahaemolyticus. The RPA (recombinase polymerase amplification) primer for detecting the vibrio parahaemolyticus comprises a forward primer with the sequence as shown in Seq ID No.1 in the specification and a reverse primer with the sequence as shown in Seq ID No.2 in the specification aiming at an irgb gene. Compared with the prior art, the kit and the method have the advantages that whether nucleic acid of the vibrio parahaemolyticus exists in a sample or not can be rapidly detected; and the operation is simple, special and expensive instruments are not needed, and important application values in the fields of disease surveillance, clinical diagnosis, food safety inspection and the like are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to RPA primers and probes for detecting Vibrio parahaemolyticus, a kit and an isothermal rapid detection method for RPA. Background technique [0002] Vibrio parahaemolyticus (Vp) is a Gram-negative halophilic bacterium belonging to the Vibrio family and one of the most important food-borne pathogens. It is widely distributed in coastal areas, Fish, shellfish and other seafood and pickled food are important pathogenic bacteria that cause food poisoning in coastal areas. They can cause typical gastroenteritis reactions such as diarrhea, intestinal cramps, nausea, vomiting and headaches in patients, and severe cases can cause sepsis. According to the data from the National Foodborne Disease Surveillance Network, food poisoning caused by Vibrio parahaemolyticus has shown an obvious upward trend in the scale of occurrence and population exposure, ranking first among microbial fo...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2522/101Y02A50/30
Inventor 刘小青陈晶黄建飞陈国培王珍妮刘斌谢光宗林振润杨国武盛司潼张晓英
Owner 深圳市计量质量检测研究院(国家高新技术计量站国家数字电子产品质量监督检验中心)
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