Kit, primer pair, probe and method for detecting vibrio parahaemolyticus

A technology of vibrio hemolyticus and primer probe, which is applied in the field of probes, primer pairs, and detection kits for vibrio parahaemolyticus, which can solve the problems of low sensitivity and low specificity of vibrio parahaemolyticus, and avoid investment in instruments , strong specificity and high sensitivity

Inactive Publication Date: 2019-06-21
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome defects such as low sensitivity and low specificity of using RPA to detect Vibrio parahaemolyticus in the prior art, and to provide a test kit, primer pair, probe needle and method

Method used

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  • Kit, primer pair, probe and method for detecting vibrio parahaemolyticus
  • Kit, primer pair, probe and method for detecting vibrio parahaemolyticus
  • Kit, primer pair, probe and method for detecting vibrio parahaemolyticus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Primer Screening

[0055] Design unlabeled upstream and downstream primers, use the RPA Basic kit to amplify separately, take uniform bands, and further label the primers with good specificity, and design a labeled probe between the upstream and downstream primers.

[0056] The inventor designed 6 pairs of primers for the toxR gene of Vibrio parahaemolyticus, but the specificity was not good, and 1 pair of primers was designed for the target gene NC_004605.1, and the specificity was good, and then the primers used in RPA-LF were designed and probes.

[0057] The primer sequences are shown in Table 1 (F refers to the upstream primer, R refers to the downstream primer).

[0058] Table 1

[0059]

[0060]

[0061] 2. The above 7 pairs of primers were amplified using the RPA Basic kit, and the DNA concentration of Vibrio parahaemolyticus was 100 ng / μL. The result is as figure 2 (F1+R1, F2+R2, F4+R4, F5+R5, F6+R6, F7+R7, F8+R8 from left to right), all pr...

Embodiment 2

[0066] Embodiment 2 is used to detect the establishment of the RPA method of Vibrio parahaemolyticus

[0067] (1) Design and synthesis of primers

[0068] The specificity of the primers is an important factor in the method established in this experiment. According to the gene sequence of NC-004605.1 registered in GenBank, primer5 software was used to design primers, and their specificity was preliminarily verified by BLAST software comparison analysis. Primer synthesis was completed by Sangon Bioengineering (Shanghai) Co., Ltd.

[0069] The nucleotide sequence of the RPA primer is:

[0070] Upstream primer P1 (RPA-154-F; primer number F4):

[0071] 5'-ATTTCTGAGCTTATTGGCGGTTTCTGTCGG-3' (SEQ ID NO. 1);

[0072] Downstream primer P2 (Biotin-RPA-154-R; primer number R4):

[0073] 5'-Biotin-TAACAGGCTCGCAAACGAATGAAAAGGTGG-3' (SEQ ID NO.2)

[0074] Probe (RPA-154-Probe):

[0075] (Carboxyfluorescein / FAM)-SEQ ID NO.3-(THF / dSpacer)-SEQ ID NO.4-SpacerC3-3'

[0076] SEQ ID NO. 3:...

Embodiment 3

[0080] Embodiment 3 reaction temperature optimization

[0081] A total of 6 temperatures of 30°C, 35°C, 37°C, 39°C, 40°C and 45°C were used to evaluate the optimal reaction temperature of RPA, and the amount of template used in each system was 100ng. Using Twist nfo kit for RPA amplification, the loading sequence and reaction conditions of the RPA amplification reaction are as follows: first, add the components in the RPA reaction system except magnesium acetate to the Twist Into the freeze-dried enzyme complex reaction tube provided by the nfo kit, then add magnesium acetate to the lid of the reaction tube, cover the lid and centrifuge for a short time, so that the magnesium acetate enters the reaction system, and then put the reaction tube into a thermostat of the corresponding temperature Incubate in the bath for 30min.

[0082] The RPA amplification reaction system is 50 μL: 2.1 μL each of 10 μM upstream primer and 10 μM downstream primer, 0.6 μL probe (concentration i...

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Abstract

The invention discloses a kit, a primer pair, a probe and a method for detecting vibrio parahaemolyticus. The kit comprises an RPA reaction system which comprises an RPA primer probe mixture, the RPAprimer probe mixture comprises the primer pair and the probe, a nucleotide sequence of an upstream primer in the primer pair is shown as SEQ ID NO.1 while that of a downstream primer in the primer pair is shown as SEQ ID NO.2, and nucleotide sequences of the probe are shown as SEQ ID NO.3 and SEQ ID NO.4. The primer is high in specificity and sensitivity and accurate in detection result. By combining RPA technology for detecting vibrio parahaemolyticus with a colloidal gold flow measuring chromatography test paper strip, quick detection of vibrio parahaemolyticus can be realized, and 10pg of vibrio parahaemolyticus DNA can be detected at least.

Description

technical field [0001] The invention belongs to the technical field of rapid diagnosis of food-borne pathogenic bacteria, and in particular relates to a kit, a primer pair, a probe and a method for detecting Vibrio parahaemolyticus. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a halophilic Gram-negative bacterium commonly found in oceans and estuaries around the world, and is the leading cause of food poisoning in coastal areas. Symptoms after human infection include diarrhea, nausea, vomiting and fever, and even dehydration coma, which brings great safety hazards to human health and aquatic product quality and hygiene. With the increasing sales of seafood, it is very important to establish a fast and accurate detection method for Vibrio parahaemolyticus for the quality detection of seafood and human health. It can buy precious time for the effective treatment and prevention of diseases, and minimize losses and the risk of disease epide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/6804C12Q1/04C12N15/11C12R1/63
Inventor 蒋蔚王权刘永杰韩先干任亚玲
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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