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High-throughput quantitative detection kit for pathogenic vibrio

A technology of pathogenic Vibrio and detection kits, applied in the direction of microbial determination/testing, microbial-based methods, and resistance to vector-borne diseases, etc., can solve problems such as economic losses in the mariculture industry, and achieve low cost and reliability Strong and specific effect

Active Publication Date: 2019-08-06
浙江正合谷生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Vibrio harveyi ( Vibrio Harvey ), and Vibrio anguillaris ( Vibrio anguillarum ) is an important pathogenic bacterium of cultured prawns, and the aquaculture animal diseases caused by it are popular in the world, and it is also a pathogenic bacterium of many cultured fish, which will cause huge economic losses in the mariculture

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  • High-throughput quantitative detection kit for pathogenic vibrio
  • High-throughput quantitative detection kit for pathogenic vibrio
  • High-throughput quantitative detection kit for pathogenic vibrio

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specific Embodiment 1

[0014] A high-throughput quantitative detection kit for pathogenic Vibrio, including 8 kinds of LAMP detection primer sets for pathogenic Vibrio, the gene sequences of which are shown in Table 1 below:

[0015] Table 1

[0016]

[0017] The design of the above-mentioned gene target site: choose inter-species specific and intra-species conservative gene design primers, because the external primer F3 also has the opportunity to combine with and extend the F3c on the template, and replace the complete complementary single strand connected by FIP. At this time, F1c on FIP can realize the complementarity with F1 on this single chain, and form a circular structure through self-base pairing. Similarly, the synthesis of downstream primers BIP and B3 is similar to that of primers FIP and F3, which provides the possibility of forming a dumbbell-shaped single-stranded structure. At this time, under the action of DNA polymerase with strand displacement ability, the F1 segment at the 3...

specific Embodiment 2

[0020] A high-throughput quantitative detection kit for pathogenic Vibrio, the LAMP reaction system and the final concentrations of the components are as follows: 1 μL sample template DNA, 2.5 μL 10× reaction buffer, 1.5 μL MgSO with a concentration of 100 mM 4 , 3.5 μL of 10 mM dNTPs, 1 μL of 8 U / μl Bst DNA polymerase, 1 μL FIP / BIP primer set solution, 1 μL F3 / B3 primer set solution and 4 μL betaine, and then make up 25 μL with double distilled water; the FIP / BIP primer set solution is Vibrio parahaemolyticus shown in Table 1 , Vibrio vulnificus, Vibrio anguillarum, Vibrio alginolyticus, Vibrio cholerae, Vibrio riverine, Vibrio harveylius and Vibrio splendidus FIP / BIP primer set solution, the end of each primer The concentration is 40 μM, and the F3 / B3 primer group solutions are Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio anguillarum, Vibrio alginolyticus, Vibrio cholerae, Vibrio riverina, and Vibrio harveyi as shown in Table 1. The final concentration of each primer...

specific Embodiment 3

[0023] A method for the high-throughput quantitative detection of pathogenic Vibrio using the detection kit of the above specific embodiment two, comprising the following steps:

[0024] Take the sample to be tested, extract the sample template DNA according to the commercial bacterial DNA extraction kit, take 1 μL of the sample template DNA, add 2.5 μL 10× reaction buffer according to the composition of the LAMP reaction system, 1.5 μL MgSO 4 , 3.5 μL dNTPs, 1 μL Bst DNA polymerase, 1 μL FIP / BIP primer set solution, 1 μL F3 / B3 primer set solution and 4 μL betaine, then make up 25 μL with double distilled water and mix well, take 20 μL of the mixture and add it to each well of the sample detection chip (Wherein the primer set solution is Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio anguillarum, Vibrio alginolyticus, Vibrio cholerae, Vibrio riverine, Vibrio harveyi and Vibrio splendidus 8 kinds of pathogenic vibrio The primer set solution of each pathogenic Vibrio is ad...

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Abstract

The invention discloses a high-throughput quantitative detection kit for pathogenic vibrio. The kit is characterized in that the kit includes a LAMP detection primer set, whose gene sequences are shown as SEQ ID NO.1-NO.32, of 8 kinds of pathogenic vibrio; and the kit also includes a sample template, 10*reaction buffer, MgSO4, dNTP, Bst DNA polymerase and double distilled water. The kit has the advantages of being strong in specificity, high in sensitivity, high in throughput, strong in reliability and low in cost, and has no false negative results.

Description

technical field [0001] The invention relates to a pathogenic Vibrio detection kit, in particular to a high-throughput quantitative detection kit for pathogenic Vibrio. Background technique [0002] Vibrio is widely present in seawater, seabed sediments, and seafood such as fish, shellfish, shrimp, and crab. Human consumption of food contaminated by such bacteria will cause typical gastroenteritis reactions such as diarrhea, vomiting, and fever. Severely ill patients will also suffer from dehydration, shock, and even death. Reports in recent years have shown that the scale of food poisoning caused by Vibrio parahaemolyticus and the scale of population exposure have shown an obvious upward trend, surpassing Salmonella and ranking first in bacterial food poisoning in my country. Therefore, inventing a set of high-throughput, rapid, accurate, and comprehensive methods for detecting common pathogenic Vibrio and typing technology is the key to ensuring food quality and safety and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12R1/63
CPCC12Q1/689C12Q1/6844C12Q2531/119Y02A50/30
Inventor 苏秀榕周君芦晨阳韩姣姣李晔
Owner 浙江正合谷生物科技有限公司
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