Loop-mediated isothermal amplification quick detecting kit for vibrio harveyi and method

A loop-mediated constant temperature, detection kit technology, applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc. Achieve shorter time, faster cost, improved accuracy and specificity

Inactive Publication Date: 2012-12-26
HUAIHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional detection methods of the bacteria include morphological detection, physiological and biochemical characteristics detection, etc. These detection methods are not only heavy in workload, but also time-consuming, which are far from meeting the diagnostic requirements of aquaculture production.

Method used

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  • Loop-mediated isothermal amplification quick detecting kit for vibrio harveyi and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1, a loop-mediated constant temperature amplification rapid detection kit for Vibrio harveyi, which consists of:

[0035] (1) Reaction solution A, 1 tube, its volume percentage composition is:

[0036] ①10×Lampbuffer, 17%; the composition of Lampbuffer is: 200mM Tris-HCl, 100mM KCl, 100mM (NH 4 ) 2 SO 4 , 20 mM MgSO 4 , Trition X-100 with a mass concentration of 1%, the pH of the Lampbuffer is 8.8;

[0037] ②dNTP, 16.5%, the concentration is 10mM;

[0038] ③ Magnesium sulfate aqueous solution, 14%, the concentration is 25mM;

[0039] ④ betaine aqueous solution, 26.5%, concentration is 8mol / L;

[0040] ⑤vh-F3, 2.5%, the concentration is 10μM,

[0041] The upstream external primer vh-F3 is: CAA GAG CGACCA CTT CAT GT;

[0042] ⑥vh-B3, 2.5%, the concentration is 10μM,

[0043] The downstream external primer vh-B3 is: TTA GCG CTG CAC GGA AAC;

[0044] ⑦vh-FIP, 10.5%, the concentration is 10μM,

[0045] The upstream internal primer vh-FIP is: TCG CGC TCA AA...

Embodiment 2

[0053] Example 2, a rapid detection method for loop-mediated constant temperature amplification of Vibrio harveyi, using the kit described in Example 1; the steps are as follows:

[0054] (1) DNA extraction: take 1 mL of the sample to be tested and centrifuge at 12,000 r / min for 1 min, discard the supernatant, suspend the bacteria in 100 μL of sterilized distilled water, boil at 100°C for 10 min, and cool in an ice bath for 12,000 r / min centrifuged for 10min, and the supernatant was taken as PCR template DNA;

[0055] (2) LAMP amplification: Add 15 μL of reaction solution A, 8 μL of ultrapure water, 1 μL of template DNA, and 1 μL of reaction solution B to the reaction tube; incubate at 65°C for 60 minutes, inactivate at 80°C for 5 minutes; carry out negative control at the same time and the amplification of positive control, wherein the negative control adopts the negative control sample of healthy fish tissue total DNA, and the positive control adopts the positive control sa...

Embodiment 3

[0057] Example 3, using the kit described in Example 1 to carry out the detection experiment of artificially contaminated aquatic products. The steps are as follows:

[0058] (1) Take the muscle tissue of the inspected variegated clams, razor clams, Chinese prawns, jade snails, cockles, sandfish, squid, small yellow croaker, and yellow anchovy, and homogenize them, and artificially stain them (add 100 μL of Vibrio harveyi S090801 meat to the tissue fluid) Soup culture) and cultured in nutrient broth for 4 h, respectively take 1 mL of homogenate enrichment solution and extract template DNA by boiling.

[0059] (2) Take the DNA sample obtained in step (1) as a template, and amplify at a constant temperature according to the following reaction system. The reaction system of 25 μL PCR is: take 15 μL of reaction solution A, 8 μL of ultrapure water, 1 μL of template DNA, and add 1 μL of reaction solution B; the reaction program of PCR is: incubate at 65°C for 60 min, inactivate at ...

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Abstract

The invention provides a loop-mediated isothermal amplification quick detecting kit for vibrio harveyi. The loop-mediated isothermal amplification quick detecting kit comprises one reactive liquid A, wherein the reactive liquid A comprises the following components: 10 * Lampbuffer, dNTP, a magnesium sulfate water solution, a glycine betaine water solution, a primer vh-F3, vh-B3, vh-FIP, vh-BIP, Bst DNA (Deoxyribose Nucleic Acid)enzyme, 10 * fluorochrome SYBRGreen I, a positive control sample, a negative control sample and ultrapure water. The invention also relates to a detecting method using the kit. The kit has the advantages of high specificity, high sensitivity, fast speed, low cost and simpler operation method, and is suitable for quickly detecting an aquaculture site. With adoption of the kit and the method, defects of long time, high workload, cross contamination, complex in operation and demands on complex instruments in the prior art can be solved; a new technical platform is provided for detecting diseases of aquatic livestock; and urgent needs on site detecting of diseases caused by the vibrio harveyi at present can be met better.

Description

technical field [0001] The present invention relates to a detection kit for Vibrio harveyi, in particular to a rapid detection kit for loop-mediated constant temperature amplification for Vibrio harveyi; the present invention also relates to a detection kit using the aforementioned kit Methods. Background technique [0002] Vibrio harveyi is a Vibrio with luminous properties, which was named Benekeella harveyi by Johnson et al. in 1936 ( Beneckea harveyi ), in 1981, Baumann et al. formally assigned it to the genus Vibrio and named it Vibrio harveyi ( Vibrio Harvey ). The bacterium is a normal member of the microflora in seawater and a common pathogenic bacterium in seawater. It can cause a variety of fish such as amberjack, perch, grouper, whitefin shark, rainbow trout and Atlantic salmon, toothfish, gilthead seabream, wolf bass, Senegal sole, and goby in mariculture. The fungus is especially harmful to prawns, and has caused a large number of deaths of cultured prawns ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/63
Inventor 张晓君陈丽毕可然闫斌伦秦国民
Owner HUAIHAI INST OF TECH
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