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Detection kit and detection method for 3 species of food-borne viruses in marine products

A virus detection and kit technology, applied in the field of detection, can solve the problems of false positives, lack of high throughput, identification of one or a few bacteria, etc., and achieve the effect of high throughput detection and high detection sensitivity

Inactive Publication Date: 2009-11-04
曹际娟 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, different from conventional virus detection, pathogenic viruses contained in food are generally diverse and small, and these viruses are often difficult to enrich and culture in vitro, which determines that the detection of foodborne viruses has always been a priority in microbial detection. Difficulties and gaps
Existing detection technologies such as microbial culture and biochemical identification, immunological technology, PCR technology, etc. have some problems: conventional biochemical identification methods are complicated to operate and time-consuming, and often only one or a few types of bacteria can be identified in one detection experiment; Although immunological technology is highly sensitive, it is prone to contamination and often has false positives; PCR technology is generally used in conjunction with gel electrophoresis technology, which can only detect viruses and does not have the characteristics of high throughput
At present, for foodborne viral diseases in aquatic products, there is still no complete pathogen detection and identification method, especially the detection technology suitable for rapid and multi-species detection at the same time, so it is urgent to develop

Method used

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  • Detection kit and detection method for 3 species of food-borne viruses in marine products
  • Detection kit and detection method for 3 species of food-borne viruses in marine products
  • Detection kit and detection method for 3 species of food-borne viruses in marine products

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1, the establishment of 3 kinds of foodborne virus detection kits and detection methods in aquatic products

[0043] (1) Establishment of the kit

[0044] The primers determined in this embodiment for detection are shown in the table below:

[0045]

[0046]

[0047] On this basis, three foodborne virus detection kits in aquatic products were designed for PCR amplification:

[0048] The kit includes reverse transcription-Tag DNA polymerase at a concentration of 5U / μL, reverse transcriptase at a concentration of 5U / μL, and RT-PCR reaction solution; the RT-PCR reaction solution contains 10mM Tris·HCl, 50mM KCl, 25mM MgCl 2 10 mM each of dNTP (dATP, dGTP, dCTP and dTTP), 40 U / μL of RNase inhibitor, and 20 μM each of the upstream and downstream primer pairs of the above three foodborne viruses;

[0049] Storage conditions of the kit: -20°C.

[0050] The mPCR-DHPLC method of using this kit to detect 3 kinds of foodborne viruses in aquatic product samples i...

Embodiment 2

[0078] Embodiment 2, specificity test

[0079] A variety of food virus standard substances were used to simulate the operation steps of daily inspection, and nucleic acid was extracted according to the method established in Example 1, and mPCR-DHPLC detection was performed.

[0080] The RNA of hepatitis A virus, Norwalk virus and rotavirus were extracted respectively, and the DNA of poliovirus (I, II, III, vaccine strain) and Coxsackie virus (type 1-6, vaccine strain) DNA, Pseudorabies virus DNA and hepatitis B virus DNA, and sterilized water as blank control. Using the specific primers of three viruses, the extracted template was amplified according to the method established in Example 1, and mPCR-DHPLC specificity test was carried out. The test results are attached figure 1 shown.

[0081] The results of the specificity test show that the detection kit and detection method established in the embodiment can specifically detect three target viruses, only hepatitis A virus,...

Embodiment 3

[0086] Embodiment 3, multiplex PCR-DHPLC sensitivity test

[0087] With the food virus standard substance of known virus amount, the kit and detection method established in Example 1 were used to make a template and mPCR-DHPLC detection to determine the sensitivity of detection. Table 1 shows the genomic nucleic acid fragments extracted from the three viruses at different inoculation amounts, the OD value was measured by a spectrophotometer, and the nucleic acid content was calculated.

[0088] In order to verify the detection limit of this detection method, the concentration of the positive plasmid pMD18-T (respectively containing the target sequences of hepatitis A virus, Norwalk virus and rotavirus) was measured with a spectrophotometer, and then diluted to 1×10 -1 ng, 1×10 -2 ng, 1×10 -3 ng, 1×10 -4 ng, 1×10 -5 ng, 1×10 -6 ng, 1×10 -7 ng, 1×10 -8 ng, extract plasmid DNA according to the method of Example 1, and perform mPCR-DHPLC detection. The following table (tab...

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Abstract

The invention discloses a detection kit and a detection method for 3 species of food-borne viruses in marine products. The kit comprises reverse transcription Tag DNA polymerase with a concentration of 5U / mu L, inverse transcriptase with a concentration of 5U / muL and an RT-PCR reaction solution, wherein the RT-PCR reaction solution contains 10 millimols of Tris.HCl, 50 millimols of KCl, 25 millimols of MgCl2, 10 millimols of dNTP, a ribonuclease inhibitor with a concentration of 40 U / mu L, and 20 mu mols of downstream primer pair and 20 mu mols of upstream primer pairs of the food-borne viruses. The kit can synchronously detect hepatitis A viruses, rotaviruses and norwalk viruses. The kit has extremely high sensitivity, can detect 1*10 nano gram of viral nuclei and is superior to the prior reported detection method for viruses in the marine products. In addition, the method is short in detection time, simple and quick, and suitable for quick detection.

Description

technical field [0001] The invention relates to a test kit for detecting three kinds of food-borne viruses in aquatic products, in particular to a test kit for detecting multiple viruses in aquatic products by using multiple PCR and denatured high performance liquid chromatography (DHPLC) techniques. Methods of detection are also contemplated. Background technique [0002] Hepatitis A virus (HAV), rotavirus, and Norwalk virus are common foodborne viruses in aquatic products. They are very easy to pollute the environment, food, water sources, seafood (such as cockles, etc.), tableware, utensils for production and processing, etc., and healthy people's hands. This can lead to infection of susceptible persons or outbreaks of regional epidemics. Due to the global development of food production, the application of new food production technology, the change of people's eating habits and other factors, to a certain extent, the incidence of foodborne viral diseases is increasing. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N30/02C12R1/93
Inventor 陈颖
Owner 曹际娟
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