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
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[0042] Example 1. Establishment of detection kits and detection methods for 3 kinds of foodborne viruses in aquatic products
[0043] (1) Establishment of the kit
[0044] The primers determined for detection in this example are shown in the following table:
[0045]
[0046]
[0047] On this basis, a test kit for three foodborne viruses in aquatic products for PCR amplification was designed:
[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 , DNTP (dATP, dGTP, dCTP and dTTP) each 10mM, RNase inhibitor 40U / μL, and the upstream and downstream primer pairs of the above three foodborne viruses 20μM each;
[0049] The storage condition of the kit: -20℃.
[0050] The mPCR-DHPLC method for detecting three foodborne viruses in aquatic product samples using this kit includes the following...
Example Embodiment
[0078] Example 2. Specificity test
[0079] A variety of food virus standard materials were used to simulate the operation steps of daily inspection, and the nucleic acid was extracted according to the method established in Example 1 for mPCR-DHPLC detection.
[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 were used. Pseudorabies virus DNA and hepatitis B virus DNA, while sterilized water was used as a blank control. Using three virus-specific primers, the extracted template was amplified according to the method established in Example 1, and the mPCR-DHPLC specificity test was performed. The test results are attached figure 1 Shown.
[0081] The specific test results show that the detection kits and detection methods established in the examples can specifically detect three target viruses. Only hepatitis A virus, norwalk virus a...
Example Embodiment
[0086] Example 3. Multiplex PCR-DHPLC sensitivity test
[0087] Using food virus standard substances with known virus levels, the kit and detection method established in Example 1 were used to make templates and mPCR-DHPLC detection to determine the detection sensitivity. Table 1 shows the extracted genomic nucleic acid fragments of the three viruses at different inoculation amounts. The OD value was measured by 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 (containing the target sequence 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, the plasmid DNA was extracted according to the method in Example 1, and detected by mPCR-DHPLC. The following table (Table 1) shows the virus concentra...
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