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Method for detecting GI type Norovirus in seafood

A virus and food technology, applied in the biological field, can solve the problems of false negatives, strong norovirus infectivity, and difficulty in detecting variants or genotypes, achieving low false positives and false negatives, wide application prospects, and sensitivity. high effect

Inactive Publication Date: 2019-05-17
山东时进检测服务有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Norovirus is highly infectious, and the infectious dose can be as low as 10-100 virions, and frequent genome recombination between different genotypes makes norovirus highly variable. Type detection methods are difficult and often lead to false negatives

Method used

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  • Method for detecting GI type Norovirus in seafood
  • Method for detecting GI type Norovirus in seafood
  • Method for detecting GI type Norovirus in seafood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, real-time fluorescent PCR primer design

[0032] The VP1 gene sequence information of all type GI noroviruses was retrieved from NCBI. After sequence analysis and comparison, the highly conserved regions of type GI noroviruses were found, 10 pairs of primers and probes were designed, and BLAST sequence comparison was performed. , screen out 4 pairs of primers and their probe combinations with better specificity, and then carry out the biosynthesis of the sequence, in which, the 5' end of each probe is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a quencher group BHQ1, each combined sequence is shown in Table 1.

[0033] Table 1. Sequences of primers and their probe combinations

[0034]

Embodiment 2

[0035] Embodiment 2, real-time fluorescent PCR detects the specificity of GI type norovirus

[0036] Use 4 pairs of primers and probe combinations thereof shown in Table 1 in Example 1 to carry out real-time fluorescent PCR detection of oyster samples infected by the following different viruses: rotavirus, astrovirus, hepatitis A virus, different subtypes GI. GI Noroviruses from 1 to 9, GII Noroviruses of different subtypes GII.1 to 21, GIII to VII Noroviruses, each sample was repeated three times, and the results were as follows:

[0037] The detection results of blank control and negative control using different primers and their probe combinations were all negative;

[0038] The detection results of primers and probe combinations 1 and 2 in Table 1 are positive to all different subtypes of GI type norovirus strain infection samples, while other samples are negative;

[0039] The primers and their probe combinations 3 in Table 1 were positive for all different subtypes of G...

Embodiment 3

[0053] Embodiment 3, real-time fluorescence PCR detects the sensitivity of GI type norovirus

[0054] With the DNA solution of the sample identified as GI type Norovirus positive in Example 2, measure the value of OD260 / OD280, and calculate the DNA concentration, use RNase-free Water to dilute to a certain concentration, and then serially dilute to a concentration of 1 pg respectively / μL, 0.1pg / μL, 0.01pg / μL, 0.005pg / μL, 0.001pg / μL of different standard samples.

[0055] Using the above-mentioned different standard samples as templates, the primers and their probe combinations 1 and 2 in Table 1 were used respectively, and the detection was carried out according to the method of step 2 in the real-time fluorescent PCR detection described in Example 2.

[0056] Result: when the standard sample concentration of the primers and probe combination 1 in Table 1 is 0.005pg / μL and above, the detection result is positive, and when the standard plasmid concentration is 0.001pg / μL, the ...

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Abstract

The invention discloses a method for detecting GI type Norovirus in seafood. The method comprises the following steps of S1, taking the seafood as a sample to be detected, and extracting virus RNA; S2, adopting RNA or cDNA of the RNA as a template, and adopting a specific PCR primer composition for detecting the GI type Norovirus for performing PCR detection; S3, detecting the PCR product, and judging whether the seafood to be detected is contaminated with the GI type Norovirus, wherein the specific PCR primer composition of the GI type Norovirus includes a primer pair for specifically amplifying a GI type Norovirus VP1 gene, a sense primer of the primer pair is shown in SEQ ID No.1, and a reverse primer of the primer pair is shown in SEQ ID No.2. The primer composition has the advantagesof being good in specificity, high in sensitivity and coverage, and has the wide application prospect in the aspect of detecting the GI type Norovirus in seafood.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting GI type norovirus in marine food. Background technique [0002] Norovirus (NoV), also known as Norwalk Viruses, is a virus that causes non-bacterial acute gastroenteritis and is highly contagious and rapidly spreading. Norovirus is a single-stranded positive-sense RNA virus with a diameter of about 26-35 nm, no envelope, rough surface, spherical shape, icosahedral symmetry, and belongs to the family Caliciviridae. The Norovirus genome is about 7.7kb in length and is divided into three open reading frames (ORFs), with 5' and 3' untranslated regions (UTRs) at both ends. Among them, ORF1 encodes 6 non-structural proteins, mainly including nucleosides Acid hydrolase, 3C-like protease, and RNA-dependent RNA polymerase; ORF2 and ORF3 encode major structural protein (VP1) and minor structural protein (VP2), respectively. VP1 consists of two regions, namely the shell...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
Inventor 沈源庆曾海英沈萍萍
Owner 山东时进检测服务有限公司
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