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Method for detecting GII type norovirus in marine food

A virus and food technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problem of norovirus highly infectious, false negative, mutant strain or genotype detection methods. and other problems, to achieve the effect of wide application prospects, low false positive and false negative, and high sensitivity

Inactive Publication Date: 2019-06-11
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 GII type norovirus in marine food
  • Method for detecting GII type norovirus in marine food
  • Method for detecting GII type norovirus in marine food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0032] The sequence information of all GⅡ norovirus VP1 genes was retrieved from NCBI. After sequence analysis and comparison, the highly conserved region of GⅡ norovirus was found, 8 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] Example 2. Specificity of real-time fluorescent PCR detection of GⅡ 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 primers and probe combinations 1 and 2 in Table 1 are positive for all different subtypes of GⅡ norovirus strain-infected samples, while other samples are negative;

[0039] The primers and their probe combinations 3 in Table 1 were positive for all different subtypes of GⅡ norovirus-infected samples...

Embodiment 3

[0053] Example 3, Sensitivity of real-time fluorescent PCR detection of GⅡ type norovirus

[0054] Measure the value of OD260 / OD280 of the DNA solution of the sample identified as GⅡ type norovirus positive in Example 2, 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] Results: The primer and probe combination 1 in Table 1 is positive when the concentration of the standard sample is 0.005pg / μL and above, and negative when the concentration of the standard sample is 0.001pg / μL;

[0057] The...

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Abstract

The invention discloses a method for detecting GII type norovirus in marine food. The method comprises the following steps: S1, taking marine food as a sample to be detected, and extracting virus RNA;S2, taking the RNA or cDNA of the RNA as a template, and carrying out PCR detection by using a specific PCR primer combination for detecting the GII type norovirus; S3, detecting the PCR product, andjudging whether the marine food to be detected is polluted by the GII type norovirus or not, wherein the specific PCR primer combination of the GII type norovirus comprises a primer pair for specifically amplifying a GII type norovirus VP1 gene, an upstream primer of the primer pair is shown as SEQ ID No.1, and a downstream primer of the primer pair is shown as SEQ ID No.2. The primer combinationused in the method disclosed by the invention has the advantages of good specificity, high sensitivity and high coverage degree, and has a wide application prospect in the aspect of detecting the GIItype norovirus in the marine food.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting GII 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 shel...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
Inventor 刘志敏毕迎斌徐娜
Owner 山东时进检测服务有限公司
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