Test and quality control kit of Norovirus in shellfish and non-diagnostic test method

A technology for virus detection and kits, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of lack of standard quality control system for norovirus detection and achieve detection results accurate effect

Inactive Publication Date: 2015-12-23
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In our country and most countries in the world, there is a lack of standard quality control system for the detection of norovirus in fo...

Method used

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  • Test and quality control kit of Norovirus in shellfish and non-diagnostic test method
  • Test and quality control kit of Norovirus in shellfish and non-diagnostic test method
  • Test and quality control kit of Norovirus in shellfish and non-diagnostic test method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: the establishment of coliphage MS2 standard curve

[0051] 1. Take the coliphage MS2 quality control sample (2.5×10 10 pfu / mL), take 100 μL to extract viral RNA, and finally dilute in 100 μL RNase-free H 2 O; take 20 μL RNA extract and dilute in 180 μL RNase-free H 2 O, gradient dilution 10 times, 100 times, 1000 times, 10000 times, 100000 times, the virus concentration represents 2.5×10 9 , 2.5×10 8 , 2.5×10 7 , 2.5×10 6 , 2.5×10 5 , 2.5×10 4 pfu / mL, respectively perform fluorescence quantitative RT-PCR reaction.

[0052] 2. Establish regression standard curve for Ct value and phage concentration. The instrument automatically generates a standard curve based on the assignment and Ct values ​​(see figure 1 and 2 ): y=58.716-3.352lnx (y is Ct value, and x is coliphage MS2 concentration (pfu / mL)), R 2 =0.982, the amplification efficiency was 98.74%.

Embodiment 2

[0053] Embodiment 2: Determination of the recovery efficiency of adding coliphage MS2 to shellfish samples

[0054] 1. Materials and methods

[0055] (1) Strains and strains

[0056] Escherichia coli phage MS2 standard sample: It comes from ATCC and is prepared in large quantities in the laboratory.

[0057] (2) Reagents

[0058] PBS buffer (pH 7.0).

[0059] Proteinase K working solution (proteinase K (30U / mg), 20±0.1mg; water, 200±2mL; dissolve proteinase K in water and mix well; store the commonly used working volume at -20°C, with a shelf life of 6 months; Once thawed, store at 4°C with a shelf life of 1 week).

[0060] Viral RNA extraction kit: QIAGENRneasyminiKit (CatNo74106).

[0061] RT‐PCR Kit: THERNACOMPANYAMBION AgPath‐ID TM One-step RT-PCRKit (P / N: AM1005).

[0062] (3) Experimental instruments and materials

[0063] Fluorescent quantitative PCR instrument: ABI7500;

[0064] Centrifuge tubes: 50mL, 2mL, 1.5mL, WHATSON Company;

[0065] DK‐8D electric heat...

Embodiment 3

[0091] Embodiment 3: the detection of norovirus in shellfish actual sample

[0092] 1. Norovirus enrichment in shellfish

[0093] a. The shellfish samples must be alive or undamaged by freezing, and the soil must be removed and cannot be soaked in water again. Operate on ice, use tweezers, a scalpel, etc. to open the shellfish shell, separate the digestive glands (digestive glands), take a sample of 10 shellfish (less than 10 are all sampled), use sterile scissors to cut up the viscera and mix; take a mixed sample Put 2g±0.2g into a 50mL centrifuge tube (or other sterile centrifuge tube) as a sample for RNA extraction, and store the rest at -80°C.

[0094] b. Add 1 mL proteinase K working solution and 100 μL process control substance E. coli phage MS2 respectively, and vortex to mix.

[0095] c. Incubate on a shaker at 37°C for 1h, 320r / min; in a water bath at 60°C for 15min.

[0096] d. Centrifuge at 3000×g for 5 minutes at 4°C, take the supernatant, and record the volume ...

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Abstract

The invention discloses a test and quality control kit of Norovirus in shellfish and a non-diagnostic test method. The kit comprises one tube of NVGI+GII parting 2*RT-PCR mix, MS2 2*RT-PCR mix, RNase free water, NV GI+GII positive control and coliphage MS2 (2.5*1010 pfu/mL) respectively. The non-diagnostic test method comprises the following steps: (1) enrichment of Norovirus in shellfish; (2) extraction of virus RNA; (3) establishment of a coliphage MS2 standard curve; (4) test of the Norovirus and the coliphage MS2; (5) determination of the recovery rate of the virus RNA; (6) determination of results. According to the test and quality control kit of the Norovirus in shellfish and the non-diagnostic test method, the quality is effectively guaranteed in the whole virus detection process of critical steps of sample pretreatment, virus concentration, quality control, test, result determination and the like, stability, accuracy and controllability of the test process are enhanced, and the detection technology blank in the field is filled.

Description

Technical field: [0001] The invention relates to a quality control kit for detecting norovirus in shellfish and a non-diagnostic non-diagnostic detection method, belonging to the technical fields of food safety, environmental monitoring and food-borne virus detection. Background technique: [0002] Norovirus is an icosahedral symmetrical spherical single-stranded positive-sense RNA virus of the Caliciviridae family, also known as Norovirus, Norwalk virus or Pyovirus, and is a major virus that causes non-bacterial acute gastroenteritis , mainly in the intestinal tract, and can be transmitted through contaminated water, food, objects, air, etc. The full length of the Norovirus genome is about 7.7kb, including 3 open reading frames (ORFs), ORF1 encodes non-structural proteins, including RNA polymerase (RNA-dependent RNApolymerase, RdRp); ORF2 and ORF3 encode the main (VP1) and minor (VP2) capsid protein; according to the homology of VP1 sequence, norovirus can be divided into ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12Q1/70
CPCC12Q1/70C12Q1/6888C12Q2561/101C12Q2531/113
Inventor 房保海岳志芹孙涛张瑾赵玉然梁成珠王宫璞
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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