Test and quality control kit and test method of Hepatitis A virus and Norovirus in vegetable

A technology for detection of hepatitis A virus and virus, applied in biochemical equipment and methods, measurement/testing of microorganisms, resistance to vector-borne diseases, etc., can solve the problems of lack of standard quality control system, etc., and achieve accurate detection results Effect

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

AI Technical Summary

Problems solved by technology

[0006] In my country and most countries in the world, there is a lack of standard quality control system for the detection of hepatitis A virus and noro

Method used

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  • Test and quality control kit and test method of Hepatitis A virus and Norovirus in vegetable
  • Test and quality control kit and test method of Hepatitis A virus and Norovirus in vegetable
  • Test and quality control kit and test method of Hepatitis A virus and Norovirus in vegetable

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1: the establishment of coliphage MS2 standard curve

[0068] 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.

[0069] 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

[0070] Example 2: Determination of virus extraction efficiency in frozen strawberry samples

[0071] 1. Materials and methods

[0072] (1) Strains and strains

[0073] Freeze-dried live attenuated hepatitis A vaccine: Changsheng Technology.

[0074] Escherichia coli phage MS2 standard sample: It comes from ATCC and is prepared in large quantities in the laboratory.

[0075] (2) Reagents

[0076] PBS buffer (pH 7.0).

[0077] Viral RNA extraction kit: QIAGENRneasyminiKit (CatNo74106).

[0078] TGBE buffer: pH 9.0.

[0079] RT‐PCR Kit: THERNACOMPANYAMBION AgPath‐ID TM One-step RT-PCRKit (P / N: AM1005).

[0080] (3) Experimental instruments and materials

[0081] Fluorescent quantitative PCR instrument: ABI7500.

[0082] Centrifuge tubes: 50mL, 2mL, 1.5mL, WHATSON Company.

[0083] Frozen Strawberry Samples: Samples for laboratory testing.

[0084] (4) method

[0085] ① Enrichment of viruses in frozen strawberries

[0086] a. Take 25g of the test sample and add it to ...

Embodiment 3

[0114] Example 3: Detection of Hepatitis A Virus and Norovirus in Frozen Strawberries

[0115] 1. Enrichment of viruses in frozen strawberry samples

[0116] a. Take 25g of the test sample and add it to the bagpage; add 35mL of TGBE buffer solution and 30U of pectinase Aspergillus niger, and add 100μL of coliphage MS2 standard sample at the same time. Incubate with constant shaking at 60 rpm for 20 min at room temperature.

[0117] Note: For acidic soft fruits, the pH of the eluate should be monitored at 10-min intervals during the incubation. If the pH is below 9.0, adjust to 9.5 with NaOH solution. If the pH value is adjusted, the incubation time should be extended by 10 min.

[0118] b. Collect the eluate in a 50mL sterile centrifuge tube, centrifuge at 10000×g for 30min, and clarify by centrifugation at 4°C. Transfer the supernatant to a 50 mL sterile centrifuge tube, and adjust the pH to 7.0 with 1 mol / L hydrochloric acid solution.

[0119] c. Add 0.25 volume of 5×PE...

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PUM

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Abstract

The invention discloses a test and quality control kit and test method of Hepatitis A virus and Norovirus in a vegetable. The kit comprises one tube of HAV 2*RT-PCR mix, NV GI+GII parting 2*RT-PCR mix, MS2 2*RT-PCR mix, RNase free water, HAV positive control, GI+GII positive control and coliphage MS2 (2.5*1010 pfu/mL) respectively. The test method comprises the following steps: (1) enrichment of virus in a vegetable sample; (2) extraction of virus RNA; (3) establishment of a coliphage MS2 standard curve; (4) test of Hepatitis A virus, Norovirus GI type and GII type and coliphage MS2; (5) determination of the recovery rate of virus RNA; (6) determination of results. According to the test and quality control kit and test method of the Hepatitis A virus and Norovirus in the vegetable, 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 and a detection method for detecting hepatitis A virus and norovirus in fruits and vegetables, belonging to the technical fields of food safety, environmental monitoring and food-borne virus detection. Background technique: [0002] Fruit and vegetable production occupies an important position in my country's import and export trade. Taking strawberries as an example, my country's strawberry planting area is about 90,100 hectares, with a total output of 2.206 million tons, accounting for 35.7% of the world's total output, making it the largest strawberry producer in the world. Domestic strawberry production areas are mainly distributed in eastern coastal areas such as Shandong, Anhui, Liaoning, Hebei, and Jiangsu. Frozen strawberries are an important export agricultural product. In 2012, 5,893 batches of frozen strawberries were exported nationwide, with a weight of 155,104.97 tons and a value of 203.791...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/70C12Q1/706C12Q2561/113C12Q2561/101C12Q2531/113Y02A50/30
Inventor 岳志芹房保海赵玉然王宫璞孙涛梁成珠
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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