Detection and quality control kit for hepatitis A viruses and noroviruses in water sample, as well as detecting method

A technology for detection of hepatitis A virus and virus, applied in the direction of biochemical equipment and methods, methods based on microorganisms, measurement/inspection of microorganisms, etc., can solve the lack of standard quality control for the detection of hepatitis A virus and norovirus System and other issues, to achieve accurate test results

Inactive Publication Date: 2015-12-16
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

[0004] In our country and most countries in the world, there is a lack of standard quality control system for the detection of hepatitis A virus and norovirus in water quality, mainly due to the lack of effective, simple, fast and reliable technology for the concentration and detection of these pathogens

Method used

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  • Detection and quality control kit for hepatitis A viruses and noroviruses in water sample, as well as detecting method
  • Detection and quality control kit for hepatitis A viruses and noroviruses in water sample, as well as detecting method
  • Detection and quality control kit for hepatitis A viruses and noroviruses in water sample, as well as detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: the establishment of coliphage MS2 standard curve

[0061] 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.

[0062] 2. Establish regression standard curve for Ct value and phage concentration. Generate a standard curve based on assignment and Ct values ​​(see figure 1 and figure 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

[0063] Example 2: The recovery verification of sample addition of hepatitis A live attenuated vaccine

[0064] 1. Materials and methods

[0065] (1) Strains and strains

[0066] Freeze-dried live attenuated hepatitis A vaccine: Changsheng Technology.

[0067] Escherichia coli phage MS2 standard sample: It comes from ATCC and is prepared in large quantities in the laboratory.

[0068] (2) Reagents

[0069] PBS buffer (pH 7.0).

[0070] Viral RNA extraction kit: QIAGENRneasyminiKit (CatNo74106).

[0071] TGBE buffer: pH 9.0.

[0072] RT‐PCR Kit: THERNACOMPANYAMBION AgPath‐ID TM One-step RT-PCRKit (P / N: AM1005).

[0073] (3) Experimental film

[0074] A positively charged nylon membrane was purchased from 3M Company (CATALOGNO: NM04711BA045, diameter 47 mm, pore size 0.45 μm).

[0075] (4) Experimental instruments and materials

[0076] Filter: Pall Corporation 300mL MagneticFilterFunnel.

[0077] Diaphragm vacuum pump: Jinteng brand, model GM‐0.33A.

[0078] Fluoresc...

Embodiment 3

[0113] Embodiment 3: the detection of hepatitis A virus and norovirus of water quality sample

[0114] 1. Positively charged membrane method to capture virus

[0115] Add 100 μL process control virus E. coli phage MS2 to the water body to be tested, adjust the pH value of the water body to be tested to 7.0, and filter all samples through a positively charged filter membrane with a diameter of 47 mm and a pore size of 0.45 μm under sterile conditions; transfer the filter membrane to Add 4mL TGBE buffer solution to 50mL sterile centrifuge tube A; add another 10mL TGBE buffer solution to the water sample bottle, shake the centrifuge tube and bottle at 500 oscillations / min for 20±5min; collect the eluate from the test tube and bottle into the B centrifuge tube; Rinse the inner wall of the centrifuge tube where the filter membrane is placed with another 2mL TGBE buffer solution, shake gently and shake upside down, and add it to the centrifuge tube A.

[0116] Adjust to pH 7.0±0.5 ...

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Abstract

The invention discloses a detection and quality control kit for hepatitis A viruses and noroviruses in a water sample, as well as a detecting method. The kit comprises a tube of HAV 2*RT-PCR mix, a tube of NV GI+GII type 2*RT-PCR mix, a tube of MS2 2*RT-PCR mix, a tube of RNase-free water, a tube of HAV positive control, a tube of NV GI+GII positive control and a tube of coliphage MS2(2.5*1010 pfu/mL). The detecting method comprises the following steps: 1, virus concentration; 2, virus RNA extraction; 3, coliphage MS2 standard curve establishment; 4, hepatitis A viruses, norovirus GI type, norovirus GII type and coliphage MS2 detection; 5, virus RNA recovery ratio determination; 6, result judgment. According to the detection and quality control kit for the hepatitis A viruses and noroviruses in the water sample, as well as the detecting method, the quality of the whole virus detection process is effectively controlled through the key steps of sample pretreatment, detection, result judgment and the like, the stability, accuracy and controllability of the detection process are ensured, 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 water samples, and belongs to the technical fields of food safety, environmental monitoring and food-borne virus detection. Background technique: [0002] Viruses such as hepatitis A virus and norovirus are the culprits of many major outbreaks of epidemiological events of waterborne non-bacterial diseases. Among them, hepatitis A virus (HepatitisAVirus, HAV) is a positive-strand RNA virus, which belongs to the family Picornaviridae and the genus Hepavirus. Although the Hepatitis A vaccine has been successfully developed to effectively control the transmission and prevalence of Hepatitis A, in places where the population immunity level is low, the population is generally susceptible, and HAV mainly pollutes water or food through the fecal-oral route, leading to outbreaks of Hepatitis A . Although there is only one serotype in the HAV...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851
Inventor 房保海岳志芹孙涛张晓文孙明君梁成珠
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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