Quantitative detection kit of hepatitis A virus

A hepatitis A virus, quantitative detection technology, applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc. Accuracy and traceability, guaranteed precision and accuracy, good detection specificity

Inactive Publication Date: 2014-05-21
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The classic cell culture method has a long detection cycle and low sensitivity, which is not suitable for the detection of hepatitis A virus in the environment and food
At present, RT-PCR (reverse transcription-PCR) method is mostly used in the world, but reverse transcription-PCR can only detect viruses qualitatively but not quantitatively, and its sensitivity is not high

Method used

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  • Quantitative detection kit of hepatitis A virus
  • Quantitative detection kit of hepatitis A virus
  • Quantitative detection kit of hepatitis A virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Fluorescent quantitative PCR primer design and specificity verification

[0033] By consulting the relevant information of hepatitis A virus, SYBR Green real-time PCR primers were designed with Beacon Designer7 software. Select the conserved region encoding the VP1-VP3 capsid protein in the hepatitis A virus genome, and design the upstream and downstream primers of the gene fragment in the conserved region encoding the VP1-VP3 capsid protein in the hepatitis A virus genome. The sequences are SEQ ID NO: 1~SEQ ID NO: 2 (Table 1), the length of the amplicon is 112bp; use the Bio-Rad CFX Manager instrument to perform temperature gradient fluorescent quantitative PCR in the range of 43.0°C-63.0°C, optimize the annealing temperature to 55°C, and determine the primer effect.

[0034] Using the hepatitis A virus genome cDNA as a template, use non-hepatitis A virus primers such as specific detection primers for influenza virus, hepatitis C virus, and HIV for fluoresce...

Embodiment 2

[0035] Embodiment 2: the drawing of standard curve

[0036] Dilute the standard with ddH 2 O diluted to 1.0 x 10 7After copy / μL, perform 10-fold dilutions sequentially, use dilutions of different concentrations as templates, and take 10 for the standard curve 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 7 spots for copies / μL. Such as figure 2 Shown, different dilutions of hepatitis A virus standard (10-10 7 copy / μL), the amplification curve is clear and the spacing is uniform. The linear relationship of the standard curve is good Y=-3.3275X+40.799, R 2 =0.9988, the amplification efficiency was 99.77%, and the detection limit was as low as 10 copies / μL.

[0037] Dilute the standard with ddH 2 O diluted to 1.0 x 10 8 After copy / μL, perform 10-fold dilutions sequentially, use dilutions of different concentrations as templates, and take 10 for the standard curve 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 8 spots for copies / μL. Such as image 3 Shown, different d...

Embodiment 3

[0038] Example 3: Extracting viral RNA and reverse transcription.

[0039] Extract viral RNA: Add Trizol and chloroform to the EP tube containing hepatitis A virus (the volume ratio of each component added is: virus liquid: Trizol: chloroform = 1:2:1), shake for 30 seconds, and let stand on ice for 5 minutes Finally, centrifuge at 12000r / min at 4°C for 15min; gently absorb the upper aqueous phase, put it in a new EP tube, and discard the rest. Add an equal volume of isopropanol and 1 μL Glyco Blue Coprecip (Invitrogen) to the EP tube containing the aqueous phase liquid, mix well, place in a -20 °C refrigerator for 2 min, centrifuge at 12000 r / min, 4 °C for 10 min; discard the supernatant Wash the precipitate with 200ml of 75% ethanol (made with RNase-free water), centrifuge at 12000r / min, 4°C for 10min; discard the supernatant, and let the precipitated RNA dry naturally at room temperature; dissolve the RNA with 20μL RNase-free water .

[0040] The reverse transcription syst...

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Abstract

The invention relates to a quantitative detection kit of a hepatitis A virus. Specifically, a primer pair which detects the specificity of the hepatitis A virus is designed and verified; the optimal fluorescent quantitative PCR (Polymerase Chain Reaction) condition suitable for qualitatively and quantitatively detecting the hepatitis A virus is found by optimizing a fluorescent quantitative PCR system in real time; a standard substance and a quality control article for preparing a standard curve are additionally arranged in the detection kit to eliminate the differential interference among samples to ensure the precision and accuracy of quantitative detection to the maximum extent, thereby breaking through the defects that the previous detection methods of the hepatitis A virus are low in precision and great in uncertainty and the like. The detection method disclosed by the invention is high in sensitivity, and the lowest limit of detection can reach up to 10 copy/microlitre. Meanwhile, the kit is good in repeatability, and no significant differences (p is less than 0.05) exist in the group and among the groups after experiments are repeated for 6 times. The method disclosed by the invention can quickly and accurately detect the precise content of the hepatitis A virus.

Description

Technical field [0001] The present invention is a biological analysis test and environmental monitoring technology field. It specifically involves a quantitative detection kit of hepatitis A virus nucleic acid, which has extremely high sensitivity and accuracy. Background technique [0002] Hepatitis a Virus (HAV) is an important food -based intestinal virus that can lead to severe hepatitis.The virus belongs to the genus of the liver virus of the small RNA virus. The virus particles have no cystic membrane and are symmetrical structures of the twenty-twenty-somatoscopy, with a diameter of 27-32nm, which contains a positive chain RNA genome with a length of about 7.5KB.HAV is mainly transmitted through the dung mouth. Direct contact between people or consumption of contaminated food and water is considered to be the main way for HAV to spread (KOOPMANS & Duizer, 2004).In the world, about 1 million people are infected with HAV each year, but 50%of them have uncertain sources of in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2563/107Y02A50/30
Inventor 陈全姣隋志伟乔煜婷高志敏赵丽华
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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