Fast and sensitive method of detecting virus infection titer of attenuated live hepatitis A vaccine

A live attenuated vaccine and sensitive detection technology, applied in biochemical equipment and methods, microbe measurement/inspection, and resistance to vector-borne diseases, etc., can solve the problems of complicated steps, long time-consuming, long vaccine storage period, etc., to achieve The effect of overcoming complicated steps, shortening the detection cycle, and reducing the probability of contamination

Active Publication Date: 2006-05-17
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the complicated and time-consuming steps of the existing cell culture method, which often lead to long vaccine storage period and shortened vaccine validity period, as well as the inability to distinguish infectious virus and

Method used

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  • Fast and sensitive method of detecting virus infection titer of attenuated live hepatitis A vaccine
  • Fast and sensitive method of detecting virus infection titer of attenuated live hepatitis A vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] 1. Virus culture

[0079] The KMB17 cells were divided into small square bottles, and after two days of culture, they grew into a fresh monolayer; the hepatitis A live attenuated vaccine sample Ref was serially diluted 10 times, and 10 -5 , 10 -6 , 10 -7 and 10 -8 Dilutions were inoculated on KMB17 cells, 4 bottles of cells were inoculated for each dilution, 1.0ml of virus solution was inoculated in each bottle of cells, after adsorption for 2 hours, maintenance solution was added, and cultured at 35°C. The other two bottles of cells were added with 1ml of phosphate buffered saline as negative control.

[0080] 2. Extraction of intracellular hepatitis A virus RNA

[0081] After culturing for 8 days, drain the maintenance solution in the culture bottle, add 2.5ml TRIzol reagent to each bottle for lysis, and divide the uniform lysate into two 1.5ml tubes, one tube is used for subsequent experiments immediately, and the other tube is stored at -70 ℃ for standby, and t...

Embodiment 2

[0125] 1,2,3 and 4 steps are the same as embodiment 1;

[0126] 5. Detection of negative-strand RNA intermediates of hepatitis A virus

[0127] (1), reverse transcription reaction

[0128] Add the following reagents in sequence to a nuclease-free 0.5ml centrifuge tube:

[0129] 10× reverse transcription buffer 5μl

[0130] dNTP mix (3mmol / L) 4μl

[0131] Forward primer N1 (5~60μmol / L) 2μl

[0132] RNase inhibitor (40u / l) 2μl

[0133] Reverse transcriptase (4u / μl) 2μl

[0134] HAV RNA 30μl

[0135] Nuclease-free water Make up to a total reaction volume of 50 μl

[0136] React at 42°C for 120 minutes; boil the obtained reverse transcription reaction product for 75 minutes;

[0137] (2), the first round of polymerase chain reaction

[0138] Add the following reagents in sequence to a nuclease-free 0.5ml centrifuge tube:

[0139] 10×PCR buffer 15μl

[0140] dNTP mix (3mmol / L) 4μl

[0141] Reverse primer N2 (5~60μmol / L) 2μl

[0142] Forward primer M1 (5~60μmol / L) 2μl ...

Embodiment 3

[0159] 1,2,3 and 4 steps are the same as embodiment 1;

[0160] 5. Detection of negative-strand RNA intermediates of hepatitis A virus

[0161] (1), reverse transcription reaction

[0162] Add the following reagents in sequence to a nuclease-free 0.5ml centrifuge tube:

[0163] 10× reverse transcription buffer 3.5μl

[0164] dNTP mix (8mmol / L) 2μl

[0165] Forward primer N1 (5~60μmol / L) 1μl

[0166] RNase inhibitor (40u / μl) 1μl

[0167] Reverse transcriptase (4u / μl) 2μl

[0168] HAV RNA 15μl

[0169] Nuclease-free water Make up to a total reaction volume of 35 μl

[0170] React at 40°C for 100 minutes; boil the obtained reverse transcription reaction product for 55 minutes;

[0171] (2), the first round of polymerase chain reaction

[0172] Add the following reagents in sequence to a nuclease-free 0.5ml centrifuge tube:

[0173] 10×PCR buffer 12μl

[0174] dNTP mix (6mmol / L) 4μl

[0175] Reverse primer N2 (5~60μmol / L) 3μl

[0176] Forward primer M1 (5~60μmol / L) 3μ...

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Abstract

The present invention provides one kind of fast and sensitive method for detecting virus infection titer of attenuated live hepatitis A vaccine. The method determines the existence of the virus via detecting the negative brand RNA intermediate of marker appearing during duplicating hepatitis A virus, and is superior to available inverse transcription PCR method and cell culturing method. The method has culture period shortened to 8 days, short detection period, no need of replacing maintaining liquid, less contamination possibility, less cell post-treating steps, high work efficiency and capacity of providing virus heredity information via sequencing the PCR product.

Description

technical field [0001] The invention relates to a method for detecting hepatitis A virus, which belongs to the field of medical biotechnology. Background technique [0002] Hepatitis A (hereinafter referred to as hepatitis A) is a common viral intestinal infectious disease prevalent worldwide, with at least 1.5 million clinical cases occurring every year. At present, the epidemic situation of hepatitis A in my country is still very serious, and new situations have emerged. On the one hand, in most areas, sanitation conditions are still backward, and they belong to areas with a high incidence of hepatitis A. Mass outbreaks of hepatitis A have been reported from time to time. The national incidence of hepatitis A is about 16 / 100,000. According to the data released by the Ministry of Health, in the first quarter of 2004, hepatitis A was one of the top five infectious diseases. On the other hand, in economically developed areas, with the improvement of sanitation conditions, t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCY02A50/30
Inventor 孙明波蒋燕军廖国阳李卫东周健姜述德
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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