Reverse transcription of attenuated live hepatitis A vaccine titre as one polymerase chain reaction detecting process

An attenuated live vaccine and polymerase technology, applied in the field of detection methods, can solve the problems of not being able to meet the needs, taking a long time, and not being able to reflect live viruses

Inactive Publication Date: 2003-05-14
ZHEJIANG PUKANG BIOTECH +1
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  • Abstract
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AI Technical Summary

Problems solved by technology

However, the immunity caused by live attenuated hepatitis A vaccine is mainly due to the number of live viruses rather than the total amount of antigens. Due to the reproduction characteristics of HAV, the content of vacuolar virus (no RNA) is relatively high, and the conventional ELISA method mainly detects antigens. The obtained results can not reflect the amount of live virus, and can only be used as a reference, and the sensitivity is poor; although the half-infectious dose (TCID50) method of tissue culture can reflect the amount of live virus, it takes a long time (about one month), and the operation It is cumbersome and cannot meet the needs, but the reverse transcription-polymerase chain reaction (RT-PCR) method can overcome the above shortcomings and is a new method of research

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Take live attenuated hepatitis A vaccine (H2) 9306, 9307, 9404, 9508, mix, precipitate with polyethylene glycol, and dissolve in phosphate buffer solution-sodium lauryl laurate (PBS-SLS). Referring to the nucleotide sequence of the H2 strain of the live attenuated hepatitis A virus vaccine, select the HAV highly conserved region VP3 base gene region as the target region, and design synthetic primers. The sequence of the primers is: positive strand primer 5'ACA GGT ATA CAA AGT CAG C 3 ', negative strand primer 5' CTC CAG AAT CAT CTC CAA C 3'. Take 4 hepatitis A vaccines and mix them, take 1ml and put it into a 1.5ml centrifuge tube, add polyethylene glycol 6000 and sodium chloride, 4 precipitation overnight, centrifuge at 12000g the next day, remove the supernatant, dissolve the precipitation with PBS-SLS, and use PBS -SLS diluted the dissolved vaccine virus to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , make 4 tubes for each dilution, the reaction conditions are 1ul sa...

Embodiment 2

[0021] Use the feces or serum samples of patients at the hepatitis A outbreak point, after separation, extraction, purification, and concentration, carry out RT-PCR reaction. 33 times, wherein the first cycle 94 was extended to 5 minutes, and the last cycle 72 was extended to 10 minutes. After the reaction was over, 10ul of the product was taken and electrophoresed on 2% agarose. There was a clearly visible electrophoresis band at about 200bp that was positive. Otherwise, it is negative.

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PUM

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Abstract

The reverse transcription-polymerase chain reaction detecting process selects the gene zone of hepatitis A virus encoding structure protein 3-carboxyl end as target zone in designing synthesis primer, and adopts strict reverse transcription-polymerase chain reaction detecting technology in the detection of live hepatitis A vaccine titre. Compared with available tissue culture process, the technology has similar sensitivity but period shortened from one month to one day as well as the features of being simple, sensitive and specific. The technology may be used in routine detection and quality control of live hepatitis A vaccine as well as in clinical diagnosis and laboratory diagnosis of live hepatitis A virus and epidemiological survey.

Description

technical field [0001] The invention is a detection method, especially a molecular biology detection method for rapidly detecting the titer of the live attenuated hepatitis A vaccine. Background technique [0002] Hepatitis A is a worldwide infectious disease, which is caused by hepatitis A virus (HAV), and about 4 billion people in the world are threatened by it. For this reason, many scientists around the world have carried out arduous work on this, and now they have successfully developed a live attenuated hepatitis A vaccine, which has effectively controlled the prevalence of hepatitis A. However, the immunity caused by live attenuated hepatitis A vaccine is mainly due to the number of live viruses rather than the total amount of antigens. Due to the reproduction characteristics of HAV, the content of vacuolar virus (no RNA) is relatively high, and the conventional ELISA method mainly detects antigens. The obtained results can not reflect the amount of live virus, and c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/06C12Q1/25G01N27/447G01N33/50
CPCY02A50/30
Inventor 陈勇罗永能洪艳杨连华毛江森
Owner ZHEJIANG PUKANG BIOTECH
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