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Method for rapidly detecting virus titer of hepatitis A vaccine based on PMA-qRT-PCR method

A hepatitis A, detection method technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc., can solve the problems of long detection cycle, heavy workload, complicated operation, etc. Test period, good linear effect

Active Publication Date: 2020-08-07
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a fast and effective hepatitis A vaccine for the existing cell culture-ELISA method to detect the hepatitis A vaccine virus titer with large workload (about 1 month), complicated operation and long detection cycle A viral titer assay that has the requisite sensitivity, specificity, and accuracy

Method used

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  • Method for rapidly detecting virus titer of hepatitis A vaccine based on PMA-qRT-PCR method
  • Method for rapidly detecting virus titer of hepatitis A vaccine based on PMA-qRT-PCR method
  • Method for rapidly detecting virus titer of hepatitis A vaccine based on PMA-qRT-PCR method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 investigates the influence of the concentration of PMA and photolysis time on detection result

[0041] Human diploid cell KMB17 was cultured to a monolayer in a cell culture flask, then poured off the excess medium, added PBS buffer to the cells to wash the cell surface, then discarded the excess PBS buffer, and then added trypsin to digest the cells , followed by diluting the cells with MEM medium to 0.5-1.0×10 6 a / ml;

[0042] Take the hepatitis A vaccine harvest liquid or finished product as the sample to be tested;

[0043] Add 1.0ml of diluted cells into a 1.5ml centrifuge tube, discard the supernatant after centrifugation;

[0044] Add 300 μl / tube of the sample to be tested to the cells in step (3), resuspend the cells, and incubate with shaking at 37°C for 2 hours;

[0045] Add PMA respectively, so that the final concentration of PMA is 25, 50, 100 μM, mix well, and incubate at 4°C for 30 minutes in the dark;

[0046] Freeze and thaw the broken ...

Embodiment 2

[0051] Embodiment 2 investigates the influence of incubation temperature and time of PMA on detection result

[0052] The same method as in Example 1 was carried out, except that after adding 50 μM PMA, the freeze-dried live attenuated hepatitis A vaccine was tested under the conditions of incubation temperature 4°C, room temperature, and 37°C, and incubation time 10, 30, and 60 minutes, respectively. 10 consecutive tests.

[0053] The result is as figure 2 As shown, the copy number decrease rate of PMA under the condition of 4°C dark-proof incubation was significantly higher than that at room temperature and 37°C (P<0.05). Among them, the average copy number decline rates of the 9 experimental groups of P1-P4 primers were 92.19%, 93.46%, 92.91%, 91.60%, 91.91%, 90.48%, 90.43%, 91.46% and 90.92%. Among them, the 4°C+30min group had the highest copy number decline rate (93.46%), which was significantly higher than that of the 4°C+10min group (P=0.019); no significant differe...

Embodiment 3

[0054] Embodiment 3 investigates the impact of different inactivation methods of hepatitis A vaccine on detection results

[0055] Carry out the same method as Example 2, the difference is that the following conditions are used to process the hepatitis A live attenuated vaccine: ① heat inactivation: incubate at 70°C for 5 minutes; ② formaldehyde inactivation: the volume ratio of vaccine sample to inactivator is 1:1250 For inactivation, incubate at 37°C for 12 days; ③β-propiolactone inactivation: inactivate according to the volume ratio of vaccine sample to inactivator 1:2000, and incubate at 4°C for 24h; ④84 disinfectant inactivation: according to the volume ratio of vaccine sample and inactivator Inactivation was carried out with a volume ratio of 1:90, and incubated at room temperature for 20 min. Six consecutive tests were performed in each group. The result is as image 3 As shown, wherein, P1-P4 primers detect heat inactivation, β-propiolactone inactivation and 84 disin...

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Abstract

The invention discloses a method for rapidly detecting virus titer of hepatitis A vaccine based on a PMA-qRT-PCR method, which comprises: incubating human diploid cells and a hepatitis A vaccine, adding PMA with a final concentration of 50 [mu]M, uniformly mixing, incubating for 30 min at a temperature of 2-8 DEG C in a dark place, placing on a photolysis instrument, carrying out photolysis for 30min, extracting nucleic acid, carrying out quantitative detection by using a qRT-PCR kit, drawing a regression curve by using a standard product result, and representing the virus titer of the sampleto be detected by using the obtained result. According to the detection method, all experiments can be completed within one day, an inspection period is greatly shortened, and the kit has good linearity, parallelism, sensitivity, repeatability, correlation, specificity and applicability, can be used for detecting samples with titer of 5.0-8.0 lgCCID50 / ml by a detection method, and meets the requirements on the virus titer detection range of hepatitis A vaccine finished products in the registration standard of vaccine production enterprises in China.

Description

【Technical field】 [0001] The invention relates to a virus titer detection method, in particular to a rapid detection method for hepatitis A vaccine virus titer based on the PMA-qRT-PCR method. 【Background technique】 [0002] Hepatitis A virus (HAV) belongs to the Picornaviridae family and belongs to the Hepacivirus genus. The genome is composed of single-stranded positive-strand RNA with a length of about 7.5kb, including an ORF and non-coding region sequences at both ends, and a small protein encoded by a viral gene is covalently linked at the 5' end, called viral genome protein ( VPg) with a Poly(A) tail at the 3' end. HAV is the main pathogen that causes viral hepatitis A (hepatitis A), mainly hosts primates such as humans, rhesus monkeys and orangutans, and is mainly transmitted by feces-oral transmission. HAV is prevalent all over the world, and the prevalence is closely related to hygiene, cleanliness and the degree of development of the country. Vaccines can effect...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/706C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114C12Q2521/107Y02A50/30
Inventor 卞莲莲高帆孙世洋梁争论毛群颖吴星
Owner NAT INST FOR FOOD & DRUG CONTROL
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