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A kind of quality control product and preparation method thereof for HCV nucleic acid detection with influenza virus as carrier

A technology for influenza virus and quality control products, applied in the field of HCV nucleic acid detection, to achieve the effect of non-biological infectivity, easy storage and transportation, and mass production and supply

Active Publication Date: 2018-11-20
上海市临床检验中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of influenza virus as a carrier for quality control at home and abroad

Method used

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  • A kind of quality control product and preparation method thereof for HCV nucleic acid detection with influenza virus as carrier
  • A kind of quality control product and preparation method thereof for HCV nucleic acid detection with influenza virus as carrier
  • A kind of quality control product and preparation method thereof for HCV nucleic acid detection with influenza virus as carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Construction of pHW-NS-HCV plasmid

[0035] 1. Primer design

[0036] According to the HCV 5'UTR gene sequence in pMD18-HCV plasmid and the NS segment sequence in pHW-NS plasmid, primers for amplifying the reverse complementary sequence of HCV 5'UTR, NS1 and NEP were designed respectively. To ensure that NEP protein is expressed independently of NS1-HCV protein, a PTV-12A peptide gene sequence (5'-gcgaccaactttagcctgctgaaacaggcgggcgatgtggaagaaaacccgggcccg-3') was inserted between the HCV-NEP genes. At the same time, in order to ensure the accurate expression of NEP protein, a site-directed mutation was performed on the splice acceptor site (base 524 to 527) of the NS gene (5'-TCCA-3' changed to 5'-CCCG-3'). The specific primer sequences and amplification products are listed in Table 1.

[0037] Table 1 PCR amplification primers

[0038]

[0039]

[0040] a The underline indicates the recognition site of the restriction endonuclease BsmB, and the bold font indic...

Embodiment 2

[0058] Rescue and Identification of Mutant Influenza Virus rPR8-NS-HCV

[0059] 1. Rescue of mutant virus

[0060] Prepare the pHW-PB2, pHW-PB1, pHW-PA, pHW-HA, pHW-NP, pHW-NA, and pHW-M plasmids for the bidirectional expression of the other seven segments of the A / Puerto Rico / 8 / 34 influenza virus. Take the recombinant plasmid pHW-NS-HCV constructed in Example 1 and 1 μg of each of the above seven plasmids and mix them evenly, and dilute them into 100 μl of Opti-MEMI. Take 20 μl of transfection reagent lipofectamine 2000 (Invitrogen) and dilute it into 100 μl of Opti-MEMI according to the instructions, and place it at room temperature for 5 minutes. Then the diluted mixed plasmids were added to the transfection reagent and combined at room temperature for 20 min. Wash the 293T cells in the 6-well plate that have been cultured for about 18-24 hours with 90% density, uniform distribution and good growth condition once with Opti-MEMI, and then add 800 μL of OPTI-MEMI to the mix...

Embodiment 3

[0068]Inactivation and purification of rPR8-NS-HCV virus

[0069] The mutant virus was inoculated into 9-11-day-old SPF chicken embryos, and the allantoic fluid of the chicken embryos was collected 72 hours later. The allantoic fluid was centrifuged at 5000g at low speed to remove cell debris, and then inactivated with 0.1% formaldehyde solution at 4°C for 7 days. Then take the original times, 10 -1 and 10 -2 The doubly diluted virus liquid was inoculated into the allantoic cavity of chicken embryos in groups, and 10 9-11-day-old chicken embryos were inoculated in each group, with 0.2ml inoculated per embryo, and cultured at 33-35°C for 72 hours. Those who died within 24 hours were not counted, and at least 80% of the chicken embryos in each group had to survive. Take 0.5ml of allantoic fluid from each embryo of the surviving chicken embryos, mix them according to the group, and then pass them blindly for one generation, inoculate 10 embryos in each group, inoculate each em...

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Abstract

The invention relates to an influenza virus carried HCV nucleic acid test quality control product and a preparation method thereof; the quality control product is prepared from HCV conserved gene carried recombinant influenza virus through enlarged culture, inactivation and purification; the preparation method comprises the following steps: (1) constructing a plasmid which is used for rescuing influenza virus and is interpolated with HCV conserved gene in PR8 virus NS gene; (2) co-transfecting 293T cell to the constructed plasmid with plasmids which are respectively used for transcriptional expression of PR8 viruses PB2, PB1, PA, HA, NP, NA and M, so as to rescue and obtain HCV 5' UTR carried recombinant influenza virus; and (3) enlarged-culturing, inactivating and purifying the recombinant influenza virus so as to obtain the quality control product. The quality control product disclosed by the invention can be used for really simulating HCV pathogens and achieving all-around monitoring on HCV detection; and the quality control product is easy in large-scale preparation, low in cost, good in stability, easy in storage and transportation, and good in application prospect.

Description

technical field [0001] The invention belongs to the field of HCV nucleic acid detection, and in particular relates to a quality control product for HCV nucleic acid detection using influenza virus as a carrier and a preparation method thereof. Background technique [0002] Hepatitis C virus (HCV) is a blood-borne RNA virus that can cause various chronic liver diseases, including cirrhosis and hepatocellular carcinoma. According to the statistics of the World Health Organization, the global HCV infection rate is about 3%. It is estimated that about 170 million people suffer from chronic hepatitis C, and about 35,000 new cases of hepatitis C occur each year. my country is one of the countries with a high incidence of HCV chronic infection, and there are about 38 million HCV infected people. Chronic HCV infection leads to chronic inflammation, necrosis and fibrosis in the liver, and some patients may develop cirrhosis or even liver cancer, which can seriously affect life and h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6806C12N15/63C12R1/93
CPCC12N15/63C12Q1/707
Inventor 王雪亮蒋玲丽肖艳群王华梁
Owner 上海市临床检验中心
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