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Fluorescent quantitative PCR kit for simultaneous detection of hepatitis B Virus, hepatitis C Virus and human immunodeficiency virus Type 1

A technology of simultaneous detection and kits, applied in the direction of microorganism-based methods, measurement/testing of microorganisms, microorganisms, etc., can solve the problems of unsuitable screening of blood donors and high cost, and achieve short detection time, low detection cost, The effect of high precision

Active Publication Date: 2019-01-11
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Although there have been reports on methods for simultaneous detection of HBV, HCV and HIV-1 at home and abroad, the required equipment is advanced, expensive, and well-trained molecular biology technicians are required to complete it, which is not suitable for large-scale screening of blood donors

Method used

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  • Fluorescent quantitative PCR kit for simultaneous detection of hepatitis B Virus, hepatitis C Virus and human immunodeficiency virus Type 1
  • Fluorescent quantitative PCR kit for simultaneous detection of hepatitis B Virus, hepatitis C Virus and human immunodeficiency virus Type 1
  • Fluorescent quantitative PCR kit for simultaneous detection of hepatitis B Virus, hepatitis C Virus and human immunodeficiency virus Type 1

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Embodiment 1

[0078] The preparation of embodiment 1 kit

[0079] According to the genome sequences of HBV, HCV and HIV-1 viruses, the sequences of detection primers and probes (SEQ ID NO.1~SEQ ID NO.12) were designed respectively, and the detection primers and probes of HBV, HCV and HIV-1 were synthesized respectively .

[0080] The primer probe sequences for detecting HBV are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3:

[0081] SEQ ID NO.1: Upstream primer: 5'-TGTCTGCGGCGTTTTATCA-3'

[0082] SEQ ID NO.2: Downstream primer: 5'-TAGTCCAGAAGAACCAACAAGAA-3'

[0083] SEQ ID NO.3: Taqman probe: 5'-ATGAGGCATAGCAGCAGGAT-3', the 5' fluorescent group of the probe is labeled with FAM, and the 3' quencher group is labeled with MGB.

[0084] The primer probe sequences for detecting HCV are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3:

[0085] SEQ ID NO.4: Upstream primer: 5'-TGGCGTTAGTATGAGTGTCGT-3'

[0086] SEQ ID NO.5: Downstream primer: 5'-GACCACTATGGCTCTCCCG-3'

[0087] SEQ ID NO...

Embodiment 2

[0098] The detection sensitivity analysis of embodiment 2 kit

[0099] Prepare a PCR reaction system in the reagent preparation room as follows, PCR reaction solution: 16 μl×n, enzyme system: 2 μl×n, primer-probe mixture: 2 μl×n. Dispense the PCR reaction liquid into PCR reaction tubes according to 20 μl / tube, and move the reaction tube containing the PCR reaction liquid to the sample processing area. Use a suction nozzle with a filter element to take 5 μl of the supernatant of the processed sample, negative control, and positive control, respectively, and add them to the PCR reaction tubes equipped with the reaction system. Close the tube cap, centrifuge for a few seconds and move to the amplification detection area. Put the PCR reaction tube into a fluorescent PCR amplification instrument for amplification detection, and the reaction program of PCR amplification is shown in Table 3.

[0100] The results of PCR amplification were analyzed according to the following criteria: ...

Embodiment 3

[0112] The detection precision analysis of embodiment 3 kits

[0113] Adopt the method described in embodiment 2, utilize the fluorescent quantitative PCR kit that embodiment 1 provides to detect HBV, HCV and HIV-1 synchronously, to HBV positive control clone plasmid (1 * 10 6 copies / μl, 1×10 4 copies / μl) as the sample to be tested for HBV, for HCV positive control virus-like particles (1×10 4 copies / μl, 1×10 2 copies / μl) as HCV samples to be tested, for HIV-1 positive control virus samples (1×10 4 copies / μl, 1×10 1 copies / μl) as the sample to be tested for HIV-1. Use a quality-tested fluorescent quantitative PCR kit for the simultaneous detection of HBV, HCV, and HIV-1 for detection, each of which is repeated 8 times. Detection was performed on an ABI7500 instrument. The results showed that the coefficients of variation of the detection Ct values ​​of different nucleic acid concentrations were all Figure 5 , HCV precision test see Figure 6 , HIV-1 precision test se...

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Abstract

The invention relates to a fluorescent quantitative PCR kit for simultaneous detection of hepatitis B virus, hepatitis C virus and human immunodeficiency virus. The kit comprises HBV, HCV and HIV-1, the sequence of which is shown in SEQ ID NO. 1 to SEQ ID NO. 9. The kit provided by the invention adopts a multiplex fluorescent PCR method, and can realize DNA virus HBV and RNA virus HCV and HIV-1 atthe same time, and the amplification of one virus nucleic acid will not affect the other two, has the advantages of high sensitivity, strong specificity, good repeatability, short detection time, lowdetection cost, low technical requirements and less pollution, etc. for blood donors in blood centers or blood stations of HBV, HCV and HIV-1 simultaneous detection of the three viruses has importantsignificance and high application value.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a fluorescent quantitative PCR kit for synchronous detection of hepatitis B virus, hepatitis C virus and human immunodeficiency virus type 1. Background technique [0002] Hepatitis B virus (HBV) belongs to the family Hepadnaviridea and the genus Orthohepadnavirus-s, and is the pathogenic factor of hepatitis B. HBV is a small enveloped DNA virus with a diameter of about 42nm. HBV genomic DNA is a partially double-stranded circular structure with a length of about 3200bp. The B and C genotypes prevalent in China are both 3215bp. According to the World Health Organization, more than 2 billion people in the world have been infected with hepatitis B virus, of which about 350 million people suffer from chronic hepatitis B (HBV), which is a partly double-stranded DNA virus that is very widespread. , is a pathogenic factor leading to acute and chronic hepatitis and hepat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/703C12Q1/706C12Q2600/16C12Q2600/166C12Q2531/113C12Q2563/107C12Q2537/143
Inventor 朱灵崔俊生朱灿灿杨柯赵俊胡安中邓国庆刘勇
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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