HBV detection method and kit, and application thereof

A detection method and kit technology, applied in the field of bacterial detection, can solve problems such as interference with HBVPgRNA concentration, inaccurate results, and quantitative contamination, and achieve the effects of good specificity, high sensitivity, and pollution prevention

Inactive Publication Date: 2017-04-05
北京旌准医疗科技有限公司
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  • Abstract
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Problems solved by technology

However, for the digestion of high-concentration HBV DNA, it is necessary to repeatedly verify the dosage of DNaseI, so it is difficult to completely digest all concentrations of HBV DNA at one time, resulting in the remaining HBV DNA content interfering with the concentration of HBV PgRNA, resulting in inaccurate results If only the HBV PgRNA template can be amplified in a targeted and selective manner, the influence of HBV DNA on HBV PgRNA can be eliminated
[0003] In the prior art, HBV DNA and RNA co-extraction kits are usually used, and then the extra

Method used

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  • HBV detection method and kit, and application thereof
  • HBV detection method and kit, and application thereof
  • HBV detection method and kit, and application thereof

Examples

Experimental program
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Example Embodiment

[0046] Example 2

[0047] Qualitative detection of HBV virus

[0048] This embodiment 2 provides a qualitative detection method of HBV virus:

[0049] 1. PCR amplification reaction of HBV virus

[0050] 1) Reagents and components used in the PCR amplification reaction of HBV virus DNA

[0051] (1) HBV virus primer probe mixture:

[0052] Specific primer sequence F1 and HBV PgRNA specific probe RT-FP were designed for HBV PgRNA. F1 and RT-FP were compared with NCBI, especially 6 types B, 7 types C and 7 types D were selected. The results of Blast comparison are shown in 1a and 1b, and the results show that the comparison results are 100% alike, and the primer design is highly specific.

[0053] For the RT-R1 (R2) primer, the 3'end of the primer is 13 poly-Ts, and at the same time, to ensure the specific introduction of the VN merging base sequence (where V represents the base ACG 3 bases, and N represents the base ACTG Four bases), a non-human and viral sequence 5'-ATTCGATATCGCATGATGACTG...

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Abstract

The invention discloses an HBV detection method. The method comprises the following steps: separating a nucleic acid extract liquid from serum to obtain a sample to be detected; detecting a reaction result by a fluorescent quantitative PCR instrument, and setting a fluorescence signal as fluorescein corresponding to the 5' end fluorescence group of a Taqman fluorescent probe PgRNA-FP when collected, and collecting the fluorescence signal at 60 DEG C; and carrying out result analysis. Specific reverse transcription is carried out according to HBV PgRNA poly A, and then amplification is carried out according to HBV PgRNA specific primers and the probe, so possible interference caused by HBV DNA is effectively eliminated, and the HBV PgRNA detection specificity is effectively improved.

Description

technical field [0001] The present invention relates to a detection method, kit and application of bacteria, in particular to a detection method, kit and application of HBV virus. Background technique [0002] HBV virus is a double-stranded DNA virus. In the blood of hepatitis B patients, there are both HBV DNA and HBV RNA. kb, of which, the length of PgRNA is 3.5kb, and its sequence is highly homologous and overlapped with HBV DNA. It is very difficult for PCR specific selective amplification. To detect the content of PgRNA in blood (serum or plasma), the current conventional method is to extract The nucleic acid extracted from the serum of the hepatitis B patient was digested with DNaseI enzyme, and after the HBV DNA was digested, the remaining HBV PgRNA was subjected to PCR amplification to obtain the quantitative detection result of HBV PgRNA. However, for the digestion of high-concentration HBV DNA, it is necessary to repeatedly verify the dosage of DNaseI, so it is di...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6806C12Q1/6851C12Q1/706C12Q2523/308C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 叶锋刘明坤陈广磊
Owner 北京旌准医疗科技有限公司
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