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Quantitative detection kit for hepatitis B virus nucleic acid

A hepatitis B virus and detection kit technology, applied in the fields of molecular biotechnology and gene detection, to achieve the effects of high-sensitivity real-time fluorescent quantitative detection, optimization of experimental system, and improvement of amplification efficiency

Active Publication Date: 2018-12-11
广州市宝创生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no way to use this method to complete the ultra-high sensitivity quantitative detection of hepatitis B virus nucleic acid

Method used

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  • Quantitative detection kit for hepatitis B virus nucleic acid
  • Quantitative detection kit for hepatitis B virus nucleic acid
  • Quantitative detection kit for hepatitis B virus nucleic acid

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1 Novel highly sensitive hepatitis B virus nucleic acid quantitative detection kit

[0031] Establish a PCR combined with nucleic acid invasion reaction method for real-time quantitative detection of HBV DNA, the principle is as follows figure 1shown. The target template is amplified by designing a pair of PCR primers. In order to increase the binding efficiency of the primers and the template to be tested, in particular, LNA base modifications are added to the primers to further improve the amplification efficiency of the HBV template. For the amplification product, use primer 2 as the upstream probe to design a fluorescent downstream probe, which can anneal and hybridize with the template to form a single-base overlapping intrusion structure, which can be specifically recognized by Afu enzyme, thereby cutting the downstream probe and The template is complementary to the chemical bond of the first base, which produces a fluorescent signal. Since the Tm val...

Embodiment 2

[0044] The sensitivity of embodiment 2 hepatitis B virus nucleic acid quantitative detection kits

[0045] In this example, the hepatitis B virus nucleic acid quantitative detection kit described in Example 1 was used to detect different concentrations of HBV DNA templates to verify the sensitivity of the detection method of the present invention.

[0046] Reaction conditions:

[0047] The 50 μL amplification system includes: 5 μL 10×buffer, 250 μmol / L dNTP, 500 nmol / L PCR upstream and downstream primers (SEQ ID NO.1 to SEQ ID NO.4), 250 nmol / L probe (SEQ ID NO.5 to SEQ ID NO. ID NO.7), 3U TaqDNA polymerase, 40U Afu enzyme and 20 μL plasma to extract HBV template DNA. HBV template concentrations were: 2×10 8 , 2×10 7 , 2×10 6 , 2×10 5 , 2×10 4 , 2×10 3 , 2×10 2 , 20, 2, 0.5 and 0IU / ml (QS quantitative template is 10 3 copy / μL), add water to make up to 50 μL. Amplification reaction program: 95°C, 3min; 95°C for 20s, 67°C, 30s, 70°C, 20s, 5 cycles of pre-amplification;...

Embodiment 3

[0049] The quantitative limit of embodiment 3 hepatitis B virus nucleic acid quantitative detection kits

[0050] This embodiment uses the hepatitis B virus nucleic acid quantitative detection kit described in Example 1 to detect HBV standard templates of different concentrations mixed in human serum samples, which is used to verify the detection method of the present invention when detecting simulated samples Quantitation limit detection situation.

[0051] Reaction conditions:

[0052] The 50 μL amplification system includes: 5 μL 10×buffer, 250 μmol / L dNTP, 500 nmol / L PCR upstream and downstream primers (SEQ ID NO.1 to SEQ ID NO.4), 250 nmol / L probe (SEQ ID NO.5 to SEQ ID NO. ID NO.7), 3U TaqDNA polymerase, 40U Afu enzyme and 20 μL plasma to extract HBV standard template DNA. HBV standard template concentrations are: 2×10 7 , 2×10 6 , 2×10 5 , 2×10 4 , 2×10 3 , 2×10 2 , 20 and 2IU / ml (QS quantitative template is 10 3 copy / μL), add water to make up to 50 μL. Amplif...

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Abstract

The invention discloses a fluorescent quantitative PCR (polymerase chain reaction) detection kit for hepatitis B virus nucleic acid. The quantitative detection kit for the hepatitis B virus nucleic acid comprises primers and probes aiming at an S gene and / or a C gene, wherein the sequences of the primers are as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 in the specification, the sequences of the probes are as shown in SEQ ID NO.5 to SEQ ID NO.7 in the specification, and the three probes are respectively marked with fluorescence. The high-sensitivity quantitative detection kit for the hepatitis B virus nucleic acid has the advantages that HBV genome DNA molecules are detected, the quantitative range is wide, the sensitivity can reach 0.5 IU / ml of HBV DNA, the quantitative limit is as low as 2 IU / ml of HBV DNA, and the detection limit is as low as 1 IU / ml of HBV DNA; and in addition, a method and the kit are adopted, by means of the multi-functional utilization of theprimers and the probes at the nucleic acid invasion reaction stage, the complexity of the probe design and a detection system can be further reduced, and the economic cost of the probe design and thedetection system can be lowered.

Description

technical field [0001] The invention belongs to the field of molecular biotechnology and gene detection, in particular to an ultra-high sensitivity quantitative detection kit of hepatitis B virus nucleic acid. Background technique [0002] HBV belongs to the family of Hepatodeoxyribonucleic Acid Viruses, and under the electron microscope, it appears as small round particles with a diameter of 42nm, called Dan's particles. Dan's granules are composed of envelope and core. The envelope contains surface protein, glycoprotein and cell fat; the core contains core protein, HBV genomic DNA and HBV DNA polymerase. The HBV genome is a partially double-stranded circular DNA, with the longer strand typically containing 3182 nucleotides and the shorter strand 1700-2800 nucleotides. The whole genome DNA of HBV contains four overlapping open reading frames, which encode four proteins with special structures and functions. [0003] After the HBV virus invades the human body, it binds to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851
CPCC12Q1/6851C12Q1/706C12Q2600/166C12Q2525/117C12Q2545/101C12Q2563/107
Inventor 王建平
Owner 广州市宝创生物技术有限公司
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