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A nucleic acid sequence and kit for detecting hepatitis B virus

A hepatitis B virus and nucleic acid sequence technology, which is applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., and can solve the problems of decreased detection sensitivity, poor stability, and performance indicators that cannot reach clinical diagnosis and treatment. , to achieve good reproducibility, high accuracy, and reduce experimental deviation

Active Publication Date: 2019-09-03
温州迪安医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The primers and probes designed by existing quantitative reagents are often based on the type and sequence information of HBV infection several years ago, which cannot meet the detection requirements of emerging subtypes, resulting in a decrease in detection sensitivity, poor stability, and performance indicators that cannot meet clinical requirements. Diagnosis and Treatment Requirements

Method used

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  • A nucleic acid sequence and kit for detecting hepatitis B virus
  • A nucleic acid sequence and kit for detecting hepatitis B virus
  • A nucleic acid sequence and kit for detecting hepatitis B virus

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: The primers and probes that detect HBV DNA and human internal reference gene ERG are synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd., and the sequences are as follows:

[0032] Primers for amplifying HBV DNA:

[0033] F1 (SEQ ID NO: 1): 5'-cywcggayggaaaytgcac-3';

[0034] R1 (SEQ ID NO: 2): 5'-aggcccachcccatagg-3';

[0035] F2 (SEQ ID NO: 3): 5'-ctctaygtwtccctchtgytgctgt-3';

[0036] R2 (SEQ ID NO: 4): 5'-cactagtaaaytgagccadgagaaa-3';

[0037] Probes for detection of HBV DNA:

[0038] PHBV (SEQ ID NO: 5): 5'-tgtattcccatcccatc-3';

[0039] Among them, w represents a degenerate base (a or t), y represents a degenerate base (c or t), h represents a degenerate base (a or t or c), and d represents a degenerate base (a or t or g); the 5' end of the HBV probe PHBV (SEQ ID NO: 5) sequence is marked with a FAM fluorescent group, and the 3' end is marked with an MGB quenching group,

[0040] Primers for amplifying the human ERG gene:

[0041] F3 (SE...

Embodiment 2

[0046] Embodiment 2: the preparation method of HBV DNA quantitative kit.

[0047] (1) PCR reaction solution: 2*supermix (purchased from U.S. Bio-rad Company, article number 1863026), which is 2*PCR reaction premix, which contains PCR reaction buffer solution of the present invention, hot-start DNA polymerase, Mg 2+ and dNTPs and other components, stored at -20°C;

[0048] (2) Primer-probe premix solution: Submit the nucleotide sequences shown in SEQ ID NO: 1 to 8 to Yingwei Jieji (Shanghai) Trading Co., Ltd. for synthesis. After getting the dry powder, use deionized water to separate the Dilute 6 primers to a concentration of 100uM, dilute two probes to a concentration of 10uM, then mix the 8 primer-probe master solutions at a ratio of 9:9:9:9:25:9:9:25, and store at -20°C ;

[0049] (3) Positive control: Serum samples containing HBV C genotype plasmid (purchased from the China Institute of Standards and diluted) and human ERG gene plasmid, wherein the concentration of HBV C...

Embodiment 3

[0052] Embodiment 3: detection method.

[0053] Instruments: Eppendorf Mastercycler pro S qualitative PCR instrument, Bio-rad QX200 droplet digital PCR system (including droplet generator, sealing device, droplet reader), BECKMAN 22R desktop micro-refrigerated centrifuge, WH-866 vortex oscillator (Taicang Hualida), low-speed plate centrifuge (Anhui Zhongjia).

[0054] (1) Preparation of HBV patient samples, positive control and negative control DNA templates: Use QIAGEN Serum Virus Extraction Kit (Column Method, Cat. No. 57704), operate according to the kit instructions, and extract patient samples into 500 μL serum in advance Add 2.5 μL of the internal reference plasmid, and then extract; take 500 μL of negative and positive control samples for extraction directly, and use 50 μL of eluent for elution, and use it as a PCR reaction template for later use.

[0055] (2) using the nucleic acid obtained in step (1) as a template, using 3 pairs of specific primers and two specific...

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Abstract

The invention discloses a nucleotide sequence and a kit for detecting HBV (Hepatitis B Virus), and belongs to the technical field of molecular diagnosis. A primer and a probe for detecting the HBV, provided by the invention, are capable of specifically amplifying and detecting virus DNA (Deoxyribonucleic Acid) of nine HBV gene type viruses from A to I. The invention also discloses the kit for detecting the HBV. The kit disclosed by the invention is capable of realizing quick, sensitive and accurate detection on accurate copy number of various sub types of HBV. The nucleotide sequence and the kit for detecting the HBV, disclosed by the invention, have the advantages of simpleness in operation, strong specificity, high sensitivity, low cost, high throughput and the like and can be used for carrying out quick hypersensitive detection on the DNA of clinical HBV, and reference basis is provided for diagnosis and treatment on clinic CHB (Chronic Hepatitis B).

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a nucleic acid sequence and a kit for detecting hepatitis B virus. Background technique [0002] Hepatitis B virus (Hepatitis B virus, HBV for short) is a DNA virus belonging to the hepadnaviridae family. According to current knowledge, HBV is only susceptible to humans and orangutans, causing viral hepatitis B disease. Chronic Hepatitis B (CHB) is a liver inflammatory disease caused by chronic infection of HBV in human liver cells and can cause multiple organ damage. It mainly affects children and young adults, and its continuous progression can lead to liver cirrhosis, liver failure and liver cancer. CHB is widely prevalent in countries all over the world. It is estimated that more than 240 million people are chronically infected with hepatitis B (hepatitis B surface antigen positive for at least 6 months) worldwide. More than 686,000 people die each ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686
Inventor 任绪义虞闰六罗英宣文静
Owner 温州迪安医学检验所有限公司
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