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Hepatitis B virus (HBV) DNA quantitative detection kit

A technology of hepatitis B virus and kit, which is applied in the direction of recombinant DNA technology, DNA/RNA fragments, microbial measurement/inspection, etc. It can solve the problems that digital PCR cannot be directly applied, and achieves the reduction of false positive detection results and high detection efficiency. Yield, simple design effect

Active Publication Date: 2019-09-17
BEIJING DAWEI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, commercial kits are all products applied on qPCR instruments and cannot be directly applied to digital PCR. Therefore, an optimized kit suitable for dPCR is needed

Method used

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  • Hepatitis B virus (HBV) DNA quantitative detection kit
  • Hepatitis B virus (HBV) DNA quantitative detection kit
  • Hepatitis B virus (HBV) DNA quantitative detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Embodiment 1: The kit that is used for quantitative determination HBV DNA in human serum or plasma

[0105] This kit is used for the quantitative determination of HBV DNA in human serum or plasma. The reagent components included in the kit are as follows:

[0106] kit components main ingredient dPCR Reaction Master Mix HBV specific primers, probes, hot start Taq enzyme, UDG enzyme, dNTPs and MgCl 2

External reference quality control Marked recombinant plasmids containing external reference gene fragments HBV Negative Quality Control Labeled inactivated HBV positive serum or plasma HBV Strong Positive Quality Control Labeled inactivated HBV positive serum or plasma HBV Weak Positive Quality Control Labeled inactivated HBV positive serum or plasma

[0107] First, sample processing and nucleic acid extraction are performed. The sample is serum or plasma; the sample volume is 0.5-2 mL, preferably 1 mL. The external refe...

Embodiment 2

[0126] Example 2: Sensitivity

[0127] The detection limit reference product is the clinical HBV DNA sample determined by the national reference product, and the HBV negative serum is used to dilute the sensitivity of the kit to 10IU / mL as the detection limit reference product (20 repetitions). According to the steps in Example 1, the detection sensitivity involved in this application was evaluated. The actual measured results are shown in Table 1 below:

[0128]

[0129] The primers and probes for this application in the table above are as follows:

[0130] Forward primers include: primers shown in SEQ ID NO.:1 and SEQ ID NO.:2,

[0131] Reverse primers include: primers shown in SEQ ID NO.:3,

[0132] Probes include: the probes shown in SEQ ID NO.:4 and SEQ ID NO.:5.

[0133] Alternative primers and probes are different from the primers and probes of the present application for the same site design of hepatitis B virus (HBV) DNA, and its specific sequence is as follows...

Embodiment 3

[0143] Example 3: Accuracy

[0144] The accuracy reference product is the clinical HBV DNA sample determined by the national reference product, which is diluted with HBV negative plasma to 1.0×10 5 IU / ml, 1.0×10 4 IU / ml, 1.0×10 3 IU / ml and 1.0×10 2 IU / ml, as a reference product for accuracy, a total of 4. Nucleic acid extraction was carried out with the accuracy reference product, HBV DNA negative quality control, HBV DNA strong positive quality control and HBV weak positive quality control. According to the steps in Example 1, the detection accuracy involved in this application is evaluated. The actual measured results are shown in Table 2 below:

[0145]

[0146] The test results show that: in the test of 4 accuracy reference products, in 10 2 -10 5 Within the detection range of IU / mL, the deviations detected by the primers and methods involved in the present application are all less than 20%, and the accuracy is high.

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Abstract

The invention provides a hepatitis B virus (HBV) DNA quantitative detection kit. The kit utilizes a digital polymerase chain reaction (dPCR) technology to quantitatively detect HBV DNA in biological samples and can be used for monitoring baseline level and change conditions of HBV DNA in patients suffering from hepatitis B and evaluating the response and therapeutic effect of antiviral therapy.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a hepatitis B virus (HBV) DNA quantitative detection kit. Background technique [0002] Hepatitis B virus (HBV) is a pathogen that can cause viral hepatitis. According to the statistics of the World Health Organization, about 2 billion people in the world have a history of HBV infection, of which 240 million people suffer from chronic hepatitis B; about 650,000 people die each year from liver failure, liver cirrhosis, and hepatocytes caused by chronic hepatitis B. cancer. HBV infection is the cause of 30% of liver cirrhosis and 45% of hepatocellular carcinoma in the world; in China, 60% of liver cirrhosis and 80% of hepatocellular carcinoma are caused by HBV infection. [0003] Serological detection is usually used as an important indicator for the diagnosis, treatment and prognosis of HBV infection, such as: HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc and anti-HBc-immunoglobulin M....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/706C12Q1/6851C12Q2563/159C12Q2545/113C12Q2537/16Y02A50/30
Inventor 马骏翁蓉蓉
Owner BEIJING DAWEI BIOTECH LTD
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