SYBR GreenI fluorogenic quantitative PCR detection method for hepatitis B virus

A hepatitis B virus, fluorescence quantitative technology, applied in the biological field, can solve the problems of high cost and unfavorable clinical specimen development

Pending Publication Date: 2019-01-22
重庆高圣生物医药有限责任公司
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Problems solved by technology

According to the different forms of fluorescent dye labeling, it can be divided into three categories: non-specific amplification-double-stranded DNA intercalation fluorescent dyes, fluorescent-labeled primers for specific amplification, and fluorescent-labeled probes for specific amplification. However, fluorescent-labeled primers or probes The cost of the method is relatively expensive, which is not conducive to the development of a large number of clinical specimens

Method used

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  • SYBR GreenI fluorogenic quantitative PCR detection method for hepatitis B virus
  • SYBR GreenI fluorogenic quantitative PCR detection method for hepatitis B virus
  • SYBR GreenI fluorogenic quantitative PCR detection method for hepatitis B virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] [Example 1] Target selection, primer design and screening

[0025] (1) Target selection and primer design:

[0026] Using the http: / / www.ncbi.nlm.nih.gov / blast website to search for the HBV C gene sequence from China, and using DNAMAN software for sequence comparison, find out the conserved region within the C gene range, design primers, Primer sequences are shown below;

[0027] Table 1 HBV primer pair and amplification length

[0028]

[0029] (2) Primer screening

[0030] Using pBS-HBV-3.6 Ⅱ plasmid as a template, PCR amplification was carried out with different primer pairs, and the amplification efficiency of the amplified products was detected by agarose gel electrophoresis. Screen out the best primer pair for subsequent experiments;

[0031] The PCR amplification system is 20 μL, and the specific components are: LA Taq MIX 10 μL, upstream primer 1 μL, downstream primer 1 μL, template 1 μL, nuclease-free water 7 μL;

[0032] The reaction program of PCR is:...

Embodiment 2

[0033] [Example 2] Hepatitis B virus SYBR Green Ⅰ fluorescent quantitative PCR detection method standard curve preparation

[0034] (1) Production of standard products

[0035] Use pBS-HBV-3.6 Ⅱ plasmid to make HBV detection standard, use NanoDrop Lite spectrophotometer to calculate the plasmid concentration, and calculate the copy number, the copy number calculation formula is: (6.02×10 23 )×(g / ml) / (DNA length×660)=copies / ml. Dilute the positive control plasmid in a 10-fold gradient to 10 3 ~10 8 Copies / ml 6 dilution gradients to make a standard curve;

[0036] (2) Standard curve creation

[0037] Take 1×10 3 ～ 1×10 8 Copies / mL 6 gradient standard products were amplified by fluorescent quantitative PCR to obtain the amplification curve, and the standard curve with the logarithm of the copy number of the initial concentration as the x-axis and the Ct value as the y-axis. The standard curve equation is y = 3.3521x +14.563, correlation coefficient R² = 0.9985.

Embodiment 3

[0038] [Example 3] Reproducibility verification of hepatitis B virus SYBR Green Ⅰ fluorescent quantitative PCR detection method

[0039] Selected recombinant plasmid standard products were divided into 1 × 10 5 ～ 1×10 7 3 concentrations of copies / mL are used to evaluate the repeatability of SYBRGreen Ⅰ fluorescence quantitative PCR. The Ct value among 3 parallel experimental tubes for each concentration. The results showed that the Ct values ​​of the three parallel tubes in the same experiment basically coincided, indicating that the system had good repeatability.

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Abstract

The invention establishes a rapid SYBR GreenI fluorogenic quantitative PCR detection method for hepatitis B virus. A primer is designed based on the conserved sequence of the C gene region of hepatitis B virus DNA published by GenBank, and the method for detecting hepatitis B virus and conducting negative / positive result determination through SYBR GreenI fluorogenic quantitative PCR detection is established. The method of the invention has the advantages of high sensitivity, good repeatability, high specificity, low cost, short time consumption and accurate detection.

Description

technical field [0001] The invention specifically relates to a detection method of hepatitis B virus SYBR Green I fluorescent quantitative PCR, which belongs to the field of biotechnology. Background technique [0002] HBV infection is the main cause of acute and chronic liver diseases. About 30% of the world's population, or 2 billion people, have ever been infected with hepatitis B virus, about 350 million chronic HBV infected people are facing the risk of developing fatal liver disease, and about 1 million chronic hepatitis B infected people die of liver cirrhosis every year and liver cancer. Among the known human carcinogens, hepatitis B virus ranks second only to tobacco. In my country, as a major hepatitis country, about 50% to 70% of the population has been infected with hepatitis B virus, and 8% to 12% of the population is HBsAg carriers. [0003] Currently, the diagnosis of HBV mainly relies on enzyme-linked immunosorbent assay and PCR detection techniques. In r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/706C12Q2531/113C12Q2545/114C12Q2563/107
Inventor 周勇项周
Owner 重庆高圣生物医药有限责任公司
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