Hepatitis B virus (HBV) fluorescent quantificationally PCR detecting kit
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A hepatitis B virus and hepatitis B virus technology, which can be used in measurement devices, material analysis by optical means, instruments, etc., can solve the problems of inconvenient detection of hepatitis B virus, long time, and heavy workload.
Inactive Publication Date: 2008-04-09
YANGTZE RIVER PHARMA GRP BEIJING HAIYAN PHARMA
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[0003] The traditional detection methods of hepatitis B virus are generally cell culture method and enzyme-linked immunoassay (ELISA),
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Embodiment 1
[0076] Embodiment 1---Hepatitis B virus real-time fluorescent PCR detection method and its kit
[0077] 1. Composition of real-time fluorescent PCR diagnostic kit for hepatitis B virus
[0078] The kit consists of hepatitis B virus lysate, PCR buffer (mix), a pair of specific primers PHBV, a specific probe FHBV, Taq enzyme, UNG enzyme, hepatitis B virus strong positive control, negative control, critical positive control Product control blood serum, HBV quantitative standard composition.
[0079] 2. The specific amplification gene sequence of hepatitis B virus is:
[0080] Through gene sequence analysis, we selected the conserved sequence to design a specific primer and a specific fluorescent probe. The Tm values designed by a pair of primers are close, with a difference of less than 2°C.
[0143] Using the above method to detect 135 cases of clinical samples, including 63 cases of hepatitis B patients, the positive rate of detection is 99%, the accuracy rate is 98.4%, and it can carry out accurate quantitative analysis of hepatitis B virus, which is far superior to ELISA and cell culture methods . The present invention has good specificity, high sensitivity, accurate quantification, fast and simple, high repeatability, can carry out rapid qualitative and quantitative detection of hepatitis B virus, and can replace the traditional culture method and ELISA (enzyme-linked immunosorbent assay) that have been used all the time diagnosis method. In addition, the alkaline lysis method of hepatitis B virus reduces the workload and cost of PCR diagnosis of clinical samples, and has potential application value.
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Abstract
The present invention relates to a real time fluorescence metered PCR test way of HBV and a reagent kit. The present way adopts alkali cracking to extract HBV DNA, gets and adds a portion of the trace lysate directly into PCR reaction liquid for augmentation after cracking procedure; the reaction liquid includes a pair of specificity primers and a probe marked with fluorescent dye, PCR method is applied to augment target genes, the intensity of the reaction system is real-time tested, and Ct value is calculated according to analysis software as well as the virus content value of specimen. Under the conditions that sensitivity and accuracy are guaranteed, the reagent kit of the present invention can be operated easily and fast, thus reducing the labor intensity of clinical test and the cost of clinical PCR diagnosis at the same time, and continuing the rapidness and easiness function of fluorescence metered PCR test way. The way which is suitable for the clinical HVB metered test can be used as an assistant diagnosis way of HBV infection and a monitor mean of clinical treatment effect in a clinical lab, has comparatively low requirements of test operator, and possesses practical clinical application value.
Description
technical field [0001] The invention relates to a fluorescent quantitative PCR detection method and a kit for rapidly quantitatively detecting serum DNA of different genotyping hepatitis B viruses (Hepatitis B virus, HBV), and belongs to the field of biotechnology. Background technique [0002] Hepatitis B is a common disease that seriously endangers the broad masses of the people, and it is still a worldwide medical problem. It is an infectious disease with high morbidity and high mortality caused by the hepatotropic HBV virus. According to statistics, there were 350,000,000 hepatitis B patients in the world in 2003, and it rose to 400,000,000 in 2004[2]. The infection rate of hepatitis B among the population in my country is 57.6%. Based on this calculation, about 690 million people in my country have been infected or are infecting with hepatitis B virus, and 120 million people are carrying hepatitis B virus. Therefore, hepatitis B has become a very serious public health pr...
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IPC IPC(8): G01N21/00C12Q1/68
Inventor 唐先兵石和鹏李晓雯肖静段清芬吕倩倩郑宜婷
Owner YANGTZE RIVER PHARMA GRP BEIJING HAIYAN PHARMA
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