Fluorescent PCR detecting method for hepatitis B virus gene parting and reagent kit
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A hepatitis B virus, genotyping technology, applied in biological testing, genetic engineering, fluorescence/phosphorescence, etc., can solve the problem that 100% typing accuracy cannot be guaranteed
Active Publication Date: 2005-03-02
GUANGZHOU HUAYIN MEDICINE SCI & TECH
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Problems solved by technology
The variation of HBV is absolute, and it is necessary to continuously produce and accumulate various variations. Currently, all methods except genome full sequence analysis cannot guarantee 100% typing accuracy
Method used
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Embodiment 1
[0054] Embodiment 1: Fluorescent PCR assay method and kit for hepatitis B virus genotyping of the present invention.
[0055] 1. Kit composition:
[0056] (1) DNA extraction reagent: including A solution: 6mol / L guanidine isothiocyanate, 30mmol / L sodium citrate, 0.58% sodium dodecylsulfonate, 1mmol / L disodium edetate, 2% Glycogen; liquid B: isopropanol; liquid C: 75% ethanol aqueous solution.
[0057] (2) PCR reagents:
[0058] Type A PCR reaction solution (dosage per person):
[0059] Adding amount Final concentration
[0060] 10×PCR buffer (100mmol / L Tris-HCl, 2.5μL 1×PCR buffer
[0061] pH 8.9, 500mmol / L KCl, 50% glycerol)
[0062] 25mmol / L MgCl 2 2.5μL 2.5mmol / L
[0063] 5mmol / L dNTP 1μL 0.2mmol / L
[0064] 100pmol / μL Type A Primer 1 0.15μL 0.6μmol / L
[0065] 100pmol / μL Type A Primer 2 0.15μL 0.6μmol / L
[0066] 100pmol / μL type A probe 0.03μL 0.12μmol / L
[0067] 5U / μL Taq Enzyme 1U
[0068] 1U / μL UNG 0.1U...
Embodiment 2
[0101] Embodiment 2: clinical experiment
[0102] A total of 103 specimens were tested in this experiment, including 1 case of type A, 1 case of type D, 46 cases of type B and 53 cases of type C. In one case, all types of reaction solutions were negative, and the reaction temperature was appropriately lowered (55°C) before PCR reaction, and it was found to be type C. In 1 case, both type B and type C reactions were amplified, and the reaction temperature was increased (61°C) before PCR reaction, and it was found that type C was also performed. Too many HBV mixed infection, or chimeric HBV infection also occurs in clinical practice, so if multiple types are amplified in the reaction solution, but the adjustment of the reaction conditions can not be clearly classified, it should be carried out. HBV full sequence analysis.
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Abstract
The invention relates to a fluorescent PCR detecting method for a HBV type seperating and the reagent box. The characteristics of the method lies in finding out the HBV DNA complete sequence which has been seperatedd from the GenBank to make a sequence; according to the result of the sequence matching, finding out the gathering zone of the type specific base of the BHA gene type, designing a probe and a pair of primers according the gathering zone. Extract HBV DNA from the blood serum sample to make PCR amplification to realize HBV gene type seperating detection in the fluorescent PCR when the probe and primer exist. The invention provides a reagent box and the type positive standard used in it. The accuracy of type parting reaches to 98% and can reach to 100% by adjusting the reaction condition., simpler and quicker, the test procedure and the time consuming is less, the cost is lower than the complete sequence analysis and more quick and accurate than the regular relevant PCR and RFLP method, besides, the closed check adopted in the invention can avoid the contamination efficiently.
Description
technical field [0001] The invention relates to a fluorescent polymerase chain reaction (PCR) detection method for genotyping of hepatitis B virus (Hepatitis B virus, HBV) and a kit thereof. Background technique [0002] Hepatitis B virus mainly causes liver damage through the host's immune mechanism. The body's cells recognize the virus antigen and attack the infected liver cells to cause inflammation. This process is affected by multiple factors including the host and the virus. Viral genetic heterogeneity affects antigen expression and may also play an important role. Different strains have different frequencies of certain mutations, different strains have different resistance to immune clearance, and other factors may lead to different genotypes having different post-infection disease spectrum. According to the current research, HBV C genotype is associated with heavier liver lesions, while B type is associated with milder lesions; A type is associated with chronic hepa...
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