文献类型：专利

英文题名：Detecting hepatitis B virus (HBV) DNA and single base mutation based on quantum dot resonance energy by preparing detecting probe and HBV DNA clinical sample and detecting the complementary target HBV DNA and single base mutation

作者：JIN Q;MAO H;WANG X;ZHAO J;ZHONG X

机构：[1]UNIV EAST CHINA SCI & TECHNOLOGY;[2]CHINESE ACAD SCI SHANGHAI INST MICROSYST

申请号：CN101993956-A

申请日：2010-01-03

公开日：2011-03-30

语种：英文

收录：DERWENT

摘要：NOVELTY - Detecting hepatitis B virus (HBV) DNA and single base mutation based on quantum dot resonance energy, comprises: (a) preparing detecting probe and HBV DNA clinical sample: and (b) detecting the complementary target HBV DNA and single base mutation by: (i) judging the result of HBV DNA wild type transferred by QDs-Cy5 fluorescence resonance energy; (ii) detecting the HBV DNA single base mutation based on QDs-Cy5 fluorescence resonance energy; and (iii) judging the mutation type of HBV single base mutation sample. USE - The method is used for detecting HBV DNA and single base mutation based on quantum dot resonance energy (claimed). ADVANTAGE - The method has increased specificity, rapid processing steps, high resolution, high flexibility, and high throughout. DETAILED DESCRIPTION - Detecting hepatitis B virus (HBV) DNA and single base mutation based on quantum dot resonance energy, comprises: (a) preparing detecting probe and HBV DNA clinical sample: (i) preparing CdSe/ZnS core/shell structure quantum dot wrapped by mercaptopropionic acid (MPA): obtaining 0.5 ml CdSe/ZnS core/shell structure quantum dot wrapped by trioctylphosphine oxide (TOPO) with fluorescence emitting peak of 575-615 mm, adding 2-4 ml chloroform, gradually adding 2-4 ml MPA solution; stirring for 30-60 minutes, adding 5-8 ml water, repeatedly shaking and removing the organic layer, adding acetone to the water layer, centrifuging and depositing, dissolving the QDs into Milli-Q super-pure water, and obtaining the CdSe/ZnS core/shell structure quantum dot wrapped by MPA; (ii) designing and compounding HBV DNA expanded primer and detecting probe: PCR expanding primer: PCR upstream primer: 5'-acgaccfaccttgaggcatacttc-3' (SEQ ID NO. not defined); PCR downstream primer: 5'-cagagcagaggcggtgtcg-3' (SEQ ID NO. not defined); detecting probe: -NH2 modified single chain DNA probe: 5'-tggatgatgtggtat(t)10(CH2)3-(NH2)-3' (SEQ ID NO. not defined); Cy5 marked signal DNA probe-1: 5'-Cy5-tggctttcagttata-3' (SEQ ID NO. not defined); Cy5 marked signal DNA probe-2: 5'-Cy5-tggctttcagttata-3' (SEQ ID NO. not defined); (iii) preparing QDs-DNA probe cross-linked product: mixing 0.5 g/L 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EGD), 0.5 g/L N-hydroxysuccinimide, and 0.05 microns of MPA wrapped QDs at a ratio of (1-2):(1-2):(2-6), activating for 0.5-2 hours, adding -NH2 functionally modified single chain DNA probe, heating for 0.5-2 hours at 37 degrees C, and obtaining the QDs-DNA probe cross-linked product; and (iv) preparing HBV DNA clinical sample: adopting cracking method to extract the HBV gene group DNA in the positive serum sample, and expanding the HBV DNA and getting the HBV DNA clinical sample; and (b) detecting the complementary target HBV DNA and single base mutation: (i) detecting the complementary target HBV DNA and single base mutation: adding 10 mu l HBV DNA of PCR sample into 115 mu l of 20 mM of Taq linking buffer solution, denaturing for 5 minutes at 95 degrees C by PCR; obtaining single chain; putting the mixed solution into the fridge, cold bathing for 5 minutes in cold water; adding 70 mu l of QDs-DNA probe, 10 mu l of Cy5 marked signal probe-1 (10 mu M) and 5 mu l Taq DNA linking enzyme; reacting 210 mu l of the mixed solution for 15-30 minutes at 42 degrees C, after the linking reaction, heating and denaturing for 5 minutes at 85 degrees C by Eppendorf PCR instrument, changing double chain into single chain; and putting the mixed solution into the cold water for 5 minutes; rapidly putting the reaction mixed solution into 96-hole microplate reader, using 485 microns of light to radiate by 1420 type microplate reader, detecting the fluorescence emitting intensity of the reaction solution in 580 microns part; and detecting the complementary target HBV DNA and single base mutation with microplate reader; (ii) judging the result of HBV DNA wild type transferred by QDs-Cy5 fluorescence resonance energy: comparing the detecting result of 670 microns of fluorescence emitting intensity and the hollow sample of PCR sample, if 670 microns of fluorescence emitting intensity is reinforced, judging whether the HBV DNA is not mutated, it is wild type; comparing the 670 microns of fluorescence emitting intensity with standard curve, ensuring the concentration of the HBV DNA in the sample to detect the wild type HBV DNA; (iii) detecting the HBV DNA single base mutation based on QDs-Cy5 fluorescence resonance energy: comparing the detecting result of the 670 microns of fluorescence emitting intensity with the control sample, if the fluorescence emitting intensity of the sample is equal to the hollow noise fluorescence emitting intensity, judging the HBV DNA is single base mutated; and (iv) judging the mutation type of HBV single base mutation sample: substituting the sample to single base mutation sample of HBV DNA, substituting the Cy5 marked single probe-1 to Cy5 marked single probe-2, detecting with the same method; comparing the detecting result of the 670 microns of fluorescence emitting intensity with the hollow sample, if the fluorescence intensity is obviously reinforced, judging the HBV DNA is YVDD mutated, if not, is another mutation.