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Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA

机译:使用高效TaqMan探针通过实时PCR快速定量乙型肝炎病毒DNA并提取病毒DNA

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摘要

Aim: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. Methods: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. Results: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 104/ mL, 6.3 × 102/mL and 1.6 × 103/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 109/mL, 2.08 × 106/mL and 4.40 × 107/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 105/ mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples, Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. Conclusion: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA. © 2006 The WJG Press. All rights reserved.
机译:目的:利用高效的TaqMan探针和病毒DNA的提取方法,通过实时PCR快速定量乙肝病毒(HBV)DNA。方法:通过将分别针对HBV基因组的S,C和X区的PCR产物克隆到pGEM-T载体中,制备了三种标准品。通过比较使用某些血清DNA衍生自三种不同标准曲线的HBV血清样品的拷贝数和Ct值,选择了一对引物和匹配的TaqMan探针。然后,通过实时PCR分析了样品A,B和C中六种HBV DNA提取方法(包括异硫氰酸胍,蛋白酶K,NaI,NaOH裂解,碱裂解和简单煮沸)的效率。同时,对8份临床HBV血清样本进行了定量。结果:源自S,C和X区标准曲线的同一HBV血清样品的拷贝数分别为5.7×104 / mL,6.3×102 / mL和1.6×103 / mL。相对Ct值分别为26.6、31.8和29.5。因此,选择来自S区的引物和匹配的探针来进一步优化六种提取方法。在NaOH裂解法中,HBV血清样品A,B和C的拷贝数分别为3.49×109 / mL,2.08×106 / mL和4.40×107 / mL,相对Ct值为19.9、30和26.2。是六种方法中效率最高的。简单沸腾显示的效率略低于NaOH裂解。异硫氰酸胍,蛋白酶K和NaI显示HBV血清样品A,B和C的拷贝数约为105 / mL,而Ct值约为30。碱性无法量化三份HBV血清样品的拷贝数,标准差所有8个临床HBV血清样本中的(SD)和系数变异(CV)都非常低,表明一式三份的HBV DNA定量是可靠且准确的。结论:基于优化的引物和来自S区的TaqMan探针结合NaOH裂解实时荧光定量PCR是一种简便,快速,准确的HBV血清DNA定量方法。 ©2006 The WJG Press。版权所有。

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