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目的 :建立简便的 HBV DNA序列中 BCP双突变的检测方法。方法 :采用终点终止法和偏振光检测技术进行点突变的检测。首先对 HBVC区基因进行 PCR扩增 ,然后用特异探针与扩增产物中待测核苷酸的下游序列杂交 ,使探针的 3’端可以在 DNA聚合酶的催化下 ,依据其互补链上的待测核苷酸连接上一个标有特定荧光素的 d NTP,然后检测该3’端带有荧光素的探针 ,根据检测到的荧光素种类和偏振光的强度可以判定待测点是何种核苷酸。结果 :该方法可以检测出 HBV基因序列中 BCP双突变核苷酸类型 ,可以检测 1个拷贝的模板 ,并且可以从 BCP野生型株 DNA序列中检出 5 % BCP双突变 DNA序列 ,对 76例 HBV DNA阳性患者进行检测 ,结果 HBV BCP基因双突变率为 5 3.9( 4 1 /76)。结论 :该技术可以对血清中 HBV DNA C区 BCP双突变。
Objective: To establish a simple method for the detection of BCP double mutations in HBV DNA sequences. Methods: Endpoint termination method and polarized light detection techniques were used to detect point mutations. Firstly, the gene of HBVC is amplified by PCR, then the specific probe is used to hybridize with the downstream sequence of the nucleotide to be detected in the amplification product, so that the 3 ’end of the probe can be catalyzed by DNA polymerase according to its complementary strand The nucleotide to be detected is connected to a dNTP labeled with a specific fluorescein, and the probe with the fluorescein at the 3 ’end is detected. Based on the detected type of fluorescein and the intensity of the polarized light, the point to be measured can be determined What kind of nucleotide. Results: The method could detect the nucleotide sequence of BCP double mutation in HBV gene sequence, detect one copy of the template, and detect 5% BCP double mutant DNA sequence from BCP wild type strain DNA. For 76 cases HBV DNA positive patients were detected, the results of HBV BCP gene double mutation rate of 5 3.9 (4 1/76). Conclusion: This technique can double-stain BCP in serum HBV DNA C region.