一种宫颈脱落细胞HPV DNA抽提的改良方法  

An improved method on DNA extraction of human palollomavius in cervical epithelium samples

作　　者：伍金华[1] 林啟新[1] 陈建勇[1] 

机构地区：[1]广东省江门市中心医院检验科,广东江门529000

出　　处：《现代预防医学》2013年第4期644-646,共3页Modern Preventive Medicine

摘　　要：目的建立一种抽提宫颈脱落细胞HPV DNA的改良方法。方法收集120例妇科门诊患者宫颈脱落细胞标本,分别采用传统方法(分离法)和改良方法(蛋白酶K消化分离法)抽提HPV DNA,用紫外分光光度法测定DNA的纯度和含量,经扩增后采用核酸分子快速导流杂交技术进行HPV DNA分型检测。结果①传统方法抽提的DNA纯度、含量分别为(1.74±0.59)和(103.97±166.32)μg/ml,改良方法抽提的DNA纯度、含量分别为(1.81±0.47)和(109.34±168.39)μg/ml,改良方法检测的DNA纯度和含量分别高于传统方法(P﹤0.05)。②传统方法检测出现PCR反应抑制物的例数为6例,而改良方法则为0例。③传统方法和改良方法HPV分型的总阳性率、单一感染率、多重感染率分别为20.0%、16.7%、3.3%和20.8%、15.8%、5.0%,两法一致性检验Kappa值为0.926,结果具有较好的一致性。结论改良方法对宫颈脱落细胞HPV DNA抽提比传统方法简单,有助于提高DNA的质量,有效去除PCR反应抑制物,适用于临床上大样本量不同状态宫颈脱落细胞HPV DNA分型的检测。OBJECTIVE To develop an improved method on DNA extraction of human papillomavirus in cervical epithelium samples. METHODS The extraction of HPV-DNA in 120 cases collected from cervical epithelium samples in a gynecology clinic was treated by traditional method （separation method） and improved method （proteinase K digestion separation method） . DNA purity and concentration of the samples were measured by the UV spectrophotometer. HPV subtypes were detected by flow-through hybridization and gene chip after PCR amplification. RESULTS （1） DNA puritify （ 1.81 ±0.47） and concentration of the HPV-DNA extraction （109.34 ± 168.39） μg/ml used by improved method were significantly higher compared with those used by traditional method DNA purity （1.74 ± 0.59） and concentration of the HPV-DNA extraction （103.97 ±166.32）μg/ml （P 〈 0.05）. （2） There were 6 cases of PCR inhibitors were detected from the samples treated by the traditional method, but no PCR reaction inhibitor was detected from the samples treated by the improved method. （3） The rates of positive, single types of infection and multiple infection in the HPV types treated by the traditional method and the improved method were 20.0%, 16.7%, 3.3% and 20.8%, 15.8%, 5.0%, respectively. The value of Kappa was 0.926, which indicates that there was a high degree of consistency. CONCLUSION The HPV DNA in cervical epithelium extraction procedure is better than the tra- ditional extraction procedure. The improved method ensures the quality of HPV DNA and is easier to get rid of the PCR reaction inhibitors. This improved method is suitable for the large quantity and different state of cervical epithelium samples.

关 键 词：HPV DNA DNA抽提 蛋白酶K 分离法 

分 类 号：R711[医药卫生—妇产科学]