宫颈癌高危人群HPV16及18感染检测方法的建立

ESTABLISHMENT OF A METHOD FOR DETECTING HPV16 AND HPV18 IN HIGH-RISK POPULATION OF CERVICAL CARCINOMA

①目的 建立宫颈癌高危人群人乳头状瘤病毒 (HPV) 1 6及 1 8感染的检测方法。②方法 对HPV1 6及 1 8E6 /E7基因序列进行序列比较分析 ,根据二者的共享序列设计通用引物 ,进行外侧扩增 ;在通用引物扩增序列之间分别设计HPV1 6和 1 8的特异性引物 ,分别进行半套式PCR扩增 ,以检测并鉴定女性宫颈分泌物中HPV1 6及 1 8的感染。对该方法的敏感度和特异度进行了测定 ,并用该法对 31例宫颈癌病人和 1 1 3例对照组妇女宫颈分泌物进行了检测。③结果 PCR扩增后HPV1 6、HPV1 8分别有 341、4 30bp和 1 0 9、4 73bp的带产生。该反应体系最低可检测浓度为 3μg/L。用该方法对人类基因组DNA、人类巨细胞病毒 (HCMV)和大肠埃希菌DNA以及其他型别的HPV进行检测 ,均未见假阳性结果产生。宫颈癌病人HPV1 6的感染率为 6 7.7% ,HPV1 8的感染率为 1 9.4 % ,明显高于对照组 (χ2 =31 .5 2、6 .2 6 ,P <0 .0 1、0 .0 5 )。④结论 HPV1 6、HPV1 8感染与宫颈癌的发生密切相关。本研究建立的方法具有特异度和敏感度高、简便、快速等特点 ,可推广应用于临床检测。

Objective To set up a method for detecting human papillomaviruses (HPV) types 16 and 18 in high-risk population of cervical carcinoma. Methods The gene sequences of HPV 16 and 18 E6/E7 were compared and analyzed by DNA Star software. The universal primers were designed according to sharing sequence. The special primers of HPV 16 and HPV 18 were then used for semi-nested PCR amplification of these types of HPV. The sensitivity and specificity of the method were tested. Cervical secretions of 31 patients with cervical carcinoma and 113 control women were detected at the same time. Results After amplification of PCR,specific fragments of 341 bp and 430 bp were amplified in HPV16 , 109 bp and 473 bp in HPV19, respectively. The minimum detectable concentration was 3 μg/L. There was no false-positive result when it was used to detect human genome DNA,HCMV,E.coli. and other types of HPV. The positive rates of HPV16,18 for patients with cervical carcinoma were 67.7% and 19.4% ,respectively,much higher than that for the control group(χ 2=31.52,6.26,P<0.01,0.05). Conclusion The occurrence of cervical carcinoma is closely related to the infection of HPV16 ,18. The method established is of specific and characteristics of high sensitivity, rapid detection and easy to use. It is worthwhile to be popularized clinically.