两种人乳头瘤病毒分型检测方法的比较

Comparison of two methods in genotyping and detecting human papillomavirus

目的通过比较PCR联合地高辛标记探针杂交法与实时荧光定量PCR法检测宫颈液标本中人乳头瘤病毒（HPV）感染的一致性,评价PCR联合地高辛标记探针杂交法的临床应用价值。方法将4型HPV（16、51、54和56）探针3′端加尾地高辛标记,检测标记探针的灵敏度和特异性。收集63例第二代杂交捕获（HC-Ⅱ）阳性和18例HC-Ⅱ阴性的宫颈液标本,采用HPV通用引物MY11/09对所有标本L1区基因序列进行PCR扩增,利用地高辛标记探针杂交法对PCR产物进行检测分型;同时利用实时荧光定量PCR对这81例标本进行检测,将两者的检测结果进行比较。结果地高辛标记探针杂交法检测出HC-Ⅱ阳性标本中HPV-16型40例,HPV-51型2例,HPV-56型2例,分别占HC-Ⅱ阳性标本的63.5%、3.2%和3.2%,HC-Ⅱ阴性标本中检测出HPV-16型1例;实时荧光定量PCR法检测出HC-Ⅱ阳性标本中HPV-16型39例,HPV-51型2例,HPV-56型2例,占HC-Ⅱ阳性标本的61.9%、3.2%和3.2%,HC-Ⅱ阴性标本中检测出HPV-16型1例;重复性检测显示两种方法检测4型HPV感染的变异系数（CV）为0-1.45%和0-1.50%,检测标本中4型感染的符合率为97.5%-100%。结论PCR联合地高辛标记探针杂交法具有较好的灵敏度和特异性,可能对于临床HPV分型检测有应用价值。

Objective To evaluate the clinical application value of PCR combined with digoxin labeled probe hybridization by comparing detection results of digoxin labeled probe hybridization with real-time PCR for human papillomavirus（HPV） infection in cervix fluid specimens. Methods Type specific probes for 4 subtypes HPV（16,51,54 and 56） were labeled by incorporating a digoxin labeled oligonucleotides tail at their 3＇-end,then sensitivity and specificity of the digoxin-labeled probes were detected.Sixty-three positive and 18 negative cervix fluid specimens identified by hybrid capture Ⅱ（HC-Ⅱ） were collected.HPV L1 gene of all specimens was amplified by primers MY11/09 mediated PCR.PCR products were detected by probes hybridization,and the 81 specimens were detected by real-time PCR simultaneously. ResultsAmong 63 HPV positive specimens identified by HC-Ⅱ,HPV-16 was detected in 40 cases,HPV-51 in 2 cases and HPV-56 in 2 cases for hybridization method.The positive rates of HPV-16,HPV-51 and HPV-56 were 63.5%,3.2% and 3.2%,respectively.Moreover,in 18 HPV negative specimens identified by HC-Ⅱ,HPV-16 was found in 1 case.For real-time PCR method,HPV-16 was detected in 39 cases,HPV-51 in 2 cases and HPV-56 in 2 cases.The positive rates of HPV-16,HPV51 and HPV56 were 61.9%,3.2% and 3.2%,respectively.In 18 HPV negative specimens identified by HC-Ⅱ,HPV-16 was also found in 1 case.The coincidence rates of probes hybridization and real time PCR for detecting HPV-16,HPV51,HPV-54 and HPV56 were 97.5%,100%,100% and 100%,respectively. Conclusion The PCR combined with digoxin labeled probe hybridization may be a sensitive and specific method in clinical detection of HPV specimens.