Zhang Ju, Chen Zhongcan, Gao Yane, Jiang Yi, Xia Lin, Guo Yanhai, Yan Xiaojun
Institute of Gene Diagnosis, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xian, Shaanxi 710032, China.
J Virol Methods. 2006 Jun;134(1-2):223-9. doi: 10.1016/j.jviromet.2006.01.008. Epub 2006 Feb 20.
A specific and practical method was developed for high throughput 14 human papillomavirus (HPV) genotypes assay in clinical samples by a single PCR. GP5+/6+ polymerase chain reaction (PCR) system was used to amplify HPV DNA in 1127 samples. The PCR product was assayed by AcycloPrime reaction with fluorescence polarization (FP). Fourteen HPV genotypes specific sequence primers designed within GP5+/GP6+ amplification polymorphism regions of L1 genes for corresponding HPV genotypes were annealed with the type specific PCR products and special fluorescent terminator was added to the end of the primer under direction of the PCR products. AcycloPrime-FP analysis showed specific anneal and incorporation without any cross-reaction. The types detected with FP showed an excellent overall agreement with sequence when the individual monotype results were taken into account. The proposed method could detect more than one type of HPV infection, but the sequence method was limited. AcycloPrime-FP could reach the detection level: 100 ag for representative phylogenetically distant HPV genotypes: HPV6, 18, 31, 39, 42, 51 and 58. The results of AcycloPrime-FP showed excellent reproducibility. The proposed method allowed an economical detection of HPV genotypes without any use of labeled probe. It is expected to be an extremely useful tool for HPV genotypes screening.
开发了一种特定且实用的方法，通过单一聚合酶链反应（PCR）对临床样本中的14种人乳头瘤病毒（HPV）基因型进行高通量检测。采用GP5 + / 6 +聚合酶链反应（PCR）系统对1127份样本中的HPV DNA进行扩增。PCR产物通过荧光偏振（FP）的AcycloPrime反应进行检测。在L1基因的GP5 + / GP6 +扩增多态性区域内针对相应HPV基因型设计的14种HPV基因型特异性序列引物与型特异性PCR产物退火，并在PCR产物的指导下在引物末端添加特殊荧光终止子。AcycloPrime - FP分析显示特异性退火和掺入，无任何交叉反应。当考虑个体单型结果时，FP检测的类型与序列显示出极好的总体一致性。所提出的方法可以检测多种类型的HPV感染，但序列方法有限。AcycloPrime - FP可达到检测水平：对于具有代表性的系统发育距离较远的HPV基因型（HPV6、18、31、39、42、51和58）为100 ag。AcycloPrime - FP的结果显示出极好的重现性。所提出的方法允许在不使用标记探针的情况下经济地检测HPV基因型。预计它将成为HPV基因型筛查的极其有用的工具。
