A novel fluorescence polarization based assay for 14 human papillomavirus genotypes in clinical samples.
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.
Institute of Gene Diagnosis, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xian, Shaanxi 710032, China.
Ju
J
Zhang
Zhongcan
Z
Chen
Yane
Y
Gao
Yi
Y
Jiang
Lin
L
Xia
Yanhai
Y
Guo
Xiaojun
X
Yan
D036022:Capsid Proteins / Q000235:genetics; D002584:Cervix Uteri / Q000821:virology; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D005454:Fluorescence Polarization; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000821:virology*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014621:Vagina / Q000821:virology
D036022:Capsid Proteins; D004279:DNA, Viral