Oligonucleotide microarray with RD-PCR labeling technique for detection and typing of human papillomavirus.
Currently, screening for high-risk human papillomavirus (HPV) infection remains an important health concern throughout the world, because of the close association between certain types of HPV and cervical cancer. In this study, we explore the possibility of using approximately 70mer oligonucleotide microarray for detection and genotyping of HPV. The approximately 70mer type-specific oligonucleotide probes of four different types HPV were designed by using biological software Array designer 2.0, which analyzed the whole genome sequences of HPV and selected optimal probes. These probes were synthesized and printed onto the surface of glass slides in order to prepare a low-density microarray. HPV samples were labeled with fluorescence dyes Cy3 using a method of restriction display polymerase chain reaction (RD-PCR). HPV plasmid DNA was restricted with Sau3A I to produce multiple fragments that were ligated to adaptors subsequently and used as PCR template. PCR labeling was performed with the fluorescently labeled universal primer (Cy3-UP) whose sequence is designed according to the adaptor of the RD-PCR approaches. The labeled samples were hybridized with the oligonucleotide microarray. The scanning results showed that HPV DNA hybridized specifically with multiple spots correspondingly to show positive signals, whereas no signals were detected of all the negative and blank controls. These results demonstrated that approximately 70mer oligonucleotide microarray can be applied to HPV detection and genotyping. The application of RD-PCR in the sample labeling can increase significantly the sensitivity of the assay and will be especially useful for the discriminate diagnosis of multiple pathogens.
Min
Wei
W
Institute of Molecular Biology, Southern Medical University, Guangzhou, 510515, P.R. China. wenli@fimmu.com
Wen-Li
Ma
M
Bao
Zhang
Z
Ling
Li
L
Zhao-Hui
Sun
S
Wen-Ling
Zheng
Z
D002232:Carbocyanines / Q000737:chemistry; D004279:DNA, Viral / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D005838:Genotype; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D002232:Carbocyanines; D004279:DNA, Viral; D015345:Oligonucleotide Probes; C089187:cyanine dye 3; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases