Quantitative screening of single copies of human papilloma viral DNA without amplification.
We describe a novel quantitative viral screening method based on single-molecule detection that does not require amplification. DNA of human papilloma virus (HPV), the major etiological agent of cervical cancer, served as the screening target in this study. Eight 100-nucleotide single-stranded DNA probes were designed complementary to the E6-E7 gene of HPV-16 DNA. The probes were covalently stained with Alexa Fluor 532 and hybridized to the target in solution. The individual hybridized molecules were imaged with an intensified charge-coupled device (ICCD) in two ways. In the single-color mode, target molecules were detected via fluorescence from hybridized probes only. This system could detect HPV-16 DNA in the presence of human genomic DNA down to 0.7 copy/cell and had a linear dynamic range of over 6 orders of magnitude. In the dual-color mode, we employed fluorescence resonance energy transfer and added YOYO-3 dye as the acceptor. The two colors from Alexa Fluor 532 and YOYO-3 were dispersed by a transmission grating located in front of the ICCD. With this reinforced criterion for identifying the hybridized molecules, zero false-positive count was achieved. We also showed that DNA extracts from Pap test specimens did not interfere with the measurements.
Li
Jiangwei
J
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
Lee
Ji-Young
JY
Yeung
Edward S
ES
D045744:Cell Line, Tumor; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D030361:Papillomavirus Infections / Q000821:virology*; D002583:Uterine Cervical Neoplasms / Q000821:virology*
D015342:DNA Probes; D004279:DNA, Viral; D005456:Fluorescent Dyes