Detection of human papillomavirus DNA by PCR/microfluorometry for screening of cervical cancer.
BACKGROUND
Cervical cancer screening is conducted by a cytological Papanicolaou (Pap) test. For screening, it is becoming increasingly important to introduce a more objective result, based on human papillomavirus (HPV) DNA test. We describe here a practical method allowing the mass detection of HPV-DNA by PCR followed by fluorogenic DNA intercalation.


METHODS
Samples used were cervical scrapes or biopsy specimens obtained from women who had undergone cytological testing for cervical cancer. Crude DNAs were extracted by a simplified proteinase K-boil method. Common and type-specific primers were newly designed for major types of high-risk HPVs. A fluorogenic DNA intercalator, SYBR Green I was directly added to the specific PCR products. The resultant fluorescence was measured by a conventional fluorometric microplate reader.


RESULTS
The proposed PCR/microfluorometry (MFL) allowed a simple, rapid and economical detection of HPV-DNA without any use of labeling primers or probes. HPV-DNAs were found in 48.2% (123/255) of the cervical scrapes. The detection rate of HPV in cervical cancer biopsy specimen was 92.4% (61/66).


CONCLUSIONS
PCR/MFL detection of HPV-DNA, followed by combined type-specific PCR, is expected to be an extremely useful tool in cervical cancer screening.
Yamaguchi
Akihiro
A
Sapporo Immunodiagnostic Laboratory, Shinkawa 2-2-12-20, Kita-ku, Sapporo 001-0922, Japan.
Hashimoto
Naoko
N
Tsutae
Wataru
W
Seino
Kuniyoshi
K
Ebina
Yasuhiko
Y
Tokino
Takashi
T
Sato
Noriyuki
N
Kikuchi
Kokichi
K
D052160:Benzothiazoles; D001706:Biopsy; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D003959:Diamines; D005260:Female; D005456:Fluorescent Dyes; D005470:Fluorometry; D006801:Humans; D009930:Organic Chemicals; D027383:Papillomaviridae / Q000737:chemistry*; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis* / Q000821:virology
D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I