Coutlée F, Bobo L, Abbass H, Dalabetta G, Hook N E, Shah K, Viscidi R P
Department of Microbiology and Infectious Diseases, Hôpital Notre-Dame, Montréal, Canada.
J Med Virol. 1992 May;37(1):22-9. doi: 10.1002/jmv.1890370105.
A gene amplification method that combines the polymerase chain reaction with detection of amplified DNA in a solution hybridization/enzyme immunoassay (PCR-EIA) was developed for HPV-16 DNA. Samples were amplified with primers for the E7-E1 region of HPV-16. Amplified DNA products were identified and quantitated by hybridization in solution with a biotinylated RNA probe. Labeled DNA/RNA hybrids were measured semiquantitatively in an enzyme immunoassay using solid phase anti-biotin antibody and liquid phase B-d-galactosidase labeled monoclonal antibody against DNA-RNA hybrids. Enzyme bound to the solid phase was quantitated with a fluorogenic substrate. The assay was linear over 2 log10 dilutions of SiHa cells and the detection limit was three copies of HPV-16 genome. The sensitivity of PCR-EIA for detection of PCR amplified products compared favorably with slot and Southern blots using a 32P-labeled RNA probe. The assay was used to assess HPV-16 infection of uterine cervix in women attending a clinic for sexually transmitted diseases. Twenty-one of the 81 specimens (25.9%), obtained by cervicovaginal lavage, were positive for HPV-16 by PCR-EIA. The assay provides a convenient means to objectively measure HPV DNA amplified with PCR.
一种将聚合酶链反应与溶液杂交/酶免疫分析（PCR-EIA）中扩增DNA的检测相结合的基因扩增方法被开发用于检测HPV-16 DNA。样本用HPV-16 E7-E1区域的引物进行扩增。扩增的DNA产物通过与生物素化RNA探针在溶液中杂交来鉴定和定量。标记的DNA/RNA杂交体在酶免疫分析中使用固相抗生物素抗体和液相β-半乳糖苷酶标记的针对DNA-RNA杂交体的单克隆抗体进行半定量测量。与固相结合的酶用荧光底物进行定量。该分析在SiHa细胞的2个对数10稀释范围内呈线性，检测限为HPV-16基因组的三个拷贝。PCR-EIA检测PCR扩增产物的灵敏度与使用32P标记RNA探针的狭缝杂交和Southern杂交相比具有优势。该分析用于评估在一家性传播疾病诊所就诊的女性子宫颈的HPV-16感染情况。通过宫颈阴道灌洗获得的81个标本中有21个（25.9%）通过PCR-EIA检测HPV-16呈阳性。该分析提供了一种客观测量经PCR扩增的HPV DNA的便捷方法。
