Isothermal Method of a Recombinase Polymerase Amplification Assay for the Detection of Most Common High-Risk Human Papillomavirus Type 16 and Type 18 DNA.
BACKGROUND
Cervical cancer is a common gynecologic malignant tumor and has a great impact on women's health. Human papillomavirus (HPV) is implicated in cervical cancer and precancerous lesions and the two are possibly two stages of disease progression. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important means to prevent cervical cancer.


METHODS
Here we present a novel, rapid, sensitive and specific isothermal method of recombinase polymerase amplification (RPA), which is established to detect the two most common high-risk human papillomavirus type 16 and type 18 DNA. In this study, we evaluate the efficacy of the RPA assay, incubating clinical specimens of HPV16 and HPV18 using plasmids standard. It operates at constant low temperature without the thermal instrumentation for incubation. The products can be detected via agarose gel electrophoresis assay, reverse dot blot assay, and quantitative real-time assay with SYBR Green I. We assess the diagnostic performance of the RPA assay for detecting of HPV16 and HPV18 in 335 clinical samples from patients suspected of cervical cancer.


RESULTS
The results revealed no cross-reaction with other HPV genotypes and the RPA assay achieve a sensitivity of 100 copies. Compared with TaqMan qPCR, the RPA technique achieves exponential amplification with no need for pretreatment of sample DNA at 37°C for 20 minutes, which reveals more satisfactory performance. The agreement between the RPA and qPCR assays was 97.6% (κ = 0.89) for HPV16 positivity and 98.5% (κ = 0.81) for HPV18 positivity, indicating very good correlation between both tests.


CONCLUSIONS
Importantly, the RPA assay was demonstrated to be a useful and powerful method for detection of HPV virus, which therefore may serve as a valuable tool for rapid diagnosis of HPV infection in both commercial and clinical applications.
Biao
B
Ma
Jiehong
J
Fang
Ye
Y
Wang
Haizhen
H
He
Mingyan
M
Dai
Wei
W
Lin
Wei
W
Su
Mingzhou
M
Zhang
D000293:Adolescent; D000328:Adult; D002138:Calibration; D004279:DNA, Viral / Q000235:genetics*; D005260:Female; D005838:Genotype; D061809:Human Papillomavirus DNA Tests / Q000379:methods* / Q000592:standards; D052162:Human papillomavirus 16 / Q000235:genetics*; D052161:Human papillomavirus 18 / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000235:genetics / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011237:Predictive Value of Tests; D060888:Real-Time Polymerase Chain Reaction; D045522:Recombinases / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D013696:Temperature; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000821:virology; D057188:Workflow; D055815:Young Adult
D004279:DNA, Viral; D045522:Recombinases