ABSTRACT
Cervical cancer is the third highest cause of death in developing countries and most commonly results from highrisk human papillomavirus (HRHPV) infection. Among HRHPV genotypes, HPV16 and HPV18 are the most prevalent in cervical cancers. Therefore, the present study aimed to develop a detection assay for HPV16 and HPV18 infection using loopmediated isothermal amplification (LAMP) with lateral flow dipstick (LFD) tests. This assay is a simplified, userfriendly method for the visual detection of HPV genotypes. DNA was extracted from clinical tissue samples, and HPV genotyping was performed using nested polymerase chain reaction (PCR). The clinical samples were demonstrated to include 44 HPV16positive, 18 HPV18positive and 80 HPVnegative samples. All DNA samples were also used as templates for a LAMP reaction (30 min at 65ËC), and subsequently, a fluorescein isothiocyanatelabelled probe was hybridized with the reaction product. Finally, the LFD test was performed. The sensitivity of the LAMPLFD test was higher than LAMPturbidity, exhibiting up to 100fold higher sensitivity for HPV16 and 10fold higher sensitivity for HPV18. All HPV16 and HPV18positive samples generated positive results in both assays; however, 22 samples detected as HPVnegative by LAMPturbidity exhibited positive results by LAMPLFD test (22 of 80 samples). Therefore, these samples were further examined using quantitative (q)PCR. The results demonstrated that 20 out of the 22 samples designated positive by LAMPLFD, but negative by LAMP turbidity, gave a positive result with qPCR, while the remaining 2 samples were negative by qPCR. The present results suggested that LAMPLFD provided higher sensitivity than LAMPturbidity and nested PCR. Thus, the LAMPLFD test developed in the present study might be useful for the detection of HPV16 and HPV18 in local hospitals.
Subject(s)
DNA, Viral/metabolism , Human papillomavirus 16/genetics , Human papillomavirus 18/genetics , Nucleic Acid Amplification Techniques/methods , Antibodies/chemistry , Antibodies/immunology , DNA Probes/chemistry , DNA Probes/metabolism , DNA, Viral/isolation & purification , Female , Genotype , Gold/chemistry , Human papillomavirus 16/isolation & purification , Human papillomavirus 18/isolation & purification , Humans , Immunoassay , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction , Uterine Cervical Neoplasms/diagnosis , Uterine Cervical Neoplasms/virologyABSTRACT
High-risk human papillomavirus (HR-HPV) causes cervical cancer. HPV16 and HPV18 are the most prevalent strains of the virus reported in women worldwide. Loop-mediated isothermal amplification (LAMP) is an alternative method for DNA detection under isothermal conditions. However, it results in a turbid amplified product which is not easily detected by the naked eye. This study aimed to develop an improved technique by using gold nanoparticles (AuNPs) attached to a single-stranded DNA probe for the detection of HPV16 and HPV18. Detection of the LAMP product by AuNP color change was compared with detection by visual turbidity. The optimal conditions for this new LAMP-AuNP assay were an incubation time of 20min and a temperature of 65°C. After LAMP amplification was complete, its products were hybridized with the AuNP probe for 5min and then detected by the addition of magnesium salt. The color changed from red to blue as a result of aggregation of the AuNP probe under high ionic strength conditions produced by the addition of the salt. The sensitivity of the LAMP-AuNP assay was greater than the LAMP turbidity assay by up to 10-fold for both HPV genotypes. The LAMP-AuNP assay showed higher sensitivity and ease of visualization than did the LAMP turbidity for the detection of HPV16 and HPV18. Additionally, AuNP-HPV16 and AuNP-HPV18 probes were stable for over 1year. The combination of LAMP and the AuNP-probe colorimetric assay offers a simple, rapid and highly sensitive alternative diagnostic tool for the detection of HPV16 and HPV18 in district hospitals or field studies.
