Detection assay for HPV16 and HPV18 by loop‑mediated isothermal amplification with lateral flow dipstick tests.
Cervical cancer is the third highest cause of death in developing countries and most commonly results from high‑risk human papillomavirus (HR‑HPV) infection. Among HR‑HPV 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 loop‑mediated isothermal amplification (LAMP) with lateral flow dipstick (LFD) tests. This assay is a simplified, user‑friendly 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 HPV16‑positive, 18 HPV18‑positive and 80 HPV‑negative samples. All DNA samples were also used as templates for a LAMP reaction (30 min at 65˚C), and subsequently, a fluorescein isothiocyanate‑labelled probe was hybridized with the reaction product. Finally, the LFD test was performed. The sensitivity of the LAMP‑LFD test was higher than LAMP‑turbidity, exhibiting up to 100‑fold higher sensitivity for HPV16 and 10‑fold higher sensitivity for HPV18. All HPV16 and HPV18‑positive samples generated positive results in both assays; however, 22 samples detected as HPV‑negative by LAMP‑turbidity exhibited positive results by LAMP‑LFD 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 LAMP‑LFD, 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 LAMP‑LFD provided higher sensitivity than LAMP‑turbidity and nested PCR. Thus, the LAMP‑LFD test developed in the present study might be useful for the detection of HPV16 and HPV18 in local hospitals.
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
Ratchanida
R
Kumvongpin
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
Patcharee
P
Jearanaikoon
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
Chotchana
C
Wilailuckana
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
Nattaya
N
Sae-Ung
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
Prinya
P
Prasongdee
Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
Sakda
S
Daduang
Ubon Ratchathani Cancer Center, Ubon Ratchathani 34000, Thailand.
Metee
M
Wongsena
Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Patcharee
P
Boonsiri
Bioengineering and Sensing Technology Laboratory, BIOTEC, National Science and Technology Development Agency, Khlong Luang, Pathum Thani 12120, Thailand.
Wansika
W
Kiatpathomchai
Gynecological Oncology Division, National Cancer Institute, Bangkok 10400, Thailand.
Sukumarn Sanersak
SS
Swangvaree
Chulabhorn Research Institute, Bangkok 10210, Thailand.
Alisa
A
Sandee
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
Jureerut
J
Daduang
D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D004279:DNA, Viral / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D005838:Genotype; D006046:Gold / Q000737:chemistry; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000302:isolation & purification; D052161:Human papillomavirus 18 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007118:Immunoassay; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis / Q000821:virology
D000906:Antibodies; D015342:DNA Probes; D004279:DNA, Viral; D011933:Reagent Kits, Diagnostic; D006046:Gold