title: High sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18.
abstract: 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.
authors.affiliation: Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Ratchanida
authors.initials: R
authors.lastname: Kumvongpin
authors.affiliation: Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Patcharee
authors.initials: P
authors.lastname: Jearanaikool
authors.affiliation: Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Chotechana
authors.initials: C
authors.lastname: Wilailuckana
authors.affiliation: Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Nattaya
authors.initials: N
authors.lastname: Sae-Ung
authors.affiliation: Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Prinya
authors.initials: P
authors.lastname: Prasongdee
authors.affiliation: Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Sakda
authors.initials: S
authors.lastname: Daduang
authors.affiliation: Ubon Ratchathani Cancer Center, Ubon Ratchathani, Thailand.
authors.forename: Metee
authors.initials: M
authors.lastname: Wongsena
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Patcharee
authors.initials: P
authors.lastname: Boonsiri
authors.affiliation: Bioengineering and Sensing Technology Laboratory, BIOTEC, National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Pathum Thani, Thailand.
authors.forename: Wansika
authors.initials: W
authors.lastname: Kiatpathomchai
authors.affiliation: Gynecological Oncology Division, National Cancer Institute, Bangkok, Thailand.
authors.forename: Sukumarn Sanersak
authors.initials: SS
authors.lastname: Swangvaree
authors.affiliation: Chulabhorn Research Institute, Bangkok, Thailand.
authors.forename: Alisa
authors.initials: A
authors.lastname: Sandee
authors.affiliation: Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand; HPV & EBV and Carcinogenesis Research Group, Khon Kaen University, Khon Kaen, Thailand. Electronic address: jurpoo@kku.ac.th.
authors.forename: Jureerut
authors.initials: J
authors.lastname: Daduang
mesh_terms: D003124:Colorimetry; D018076:DNA, Complementary / Q000737:chemistry; D005260:Female; D005838:Genotype; D006046:Gold; D052162:Human papillomavirus 16 / Q000235:genetics / Q000302:isolation & purification*; D052161:Human papillomavirus 18 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D030361:Papillomavirus Infections / Q000175:diagnosis / Q000821:virology; D012680:Sensitivity and Specificity; D013696:Temperature; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis / Q000821:virology
chemical_list: D018076:DNA, Complementary; D006046:Gold
keywords: AuNPs; HPV16; HPV18; LAMP