Visual Detection of High-Risk Human Papillomavirus Genotypes 16, 18, 45, 52, and 58 by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye

Abstract

A simple, rapid, sensitive, qualitative, colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB) was established to detect high-risk human papillomavirus (HPV) genotypes 16, 18, 45, 52, and 58. All initial validation studies with the control DNA proved to be type specific. The colorimetric type-specific LAMP assay could achieve a sensitivity of 10 to 100 copies at 63°C for 65 min, comparable to that of real-time PCR. In order to evaluate the reliability of HPV type-specific LAMP, the assay was further evaluated with HPV DNAs from a panel of 294 clinical specimens whose HPV status was previously determined with a novel one-step typing method with multiplex PCR. The tested panel comprised 108 HPV DNA-negative samples and 186 HPV-DNA-positive samples of 14 genotypes. The results showed that the sensitivity of HPV type-specific LAMP for HPV types 16, 18, 45, 52, and 58 was 100%, 100%, 100%, 100%, and 100%, respectively, and the specificity was 100%, 98.5%, 100%, 98.8%, and 99.2%, respectively, compared with a novel one-step typing method with multiplex PCR. No cross-reactivity with other HPV genotypes was observed. In conclusion, this qualitative and colorimetric LAMP assay has potential usefulness for the rapid screening of HPV genotype 16, 18, 45, 52, and 58 infections, especially in resource-limited hospitals or rural clinics of provincial and municipal regions in China.It is well established that virtually all cervical cancers are caused by persistent high-risk oncogenic human papillomavirus (HPV) infections in the cervix. Thus, cervical screening could be improved by testing for the DNA of high-risk types of HPV as a primary screening tool (3, 9, 24). So far, at least 14 high-risk HPV genotypes have been shown to cause cervical cancer; those most important for cervical cancer are HPV types 16, 18, 45, 31, 33, 52, 58, and 35 (6, 7, 26). HPV16 is by far the predominant type, causing more than 50% of cancers; HPV18 follows, causing 14%; and HPV45 causes approximately 2.8%. These genotypes are highly prevalent in all regions of the world and account for approximately 70% of all cancers of the cervix (2, 13, 25). HPV types 52 and 58 are rare in Western countries; however, they are prevalent in Asian populations, especially in China (5,14,15,28,29).Technologies available for HPV genotyping vary by method and platform and may offer type-specific characterization for a multitude of different high-and low-risk HPV types. For example, The Qiagen Hybrid Capture 2 HPV DNA (HC2) test and the Roche Amplicor HPV test (Amplicor) detect the DNA of 13 high-risk HPV types. The Linear Array HPV genotyping test allows for the discrimination of 37 HPV genotypes. The PapilloCheck HPV screening test (Greiner Bio-One GmbH, Frickenhausen, Germany) is a PCR-based DNA microarray system for the detection and identification of 24 HPV genotypes (4, 7, 13, 25). However, these methods might not be suitable in primary clinical settings in developing countries or for field use, because of the sophisticated ...

Improved Detection Limit in Rapid Detection of Human Enterovirus 71 and Coxsackievirus A16 by a Novel Reverse Transcription–Isothermal Multiple-Self-Matching-Initiated Amplification Assay

“…Two visual RT-IMSA assays were established individually by adding (i) a 1.0-l hydroxynaphthol blue (HNB) dye (3.0 mmol/liter; Lemongreen, Shanghai, China) to the mixture before amplification, according to reported visual RT-LAMP assays using HNB dye (17)(18)(19), and (ii) a 1.0-l modified HNB (mHNB) dye that consisted of 1.5 mol HNB and 1,000ϫ GeneFinder (Zeesan Biotech, Xiamen, China) after amplification, termed visual RT-IMSA (HNB) and RT-IMSA (mHNB) assays, respectively. The GeneFinder is a nucleic acid dye that was used to establish visual detection in the RT-LAMP assay (27,28).…”

“…Further-more, products can be detected with the RT-LAMP assay through various methods. Apart from traditional gel electrophoresis, the RT-LAMP products can be detected through spectrophotometric equipment to measure turbidity (15) or through direct a visual inspection of turbidity (16) and color changes (17)(18)(19). To date, several RT-LAMP-based detection methods for EV71 and CVA16 have been established in our laboratory and with other groups (18,(20)(21)(22).…”

Sensitivity Evaluation of SYBR Green I, SYBR Safe and Calcein Dyes for Detection of Human Papillomavirus 16 by Loop-Mediated Isothermal Amplification

“…It is a small-sized DNA virus and comprises 8 kb, circular, double stranded DNA genome (Ramqvist & A study conducted by Siti Aishah et al (2006) reported two cases of juvenile laryngeal papillomatosis containing HPV viral genomes in Malaysia. Among all, HPV genotype 16 (HPV 16) is one of well-knowned risk factors for the development of oral cancer, representing >95% of all HPV positive squamous cell carcinoma (OSCC) (Luo et al 2011). Several molecular methods, including loop-mediated isothermal amplification (LAMP), have already been introduced for the detection of HPV-16 (Hagiwara et al 2007;Luo et al 2011;Livingstone et al 2016).…”

“…Among all, HPV genotype 16 (HPV 16) is one of well-knowned risk factors for the development of oral cancer, representing >95% of all HPV positive squamous cell carcinoma (OSCC) (Luo et al 2011). Several molecular methods, including loop-mediated isothermal amplification (LAMP), have already been introduced for the detection of HPV-16 (Hagiwara et al 2007;Luo et al 2011;Livingstone et al 2016). LAMP is a gene amplification method that can amplify nucleic acids in less than 60 mins under isothermal conditions without a thermocycler which provides highly sensitive and specific results (Notomi et al 2000).…”

Direct Blood PCR in Combination with Nucleic Acid Lateral Flow Immunoassay for Detection of Plasmodium Species in Settings Where Malaria Is Endemic

“…Several attempts have been made to simplify molecular tools such as the amplification of DNA in an isothermal manner (7), adding the specimen without complete DNA isolation (4), or by adding colorimetric substances that allow the PCR result to be interpreted visually (8). In the present study, a direct blood PCR (db-PCR) combined with a rapid readout system, nucleic acid lateral flow immunoassay (NALFIA), is described.…”