The Evaluation on Molecular Techniques of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP), Reverse Transcription Polymerase Chain Reaction (RT-PCR), and Their Diagnostic Results on MinION TM Nanopore Sequencer for the Detection of Dengue Virus Serotypes

Abstract

Background and aims:. Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay is a method described as a simple, rapid, and cost-effective, and a powerful gene amplification technique for rapid identification of microbial infection. The aims of this study were to validate a reverse-transcription LAMP method (RT-LAMP) with a reverse-transcription PCR technique (RT-PCR) for DENV serotype detection, and to evaluate the potential of this RT-LAMP method from which its amplification product can be applied for MinIONTM Nanopore sequencing. Methods: This study was performed by using 26 patient serum samples from the Confirmed group and 23 sera from the Suspected group, and 10 sera from Healthy volunteer group. The same validation study to confirm the sensitivity and specificity between the RT-LAMP assay and RT-PCR was also conducted by making a serial dilution of template as well as dengue serotype detected sera. Furthermore, experiments of MinIONTM Nanopore sequencer using the amplification product (amplicon) of RT-LAMP assay from patient serum samples and its comparative diagnostics with RT-PCR and Sanger’s Sequencing were also done. Results: From the validation study, 17 (65.4%) out of the 26 sera of Confirmed group were serotype detected by RT-LAMP assay, compared to that of RT-PCR which was only 10 (38.4%). Same result in Suspected group, there were 11 (47.8%) out of 23 sera of Suspected group were serotype detected by RT-LAMP assay, compared to that of RT-PCR which was only 1 (4.3%). For the Healthy group, the result was somewhat comparable between the two methods. In the serial dilution study, RT-LAMP was superior than that of the RT-PCR technique, particularly seen in the serial dilution of clinical serum samples. The limit of detection of RT-LAMP was down to the titer of 1/1000x, while RT-PCR was only 1/10x. Secondly, amplicon of RT-LAMP assay from patient serum samples fits to be sequenced by the MinIONTM. In the comparative study, the results showed that RT-LAMP was more sensitive and specific compared to that of the RT-PCR technique and Sanger’s Sequencing. Conclusions: RT-LAMP assay was more sensitive to that of RT-PCR technique. In the comparative study using MinIONTM Nanopore sequencer for the detection of dengue virus serotypes, the RT-LAMP was more sensitive and specific to that of RT-PCR and Sanger’s Sequencing. The method of RT-LAMP should be applied more because its sensitive and specific, simple, rapid, cost-effective, and also can be a tool of point of care testing for the detection of dengue virus serotypes.