Development of a dry-reagent-based nucleic acid-sensing platform by coupling thermostabilised LATE-PCR assay to an oligonucleotide-modified lateral flow biosensor.
In this study, we report for the first time the development of a dry-reagent-based nucleic acid-sensing platform by combining a thermostabilised linear-after-the-exponential (LATE)-PCR assay with a one-step, hybridisation-based nucleic acid lateral flow biosensor. The nucleic acid-sensing platform was designed to overcome the need for stringent temperature control during transportation or storage of reagents and reduces the dependency on skilled personnel by decreasing the overall assay complexity and hands-on time. The platform was developed using toxigenic Vibrio cholerae as the model organism due to the bacterium's propensity to cause epidemic and pandemic cholera. The biosensor generates result which can be visualised with the naked eyes and the limit of detection was found to be 1pg of pure genomic DNA and 10CFU/ml of toxigenic V. cholerae. The dry-reagent-based nucleic acid-sensing platform was challenged with 95 toxigenic V. cholerae, 7 non-toxigenic V. cholerae and 66 other bacterial strains in spiked stool sample and complete agreement was observed when the results were compared to that of monosialoganglioside (GM1)-ELISA. Heat-stability of the thermostabilised LATE-PCR reaction mixes at different storage temperatures (4-56°C) was investigated for up to 90days. The dry-reagent-based genosensing platform with ready-to-use assay components provides an alternative method for sequence-specific detection of nucleic acid without any cold chain restriction that is associated with conventional molecular amplification techniques.
Division of Genetics & Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia; School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. Electronic address: geikyong@gmail.com.
Geik Yong
GY
Ang
Division of Genetics & Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia; School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
Choo Yee
CY
Yu
Division of Genetics & Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Kok Gan
KG
Chan
School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
Kirnpal Kaur Banga
KK
Singh
School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. Electronic address: yeancyn@yahoo.com.
Yean
Y
Chan Yean
D015374:Biosensing Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature; D014734:Vibrio cholerae / Q000235:genetics*
D004269:DNA, Bacterial
DNA biosensor; Dipstick; Dry-reagent; PCR; Sequence-specific detection; Vibrio cholerae