Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor.
In the field of diagnostics, molecular amplification targeting unique genetic signature sequences has been widely used for rapid identification of infectious agents, which significantly aids physicians in determining the choice of treatment as well as providing important epidemiological data for surveillance and disease control assessment. We report the development of a rapid nucleic acid lateral flow biosensor (NALFB) in a dry-reagent strip format for the sequence-specific detection of single-stranded polymerase chain reaction (PCR) amplicons at ambient temperature (22-25°C). The NALFB was developed in combination with a linear-after-the-exponential PCR assay and the applicability of this biosensor was demonstrated through detection of the cholera toxin gene from diarrheal-causing toxigenic Vibrio cholerae. Amplification using the advanced asymmetric PCR boosts the production of fluorescein-labeled single-stranded amplicons, allowing capture probes immobilized on the NALFB to hybridize specifically with complementary targets in situ on the strip. Subsequent visual formation of red lines is achieved through the binding of conjugated gold nanoparticles to the fluorescein label of the captured amplicons. The visual detection limit observed with synthetic target DNA was 0.3 ng and 1 pg with pure genomic DNA. Evaluation of the NALFB with 164 strains of V. cholerae and non-V. cholerae bacteria recorded 100% for both sensitivity and specificity. The whole procedure of the low-cost NALFB, which is performed at ambient temperature, eliminates the need for preheated buffers or additional equipment, greatly simplifying the protocol for sequence-specific PCR amplicon analysis.
Department of Medical Microbiology & Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. geikyong@gmail.com
Geik Yong
GY
Ang
Choo Yee
CY
Yu
Chan Yean
CY
Yean
D001483:Base Sequence; D015374:Biosensing Techniques / Q000191:economics / Q000379:methods*; D002771:Cholera / Q000175:diagnosis / Q000382:microbiology*; D002772:Cholera Toxin / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D006046:Gold / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D011934:Reagent Strips / Q000032:analysis; D012680:Sensitivity and Specificity; D013696:Temperature; D014734:Vibrio cholerae / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D011934:Reagent Strips; D006046:Gold; D002772:Cholera Toxin