Objectives: We have developed a one-step nucleic acid dipstick assay (NADA) for visually detecting polymerase chain reaction (PCR) products within 3min. "One-step" means that there were no additional procedures between amplification and detection.
Methods: This method was achieved through the use of asymmetric PCR and specially designed probes with appropriate melting temperature values. We initially combined one-step NADA with asymmetric capillary convective PCR (ACCPCR), an easy and rapid nucleic acid amplification technique, to construct an on-site nucleic acid diagnostic platform.
Results: We developed a diagnostic assay for the hepatitis B virus based on the ACCPCR-NADA platform to verify its feasibility. It exhibited an analytical sensitivity of three copies per test and a broad detection spectrum including genotype A-I. It also showed 97.9% sensitivity and 100% specificity based on the results observed using 67 serum samples with the Roche COBAS AmpliPrep/COBAS TaqMan (COBAS) system as the standard for comparison.
Conclusion: The results provide evidence for the feasibility of using an ACCPCR-NADA platform in practical applications, especially in on-site test.
Keywords: 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; Asymmetric capillary convective PCR; CCPCR; COBAS; EDC; EDTA; EV71; FITC; GAM; HBV; HCV; HEV; HIV; HPV; NADA; NCBI; NIDVD; National Center for Biotechnology Information; National Institute of Diagnostics and Vaccine Development in Infectious Diseases; Nucleic acid on-site testing; One-step nucleic acid dipstick assay; PCR; PVC; Roche COBAS AmpliPrep/COBAS TaqMan; SA; TAE; Tm; capillary convective PCR; ethylenediaminetetraacetic acid; fluorescein isothiocyanate; goat anti-mouse; hepatitis B virus; hepatitis C virus; hepatitis E virus; human enterovirus 71; human immunodeficiency virus; human papillomavirus; melting temperature; nt; nucleotide; one-step nucleic acid dipstick assay; polymerase chain reaction; polyvinylchloride; streptavidin; tris-acetate-EDTA.
© 2013.