A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis

• Published in: Biosensors & bioelectronics 2016-12, Vol.86, p.41-47
• Main Authors: Sonkar, Subash C., Sachdev, Divya, Mishra, Prashant K., Kumar, Anita, Mittal, Pratima, Saluja, Daman
• Format: Article
• Language: English
• Subjects: Adolescent; Adult; Amplification; Assaying; Asymmetric-PCR; Biosensors; Colorimetry - instrumentation; Colorimetry - methods; Deoxyribonucleic acid; Diagnostic evaluation; DNA Probes - genetics; Equipment Design; Equipment Failure Analysis; Female; Fluorescence; Hand-held-dark-reader; Humans; Middle Aged; Molecular Diagnostic Techniques - instrumentation; Molecular Probe Techniques - instrumentation; Molecular-beacon; Nucleic acids; Polymerase Chain Reaction - instrumentation; q-PCR; Readers; Reproducibility of Results; Sensitivity and Specificity; T. vaginalis; Trichomonas vaginalis; Trichomonas vaginalis - genetics; Trichomonas vaginalis - isolation & purification; Trichomonas Vaginitis - diagnosis; Trichomonas Vaginitis - parasitology; Visualization; Young Adult
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2016.06.025

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3–4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management. •First Molecular-beacon-based-PCR diagnostics assay developed for T. vaginalis.•Highly accurate, sensitive, specific and inexpensive assay for on-site diagnosis.•Assay is highly reproducible and has a short turnaround time of 100 min.•Uses a low cost, hand-held device for easy visualization of fluorescent amplicons.