UNCORRECTED PROOF ARTICLE INFO Article history: Received 17 March 2016 Received in revised form 18 May 2016 Accepted 9 June 2016 Available online xxx Keywords: T. vaginalis Asymmetric-PCR Hand-held-dark-reader Molecular-beacon q-PCR Diagnostic evaluation ABSTRACT 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 de- tection 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 pre- sent study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20 fg 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. © 2016 Published by Elsevier Ltd. Biosensors and Bioelectronics xxx (2016) xxx-xxx Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis Subash C. Sonkar, a Divya Sachdev, a Prashant K. Mishra, a Anita Kumar, b Pratima Mittal, b Daman Saluja a, ⁎ a Medical Biotechnology Laboratory, Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India b Department of Obstetrics & Gynecology Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi 110029, India 1. Introduction According to WHO, trichomoniasis, caused by parasitic proto- zoan Trichomonas vaginalis, constitutes more than 50% of all curable sexually transmitted infections (STIs). Trichomoniasis causes mul- tiple reproductive health complications including vaginitis, cervici- tis, pelvic inflammatory disease (PID), cervical cancer and infertil- ity (Harp and Chowdhury et al., 2011; Bachmann et al., 2011). In- fection causes considerable discomfort with mental distress and ac- celerated transmissions (2–4 folds) of human immunodeficiency virus (Moodley et al., 2002; Marquardt et al., 2003; Swygard et al., 2004; Scott McClelland et al., 2007). Surveillance data indicates that about 170–190 million including 76.5 million new cases are reported an- nually in South East Asia (WHO, 2012). In India, trichomoniasis ac- counts for 1.2–28.5% amongst commercial sex workers, general com- munity and migrated population and 61.7% in women attending STD clinics (WHO, 2005; Kumarasamy et al., 2008; Kaur et al., 2008; Sood et al., 2008; Shethwala et al., 2009; Shahmanesh, et al., 2009; Madhivanan et al., 2009; Preethi et al., 2011; Paul et al., 2012; Fule et al., 2012; Dave et al., 2012; Dharma et al., 2013; Desai et al., 2013; Arora et al., 2014). One of the reasons for this wide variation in prevalence could be use of diagnostic assays that are highly variable in sensitivity and specificity (Schwebke et al., 2004; Radonjic et al., 2005; Nye et al., 2009; Unemo et al., 2013; Sonkar et al., 2016). The ⁎ Corresponding author. Email address: dsalujach59@gmail.com (D. Saluja) confirmatory diagnostics assays are not carried out because of lack of resources, simplicity and/or their high cost. Routine clinical laborato- ries in low resource settings, therefore, rely on syndromic case man- agement (SCM) which is unreliable because of lack of specificity in clinical presentation (Sonkar et al., 2016). All these factors have con- tributed to insufficient and unreliable information on prevalence and incidence of trichomoniasis in India. Hence, there is an urgent need for development of well-validated, sensitive, specific and affordable PCR based assay for use in countries with poor resource settings. In the present study, we developed an in-house molecular-beacon based PCR assay using pfoB gene. Molecular-beacon is a single stranded stem-loop structure that encompasses a probe region (loop) comple- mentary to the target DNA (amplicon). The beacon is labeled at 5'end with a fluorophore and at 3'end it carries a quencher. In stem-loop con- formation, energy from the excited fluorophore is transferred to the quencher and released as heat instead of light. When the loop region of the beacon hybridizes to the amplicon, the 5' and 3' ends of the beacon molecules are separated and the energy from the excited fluo- rophore is emitted as light, generating a detectable signal (Tyagi et al., 1996). The in-house PCR based detection method of T. vaginalis de- scribed here was evaluated by composite reference method (CRS) for its sensitivity, specificity, positive and negative predictive value (PPV & NPV respectively). In addition, use of molecular-beacon resulted in easy visualization of amplified product with minimum risk of cross contamination, a perpetual problem in diagnostic laboratories. http://dx.doi.org/10.1016/j.bios.2016.06.025 0956-5663/© 2016 Published by Elsevier Ltd.