PCR based diagnostic assay targeting the beta tubulin gene for the detection of Trichomonas vaginalis infection in vaginal swab samples of symptomatic and asymptomatic women in India

Abstract

ObjectiveTo develop an in-house PCR based diagnostic assay for identification of strains isolated from symptomatic and asymptomatic subjects of India, targeting the β-tubulin gene using specific primers. MethodsIn the present study a primer set is designed to target a well-conserved region in the beta-tubulin gene of Trichomonas vaginalis (T. vaginalis). All strains of T. vaginalis were tested and successfully detected by PCR yielding a single predicted product of 198 bp in gel electrophoresis, while there was negative response with DNA from Giardia lamblia, Toxoplasma gondii, Leishmania donovani and Entamoeba histolytica. The sensitivity and specificity for a single T. vaginalis cell per PCR was achieved. Axenic Culture, performed with long term axenized T. vaginalis culture system, was routinely examined to identify T. vaginalis. ResultsThe PCR based investigations with 498 vaginal swab samples from women attending OPD clinics of Halberg Hospital Moradabad and Queen Mary's Hospital, Lucknow, India and 17 long term axenic cultures maintained at PGIMER, Chandigarh, India using primer set BTUB 1 & BTUB 2 showed sensitivity and specificity response of 98% and 100%, respectively, while wet preparation in clinically isolated samples responded up to 62.5%. The PCR product sequencing result of symptomatic strains (SS1) of T. vaginalis (744 bp long) was submitted to NCBI (Accession No: JF513200). It shows maximum identity 98 % with XM_001284521 Trichomonas vaginalis G-3 beta-tubulin (btub) putative partial mRNA. ConclusionsThe data gathered in the present study entail that the diagnosis of T. vaginalis infection by PCR may be established as a sensitive and specific protocol, to be incorporated into a joint strategy for the screening of multiple STDs by employing molecular amplification technique. The merits and precautions of the protocol have been discussed.