DEVELOPMENT OF A MULTIPLEX REAL TIME TAQMAN ASSAY FOR SIMULTANEOUS DETECTION OF TWO MAJOR DIARRHEAL PARASITES CRYPTOSPORIDIUM AND GIARDIA

Abstract

Aims: Development of TaqMan Assay qPCR for multiplexing Giardia spp targeting GDH and Cryptosporidium spp targeting 18s rRNA. Multiplex SYBR-Green Assay qPCR as an alternative. Methods and Results: Specific primers and TaqMan probes were designed for Cryptosporidium 18S rRNA and Giardia spp GDH genes. The primers and probes for TaqMan qPCR were designed by using ‘Primer Express’ software commercially with help of value added services from the supplier. Probes were conjugated with FAM and HEX respectively; this combination was appropriate for multiplexing the real-time PCR reactions for two targets. Standard curve for each of the real-time qPCR assay was constructed with DNA concentrations ranging from 101 to 10-5 ng/μl. For each assay, the cycle threshold values (Ct) of dilution points were plotted as a function of the logarithm of the input DNA quantity. Analytical sensitivity or detection limit was calculated to be 0.0004 ng/ul for both TaqMan assays when standardized with standard positive DNA templates in contrast to 0.4 ng/ul as in case of conventional PCR. Diagnostic sensitivity and specificity of the real time PCR assays were calculated based on the results while testing against confirmed positive and negative control DNA templates. Both diagnostic sensitivity and specificity were 100%. Conclusions: The original objective of developing a multiplex real-time TaqMan assay to detect Cryptosporidium and Giardia DNA simultaneously is successfully achieved. In this study 18s rRNA gene of Cryptosporidium spp and GDH gene of Giardia spp were observed to be diagnostic as seen in conventional PCR, SYBR green qPCR and TaqMan qPCR assays in this study. The designing of the above two genes specific primers as well as probes, the methodologies choosen, and the appropriate control materials employed in the development and validation of the TaqMan qPCR assays for individual detection of either pathogens are justified to be appropriate. Extended studies on testing diagnostic efficiencies of these real time multiplex tests on clinical and/or environmental samples are recommended. Significance and Impact of the Study: The multiplex TaqMan assay developed in the present study is beneficial in terms of a single step detection of two most important protozoan pathogens known to cause diarrhoea in both human and animals. Hence it is advantageous over traditional detection techniques being in practice till date. Future development should be screening application of this multiplex assay in both hospitals as well as in epidemiological studies. Specific diagnostic genetic markers should be identified for other important diarrheogenic pathogens (including bacteria, viral, parasitic agents) for which if a panel of such targets are tested by multiplexing highthroughput detection systems, then a faster diagnosis of the accurate etiological agent as well as possible concurrent infections could be identified in one step. Success on multiplexing highly sensitive technique using specific TaqMan probes as experienced in the present study is certainly tempting to add other pathogenic targets to existing two pathogens Cryptosporidium and Giardia for their simultaneous detection.