Comparative Sensitivity and Specificity of Polymerase Chain Reaction Assays for the Detection of Theileria equi Coupled With Three DNA Template Extraction Methods

Abstract

The present study demonstrates the relative sensitivity and specificity of two 18S rRNA gene-based polymerase chain reaction (PCR) assays (designated as PCR1 and PCR2) coupled with three genomic DNA extraction methods viz., via Whatman filter paper#1 method (Ext 1), Whatman Flinders Technology Associate (FTA) elute microcards method (Ext 2), and Qiagen DNeasy Blood & Tissue Kits (Ext three3) for the detection of Theileria equi. Both PCR assays on the nucleic acid of 72 field blood samples extracted by Ext 1 and Ext 3 revealed an prevalence rate of 2.77 and 18.56%, respectively; whereas in case of Ext 2, PCR1 and PCR2 revealed 16.67% and 18.56% prevalence, respectively. Ext 1 showed only 15.38% sensitivity and 100% specificity, whereas Ext 2 displayed 92.3% to 100% sensitivity and 100% specific results with respect to Ext 3. The amplified products were clearly positive with a diminishing signal up to 10−6 DNA dilutions in Ext 2 and Ext 3 method in both PCRs. Ext 1 was able to detect only 10−3 and 10−4 DNA dilution. Based on these results, it was concluded that blood sampled on FTA cards for DNA extraction is the recommended approach with detection ability up to 10−6 dilution.