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

• Assessment of specificity and sensitivity of PCRs for detection of T. equi.
• Comparison of different DNA template extraction methods.
• Outlining of some critical measure regarding the selection of primers for PCR assay.

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.