Abstract

Genomic DNA serves as the foundation for molecular research and advancements including disease sureveillance, biodiversity conservation and environmental monitoring etc. Cost-effectiveness coupled with DNA quality and quantity are prerequisites for molecular tools, which depend on extraction protocols. The current study compared four DNA extraction protocols to evaluate DNA quantity, integrity, purity, cost-effectiveness and suitability for detecting theileriosis. The DNA concentration with 100 µL of blood was highest for Tris-HCL EDTA protocol (A) 209.6 ng/µL, followed by phenol-chloroform protocol (B) 62.58 ng/µL, modified salting out protocol (C) 27.34 ng/µL and Kit protocol (D) 25.69 ng/µL. Electrophoresis analysis-based DNA integrity was highest for protocol D, followed by protocols A, C and B, while DNA purities in terms of 260/280 and 260/230 ratios were highest for protocol A (1.85; 2.03), followed by protocol D (1.8; 1.94), protocol C (1.81;1.810 and protocol B (1.77; 1.78), respectively. In comparison, the time and cost analysis depicted the highest time consumed for protocol A (4:30 h) and the lowest for protocol D (45 minutes). In contrast, the cost expenditure was highest for protocol D, US $ 2.14 and lowest for protocol B, US $ 0.76. Protocol B involved the use of highly toxic substances (phenol and chloroform). The 18S rRNA amplification results (band length 540 bp) elaborate the higher suitability for protocols A and D and the lower suitability for protocols B and C. Sanger's sequencing analysis shows the equal value of homogeneity and heterogeneity for both A and D protocols and elaborates on their equal suitability for detecting theileriosis. Protocol A was based on the cost-effectiveness, quality analysis and effectiveness for detecting theileriosis best-suited protocol. Such a protocol can be used in the future to apply molecular tools for detecting blood-borne pathogens of veterinary and public health significance.