Application of Multiplex PCR Assay for Campylobacter fetus Sub sp. Detection and Differentiation in Bovine Preputial Wash

Abstract

The aim of this research was to evaluate the detection and differentiation of C. fetus subspecies through multiplex PCR (mPCR) method from the bovine preputial wash. The evaluation was done by comparing 2 extraction methods, which was by commercial kit extraction and boiling method, and also by measuring the effect of Transport Enrichment Media (TEM) addition as bacterial enrichment. Spiked bovine preputial washes with 2 extraction methods (commercial kit and boiling), with and without TEM addition were used as a sample. The results showed that the highest sensitivity/specificity was by boiling extraction without TEM addition, which reached 83.3%/100% for all or grouped sample. The results also supported by Poisson regression analysis which showed the comparison between sample with and without TEM addition which had 1.3 Incidence Rate Ratio (IRR). The obtained IRR score indicated that sample without TEM addition had 30% better result compared to the sample with TEM addition. Furthermore, a comparison between commercial kit and boiling extraction showed no significant difference (1.029 IRR). The research concluded that boiling extraction without TEM addition is the most accurate, fast and economical extraction method regarding its high sensitivity and specificity value, with less than 24 hours testing time and without any commercial kit requirements. However, a validation and verification with field sample were still needed before applied in the laboratory.