Evaluation of four methods of DNA recovery from Mycobacterium avium subspecies paratuberculosis present in intestine tissue of goats and comparative sensitivity of IS900 PCR with respect to culture for diagnosis of Johne's disease.

ABSTRACT

Low sensitivity of PCR reaction for detection of Mycoobacterium avium subspecies paratuberculosis (MAP) in tissues and fecal samples is mainly attributed to false negative results. Present study was undertaken to compare four methods of DNA isolation from tissues of infected animals and to determine most sensitive protocol for the recovery of DNA, suitable for IS900 PCR based detection of Johne's disease infection. Method I, the traditional van Soolingen2 method of DNA isolation was adopted for the isolation of DNA from tissues. Method II was modification (hexadecyl pyridinium chloride-HPC treatment) of van Soolingen2 method. Method III was traditional tissue DNA isolation method based on tissue lysis buffer. Method IV was modification of method III (HPC treatment). Using four methods of DNA isolation from 25 intestinal tissues of clinically infected goats, DNA was isolated from 15 (60.0%), 18 (72.0%), 13 (52.0%) and 13 (52.0%) tissues using method I, II, III and IV, respectively. All isolated DNA preparations were positive for MAP in IS900 PCR. HPC treatment enhanced the recovery of DNA from tissues of infected animals using method II. Therefore, method II can improve the diagnosis MAP infection using IS900 PCR.