‘Single Colony PCR’ using physical method of DNA recovery for the characterization of tiny colonies of Mycobacterium avium subspecies paratuberculosis

INTRODUCTION Johne’s disease (JD) is a serious infection of animals caused by Mycobacterium avium subspecies paratuberculosis (MAP). Johne’s is not a priority in India. Therefore, information on prevalence in 465.50 million domestic ruminants have not been estimated nor realized, mainly due to lack of indigenous diagnostic kits. Diagnosis is difficult due to long incubation, absence of characteristic symptoms, problems in cultivation of MAP and non-specific results in Johnin test. Until 1989, when IS900 was discovered in the MAP genome, characterization was difficult. IS900 is specifically present in 14-18 copies in MAP (Green et al., 1989) facilitating specific identification in PCR. However, PCR is still not a convenient test to standardize mainly due to difficulties in isolation of genomic DNA. MAP has rigid cell wall which is very difficult to break (Garrido et al., 2000; Hammer et al., 2000). Therefore, MAP cells must be primarily prepared before DNA isolation and selection of method of isolation of mycobacterial DNA essentially determines success of PCR. The best method for isolation of mycobacterial DNA is a combination of physical, chemical and enzymatic methods (Fus et al., 2003; Hosek et al., 2006), successfully employed by van Soolingen et al. (1993). However, a loopful of culture as starting material is required. But minute colonies of MAP were usually lost on prolonged incubation / contamination / drying of media and desired loopful growth has rarely been available (Kumar et al., 2007). Attempts to isolate DNA from a few colonies were consistently negative. The current study aimed to compare routinely used a standard DNA isolation method with a new non-chemical (physical) method and further characterization by IS900 PCR.