CPB 697 RESEARCH SEMINAR
Peetambar Kushwaha, BS
Graduate Student in Microbiology
Department of Comparative Pathobiology
“Concentration And Separation Of Mycobacterium avium subsp. paratuberculosis
By Hydroxyapatite For
PCR Detection”
Thursday, March 29, 2007
VPTH 112
3:30 pm
Abstract:
Long incubation time required by Mycobacterium avium subsp. paratuberculosis (Map), causative agent of Johne’s disease in ruminants, to grow on culture media delays detection of Map in feces. PCR is a rapid detection method but its sensitivity is compromised due to the presence of various inhibitors in feces. A method to concentrate and separate Map, free of PCR inhibitors, from bovine feces is highly desirable to enhance the sensitivity of PCR. The objective of the present study was to concentrate and separate Map using Hydroxyapatite (HA) (Ca10(PO4)6(OH)2), a compound shown to adsorb bacteria. A series of experiments were carried out to determine the HA to Map suspension ratio and the pH that facilitates the maximum adsorption and release of Map. Subsequently, serially diluted Map was processed with the same procedure to determine the sensitivity by PCR. The volume of HA to 107/ml Map suspension in pH 7.2 for maximum adsorption of Map was found to be 1 ml of HA for 10 ml of Map suspension. Maximum adsorption and release was observed at pH 5.0 and pH 11.0 respectively when 1 ml. of HA was applied to 10 ml of 108/ml of Map suspension in adsorption buffer. As low as 10 Map/ml, adsorbed at pH 5.0 and released at pH 11.0, could be detected by PCR from extracted DNA. The result indicated that HA can be used to concentrate and separate as low as 10 Map/ml detected by PCR. The method has the potential to concentrate and separate Map from fecal samples in a form that can be used for direct fecal detection of Map by PCR to bypass the lengthy culture and isolation procedures.