Detection and Differentiation of Listeria monocytogenes and Listeria innocua by Multiplex PCR

An important and emerging food bornepathogen, Listeria monocytogenes, is a Grampositive facultative intracellular organism,causing many serious illnesses includingabortion, meningitis, septicemia and gastro-enteritis in human beings and animals(Vazquez-Boland et al., 2001). In ruminants,the organism has been reported to cause‘circling disease’, due to the attack on centralnervous system (CNS) (Vishwanathan AJeyasekaran et al., 2003). A ‘zero tolerance’policy for L. monocytogenes has been adoptedby most of the regulatory bodies includingUnited States Food and Drug Administration(USFDA) (Hitchins, 1998).In contrast, another species of Listeriai.e. L. innocua is a totally non-pathogenicorganism (Rocourt, 1999). Hence, it is veryimportant to differentiate the potentialpathogen L. monocytogenes from theapathogenic species L. innocua. Both thespecies share common biochemical charac-teristics and the conventional method fordifferentiation of these two species is basedon the expression of hemolytic activity by L.monocytogenes on sheep blood agar (Hitchins,1998). But the detection of hemolysis is veryoften ambiguous (Skalka et al., 1982; 1983).Although ELISA has been successfully usedfor detection of Listeria spp., this test can notdifferentiate L. monocytogenes from L. innocua(Lovett et al., 1987; Mattingly et al., 1988).Polymerase chain reaction (PCR) has beenfound to be a very sensitive and specific toolfor detection of L. monocytogenes as well asL. innocua (Border et al., 1990; Herman et al.,1995; Liu et al., 2003; Rawool, 2005). Keepingthese in view, the present study has beendesigned to differentiate L. monocytogenesfrom L. innocua in a single tube MultiplexPCR reaction.The bacterial strains used in the studyhave been listed in Table 1. All the strainsused with the exception of Lactobacillusacidophilus were grown on Tryptic soya broth(Difco) and confirmation of the Listeriastrains was done by standard biochemicaltests and hemolysis (Hitchins, 1998). L.acidophilus was grown in MRS broth inpresence of 5% CO