DETECTION OF LISTERIA-MONOCYTOGENES INOCULATED IN DAIRY-PRODUCTS BY AMPLISCRIPT(TM)

An ultrasensitive method for the detection of Listeria monocytogenes i n dairy foods was developed using the AmpliScript(TM) technique. Ampli Script(TM) is a system that enhances detection of PCR products by tran scription of amplicons and subsequent immunoenzymatic detection of RNA :DNA hybrids in a microtiter plate. PCR primers specific for the L. mo nocytogenes hlyA gene were used to generate an amplicon containing a b acteriophage T7 promoter sequence. When the AmpliScript(TM) system was applied in the assay of various Listeria and non-listeria bacteria, s pecific signals were obtained to L. monocytogenes upon hybridization o f transcripts with a microtiter plate-immobilized DNA probe. This syst em provided significant improvements in the detectability of amplicons compared with the traditional agarose gel electrophoresis approach, a nd greatly helped to overcome the inhibitory effects of components fro m food matrices or enrichment broths on the PCR. AmpliScript(TM) provi ded important advantages for routine food analysis by using a convenie nt microtiter plate format, and eliminated the need for expensive samp le preparation techniques by allowing direct detection of L. monocytog enes cells in enrichment cultures. (C) 1997 Academic Press Limited.