Rapid Diagnostic Kits for detection of Food-Borne Pathogens

E. coli 0157:H7 and L. monocytogenes are the two high risk food pathogens associated with food borne disease outbreaks. (E. coli 0157:H7 responsible for Haemorrhagic Colitis(HC), Haemolytic Uremic Syndrome (HUS),Thrombotic Thrombocytopenic Purpurea (TTP) and L. monocytogene responsible for Septicaemia, Meningitis, Meningioencephalitis, Abortion and even death)

A highly sensitive and specific multiplex PCR assay has been developed which can unequivocally detect the presence of E. coli 0157:H7 directly from naturally contaminated milk and milk products within 10 hrs. The multiplex PCR assay developed was found to be highly specific as it produced PCR products of 152 bp (E. coli specific) and 625 bp (E. coli 0157:H7). The specificity and sensitivity of the assay was further enhanced (1-10 cells) using IMS. The assay could be successfully applied on a variety of dairy products. E. coli 0157:H7 could be detected in milk and kulfi samples as well as water samples collected from local vendors.

A multiplex PCR based on two sets of primers i.e. one genus specific targeted against 16S rRNA (genus specific 1200 bp PCR product) and `hly' (Listeria monocytogenes specific, 713 bp PCR product) has been developed since these two primer pairs consistently amplified the specific products. The specificity of the multiplex PCR assay was also checked in this study to rule out the possibility of false positive results. The sensitivity of multiplex PCR was limited to 10 ng of the pure DNA from L. monocytogenes when used as template. The multiplex PCR was able to detect as low as 1 to 10 cells of L. monocytogenes after 4 to 6 hrs enrichment in BHI broth. The multiplex PCR could be successfully applied to some natural samples of milk and milk products.

The multiplex PCR for both the targeted organisms is now available in the form of a ready to use kit that can be used in Quality Assurance and Food Microbiology laboratories for monitoring dairy foods for E. coli 0157:H7 and L. monocytogenes on routine basis. The kit was released by Union Minister for Science and Technology and Earth Sciences, Shri Kapil Sibal at a press conference in New Delhi today. The kit also includes the reagents for extraction of template DNA from milk and milk products and other foods which is a very crucial step by using a simple and effective protocol named “NDRI method” developed at NDRI, Karnal for application in set up of the PCR assays. The aforesaid multiplex PCR assays were initially standardized in the laboratory and were cross-validated in other labs.

The PCR based kits / assays are extremely sensitive and specific and can detect the targeted food pathogens directly in milk and milk products in less than ten hours as compared to conventional culture based methods and other miniaturized tests based on biochemical characteristics. Dairy Industry and Food Labs are the potential beneficiaries of these PCR kits / assays which can be integrated into the Quality Assurance and HACCP programmes for monitoring the safety of dairy foods in relation to two high risk food pathogens E. coli 0157:H7 and Listeria monocytogenes. These kits can also find application in the research laboratories involved in the area of diagnostics and other related subjects.

The PCR kits have a shelf life of 15 days at ambient temperature, 45 days at refrigeration temperature and can stay active even after six months at – 20oC

Source: Press Inormation Bureau
Date: April 07, 2008