DEVELOPMENT OF MOLECULAR TECHNIQUE FOR RISK ASSESSMENT AND RISK MANAGEMENT OF Listeria monocytogenes AND L. innocua IN FROZEN COOKED CHICKEN PLANT

Abstract

Thailand is one of the largest cooked chicken meat exporting country in the world. Unfortunately, these products can be contaminated with L. monocytogenes causes the disease Listeriosis which carries a fatality rate of 30 to 40 percent. Therefore, the various importing countries including Japan and the European countries require a zero tolerance for L. monocytogenes in the cooked chicken products. Besides, all Listeria spp. occurrence, and not only L. monocytogenes, in these products is also unacceptable to the importers. Rejection of Listeria contaminated product can reach over 5 to 20 million baht per factory per year. Hence, the aim of this study was to introduce a cost-effective molecular approach to detect, identify, and subtype L. monocytogenes and L. innocua for tracking sources of contamination. Firstly, a new comprehensive BE-LisAll biomarker for Listeria detection using in silico scheme was developed. The results showed that biomarker has a 100% specificity for Listeria species detection and could differentiate Listeria species from a variety of non-Listeria bacteria. Secondly, HRMA of rarA and ldh method which identified 9 species belonging to the genus Listeria was established. The method can be considered sufficiently applicable as method for identifying the species of Listeria isolates from the food factory with a success rate of 92.6%. Thirdly, a recently validated PCR primer set targeting the VNTR of L. monocytogenes of Chenal-Francisque et al. and a novel PCR primer set targeting the VNTR designed based on completed genome sequence of L. innocua CLIP 11262 were validated and verified. The CE-based MLVA protocols provided higher discriminatory power for subtyping of L. monocytogenes and L. innocua than RAPD method. Fourthly, a cost-effective NGS-based MLVA by PGM using 9 adopted (JLR1, JLR2, JLR4, LisTR1317, LisTR881, LMTR4, LMV1, LMV6, and LMV9) and 6 novel (TR1, TR3, TR5, TR6, TR10, and TR13) VTNR loci was developed. This method provided higher discriminatory power for differentiating between L. monocytogenes and L. innocua strains than CE-based MLVA. Finally, the developed MLVA was applied to investigate sources and routes of Listeria contamination for reducing the risk of L. monocytogenes and L. innocua contamination in cooked frozen chicken meat process. The relationships of the L. monocytogenes and L. innocua in the final products and those in the environment were evaluated. The results showed that L. monocytogenes and L. innocua in finished products can be contaminated from various sources such as dicer, floor, and cart wheel. After revised the suitable procedures, the prevalence of Listeria spp. in the finished product and processing environment were decreased, significantly. These demonstrated that the developed techniques and approaches have the potential to provide an efficient tool for investigating and tracking the routes and sources of contamination of L. monocytogenes and L. innocua in frozen cooked chicken plant.