Exploring specific primers targeted against different genes for a multiplex PCR for detection of Listeria monocytogenes

The efficacy of six different sets of primers targeted against 16S rRNA and virulence genes such as ‘iap’, ‘hly’ and ‘prf’ was evaluated in separate PCR assays. The primer pairs targeted against 16S rRNA resulted into amplification of 1.2 kb PCR product. However, sets of primers targeted against different regions of ‘iap’ produced 371 and 660 bp PCR products, respectively. The primer pair targeted against ‘prf’ gene could produce 508 bp product. Three primer pairs targeted against different regions of ‘hly’, i.e., ‘hly’, ‘hly A’ and ‘hly K9’ were able to amplify 713, 276 and 384 bp products, respectively. The PCR conditions were also optimized in respect of two internal sets of primers falling within ‘iap’ and ‘hly’ genes that amplified 119 and 188 bp products to verify the PCR results obtained with respective external sets of primers. Three different combinations involving four sets of primers based on 16S rRNA, ‘iap’, ‘hly’ and ‘prf’ were explored in respective multiplex PCR assays in order to select a suitable combination. Combination 1 and 3 worked successfully as revealed by amplification of all the four bands of expected sizes on agarose gel. However, while optimizing the different parameters for developing a functional multiplex PCR, it was observed that in both these combinations, only two of the amplified products, i.e., 1.2 kb and 713 bp could be invariably detected. Hence, these two primers were combined in the multiplex PCR and the conditions were optimized for application in dairy foods for detection of Listeria monocytogenes.