A novel typing method for Listeria monocytogenes using high-resolution melting analysis (HRMA) of tandem repeat regions.

Listeria monocytogenes, which is responsible for causing food poisoning known as listeriosis, infects humans and animals. Widely distributed in the environment, this bacterium is known to contaminate food products after being transmitted to factories via raw materials. To minimize the contamination of products by food pathogens, it is critical to identify and eliminate factory entry routes and pathways for the causative bacteria. High resolution melting analysis (HRMA) is a method that takes advantage of differences in DNA sequences and PCR product lengths that are reflected by the disassociation temperature. Through our research, we have developed a multiple locus variable-number tandem repeat analysis (MLVA) using HRMA as a simple and rapid method to differentiate L. monocytogenes isolates. While evaluating our developed method, the ability of MLVA-HRMA, MLVA using capillary electrophoresis, and multilocus sequence typing (MLST) was compared for their ability to discriminate between strains. The MLVA-HRMA method displayed greater discriminatory ability than MLST and MLVA using capillary electrophoresis, suggesting that the variation in the number of repeat units, along with mutations within the DNA sequence, was accurately reflected by the melting curve of HRMA. Rather than relying on DNA sequence analysis or high-resolution electrophoresis, the MLVA-HRMA method employs the same process as PCR until the analysis step, suggesting a combination of speed and simplicity. The result of MLVA-HRMA method is able to be shared between different laboratories. There are high expectations that this method will be adopted for regular inspections at food processing facilities in the near future.

A rapid typing method for Listeria monocytogenes based on high-throughput multilocus sequence typing (Hi-MLST).

Listeria monocytogenes infects humans via food products, causing listeriosis. Consequently, food companies pay meticulous attention to the risk of contamination of their products by this bacterium. While fragment analysis methods such as pulsed-field gel electrophoresis (PFGE) are used to trace the sources of contamination for this bacterium, some drawbacks have been identified, namely the complexity of the methods and the difficulty of making data comparisons. As an alternative, multilocus sequence typing (MLST) is now seeing widespread use; however, owing to its cost, time, and labor requirements, its diffusion into the food industry has been slow. Thus, in the present study, a High-throughput MLST (Hi-MLST) method, which can rapidly, simply, and cheaply perform MLST analyses using a next-generation sequencer (NGS) that can analyze a large volume of base sequences at once was developed. Firstly, a multiplex PCR method designed to amplify seven genes for use in MLST was developed. The discriminatory potential of the developed method was confirmed in silico, and was verified that it has the same discriminatory potential as conventional methods. Next, MLST analysis using multiplex PCR and NGS was performed for 48 strains of L. monocytogenes. The sequences obtained from this analysis have sufficiently reliable quality for all of the genes from of all the strains. Thus, this method could classify the 48 strains into 39 sequence types (ST) with a Diversity index (DI) of 0.989. In summary, using the Hi-MLST method developed in the present study, which combined multiplex PCR and NGS, cut the costs to 1/6th and the time to 1/20th that of conventional MLST methods.