Development of polymerase chain reaction and multiplex polymerase chain reaction for simple identification of thermoanaerobic spore-forming bacteria

Thermoanaerobic spore-forming bacteria such as Thermoanaerobacter, Moorella, Thermoanaerobacterium, and Caldanaerobius produce spores with extremely high heat resistance. They are known to spoil various sealed, sterile drinks; in particular, low-acid drinks distributed at high temperatures, such as canned coffee containing milk. These bacteria are difficult to culture and identify on the basis of traditional biochemical characteristics. We developed novel primers for single and multiplex PCR methods for simple identification of these bacteria at the genus level. Bacteria were correctly identified approximately 2 h after DNA extraction among 86 strains of 35 species of Gram-positive and -negative bacteria including various spore-forming bacilli. Furthermore, new Loop- Mediated Isothermal Amplification (LAMP) primers were designed to develop a specific detection method for Thermoanaerobacter mathranii and Thermoanaerobacter thermocopriae, highly problematic microbes in the food industry due to their extremely high resistance to heat and various antibacterial agents. Our LAMP method using the novel primers was able to easily detect these microbes. Our present methods effectively improve upon the complicated procedures employed in the quality control of raw materials and products in the food industry.