Abstract: A multiplex polymerase chain reaction (mPCR) assay for the rapid identification of Lactobacillus acidophilus- Bacillus natto fusant was developed in this paper. The primer pair P1/P2 was designed based on the spo0A gene encoding the stage 0 sporulation protein A of Bacillus subtilis, as well as the primer pair P3/P4 according to the conserved sequence of the β-galactosidase gene of 13 lactic acid bacterial (LAB) strains. Using the genomic DNA of the parental strains as template, PCR systems for each primer pair were optimized under the predetermined annealing temperature. The specificity analysis showed that P1/P2 could amplify the spo0A fragment (308 bp) but all seven LAB strains gave negative results; P3/P4 could amplify the target fragment (576 bp) from all LAB and five identified positive fusants but not from Bacillus subtilis. Results of multiplex PCR demonstrated that the optimal PCR system for P1/P2 could only amplify its own 308 bp target DNA, while in the optimal system for P3/P4 both target DNA fragments could be obtained. The results of identification of 50 fusant strains by multiplex PCR showed 100% consistency with that of traditional PCR and phenotype identification. The mPCR system contained 1 ng/μL template DNA, 0.4 μmol/L primer (each), 0.25 mmol/L dNTP, 0.06 U/μL Taq polymerase, and the PCR conditions were as follows: 5 min predenaturation at 94 ℃, 30 cycles of denaturation at 94 ℃ 30 s, annealing at 53 ℃ for 30 s, and extension at 72 ℃ for 60 s. This method proved to be a rapid, convenient method with high specificity for the efficient identification of spore-forming bacteria, LAB and their fusant.

Key words: stage 0 sporulation protein A gene (spo0A), β-galactosidase gene, fusant, multiplex polymerase chain reaction