Establishment of Specific Detection Methods by Nested PCR and qPCR for Bacillus firmus Based on the hag Gene

Abstract:

Hag, a Flagellin coding gene, has conserved domains on the ends and a central domain that is highly variable in the length and the sequence, and hence has been used as a molecular marker in bacterial classification and identification. In this study, the hag gene was amplified from Bacillus firmus with the primer set Bhag, which was designed based on the conserved regions of the hag gene of B. subtilis and other bacteria. Two primer sets, Bfho and Bfhi, were designed based on the obtained sequence of hag of B. firmus and were used in the nested PCR for the species-specific detection of B. firmus. Furthermore we developed another specific detection method using fluorescent quantification PCR based on the inner primer set Bfhi, and tested the detection limit. Some simulated samples were tested with this FQ-PCR method and in the positive samples the concentrations of B. firmus were determined with the same method. The sequence of hag cloned from B. firmus had 1213 bp and was only 13%15% similar to that of B. subtilis according to NCBI BLAST, and most differences existed in the variable region. The detection limits of the FQ-PCR method were 17.3×103 CFU/ml and 19.7×103 CFU/ml for B. firmus strains PC004 and PC024 respectively. In 15 simulated samples, 7 were detected positive with the FQ-PCR method, and the concentrations of B. firmus in the samples were determined as well. The detection methods developed in our study have technical advantages such as high specificity, time-saving, and high sensitivity, and thus may become valuable tools in the practical application.