Development And Application Of Specific PCR Methods For Two Silicate Bacteria, Paenibacillus Mucilaginosus And P. Edaphicus

Paenibacillus mucilaginosus and Paenibacillus edaphicus, the two typical silicate bacteria, have long been used as biofertilizers in agriculture, and have shown a good potential for applications in bioleaching and wastewater engineering in recent years. There have been considerable research involving screening of P. mucilaginosus and P. edaphicus for various utilizations at present; therefore, development of rapid identification methods for the two silicate bacteria is of great interest for related studies.In this paper, we developed specific PCR detection methods for P. mucilaginosus and P. edaphicus and the methods could be used successfully for rapid identification of P. mucilaginosus and P. edaphicus, which provide brief methods for application and exploitation of P. mucilaginosus and P. edaphicus.Firstly, four P. mucilaginosus gyrB-targeted specific primer pairs (F1-R5, F1-R8, F2-R5, F2-R8) were designed and one primer pair, F2 (5'- ACG GAT ATC TCC CAG ACG TTC AT -3') and R5 (5'- ACG GGC ACG CTG CGC CTG TAC G -3') showed good specificity according to the results of BLASTN search, analysis of secondary structures and preliminary PCR detection. This primer pair could effectively discriminate P. mucilaginosus and the most closely related P. edaphicus and selectively amplify a single 519-bp amplicon from P. mucilaginosus. A cell-PCR method was developed for P. mucilaginosus with primer pair F2 and R5. Good specificity and sensitivity were demonstrated for the method with a detection limit of 1-10 cells.Secondly, five primer pairs (F11-R13, F11-R31, F22-R2, F22-R4 and F22-R43) were designed for P. edaphicus using the same methods. Four of the five primer pairs (F11-R13, F11-R31, F22-R2 and F22-R4) showed good specificity according to the results of BLASTN search, analysis of secondary structures and preliminary PCR detection. The four primer pairs could successfully discriminate P. edaphicus and P. mucilaginosus, and gave single amplification products of a 223-bp, 124-bp, 235-bp, 232-bp from P. edaphicus, respectively. Cell-PCR methods were developed for P. edaphicus with the four primer pairs, respectively. Good specificity and sensitivity were demonstrated for these methods with detection limits of 1-10 cells.Finally, the PCR methods were used to detect total soil genomic DNA and silicate bacteria isolates from soil. The specific PCR method for P. mucilaginosus could successfully used to amplify total genomic DNA of an agricultural soil sample and produced the predicted amplicons. The sequences of 20 clones of the amplification products were very similar to the gyrB gene of P. mucilaginosus. Using the culture-PCR method, 25 isolates from soil were detected and 19 of the 25 isolates gave positive results in the PCR amplification and the 19 isolates belonged to P. mucilaginosus by sequencing of the gyrB gene and sequence analysis. The remaining 6 isolates, which gave negative results in the PCR amplification, belonged to others species of Paenibacillus. For the specific PCR detection method for P. edaphicus, positive amplification results of total soil genomic DNA could only be obtained when primer pairs of F22-R2 or F22-R4 were used and the sequences of amplified products showed a similarity of only 78.6-83.2% with gyrB gene of P. edaphicus after cloning, sequencing and sequence analysis. No positive amplification results of total soil genomic DNA was obtained with primer pairs of F11-R13 or F11-R31. No positive amplification results were obtained with all the four primer pairs when the 25 isolates were subjected to PCR detection, showing no P. edaphicus presented in the 25 isolates. So the specific PCR method for P. mucilaginosus could be effectively used for research on the application of P. mucilaginosus and the specific PCR method for P. edaphicus with primer pairs F11-R13 or F11-R31 also had the same potential for applications, but further verification would be needed.In conclusion, specific PCR methods were successfully developed for P. mucilaginosus and P. edaphicus in this paper. These methods will contribute to the application and exploitation of P. mucilaginosus and P. edaphicus.