BACKGROUND: The objective of this study was optimisation of multiplex polymerase chain reaction (PCR) by a new primer set for simultaneous detection of ropiness agents as the main bacterial spoilage of Iranian bread. After inoculation of bread dough with activated Bacillus licheniformis (ATCC 9789) and Bacillus subtilis (ATCC 6633), DNA was extracted from the dough and subjected to PCR. Then simplex and multiplex PCR tests were optimised.
RESULTS: From the results obtained, the optimum PCR conditions for simultaneous detection of the target genes in Bacillus species were an annealing temperature of 59 °C and an MgCl2 concentration of 2.5 mmol L−1. To design primers for these two bacteria, owing to close homology and the existence of similar conserved sequences in their 16S rRNA genes, lpaL and aprE genes (497 and 744 bp target sequences) respectively were chosen. Finally, by sequencing of PCR products, accurate and specific detection of the two desired Bacillus species was confirmed. The results were registered with GenBank under accession numbers HQ877873 and HQ871154.
CONCLUSION: Compared with culture-dependent methods, this procedure offers significantly higher accuracy and speed, which are crucial criteria when it comes to food safety and high volumes of referred samples respectively. Copyright © 2012 Society of Chemical Industry