Design of multiplex PCR for simultaneous detection of rope-forming Bacillus strains in Iranian bread dough

Authors

  • Alireza Sadeghi,

    Corresponding author
    1. Department of Food Science and Technology, Ferdowsi University of Mashhad, Mashhad 91775-1163, Iran
    • Department of Food Science and Technology, Ferdowsi University of Mashhad, Mashhad 91775-1163, Iran.
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  • Seyed Ali Mortazavi,

    1. Department of Food Science and Technology, Ferdowsi University of Mashhad, Mashhad 91775-1163, Iran
    2. Cellular and Molecular Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad 91775-1163, Iran
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  • Ahmad Reza Bahrami,

    1. Cellular and Molecular Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad 91775-1163, Iran
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  • Balal Sadeghi

    1. Department of Animal Genetics and Breeding, Ferdowsi University of Mashhad, Mashhad 91775-1163, Iran
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  • This paper was presented at the 2nd International ISEKI_Food Conference held in Milan, Italy on 31st August - 2nd September 2011.

Abstract

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

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