Author/Authors :
Sadeghi، Alireza نويسنده Department of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran , , Mortazavi، Seyed Ali نويسنده Professor of food Science & Technology Department, Ferdowsi University of Mashhad , , Bahrami، Ahmad-Reza نويسنده , , Sadeghi، Balal نويسنده Department of Animal Science, and Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran , , M. Matin، Maryam نويسنده Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran ,
Abstract :
In this present study, a SYBR green based real time PCR assay has been developed for specific detection and
quantification of Bacillus subtilis in dough used for bread making. New primer pairs were designed to amplify a
212 base pair fragment of the aprE gene. Specificity of these primer pairs was confirmed with conventional and
real time PCR methods. Standard curves constructed using the threshold cycle (CT) versus copy numbers of B.
subtilis showed good linearity for reference standards of cloned insert (R2=0.999, slope=-3.035) and also induced
contaminated dough (R2=0.988, slope=-3.142), and the melting temperature (Tm=82.2 oC) was consistently specific
for the amplicon. Limits of detection were 200 and 2000 colony forming units (CFUs) per ml or g of these samples,
respectively. This real time PCR offers a fast tool with high sensitivity and specificity for detection and
quantification of this rope-forming pathogen in dough used for bread making.
