Designing a SYBR Green Absolute Real time PCR Assay for Specific Detection and Quantification of Bacillus subtilis in Dough Used for Bread Making

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 a212 base pair fragment of the aprE gene. Specificity of these primer pairs was confirmed with conventional andreal 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 specificfor 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.

Keywords:

Real-time PCR , Bacillussubtilis , new primer pairs , contaminated dough