Use of touch-down polymerase chain reaction to enhance the sensitivity of Brucella melitensis detection in raw milk

Abstract

Brucellosis is a highly contagious bacterial zoonotic infectious disease severely affecting the public health and economic features of endemic and non-endemic countries. The present study assessed the potentials of using the touch-down polymerase chain reaction (TD-PCR) compared to the conventional PCR and culture methods in order to detect Brucella melitensis in raw milk samples of 55 sheep and 45 goats through deriving the primers from the omp31 element of the Brucella genome. In addition, nine isolates of B. melitensis were identified using the culture method. No positive cases were found in sediment samples, while the fatty tap layer test by conventional PCR and TD-PCR revealed 6 and 16 positive samples, respectively. Based on the survey of the limits of detection by TD-PCR and conventional PCR, TD protocol had a detection threshold of three logs higher than the conventional protocol under the experimental condition. The developed protocol of this study was highly sensitive and extremely fast. Therefore, this TD-PCR protocol could detect even a very low number of bacteria in milk samples. To our best knowledge, this is the first report on the use of the TD-PCR method to identify B. melitensis in milk.