Using real-time polymerase chain reaction as an alternative rapid method for enumeration of colony count in live Brucella vaccines

Brucellosis is a major bacterial zoonosis of global importance affecting a range of animal species and man worldwide. It has economic, public health, and bio-risk importance. Control and prevention of animal brucellosis mainly depend on accurate diagnostic tools and implementation of effective and safe animal vaccination program. There are three types of animal live vaccines - Rev-1 vaccine, S19, and . RB51. Evaluation of these vaccines depends mainly on enumeration of viable count. At present, used colony count method is time consuming, costly and requires especial skills. Hence, the aim of this study is to use and standardize real-time polymerase chain reaction (RT-PCR) as an alternative, quantitative, sensitive, and rapid method to detect the colony count of in live vaccine. Four batches of different live vaccines were evaluated using of conventional bacterial count and RT-quantitative PCR (RT-qPCR) using BSCP31 gene specific primers and probe. Standard curve was generated from DNA template extracted from 10-fold serial dilution of living RB51 vaccine to evaluate the sensitivity of RT-qPCR. Results revealed that three batches of living vaccines were acceptable for colony count when traditional bacterial enumeration method was used. Results of RT-qPCR were identical to that of conventional bacterial count. Results concluded that RT-qPCR was relatively sensitive compared to traditional bacterial colony count of these vaccines.