validation of rt-qpcr technique for detection of brucella genome in milk sheep and goat in West Bank part of Palestine

Abstract

Brucella melitensis is a severe pathogen for human and animals, even at low concentrations. The milk of sheep and goat and the fresh dairy products, including white cheese, are the main source of consumers’ contamination. Early detection, using reliable validated diagnostic tools, is crucial for the control and eradication of the disease. The aim of this study was to develop fast molecular in-house techniques, such as RT-qPCR, to detect Brucella genome in milk. The validation of the method was carried out according to the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health OIE in chapter 1.1.5: Validation and quality control of polymerase chain reaction methods used for diagnostic of infection disease and with the requests of the ISO/IEC 17025:2005: General requirements for the competence of testing and calibration laboratories. Tests for the RT-qPCR validation have proved his ability to detect 3.4 copies of Brucella's genome, into 5µl amplification product. Furthermore, DNA from non-Brucella microorganisms was not detected by developed method. While the identification of Brucella melitensis by traditional methods is time-consuming and may impair the outbreaks control, the RT-qPCR proposed can be used as a complementary, rapid and sensitive diagnostic tool for Brucella spp in Palestine, contributing to properly implement the control policy of authorities.