Development of probe‐based real‐time loop‐mediated isothermal amplification for detection of<i>Brucella</i>

Abstract

AIM: To develop a rapid, sensitive and specific diagnostic assay for the detection of Brucella. METHODS AND RESULTS: The probe-based RT-LAMP was carried out by using a set of four or six primers and different LAMP chemicals to compare its results with real-time PCR. Detection of gene amplification is done within 40 min and can be seen by amplification curve, turbidity and addition of DNA-binding dye at the end of the reaction results in colour difference under normal day light and in UV. The sensitivity of probe-based real-time LAMP assay was found 10-fold higher than Taqman-based qPCR. The specificity of the developed assay was validated by the absence of any cross-reaction with other pathogenic bacteria. CONCLUSION: The developed probe-based RT-LAMP assay is extremely rapid, cost effective, highly specific and sensitive, and has potential usefulness for rapid Brucella surveillance. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed probe-based RT-LAMP is a powerful gene amplification technique which is a specific, fast diagnostic tool for early detection and identification of Brucella.