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Development of probe‐based real‐time loop‐mediated isothermal amplification for detection of Brucella
Author(s) -
Bhat I.A.,
Mashooq M.,
Kumar D.,
Varshney R.,
Rathore R.
Publication year - 2019
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.13938
Subject(s) - loop mediated isothermal amplification , taqman , brucella , microbiology and biotechnology , real time polymerase chain reaction , hybridization probe , biology , dna , chemistry , gene , virology , genetics , brucellosis
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 .