Development and evaluation of probe based real time loop mediated isothermal amplification for Salmonella: A new tool for DNA quantification

• The probe based RT LAMP is a novel method of detection of Salmonella with high specificity and rapidity.
• It uses 4-6 primers and a double stranded probe labelled with Cy5 dye and IB quencher on different strands.
• It is based on auto cycling strand displacement DNA synthesis leading to separation of fluorophore and quencher.
• Real time LAMP can be done in a QPCR instrument in which the generation of fluorescence can be monitored in real time manner.

A one step, single tube, accelerated probe based real time loop mediated isothermal amplification (RT LAMP) assay was developed for detecting the invasion gene (InvA) of Salmonella. The probe based RT LAMP is a novel method of gene amplification that amplifies nucleic acid with high specificity and rapidity under isothermal conditions with a set of six primers. The whole procedure is very simple and rapid, and amplification can be obtained in 20 min. Detection of gene amplification was accomplished by amplification curve, turbidity and addition of DNA binding dye at the end of the reaction results in colour difference and can be visualized under normal day light and in UV. The sensitivity of developed assay was found 10 fold higher than taqman based qPCR. The specificity of the RT LAMP assay was validated by the absence of any cross reaction with other members of enterobacteriaceae family and other gram negative bacteria. These results indicate that the probe based RT LAMP assay is extremely rapid, cost effective, highly specific and sensitivity and has potential usefulness for rapid Salmonella surveillance.