Rajesh Rathore

New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination

Graphical abstract

Loop-mediated isothermal amplification (LAMP) test for specific and rapid detection of Brucella abortus in cattle

Background: Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen, needs to be diagnosed at an early stage. Loop-mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level. Objective: To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle. Methods: LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non-Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with polymerase chain reaction (PCR). Results: The developed LAMP assay works at 61 °C for 60 min and the detection limit was observed to be 100-fold more than the conventional PCR that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with Rose Bengal plate test and standard tube agglutination test. SYB® green dye I was used to visualize the result with naked eye. Conclusion: The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.

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

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 20min. 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.

Development of probe based real time loop mediated isothermal amplification for detection of Brucella

To develop a rapid, sensitive and specific diagnostic assay for the detection of Brucella.

Prevalence and Distribution of Salmonella Faecal Carriage in Small Ruminants

To determine the prevalence and distribution of Salmonella in sheep and goats, a total of 312 faecal samples was collected aseptically from healthy sheep and goats from Uttar Pradesh, Himachal Pradesh and Jammu & Kashmir states. Samples were examined by cultural and molecular methods (real-time polymerase chain reaction, qPCR) for presence of Salmonella. Out of 312 faecal samples 22 (7.05%) and 26 (8.33%) were positive for Salmonella detected by cultural method and qPCR, respectively. The results revealed 6.74% (12) and 8.42% (15) goats compared to 7.46% (10) and 8.2% (11) sheep positive for Salmonella identified by cultural method and qPCR, respectively. The study revealed that Salmonella is commonly present in small ruminants and its prevalence varies from place to place. The qPCR is a sensitive and specific tool to detect the presence of Salmonella in faecal samples.