Browsing by Author "Archana Sharma)"
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ThesisItem Restricted Development of loop-mediated isothermal amplification of DNA (LAMP) for bovine haemorrhagic septicaemia caused by Pasteurella multocida B:2(LUVAS, 2014) Varshney, Rajat; Archana Sharma)Haemorrhagic septicaemia, caused by P. multocida serotype B:2 or E:2 strains is an acute septicaemic disease with high morbidity and mortality of buffaloes and cattle in Asia and Africa. Accurate, sensitive and rapid diagnostic methods are required for early management of outbreaks and for prevention and control of the disease. The objectives of this study were to develop an isothermal loop-mediated amplification of DNA (LAMP) assay for P. multocida B:2 and to check its specificity, sensitivity and other features in comparison to conventional PCR. P. multocida B:2 P52 strain was used for standardization of various parameters and conditions of the LAMP assay. LAMP primers targeting P. multocida B:2 specific gene (GenBank accession no: AF016260.1) were designed and used in various combinations of LAMP components at different reaction temperature for different reaction time. Six different protocols were used and finally a standard protocol was devised to obtain optimum LAMP results. The LAMP reaction gave consistent results when the reaction mixture contained 4 mM additional Mg2SO4, 0.8M betaine and loop primers at 630C for 75 min in a water bath or a thermocycler. Positive LAMP products were detected as a ladder like pattern on AGE and also by picogreen dye method. The sensitivity of the LAMP (1pg of original DNA) was about ten times higher than conventional PCR in agarose gel electrophoresis. The developed assay was specific for P. multocida B:2 and no cross reactions were found against P. multocida type A and other bacteria. LAMP was more rapid than the conventional PCR. Thus, a rapid, highly sensitive and specific LAMP assay for HS causing P. multocida B:2 has been developed for the first time. Further studies are however required to validate this LAMP assay for performance in the field conditions as a ‘penside’ test.