Rapid detection of infectious bursal disease by loop-mediated isothermal amplification for field analysis

Infectious bursal disease (IBD) is an immunosuppressive, acute and highly contagious illness of growing-poultry stock infected with infectious bursal disease virus (IBDV). It is common in Pakistan, causing potential economic losses throughout the year. The objective of the study is to propose a rapid, sensitive and specific diagnostic tool, and compare it with existing commonly used reverse transcriptase polymerase chain reaction (RT-PCR) method for IBDV. Different primers were used for RT-PCR and reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) to target the IBD virus. RT-LAMP primers showed prodigious specificity without cross reaction to the other animal pathogens. Moreover, RT-LAMP was found to have 10 times higher selectivity for IBDV identification as compared to RT-PCR. RT-LAMP detected 9.2% more field samples than RT-PCR. Sequences of PCR products were determined and phylogenetic analysis of research isolates revealed its maximum similarity with indigenous and Indian IBDV isolates. RT-LAMP was found to be simple, specific, less laborious and a better technique as compared to RT-PCR for quick analysis. In general, RT-LAMP was declared positive on observing turbidity or adding fluorescence staining reagent such as SYBR Green I. The options of direct use of field sample homogenate and viewing directly the peaks in the graph shown on a monitor/laptop have made it much more convenient and time saving than gel based RT-PCR.