An accurate, rapid and simple loop-mediated isothermal amplification method for Explanatum explanatum detection in animals

Paramphistomosis is an infectious disease caused by the liver flukes and it is associated with heavy loss of ruminant's population. Explanatum explanatum is a digenetic trematode commonly affecting domesticated ruminants. The available methods for pathogen detection are laborious and expensive and offer limited specificity; thus, considered not suitable for post mortem pathogen detection, surveillance and prevalence studies. New detection techniques offering simplicity, specificity and rapidity are absolutely needed. We have designed a loop-mediated isothermal amplification (LAMP) based polymerase chain reaction method, targeting a sequence of the explanatum species, using a primer pair from the internal transcribed spacer-2 region. The DNA from adult flukes belonging to explanatum species was isolated from infected livers and used to optimize the LAMP assay. The specificity and sensitivity of the LAMP assay were evaluated and found highly efficient in species-specific DNA detection with the sensitivity to detect 50.00 pg DNA in a 25.00 µL reaction mix. The procedure has the potential to be adapted for stool samples for field detection and disease surveillance/prevalence in rural and unprivileged areas.