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PROOF OF CONCEPT RECOMBINASE POLYMERASE ASSAY (RPA) FOR THE DETECTION OF HAEMONCHUS CONTORTUS IN RUMINANTS (ID 1269)
Abstract
Introduction
Haemonchus contortus is a blood-feeding parasite which is considered one of the greatest health concerns in small ruminants. Available molecular diagnosis are laboratory-dependent and variations of the polymerase chain reaction (PCR). Recombinase polymerase amplification (RPA) is a technique suitable for a point of care diagnosis operating at a temperature of 36°C to 42°C and results within 20-30 minutes. The work presented is a qualitative proof of concept RPA for the detection of H. contortus.
Methods
The ITS2 gene of H. contortus was targeted. The time-temperature conditions were optimised by performing the reaction in combinations in a portable dry block heater using the TwistAmp Basic kit. The amplicons were analysed in 2.5% agarose gel electrophoresis. Additional investigations were conducted to reduce the non-specific amplification and validation of the assay was done.
Results
The sensitivity was recorded up to 100 fg of the serially diluted template DNA. There was no amplification when DNA from Teladorsagia circumcincta, a closely related species, was used. The specificity was validated using a proven single-species ITS2 gene PCR and loop-mediated isothermal amplification (LAMP) assay. Using 0.8M of betaine reduced the non-specific amplification. The best time-temperature factor was at 20 min at 37°C.
Conclusions
Our proof of concept study showed faster assay design by adapting the available PCR primers for RPA with minimal equipment. Our study was aimed at naked-eye detection of results but with the unavailability of the kit in the market, the methodology had to be modified. At the time of writing this abstract, we were working on improving the result detection system with the possibility of quantification for a point of care assay .