Polymerase Spiral Reaction (PSR) as a novel rapid colorimetric isothermal point of care assay for detection of Trypanosoma evansi genomic DNA

Abstract

Polymerase spiral reaction (PSR) opens new avenues for specific diagnosis of pathogens known for cryptic infection at field level and its application is still unexplored in the field of parasitology. The present study aimed to explore and optimize colorimetric based PSR technique for the detection of Trypanosoma evansi in the blood of the host by targeting the 196bp Invariable Surface Glycoprotein (ISG) gene of Trypanosoma evansi. The specificity of the test was determined against Theileria equi, Theileria annulata, Babesia caballi, Burkholderia mallei and Equine herpes virus. The T. evansi DNA was extracted from purified parasites and serially diluted from 2.8ng to 2.8 × 10−8 pg. The detection limit of PSR was found to be as low as 2.8 × 10-6 pg of T. evansi DNA, which will aid in detection of Surra infection. The duration of reaction for determination of result of field sample is 1h and result can be read by naked eyes. In addition, PSR assay was also performed on DNA extracted from 28 field equine samples; out of which 1 was found positive by microscopy and ISG-196 targeted PCR assay and 2 were recorded positive by PSR assay. Data generated shows colorimetric PSR is convenient, rapid, sensitive and specific tool for diagnosis and monitoring of Surra infection in livestock at field level. Further, visual PSR assay has wide scope for application in government policies aimed at detection of early infection, sub-clinical cases, drug-efficacy studies, control and elimination of Surra organism from livestock animals.