Loop-mediated isothermal amplification assay for the rapid detection of Anaplasma marginale in cattle based on major surface protein 5 gene

Abstract

Background & objectives: Timely intervention is needed to minimize the economic losses of vector-borne bovine anaplasmosis which can be possible by the isothermal amplification assay. Methods: Anaplasma marginale in the cattle of south Gujarat, India was detected in the PCR and LAMP by amplifying the fragment of msp5 gene. The PCR product was digested with EcoRI, and sequenced to confirm its pathogen specific detection. Results: Species specific PCR observed a band of 457 bp of msp5 DNA following 1% agarose gel electrophoresis. Positive LAMP reaction turned into yellow colour while negative sample depicted original pink colour. A detection limit of PCR and LAMP was up to 10-6 and 10-8 of the original genomic DNA of A. marginale, respectively. A single cut site of EcoRI was observed in the PCR product. Current msp5 DNA sequences of A. marginale (MW538962 and MW538961) showed 100% homology with the published sequences. Monophyletic lineage type relationship was observed with high bootstrap proportion among the msp5 DNA sequences of A. marginale in the phylogram. Prevalence rate of A. marginale was significantly higher (p<0.05) in the PCR [43/280 (15.36%)] and LAMP [62/280 (22.14%)] than the microscopic technique [17/280 (6.07%)]. Diagnostic sensitivity, specificity, positive and negative predictive values at 95% CI for LAMP assay with respect to PCR were 93.02%, 90.72%, 64.52% and 98.62%, respectively. Interpretation & conclusion: Thus LAMP can be a practical alternative to the PCR for the diagnosis of A. marginale infection in the cattle even in field condition.

Keywords

• anaplasma marginale; pcr; lamp; isothermal amplification; msp5