Loop-Mediated Isothermal Amplification-Based Detection of Canine Parvoviral DNA in Faecal Samples of Diarrhoeic Dogs

Abstract

Listen

Translate article

The present study was undertaken to validate loop-mediated isothermal amplification (LAMP) assay based on PCR (Polymerase Chain Reaction)for the detection of canine parvovirus (CPV) and to compare the diagnostic potential of LAMP. A total of 54 faecal samples collected from diarrheic dogs suspected for CPV infection were subjected to PCR, using the primer amplifying the gene coding for the VP2 protein. These samples were also tested by LAMP assay using primer sets, and the results were statistically analysed using McNemar test. The sensitivity and specificity of LAMP assay based on PCR assay was analysed. The specificity of the LAMP primers was determined by cross-examination of templates extracted from canine adenovirus 1, canine coronavirus and canine distemper virus and Escherichia coli. Sensitivity of both assays was identified by subjecting a serial ten-fold dilution of the DNA (Deoxyribo Nucleic Acid) of CPV-2 vaccine strain (Megavac-P) to LAMP and PCR assays for the determination of their detection limits. A total of 32 samples (59.25 per cent) were detected as positive by PCR, which yielded a specific amplicon of 699 bp. In LAMP assay, 39 (72.2 per cent) samples were found to be positive. The sensitivity of the test as compared with PCR was found to be 100 per cent and specificity was 68.2. Statistical analysis of the results of PCR and LAMP assay using McNemar test revealed that there exists significant difference between these two tests (P < 0.05). The specificity determination of the LAMP primers showed that no amplification products were detected in the template DNA extracted from canine adenovirus 1, canine coronavirus and canine distemper virus vaccines and E. coli, whereas only CPV-2 vaccine strain showed positive reactions. The detection limit of LAMP was up to 10-7 in the place of 10-3 for PCR. This study showed that LAMP assay could be employed as a rapid field level diagnostic tool for the diagnosis of canine parvoviral diarrhoea.