Porownanie klasycznej metody PCR oraz Real-Time Sybr-Green HRM PCR w rozpoznawaniu zakazen parwowirusowych u psow

The aim of this study was to compare a standard PCR and Sybr-Green HRM PCR in the diagnosis of canine parvoviral infections. A total of 22 feces samples were collected from dogs suspected of parvovirosis. The entire DNA for standard PCR and Real-Time PCR was isolated from the feces. In both methods this same pair of primers that allow the amplification of a fragment of VP 2 gene with a length of 1278 bp were used. The specificity of the obtained PCR products in the classical method were established based on the results of sequencing 8 out of 22 DNA probes and based on the comparison of their sequences with a CPV VP2 FJ 222823 sequence taken from the GeneBank. The specificity of Real-Time PCR products were established based on the analysis of their melting curve. In both standard and Real-Time PCR CPV DNA was detected in all 22 feces samples. The length of the obtained products was 1190 bp. To obtain a positive result in Real-Time PCR it was required to increase the number of the cycles from 30 to 60. The Ct values were between 43-53, and the analysis of the melting curve revealed that the Tm of Real-Time PCR products ranged between 80.5-85°C. Despite the results of this study indicating that both of these techniques are specific, sensitive, and repeated methods for detection of the CPV DNA, to shorten the time of Real-Time PCR the application of appropriate primers is required, which enables the amplification of shorter fragments of the DNA than those obtained in our study.