Canine Parvovirus Moleküler Tanısında Primer Seçimi ve Önemi

Abstract

Canine parvovirus (CPV), also known as bloody diarrhea in dogs, is associated with high mortality, particularly in unvaccinated animals. The causative agent is Canine Parvovirus-2, which has a single-stranded DNA genome. The most affected group is puppies (1 month and under) that have not yet been vaccinated or that have been exposed to other animals before sufficient immunity has developed after vaccination. Although there is no specific treatment, rapid diagnosis is important in order to differentiate it from other diseases it may be confused with and to guide the necessary precautions. Since clinical symptoms are not sufficient for diagnosis, diagnosis supported by rapid tests is the most commonly used approach. Alternatively, molecular methods such as polymerase chain reaction (PCR) provide a definitive diagnosis. However, the most important parameter for accurate diagnosis in PCR tests is the selection of the gene region targeted for amplification and the appropriate oligonucleotide primers. If the selected gene region is specifically chosen for diagnosis, it should generally be chosen from regions that are resistant to mutation. In this study, blood samples from 12 dogs suspected of having CPV were examined using 3 different primers. Blood samples were centrifuged at 2000 rpm to remove the leukocyte layer, and the collected middle layer was transferred to 1.5 ml sterile polystyrene tubes using a pipette. The phenol/chloroform method was used for viral DNA extraction. Optimizations were first performed with three different primer pairs targeting the capsid-encoding protein: HFor/HRev (target location 3556-3575/4185–4166), Pbs/Pbas (target location 4043–4062/4470–4449), and Pabs/Pabas (target location 3025–3045/3706–3685), and then the samples were tested with different PCR reactions for each primer pair. 7, 5, and 12 positive results were obtained from each primer pair, respectively. This situation reflects the mutations occurring in the CPV genotype variants (2a, 2b, and 2c) present in the field. Consequently, this shows us that even in viral diseases commonly diagnosed in the field, mutations occurring over time can cause difficulties in diagnosis. The results of our study emphasize the importance of always working with multiple primers.