The aim of this study was to use the real time polymerase chain reaction in the detection of Babesia canis subclinical infestations in dogs and to compare the different DNA isolation methods on PCR sensitivity. The study included 6 dogs with suspected subclinical babesiosis. DNA for real time polymerase chain reaction were isolated by the phenol method as well as by Micro AX Gravity (A & A Biotechnology, Gdynia, Poland) and Blood mini (A & A Biotechnology, Gdynia, Poland) commercial kits. In the blood of all six specimens PCR demonstrated the presence of Babesia canis DNA. The most efficient proved to be a reaction to which the genetic material was isolated by the phenol method. The amount of total DNA obtained in this way, determined spectrometrically, ranged 43.7-54.3 ng/µl. Ct value in real-time PCR for DNA samples isolated in this manner was the lowest in comparison with other isolation methods, and averaged 22.5. Similar results were obtained when DNA was isolated from the blood with the Micro AX Gravity kit, while the least efficient was the Blood Mini Kit (amount of total DNA, depending on the sample was 14.0-25.1 ng/µl, amplification in real time occurred the slowest - average Ct value = 28). Readable sequences were obtained for all PCR products where DNA was isolated using the phenol method or by Micro AX Gravity. In the case of PCR products where DNA was isolated by the Blood Mini Kit, readable sequences were obtained only for 3 out of 6 tested samples. All sequences received in our study of the 18S RNA gene fragment showed a high 99.9-100% homology with the sequence of Babesia canis EU622792 These results confirm the usefulness of the real time PCR in the diagnosis of subclinical canine babesiosis and indicate the need for choosing such a DNA isolation method for this reaction that will guarantee the highest efficiency of amplification.
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