Application of the SYBR Green real-time HRM PCR technique in the differentiation of the Babesia canis canis protozoa isolated in the areas of eastern Poland.
The aim of this study was to determine the usefulness of the real-time polymerised chain reaction (PCR) high-resolution melting (HRM) method in the differentiation of the Babesia canis canis protozoa isolated from dogs in the areas of eastern Poland. The studies involved 20 isolates of B. canis canis qualified depending on the analysis of the 18S RNA gene sequence to group A (EU 622792) and 20 isolates qualified to group B (EU 622793). It was proven with the real-time PCR technique that the melting temperature (Tm) of the obtained products of amplification was 78 degrees C for the representatives of group A and 81 degrees C for the representatives of group B, which proves that the real-time SYBR Green HRM PCR method is a technique allowing for the differentiation of the B. canis isolates which are slightly different with respect to the genetic structure, without the necessity to carry out time-consuming studies, i.e., sequencing and restriction fragment length polymorphism.
Department of Epizootiology and Infectious Diseases, Faculty of Veterinary Medicine, University of Life Sciences Lublin, 30 Głeboka St, 20-612, Lublin, Poland. ukaszek0@wp.pl
Lukasz
L
Adaszek
Stanisław
S
Winiarczyk
D000818:Animals; D001403:Babesia / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D001404:Babesiosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D052160:Benzothiazoles; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D005456:Fluorescent Dyes; D020459:Genes, rRNA; D009930:Organic Chemicals; D011044:Poland; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling / Q000379:methods; D044366:Transition Temperature
D052160:Benzothiazoles; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S; C098022:SYBR Green I