Borrelia DNA

Pukhovskaya, Natalia M., Morozova, Olga V., Vysochina, Nelya P., Belozerova, Nadejda B. & Ivanov, Leonid I., 2019, Prevalence of Borrelia burgdorferi sensu lato and Borrelia miyamotoi in ixodid ticks in the Far East of Russia, International Journal for Parasitology: Parasites and Wildlife 8, pp. 192-202 : 193-194

2.2. Borrelia DNA detection

Arthropod suspensions were prepared from individual ticks in disposable tubes with pestles in liquid nitrogen. Since 2013 «TissueLyser LT» (Qiagen, Germany) kit was used.

Total nucleic acids were isolated from individual tick suspensions using phenol-chloroform deproteinization with subsequent alcohol precipitation (“Vector Best”, Russia) during 1999–2009 and later in 2010–2014 it was replaced with adsorption of nucleic acids on silica by using “Ribosorb” (“InterLabService”, Russia).

PCR with primers SL ( Demaerschalck et al., 1995) and mastermix (“Syntol”, Russia) with subsequent electrophoresis was performed in 1999–2006; test-system for Borrelia burgdorferi s.l. detection (“IzoGen”, Russia) was used during 2007–2009. Real time PCR detection and quantitation were began to use in 2010 initially with the kit “AmpliSens B. burgdorferi sensu lato - FL” and later in 2011–2014 – with “Amply- Sens TBE, B. burgdorferi , A. phagocytophila, E. muris/E. cha ff ensis - FL” (“InterLabService”, Russia). Borrelia miyamotoi DNA was detected by PCR with subsequent electrophoresis using primers specific to p66 gene ( Fomenko et al., 2010) in 2011–2013 and by real time PCR in 2014 with the kit “Vecto Borrelia miyamotoi - FL” (Vector Best, Russia) according to the manufacturer's instructions.