Woźniakowski et al. Acta Veterinaria Scandinavica 2011, 53:10 http://www.actavetscand.com/content/53/1/10 the chicken ovotransferrin gene was also amplified for each sample. Each sample was run in duplicate. Reaction mixture, standards and conditions of real-time PCR has been described elsewhere [2]. As positive controls, HPRS16 and Rispens strains as well as MDV BAC-clones were used. PCR PCR with primers complementary to meq was applied as previously described [10]. The PCR products were puri- fied using Nucleospin Kit (Macherey-Nagel, Düren, Germany). PCR for LTR The sequences of primers specific for LTR-REV were: LTR U3 REV: 5’-CCG AGA AAT GAT ATC AGC-3’ and U5 REV: 5-GGT GGG GTA GGG ATC CG-3’ [9]. The reaction volume was 25 μL and contained: 2.5 μL of 10-fold concentrated PCR buffer (1.5 mM MgCl2), 1 μL of dNTPs (0.2 μM of each dNTP), 0.5 μL HotStar Taq Polymerase (2.5 U) (Qiagen), 5 μL of 1M betaine solution (0.2 μM) (Sigma-Aldrich, Munich, Germany), 1 μL of each primer (0.4 μM), 1 μL of total DNA and 13 μL of deionised water. PCR conditions were as fol- lowing: initial denaturation - 95°C for 5 min., following 35 cycles of: exact denaturation 94°C for 1 min., primers annealing 62°C for 1 min., products extension 72°C - 1 min., final extension of the products 72°C for 10 min. Positive control for PCR was extracted from CEKs inoculated with the REV-T strain while a negative con- trol was extracted from non-inoculated cell cultures. DNA sequencing PCR products of meq from 24 MDV strains and 10 pro- ducts for the LTR region were sequenced from forward and reversed primers on GS FLX/Titanium sequencer (Roche, Branford, Connecticut, USA) by GENOMED (Warsaw, Poland). Each amplicon was run 2 times to ensure the reliability of the results. The meq and LTR sequences were assembled into single contig in Bioedit ver. 7.0.9.0 then submitted to NCBI Genebank and aligned using Geneious ver. 5.1 [12]. PCR products were separated in 1.5% ethidium bromide stained agarose gels. Results As the first step of this study, the examined MDV field strains were propagated in CEKs. Three passages of these strains were conducted. CPE was observed in all infected cell cultures starting from the 96th h of 3rd pas- sage. An early CPE was seen as small round cells reflect- ing light waves. These cells formed foci and syncytias that detached from the wall of cell culture flask causing formation of plaques. From these passaged MDV strains, total DNA was extracted. Similarly, DNA extracted from Page 3 of 7 CEKs infected with REV-T was used as a positive con- trol. There was no CPE observed in CEKs inoculated with filtered supernatants taken from cell cultures infected with MDV thus confirming the purity of the used strains. Similarly, the results of real-time PCR for the detection of gag gene of REV in supernatants were negative. Next, real-time PCR was conducted on all DNA sam- ples extracted from infected CEKs. The specific fluores- cent signal observed as curves for ICP4, pp38 and meq genes were detected in all samples. On the basis of quantification of ICP4, pp38 and meq, the number of viral copies was determined as ranging from 63 ± 2 to 12123 ± 587 copies per 104 infected cells. Meq analysis By standard PCR, the presence of an approximately 1062 bp long product for the meq oncogene was found in samples except sample 73_08 (Figure 1, lane 14) and BAC_31_07Δmeq (Figure 1, lane 30). In 5 samples (Figure 1, lanes: 1, 18, 19, 25 and 30) the PCR products were weak and for 7 samples, additional smears in the background were observed (Figure 1, lanes: 10, 11, 12, 16, 25, 26, 30) as fine bands ranging from 210 to 550 bp. PCR products for meq oncogene were sequenced and submitted to NCBI Genebank (Table 2). Analysis of meq showed a 68 bp insertion in position 566 of meq in 12 of 24 strains (Figure 2). These inser- tions resulted in a frame shift in Polish field strains iso- lated in 2009 and 2010. Other minor single nucleotide Figure 1 PCR results for the detection of the meq oncogene in Marek’s disease virus. M-Mass Ruller 100 kb Plus (80 bp-10 000 bp), Fermentas, 1 - 23_07_PL, 2 - 25_07_PL, 3 - 29_07_PL, 4 - 30_07_PL, 5 - 31_07_PL, 6 - 32_07_PL, 7 - 2_08_PL, 8 - 3_08_PL, 9 - 7_08_PL, 10 - 8_08_PL, 11 - 45_08_PL, 12 - 48_08_PL, 13 - 56_08_PL, 14 - 73_08_PL, 15 - 8_09_PL, 16 - 23_09_PL, 17 - 26_09_PL, 18 - 40_09_PL, 19 - 42_09_PL, 20 - 45_09_PL, 21 - 51_09_PL, 22 - 81_09_PL, 23 - 82_09_PL, 24 - 86_09_PL, 25 - 8_10_PL, 26 - 10_10_PL, 27 - 11_10_PL, 28 - 30_10_PL, 29 - 61_10_PL, 30 - BAC_31_07Δmeq, 31 - BAC_7_08, 32 - HPRS16, 33 - Rispens, 34 - negative DNA control extracted from non-infected chicken embryo kidney cells. Smeared products are marked with a white box.