First, PLG-containing tubes (Qiagen Sciences, MD) were used for phenol and chloroform extraction, since they allow clean separation of the top aqueous layer by decantation after centrifugation. Second, a final step of passing the DNA through DNeasy kit columns (Qiagen Sciences, MD) was included to obtain good quality DNA for real-time PCR. B. microti and A. phagocytophilum plasmid construction Thiamine pyrophosphokinase gene of C59 wnt manufacturer B. microti (BmTPK) and APH1387 gene of A. phagocytophilum were amplified from B. microti strain RM/NS and A. phagocytophilum strain HZ, respectively, using primers listed in Table 1, which are designed specifically for RM/NS and HZ
strains genes, respectively. Table 1 Sequence of PCR primers and molecular beacon probes PCR primers/Probes/Oligos Sequence* Length Tm (°C) Size of PCR amplicon Fluorophore/Quencher RecF primer 5’ GTG GAT CTA TTG TAT TAG ATG AGG CTC TCG 3’ 30
66.1 222 bp RecR primer 5’ GCC AAA GTT CTG CAA CAT TAA CAC CTA AAG 3’ 30 67.3 RecF3 primer 5’ GCA AGA GTT CAA ATA GAA AA 3’ 20 53.7 287 bp RecR3 primer 5’ AAA GCT TTT GCA TAA ACA G 3’ 19 54.7 RecA3 probe 5’ CTG GCG GAT ATC CTA GGG GG CGC CAG 3’ 26 CT99021 in vitro 67.9 FAM/ BHQ-1 5BmicrotiTPK primer 5’ AAT ATT GTT GAA TGG GGA TAT TTG TG 3’ 26 64.2 600 bp 3BmicrotiTPK primer 5’ AAT AAT ATA GCT TTT CCA AAA
TAT AAC TGA C 3’ 31 60.2 5BmTPK primer 5’ Phosphatidylinositol diacylglycerol-lyase TGA GAG GAA CGA CCA TAG C 3’ 19 61.4 141 bp 3BmTPK primer 5’ CCA TCA GGT AAA TCA CAC GAA A 3’ 22 61.6 BmTPK probe 5’ CGC GTC GGT GTT GTT GAC CAG CGG CCG CG GAC GCG 3’ 35 61.5 CAL Fluor Orange 560/ BHQ-1 5ApAPH1387 primer 5’ ATG TAT GGT ATA GAT ATA GAG CTA AGT GA 3’ 29 57.8 1737 bp 3ApAPH1387 primer 5’ CTA ATA ACT TAG AAC ATC TTC ATC GTC AG 3’ 29 62.2 5Aphagocyt primer 5’ ATG GCT ACT ACG AAG GAT 3’ 18 57.9 152 bp 3Aphagocyt primer 5’ CGA AGC AAC ATC TCT ACA T 3’ 19 58.0 Aph1387 probe 5’ CGG TGC GAC AAA GAT GCC AGC ACT AAT GCG GCA CCG 3’ 36 61.9 CAL Fluor Red 610/ BHQ-2 5ACTA1 primer 5’ AGA GCA AGA GAG GTA TCC 3’ 18 58.0 104 bp 3ACTA1 primer 5’ CTC GTT GTA GAA GGT GTG 3’ 18 57.7 ACTA1 probe 5’ CGC TGC CCT ATC GAG CAC GGC ATC ATC AC GCA GCG 3’ 35 62.4 Quasar 670/ BHQ-2 RecA3MB-com oligo 5’ ttG CGC CCC CTA GGA TAT CCG Ctt 3’ 24 67.9 TPKMB-com oligo 5’ tt tCG CGG CCG CTG GTC AAC AAC ACC ttt 3’ 29 61.5 AphMB-com oligo 5’ ttt CGC ATT AGT GCT GGC ATC TTT GTC ttt 3’ 30 61.9 ActinMB-com oligo 5’ tt tGT GAT GAT GCC GTG CTC GAT AGG ttt 3’ 29 62.4 *Italicized molecular beacon sequence depicts the arm sequences whereas the sequences marked by bold letters indicate probe region of molecular beacons complementary to the target sequence.