abortus or Cp. pecorum were first diluted to 1:10 and subsequently used in a plaque assay. Furthermore, 500 μl of this suspension was added to McCoy cell monolayers in 25 cm2 flasks to perform the blind passage assay. The positive culture and plaque cloned Chlamydophila were then grown in specific pathogen-free eggs, the yolk sacs were harvested one week later and the bacteria were purified and stored at -80°C. C. burnetii strains were isolated by intraperitoneal inoculation of OFI mice then
on embryonated hen eggs . Briefly, 3 OF1 mice (8 weeks old) were inoculated with 0.2 mL of vaginal swab extract or milk sample tested positive in PCR. The mice PI3K inhibitor were killed nine days post inoculation and the spleens were sampled and reinoculated into 6-days-old, specific pathogen-free embryonated hen eggs. The infected yolk sacs of dead and viable embryos were harvested between 8 and 10 days after inoculation, aliquoted and frozen at -80°C. Genomic DNA of isolated chlamydophila and Coxiella was prepared using a QIAmp DNA mini Kit (Qiagen, Courtaboeuf, France) following the manufacturer’s
recommendations and characterized using RFLP-PCR method of 16S–23S rRNA intergenic region . Results Initial set-up and optimization The primer sets pmpF/pmpR821, CpcF/CpcR and Trans-1/Trans-2 designed in this study, challenged 3-MA solubility dmso simultaneously with DNA extracts of AB7, iB1 and Nine-Miles reference strains of Cp. abortus, Cp. pecorum, and C. burnetii resulted in a micro-organism-specific identification of the target sequence. The amplification conditions and master mixture components were optimized to amplify all DNA as singlet, in different combinations as duplexes or as triplex of three target sequences (Figure 1). With a primer concentration of 0.8 μM, 1.5 U of Taq polymerase, 3 mM of MgCl2 and an annealing temperature of 61°C, m-PCR produced simultaneously in one tube reaction, three specific fragments of 821, 526 and 687-bp long for Cp. abortus, Cp. pecorum and for C.
burnetii, respectively. No m-PCR product was generated using water instead of target DNA (Figure 1) Figure 1 learn more Multiplex PCR amplification of Cp. abortus, Cp. pecorum and C. burnetii references strains individually, and in all possible combinations. Lane 1: 100-bp ladder; lane 2: Cp. abortus AB7; Ketotifen lane 3: Cp. pecorum iB1; lane 4: C. burnetii Nine Miles; lane 5: Cp. abortus and Cp. pecorum; lane 6:Cp. abortus and C. burnetii; lane 7: Cp. pecorum and C. burnetii; lane 8: Cp. abortus, Cp. pecorum and C. burnetii; lane 9: Negative control without DNA. The sizes of the three different PCR products are shown on the left. Sensitivity and specifiCity of PCR m-PCR, as well as duplex or single PCR performed on reference strain (AB7, iB1 and Nine-Miles) purified DNA with the same primers, detected as little as 50 genome copies per PCR reaction (Figure 2).