Plasmid based differentiation and detection of Coxiella burnetii in clinical samples

Abstract

A "nested" PCR approach with primers based on conserved plasmid sequences was used for the highly sensitive and specific detection of Coxiella (C.) burnetii in clinical samples collected from cattle, dogs, cats and humans. Results were in good agreement with those obtained from Capture-ELISA and isolation of the organism in BGM cell culture. We also tested primers with sequences derived from genomic DNA and sequences based on 16S rRNA. In addition, we applied PCR for the differentiation of C. burnetii plasmid types from 28 isolates originating from the USA, Europe and South Africa. Reference isolates Nine Mile RSA493, Dugway 5J108-111 and all European isolates tested were recognized only by primers specific for the QpH1 plasmid. One isolate from a goat abortion in Namibia reacted identically to the reference isolate Priscilla Q177 bearing the QpRS plasmid. Reference isolate S Q217 with plasmid sequences integrated into the genome reacted with none of the plasmid-specific primer pairs.

Isolation of Coxiella burnetii by a centrifugation shell-vial assay from ticks collected in Cyprus: detection by nested polymerase chain reaction (PCR) and by PCR-restriction fragment length polymorphism analyses.

“…For direct detection of C. burnetii in triturated ticks, a nested PCR assay was performed by the primers Hfrag1/Hfrag2 in the first PCR and the primers HF1/ HF2 for the nested PCR. 5,27 The specificity of these primers was confirmed after testing 42 different bacteria isolates by nested PCR. 27 To perform DNA amplification of the isolates from Cyprus and compare them with the reference strains Nine Mile and Q212, the genomic primers CB1/CB2 were used.…”

“…5,27 The specificity of these primers was confirmed after testing 42 different bacteria isolates by nested PCR. 27 To perform DNA amplification of the isolates from Cyprus and compare them with the reference strains Nine Mile and Q212, the genomic primers CB1/CB2 were used. 28 The specificity of these 2 primers were tested in PCR with purified DNA from 25 other bacteria species.…”

Fourteen-year seroepidemiological study of zoonoses in a Greek village.

“…For direct detection of C. burnetii in triturated ticks by PCR, a nested PCR assay was performed with the primers Hfrag1/Hfrag2 in the first PCR and the primers HF1/HF2 for the nested PCR. 12,13 For R. conorii, the PCR assay was performed by 10 L of the boiled extract of the triturated ticks as sample DNA. Amplification, digestion, and electrophoresis were carried out as described previously.…”

Coxiella burnetiiin Central Italy: Novel Genotypes Are Circulating in Cattle and Goats

“…For this reason, several genotyping methods have been described thus far. Until 2005, these techniques were based on plasmid typing (Willems et al 1993, Valkovà and Kazàr 1995, Jäger et al 2002, restriction fragment length polymorphism and pulsed-field gel electrophoresis (RFLP-PFGE) analysis (Vodkin et al 1986, Hendrix et al 1991, Jäger et al 1998, and sequence analysis of individual genes such as16S, 23S (Stein et al 1997), com1, mucZ, and icd (Zhang et al 1997, Nguyen and Hirai 1999, Sekeyovà et al 1999. Nevertheless, some of these methods exhibited limitations on inter-and intralaboratory reproducibility that hindered their widespread use (Massung et al 2012).…”