Protocol for the rapid detection of the urogenital tract mollicutes and Chlamydia with concomitant LGV-(sub)typing.
Urogenital tract infections can be caused by a number of pathogens, some of which, like the obligate intracellular Chlamydia trachomatis, are difficult to culture, or the cell wall-less mollicutes, like M. hominis or Ureaplasma spp. Real-time PCR (qPCR) has become an important diagnostic tool as it enables not only the species-specific detection of the organism but also the quantification essential to define the etiological relevance of a facultative pathogenic bacterium. We developed a set of TaqMan qPCRs for the detection of the species M. genitalium and M. hominis (Mh/Mg-duplex qPCR), U. parvum and U. urealyticum (Uu/Up duplex-PCR), and C. trachomatis (CT-qPCR), and for typing of lymphogranuloma venereum-associated L-serovars of C. trachomatis (LGV-qPCR) as well as a sub-typing of L1, L2, and L3. In addition, the human gap-gene was amplified as quality control of the specimen, and a cryptic plasmid co-amplified in CT-qPCR as an inhibition control. The present protocol focuses on the step-by-step description for the establishment of these TaqMan multiplex qPCRs.
Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University, Düsseldorf, Germany.
Nadine
N
Möbius
Wibke
W
Brenneisen
Anke
A
Schaeffer
Birgit
B
Henrich
D015373:Bacterial Typing Techniques / Q000379:methods*; D002692:Chlamydia trachomatis / Q000145:classification* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D008973:Tenericutes / Q000145:classification* / Q000235:genetics*; D013997:Time Factors; D014566:Urogenital System / Q000382:microbiology*
D017931:DNA Primers; D004269:DNA, Bacterial