PCR Detection and Molecular Identification of Chlamydiaceae Species

Abstract

Recent taxonomic developments, based on 16s and 23s rRNA gene sequences, have divided the family Chlamydiaceae into two genera and nine species, of which five have been found to infect humans. Few simple methods are available to detect and identify all species sensitively and specifically. In this study the suitability of the omp2 gene as a target for molecular identification of Chlamydiaceae is demonstrated. Phylogenetic analysis of partial omp2 gene sequences from all nine species agrees with the recently published taxonomic changes based on the ribosomal genes. The use of a family-specific PCR primer pair, which is able to amplify the 5 end of the omp2 gene from all Chlamydiaceae except some Chlamydophila pecorum strains, is described. Identification of all nine species was achieved using restriction fragment length polymorphism analysis with a single enzyme, AluI, confirmed by DNA sequencing. A PCR enzyme-linked oligonucleotide assay was developed which can detect a single chlamydial genome and may be applied to DNA extracts from any specimen or culture for the detection of single or mixed human chlamydial infection.The family Chlamydiaceae has recently been reclassified into two genera and nine species (4). Current human infection by Chlamydiaceae is frequently diagnosed as Chlamydia trachomatis, but less frequently as Chlamydophila pneumoniae (formerly Chlamydia pneumoniae), and rarely as Chlamydophila psittaci (formerly Chlamydia psittaci avian group), Chlamydophila abortus (formerly Chlamydia psittaci abortion group), or Chlamydophila felis (formerly Chlamydia psittaci feline pneumonitis agent). No observation has been made of human infection with Chlamydophila caviae (formerly Chlamydia psittaci guinea pig inclusion conjunctivitis agent), Chlamydophila pecorum (formerly Chlamydia pecorum), Chlamydia suis (formerly porcine Chlamydia trachomatis), or Chlamydophila muridarum (formerly Chlamydia trachomatis of mice).Other than for C. trachomatis, laboratory diagnostic methods are poorly developed and not standardized. Serology is commonly used for diagnosis (6,15), but the immune response to chlamydial infection is often delayed and variable. Microimmunofluorescence, the reference serology method, which was designed for the serotyping of C. trachomatis, is difficult to perform and not fully standardized and does not always correlate with cell culture or nucleic acid detection (8,12,15,16,20). Commercial reagents available for the detection of Chlamydiaceae by direct immunofluorescence (DIF) include the family-specific antibody against the lipopolysaccharide (e.g.,

Detection of Pathogens in Ovine and Caprine Abortion Samples from Sardinia, Italy, by PCR

“…2,5,6,9 In the present study, PCR assay was applied in routine detection of C. burnetii, C. abortus, Salmonella enterica Serovar abortusovis, T. gondii, and Neospora caninum in fetal tissues from naturally aborted ewes and goats in 2003-2005. A total of 315 fetuses (292 ovine and 23 caprine) and 84 placentae (including 76 ovine and 8 caprine samples) were analyzed during [2003][2004][2005] (Table 1). Following macroscopic examination, the brain, skeletal muscle, liver, spleen, and abomasum were removed from each fetus.…”

Chlamydia trachomatis: avances y perspectivas

“…Las bases de nucleótidos están numeradas desde el inicio con 22 bases púricas repetidas en tandem. Todos los plásmidos de C. trachomatis aislados de humanos son extremadamente similares con menos de un 1% de variaciones en la secuencia de nucleótidos, todos tienen alrededor de 7.500 nucleótidos, con 8 marcos abiertos de lectura (10,18).…”

Validation of a PCR Assay for Chlamydophila abortus rRNA gene detection in a murine model

“…The PCR was carried out using 5 µL of extracted DNA and 45 µL of PCR-mix consisting of 20 pmol of each primer, 0.2 mM of each dNTP, 2.5 units of Platinum Taq (5'-CCTTCTTTAAGAGGTTTTACCCA -3', negative sense, nt 1093-1115), which amplify a 587-bp product from the Omp2 gene of Chlamydiaceae family representatives. The reaction was performed according to Hartley et al (2001). Aliquots of 10 µL of the PCR products were analyzed by electrophoresis on a 2% agarose gel.…”

“…The bovine abortion was previously known to be negative for C. abortus infection by inoculation in an embryonated chicken egg and by rRNA and Omp2 PCR assays. A pool of suspensions was filtered using a 45 µm membrane and was submitted to counting of inclusion-forming units (IFU) according to Hartley et al (2001). After determination of the IFU/mL, DNA was extracted from 10-fold serial dilutions using the silica/guanidine isothiocyanate method (Boom et al, 1990).…”

“…This feature has allowed the use of the PCR assay for proposing a new taxonomy, for identification of new species, and for diagnosis of infection (Everett et al, 1999;Kaltenboeck et al, 2005). Differentiation among species of the Chlamydiaceae family can be achieved by restriction fragment length polymorphism analysis of the generated amplicons (RFLP-PCR) or through direct sequencing of the PCR-amplified products (Sheehy et al, 1996;Everett, 2000;Hartley et al, 2001). The aim of this study was to develop a PCR assay to amplify the intergenic space in the 16S-23S rRNA sequence of the Chlamydiaceae family in order to detect and identify C. abortus in organs of experimentally infected mice.…”