Development of a rapid real-time PCR assay for detection and quantification of four familiar species of Chlamydiaceae.
Chlamydiae are one of the causative agents of various diseases in animals and human beings, which include abortion, pneumonia, gastroenteritis, encephalomyelitis, conjunctivitis, arthritis and sexually transmitted diseases. Much work has been carried out to attempt to develop an efficient pathogen detection strategy. Here, we presented a Chlamydiaceae-specific 23S rRNA-based real-time PCR assay for simultaneous detection and quantification of four members of Chlamydiaceae family, C. trachomatis, C. psittaci, C. pneumoniae and C. pecorum, using SYBR Green and Lightcycler. The assay was characterized using plasmid constructs of the bacteria and verified on standard strains of all four species of the Chlamydiaceae and a large cohort of clinical samples collected from human and animals by comparison with fluorescence immunohistochemistry method. The results showed that the present real-time PCR assay was of high specificity and sensitivity. It was capable of detecting as few as 250fg of chlamydial DNA (equivalent to 10(-1)IFU) and was applicable to both liquid cultures and clinical samples. This assay may therefore offer a rapid, economic and reliable means for screening of the chlamydiaceae pathogens.
National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100094, PR China.
Jian-Min
JM
Yang
Hong-Xiang
HX
Liu
Yong-Xin
YX
Hao
Cheng
C
He
De-Ming
DM
Zhao
D000818:Animals; D002690:Chlamydia Infections / Q000175:diagnosis* / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000235:genetics / Q000302:isolation & purification; D002693:Chlamydiaceae / Q000235:genetics / Q000302:isolation & purification*; D023501:Chlamydophila / Q000235:genetics / Q000302:isolation & purification; D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000302:isolation & purification; D002691:Chlamydophila psittaci / Q000235:genetics / Q000302:isolation & purification; D005069:Evaluation Studies as Topic; D005455:Fluorescent Antibody Technique; D005798:Genes, Bacterial; D006801:Humans; D007150:Immunohistochemistry; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids / Q000235:genetics; D011200:Poultry; D012329:RNA, Bacterial / Q000032:analysis; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D013045:Species Specificity
D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S