| Chlamydia psittaci(Cps), a member of Chlamydia family, whose biological characteristics between bacteria and virus, has a strict intracellular parasitism and gram’s stain negative. Psittacosis chlamydia is a zoonosis caused by Cps in multiple animals. The clinical manifestations of infection by this organism in pigs, cattle, sheep and horses include abortion, stillbirth, lack of appetite, cough and diarrhea, while in pigeons, turkeys and other poultry including pneumonia, dyspnea, depressed spirit and death. In addition, the main manifestations in human are ornithosis and Reiter’s syndrome. Chlamydia psittacosis which has extensive hosts and complex route of transmission, has been found in many countries and regions around the world. According to the movement of animals and increasing of pet bird raise, there is no effective control on chlamydia psittacosis disease. The reports of cases relating to Cps at home and abroad are growing year by year.There is no rapid and accurate diagnostic method of Chlamydia psittaci in clinic. Because the clinical symptom have no differentce what is caused by Chlamydia psittaci or other Chlamydias. Therefore, establishing a rapid and accurate diagnostic method is necessary. The Taqman real-time fluorescent PCR technique, a relatively new technology, is rapid, sensitive, accurate, and quantitative. This method shows great significance in prevention, rapid detection and diagnosis of Cps.The Yl and Y2, Y3Taqman real-time fluorescent PCR and C1and C2conventional PCR for detecting were designed according to the chlamydia sequence of16S-23S rRNA and the main outer membrane protein (MOMP). X1and X2conventional PCR for sequencing were designed according to the chlamydia sequence of16S-23S rRNA and MOMP. Chlamydia psittaci nucleic acid and water regard as control. The result of Taqman real-time fluorescent PCR show that the fluorescen curve of Y1and Y2were better than the fluorescen curve of Y3. The reaction conditions and system of Taqman fluorescent real-time PCR were optimized. This two kinds of Taqman real-time fluorescent PCR, Yl, Y2, were found having good specificity which is proved by using comparison amplification test in Chlamydia psittaci and Mycoplasma hyorhinis, Mycoplasma Pneumonia, Mycoplasma synoviae, Mycoplasma gallisepticum, Escherichia coli, Shigella dysenteriae, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa, Vibrio cholerae, Vibrio parahaemolyticus, Listeria monocytogenes, Bacillus cereus, Alpha hemolytic streptococcus, Enterobacter Sakazakii. The detecting sensitivity result of the double-stranded DNA show that the sensitivity of Y1and Y2real-time fluorescent PCR were about130and5000times higher than conventional PCR for detection. The detecting sensitivity result of chlamydia genomes show that the sensitivity of two real-time fluorescent PCR were both about1000times higher than two conventional PCR for detection. Three specimens which were proved positive by real-time fluorescent PCR were amplification by X1and X2sequencing conventional PCR. The result of outputs sequencing shows that three specimens all were Chlamydia psittaci. Thus, the established method of Y1and Y2Taqman real-time fluorescent PCR are rapid, efficient, specific and sensitive,and they can be used in the detection of chlamydia psittacosis samples. |
