The Development a Polymerase Chain Reaction (PCR) to detect C.Pneumoniae and C.Psittaci

Fatemeh Sabagh, Christopher C Storey

Abstract


94

     C.pneumoniae and C.psittaci both cause respiratory infections in human. detection  of  these  organisms  in  tissue  culture  is  difficult  and serological  testing  is unreliable. There  are  no  sensitive  and  reliable  tests  for  the  detection  of  these  organisms. The  polymerase  chain reaction  (PCR  )  has  provided  an alternative  diagnostic  method  for the detection of these fastidious  organisms. The aim of this study was to develop a PCR to detect C.pneumoniae and C.psittaci from clinical samples. The PCR was optimized in a series of experiments. To determine  if  the  optimized  PCR  could  be applied  to  clinical samples, mock  positive specimen  were  produced by  adding  chlamydiae  to  throat  swab  from  healthy  adults. The DNA was extracted by phenol/chloroform. When tested by PCR all the throat swabs were negative. However,  when  diluted  and  retested,  many  of the  swabs  were  positive  and 10 IFU  of C.psittaci  and  100  IFU  of C.pneumoniae  could  be   detected.  This  experiment  indicates  that  inhibitor  to  PCR  are  found  in  throat  swab  and  further  work  is  needed   on  specimen  preparation.   The PCR was optimized in a series of experiments.    The  optimal  conditions  were  to  use  a two  segment  PCR  with  68°c  annealing  and  polymerization  temperature, 2.0mM  Mgcl2,  0.2μM  primers  and  40  cycles. The  PCR   was  highly  sensitive  and  could  detect  one  inclusion  forming  unit  with  both  C.pneumoniae   and  C.psittaci  strains.  Two  human  strains  and  one   nonhuman   strain  of  C.pneumoniae   and   two  avian   strains   and  three  mammalian   strains   of C.psittaci   were   used   to  determine   the specificity  of PCR. The PCR detected all these different strains.   C.trachomatis   strains   were not detected.  Various bacterial  strains, fungi  DNA,  and  human  DNA  were  negative  in  PCR  and no  amplification  DNA  was  found   in  negative  controls.


Keywords


PCR; Chlamydia; IFU; Pneumoniae; Psittaci

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DOI: https://doi.org/10.22037/jps.v1i4.2260

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"Journal of Paramdedical Sciences", is a publication of "School of Paramedical Sciences, Shahid Beheshti University of Medical Sciences" and "Iranian Society of Medical Proteomics".

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EISSN: 2008-4978

PISSN: 2008-496X