Development of a real-time PCR using SYBR Green and melting curve analysis to identify Bordetella pertussis and Bordetella parapertussis in clinical specimens, and evaluation of its effectiveness in current practice
Abstract number: p967
Lehours P., Menard A., Sarlangue J., Laguente C., Bebear C.H., Megraud F., De Barbeyrac B.
Whooping cough affects now young adults and teenagers, thus being able to contaminate infants unvaccinated or incompletely vaccinated. In this population, it evolves readily into atypical or severe forms difficult to diagnose and a biological confirmation is necessary. Thus molecular techniques represent invaluable tools. Here, we describe a real-time PCR assay for the identification of B. pertussis and B. parapertussis in clinical specimens
The available IS481 sequences of B. pertussis were aligned to identify conserved regions for the primer design. Primers for the detection of B. parapertussis were designed on the IS1001 sequence. A universal PCR targeting the 16S rDNA gene was also developed to be used as a control for extraction and for absence of PCR inhibitors. The PCR primers sets were tested on DNA extracted from the Bordetella species, and from diverse bacterial species commonly found in humans. The SYBR Green® real-time PCR amplification and melting curve (Tm) analysis were carried out in an ABI PRISM® 7000 thermocycler (Applied Biosystems), and followed by a melting program. A retrospective study was performed on positive (n = 19) and negative (n = 13) nasopharyngeal aspirates (NPA). We tested prospectively 131 NPA received between April 2004 and November 2005.
When using the control strains, only DNA extracted from B. pertussis and B. parapertussis led to amplification, demonstrating the specificity of these two PCRs. Melting curve analysis of the amplicons from B. pertussis and B. parapertussis control strains exhibited Tm of approximately 79°C and 86°C, respectively. Serial dilutions of the extracted DNA from bacterial suspensions were tested using this assay and the regression curve was linear from 105 to 1 bacterial genome with slopes closed to that of the maximum efficiency. For the retrospective analysis, the 19 positive samples were all positive for B. pertussis, and no peak appeared for the 13 negative samples. Prospectively, this assay allowed the detection of 21 positive clinical samples (18 B. pertussis, 3 B. parapertussis), among these 14 could not have been isolated by culture.
We have developed a new real-time PCR for the identification of B. pertussis and B. parapertussis targeting the IS sequences. This method, which uses the SYBR green chemistry and analysis of the melting point, is rapid, sensitive and specific, and can be easily implemented in a setting where a real time PCR apparatus is available.
|Session name:||XXIst ISTH Congress|
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