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Detection of Pseudomonas (Burkholderia) cepacia using PCR
P W Campbell1, J A Phillips, G J Heidecker
1Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2586, USA.
Abstract:
Pseudomonas cepacia colonization of the lung is associated with increased morbidity and mortality for cystic fibrosis (CF) patients. The lack of a sensitive detection method for Pseudomonas cepacia in CF sputum has resulted in controversy regarding its epidemiology. We designed a PCR method to detect P. cepacia using P. cepacia 16 S rRNA sequences as the amplification target region. The PCR amplification with purified DNA as template yielded the expected 209-bp products from P. cepacia, but not from related Pseudomonas species of medical importance or other bacteria which have been reported to colonize CF patients. In serial dilution experiments as few as 10(2) P. cepacia CFU were detectable. When sputum samples from three CF patients chronically colonized with P. cepacia and P. aeruginosa were analyzed, P. cepacia was detected in all three specimens by PCR, but only in two when selective culture was performed. Our data support the potential role of PCR technology in the rapid, sensitive, and definitive detection of P. cepacia in CF sputum samples, even in the context of concomitant P. aeruginosa colonization.
Insights
A new PCR method accurately detects Pseudomonas cepacia in cystic fibrosis (CF) sputum. This sensitive technique improves diagnosis and understanding of CF lung infections, even with other bacteria present.
Area of Science:
- Medical Microbiology
- Molecular Diagnostics
Background:
- Pseudomonas cepacia lung colonization increases morbidity and mortality in cystic fibrosis (CF) patients.
- Current detection methods for P. cepacia in CF sputum lack sensitivity, leading to epidemiological uncertainty.
Purpose of the Study:
- To develop and validate a sensitive Polymerase Chain Reaction (PCR) method for detecting Pseudomonas cepacia in CF sputum.
Main Methods:
- A PCR assay was designed targeting the 16S rRNA gene sequences of P. cepacia.
- The PCR method's specificity was tested against related Pseudomonas species and other common CF colonizers.
- Sensitivity was assessed through serial dilution experiments.
- The PCR assay was applied to sputum samples from CF patients.
Main Results:
- The PCR method specifically amplified a 209-bp product from P. cepacia, with no amplification from related species or other bacteria.
- As few as 10^2 colony-forming units (CFU) of P. cepacia were detectable.
- PCR detected P. cepacia in all three CF sputum samples analyzed, whereas selective culture detected it in only two.
Conclusions:
- PCR technology offers a rapid, sensitive, and definitive method for detecting P. cepacia in CF sputum.
- This molecular approach can aid in understanding the epidemiology of P. cepacia infections in CF patients, even with co-colonization.