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Detection of Pseudomonas pseudomallei by PCR and hybridization
1Department of Microbiology, University of Queensland, St Lucia, Australia.
Abstract:
A molecular method for the detection of Pseudomonas pseudomallei was developed on the basis of the differences in the 23S rRNA sequences of related species of the genus Pseudomonas. An 18-base oligonucleotide probe, designed following partial sequencing of 23s ribosomal DNA (rDNA), was used for the identification and detection of P. pseudomallei either by hybridization or by direct PCR. Optimal detection was obtained by hybridization of the probe with PCR-amplified rDNA rather than with total genomic DNA or colony blots. One nanogram of template DNA amplified in a PCR mixture containing 14% glycerol could be detected in slot blots hybridized with the digoxigenin-labelled probe and the lumigen PPD detection system. Amplified rDNA sequences from 41 P. pseudomallei strains of various origins hybridized with the probe. The probe also hybridized with three Pseudomonas mallei reference strains under conditions of high stringency but failed to hybridize with amplified rDNA sequences from other closely related Pseudomonas spp. PCR with a conserved primer and the 18-base oligonucleotide probe (direct PCR) specifically amplified P. pseudomallei and P. mallei. By using these methods, approximately 10(4) P. pseudomallei cells per ml could be detected in artificially inoculated blood samples and in blood dried on filter paper following Chelex extraction. The detection limit in blood was increased to 10(2) cells per ml by concentration of bacteria from 0.5 ml of blood or by a 24-h blood culture enrichment prior to PCR. Approximately 10(3) cells per ml were detected in seeded sputum samples. The detection times by direct PCR and indirect PCR and then probe hybridization were approximately 5 h and 24 h, respectively. These results indicate that amplification of conserved rDNA sequences by PCR directly or by hybridization with a probe to PCR fragments offers promise for the detection of P. pseudomallei and P. mallei.
Insights
A new molecular method accurately detects Pseudomonas pseudomallei using 23S ribosomal DNA (rDNA) sequencing and a specific oligonucleotide probe. This approach offers rapid and sensitive identification of P. pseudomallei in clinical samples.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Pseudomonas pseudomallei is a significant human pathogen.
- Accurate and rapid detection methods are crucial for timely diagnosis and treatment.
- Existing diagnostic methods may have limitations in sensitivity or speed.
Purpose of the Study:
- To develop and validate a novel molecular detection method for Pseudomonas pseudomallei.
- To utilize differences in 23S ribosomal DNA (rDNA) sequences for specific identification.
- To assess the sensitivity and specificity of the developed method in various sample types.
Main Methods:
- Partial sequencing of 23S ribosomal DNA (rDNA) from Pseudomonas species.
- Design of an 18-base oligonucleotide probe for P. pseudomallei detection.
- Application of the probe in hybridization assays and direct Polymerase Chain Reaction (PCR).
- Optimization of PCR conditions, including the use of glycerol, for enhanced sensitivity.
Main Results:
- The oligonucleotide probe specifically hybridized with amplified rDNA from 41 P. pseudomallei strains.
- High-stringency hybridization confirmed specificity, with cross-reactivity only to three P. mallei strains.
- Direct PCR using a conserved primer and the probe specifically amplified P. pseudomallei and P. mallei.
- Detection limits of approximately 10^4 cells/ml in blood and sputum were achieved, improved to 10^2 cells/ml with enrichment.
- Detection times were significantly reduced with direct PCR (approx. 5 hours) compared to hybridization (approx. 24 hours).
Conclusions:
- The developed molecular method based on 23S rDNA sequencing and a specific probe is a promising tool for P. pseudomallei detection.
- The method demonstrates high specificity and sensitivity in various sample types, including blood and sputum.
- Direct PCR offers a rapid and efficient approach for the identification of P. pseudomallei and P. mallei.