Use of a multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay for the rapid identification of bacterial pathogens

• Published in: Clinical microbiology and infection Vol. 14; no. 2; pp. 155 - 160
• Main Authors: Wang, Y., Kong, F., Gilbert, G.L., Brown, M., Gao, W., Yu, S., Yang, Y.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Elsevier Ltd 01.02.2008; Blackwell Publishing Ltd; Blackwell
• Subjects: Bacteria - classification; Bacteria - genetics; Bacterial Infections - diagnosis; Bacterial pathogens; Bacteriological methods and techniques used in bacteriology; Bacteriological Techniques - methods; Bacteriology; Base Sequence; Biological and medical sciences; Blotting, Southern - methods; diagnosis; DNA Gyrase - genetics; DNA, Bacterial - analysis; Escherichia coli; Fundamental and applied biological sciences. Psychology; hybridisation assay; identification; Microbiology; Molecular Sequence Data; multiplex PCR-based reverse line blot (mPCR/RLB); Polymerase Chain Reaction - methods; probes; Sensitivity and Specificity; Species Specificity; Streptococcus pneumoniae; diagnosis; probes; identification; hybridisation assay; multiplex PCR-based reverse line blot (mPCR/RLB); Bacterial pathogens; Performance evaluation; Molecular hybridization; Multiplex polymerase chain reaction; Identification; Infection; Sensitivity; Specificity; Rapid technique; Bacteriosis; Genetics; Bacteria; Diagnosis; Molecular biology
• ISSN: 1198-743X; 1469-0691
• DOI: 10.1111/j.1469-0691.2007.01890.x

The aim of this study was to develop a sensitive and reliable method for the molecular identification of pathogenic bacteria. A multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay was developed and evaluated for the rapid identification of 24 systemic and respiratory bacterial pathogens in routine diagnosis. All species-specific probes designed for the RLB hybridised with amplified DNA only from the corresponding species. Sensitivity limits of the mPCR/RLB assay varied among the 24 target organisms from 0.05 pg to 0.5 ng of genomic DNA. The sensitivity of the assay was 2 × 102 CFU/mL for Streptococcus pneumoniae and 6 × 102 CFU/mL for Escherichia coli. The specificity of each probe was tested against 24 species. There were no cross-reactions among any of the 43 probes. The mPCR/RLB assay appeared to be a useful alternative tool for the molecular identification of common pathogens.