Rapid diagnosis of acute pyogenic meningitis by a combined PCR dot-blot assay.
A multiplex PCR was employed to amplify unique conserved sequences of DNA from the pathogens Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae from cerebrospinal fluid samples of patients suffering from acute pyogenic meningitis. The accurate identification of the PCR amplified product was achieved by hybridizing dot-blots of the PCR products to probes which were specific, biotinylated internal sequences of the amplified target DNA. Detection of the hybrids was done in a colour reaction using streptavidin-alkaline phosphatase conjugate and BCIP/NBT substrates. The entire protocol took only 7 h for the correct identification of the pathogen present in clinical samples of cerebrospinal fluid. The sensitivity and specificity were >95%.
Balganesh
M
M
Astra Research Centre India, 18th Cross, Malleswaram, Bangalore-560003, India. meenakshi.balganesh@astrazeneca.com
Lalitha
M K
MK
Nathaniel
R
R
D001710:Biotin / Q000737:chemistry; D002555:Cerebrospinal Fluid / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis; D004311:Double-Blind Method; D005069:Evaluation Studies as Topic; D006193:Haemophilus influenzae / Q000302:isolation & purification; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods; D016920:Meningitis, Bacterial / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000382:microbiology; D009345:Neisseria meningitidis / Q000302:isolation & purification; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000302:isolation & purification
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D001710:Biotin