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 f rom the pathogens Haemophilus influenzae, Neisseria meningitidis and Strept ococcus pneumoniae from cerebrospinal fluid samples of patients suffering f rom acute pyogenic meningitis. The accurate identification of the PCR ampli fied product was achieved by hybridizing dot-blots of the PCR products to p robes which were specific, biotinylated internal sequences of the amplified target DNA. Detection of the hybrids was done in a colour reaction using s treptavidin-alkaline phosphatase conjugate and BCIP/NBT substrates. The ent ire protocol took on ly 7 h for the correct identification of the pathogen present in clinical samples of cerebrospinal fluid. The sensitivity and spe cificity were >95%. (C) 2000 Academic Press.