False-negative results in Real-Time PCR (RT-PCR) for meningococcal disease diagnosis

Abstract

Factors responsible for false-negative results (F-N) in RT-PCR assay for detecting N. meningitidis in serum and CSF samples were investigated. As the meningococcal disease should be rapidly treated because of its high mortality and epidemic potential, the F-N in diagnostic testing may cause treatment failures and/or on disease restraint in community. Thus, it is crucial to learn the factors which cause F-N in RT-PCR assays. The F-N were induced by inhibition, low quantity of target DNA in extracted samples, and inadequate temperature employed at PCR annealing procedure. As bacterial DNA concentration in samples might be highly variable, the ideal sample volume to be extracted could not be defined. As previously recommended for N. meningitidis gene-grouping by RT-PCR assay, the annealing temperature at 60 °C was not suitable for B and W135 genogroups. Altogether, these factors induced F-N in 31 samples; therefore, 30 % of N. meningitidis detected by RT-PCR were classified as non-genogrouped. The inhibitors and/or the low amount of target DNA induced F-N on RT-PCR, independently of the specimen volume used for extracting DNA. However, adjustments on the PCR annealing temperature and amount of extracted DNA added into the reaction might avoid the occurrence of the majority of F-N.