Triplex Real-Time PCR without DNA Extraction for the Monitoring of Meningococcal Disease

Abstract

Detection of Neisseria meningitidis has become less time- and resource-intensive with a monoplex direct real-time PCR (drt-PCR) to amplify genes from clinical specimens without DNA extraction. To further improve efficiency, we evaluated two triplex drt-PCR assays for the detection of meningococcal serogroups AWX and BCY. The sensitivity and specificity of the triplex assays were assessed using 228 cerebrospinal fluid (CSF) specimens from meningitis patients and compared to the monoplex for six serogroups. The lower limit of detection range for six serogroup-specific drt-PCR assays was 178⁻5264 CFU/mL by monoplex and 68⁻2221 CFU/mL by triplex. The triplex and monoplex showed 100% agreement for six serogroups and the triplex assays achieved similar sensitivity and specificity estimates as the monoplex drt-PCR assays. Our triplex method reduces the time and cost of processing CSF specimens by characterizing six serogroups with only two assays, which is particularly important for testing large numbers of specimens for N. meningitidis surveillance.

Keywords: Neisseria meningitidis; direct multiplex real-time PCR; meningococcal meningitis; meningococcal serogroups.

Figure 1

Bland-Altman Plots resulting from the pairwise comparison of C_t values between triplex and monoplex direct real-time PCR assays by meningococcal serogroups: (a) NmA-csaB; (b) NmB-csb; (c) NmC-csc; (d) NmW-csw; (e) NmX-csxB; and (f) Nm-csy. Level of agreement between the two assays by serogroup (C_{t Triplex} − C_{t Monoplex}), also known as observed mean difference (small dash line), is presented as a function of the average C_t values of the two assays [(C_{t Triplex} + C_{t Monoplex})/2]. Reference lines (solid line) indicate the ideal zero difference. Upper and lower limits of agreement (large dash lines) defined by mean C_t difference +/− 1.96 × SD.