Application of TaqMan low-density arrays for simultaneous detection of multiple respiratory pathogens

Abstract

The large and growing number of viral and bacterial pathogens responsible for respiratory infections poses a challenge for laboratories seeking to provide rapid and comprehensive pathogen identification. We evaluated a novel application of the TaqMan low-density array (TLDA) cards for real-time PCR detection of 21 respiratory-pathogen targets. The performance of the TLDA was compared to that of individual real-time PCR (IRTP) assays with the same primers and probes using (i) nucleic acids extracted from the 21 pathogen strains and 66 closely related viruses and bacteria and (ii) 292 clinical respiratory specimens. With spiked samples, TLDA cards were about 10-fold less sensitive than IRTP assays. By using 292 clinical specimens to generate 2,238 paired individual assays, the TLDA card exhibited 89% sensitivity (95% confidence interval [CI], 86 to 92%; range per target, 47 to 100%) and 98% specificity (95% CI, 97 to 99%; range per target, 85 to 100%) overall compared to IRTP assays as the gold standard with a threshold cycle (C(T)) cutoff of 43. The TLDA card approach offers promise for rapid and simultaneous identification of multiple respiratory pathogens for outbreak investigations and disease surveillance.

Fig. 1.

TaqMan low-density array (TLDA) card layout including the respiratory pathogens. TLDA cards were customized to include singleplex real-time PCR assays for 13 viruses and 8 bacteria known to cause acute respiratory infections. These microfluidic cards contain 384 individual wells separated into eight loading ports with 48 assay wells each (− ctrl, negative control; S1 to S7, samples 1 through 7). This format allows for easy loading of eight specimens and for the performance of each assay in duplicate. Upon completion of specimen loading, all wells are sealed individually for singleplex reactions. Each CDC respiratory panel TLDA card has built-in controls, including the internal positive control (IPC) and two human DNA/RNA controls, GAPDH and RNP3, respectively.