Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays

Abstract

In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators.

Keywords: Analytical techniques; Comparative studies; Microfluidics; PCR; Respiratory pathogens.

Fig. 1

TAC layout for CDC respiratory assays including 16 viral and 7 bacterial targets. At maximum density the cards are arranged in an 8 specimen × 48 assay array for a maximum total of 384 individual real-time PCR reactions. TACs can also be manufactured in other formats. In this study the cards were partitioned into 8 specimen × 24 assay sub-arrays where each assay was performed in duplicate in adjacent wells (squares A and B). Each column represents a single assay (abbreviation shown in parenthesis), with the exception of the internal positive control (IPCO), manufacturing control (GAPDH), and human coronavirus assays which were spotted in single wells within a column (columns 11, 13, and 14). Each card also includes an RNA control (RNP3). Sample port one was reserved for use as a negative control, permitting seven samples to be analyzed on each TAC.