Engineered Combined-Positive-Control Template for Real-Time Reverse Transcription-PCR in Multiple-Pathogen-Detection Assays

Abstract

Recently we evaluated a custom TaqMan array card (TAC) detection system, formerly known as a TaqMan low-density array (TLDA) card, for simultaneous real-time PCR identification of 21 pathogens and three control targets in duplicate from respiratory specimens (M. Kodani et al., J. Clin. Microbiol. 49:2175-2182, 2011). We engineered an adaptable and expandable system of in vitro RNA transcripts to serve as a combined positive control for both DNA and RNA targets in multiple-pathogen-detection technologies based on real-time reverse transcription-PCR. Apositive-control template for singleplex real-time PCR can be easily obtained by purifying nucleic acids from a target organism. This is also feasible when a few targets are combined in the same multiplex reaction. However, in the case of multiplepathogen-detection assays, such as the TaqMan array card (TAC) detection system, formerly known as TaqMan low-density arrays (TLDA), where 24 to 48 targets are amplified simultaneously from either RNA or DNA templates (6), obtaining a combined positive control (CPC) becomes more cumbersome. One major advantage of the TAC arrays is that total nucleic acids from one clinical specimen can be combined with PCR mix, loaded once into a TAC port, and separated by microfluidics into 48 separate reactions capable of amplifying either DNA or RNA (6). Thus, we attempted to engineer a positive control that can be loaded into one port on every card and used as a template for all assays in the panel.The design of our synthetic combined positive control originated with a compilation of ideas used to make positive-control templates for various PCR assays, including using a series of genomic regions cloned into multiple plasmids as a multiplex control (7), synthetic oligonucleotide templates (10), and runoff RNA transcript templates (9). Some alternative positive-control designs utilized a separate fluorescent channel to monitor amplification efficiency and inhibition of PCR (2), but due to the nature of TAC cards, we were able to use the same channel and the exact primer and probe sequences to generate the positive control for all assays. The advantage of this CPC is its ability to monitor reagent quality, amplification efficiency, and inhibition, as well as monitoring homogeneity of assays over time and location. In addition, this design can be applied to both viral and bacterial targets. We evaluated our CPC design for flexibility to address any observed inconsistency in amplification, potential interference between oligonucleotide sets, theoretical RNA folding, specificity, and stability.The first step in the design of the CPC system for real-time reverse transcription-PCR (rtRT-PCR) involved engineering a series of long synthetic oligonucleotides. For both DNA and RNA targets, we combined a forward primer sequence, followed by the probe sequence, and the reverse complement of the reverse primer sequence into an artificial template for each assay (see Fig. S1 in the supplemental material). The final construct was inserted into a plasmid....

Optimization of Multiple Pathogen Detection Using the TaqMan Array Card: Application for a Population-Based Study of Neonatal Infection

“…In our experience, simply combining DNA or RNA from each organism is not a reliable or feasible option for use as a positive control with the TAC method. To solve this problem, our laboratory previously reported design and performance of a plasmid that can be used to generate RNA transcripts to serve as a positive control for all RT-qPCR reactions on the TAC [17]. A similar solution may be applied to other multiple pathogen detection assays.…”

“…All TACs were run on the Applied Biosystems ViiA7 Real-Time PCR system (Life Technologies, Foster City, CA, USA) using the same cycling conditions as used for individual RT-qPCR reactions. A no template control (NTC) and a positive control consisting of combined RNA transcripts generated as previously described [17] were included on each TAC.…”

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

“…1). TACs and associated positive control materials (Kodani and Winchell, 2012) have been designed for the detection of respiratory pathogens (Kodani et al, 2011;Waller et al, 2014;Steensels et al, 2015), hepatitis genomes , enteropathogens (Liu et al, , 2013Platts-Mills et al, 2014), and bioterrorism agents Rachwal et al, 2012). Assays are independent of each other, so that modification of one primer/probe set will not require revalidation of the entire TAC.…”

Low-Density TaqMan® Array Cards for the Detection of Pathogens

“…Known standards (such as viruses and diluted nucleic acid extracts), United Kingdom National External Quality Assessment (NEQAS) and Quality Control for Molecular Diagnostics (QCMD) (www.qcmd.org), external quality assurance panels, patient specimens derived from different sample types and engineered synthetic plasmid controls can be used for validation (Kodani & Winchell, 2012). Employing such a combination allows for optimal assay scrutiny, providing information on LOD, sensitivity, specificity and any reaction inhibitors from different sample types.…”