Abstract
The TaqMan Array Card architecture, normally used for gene expression studies, was evaluated for its potential to detect multiple bacterial agents by real-time PCR. Ten PCR assays targeting five biological agents (Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis) were incorporated onto Array Cards. A comparison of PCR performance of each PCR in Array Card and singleplex format was conducted using DNA extracted from pure bacterial cultures. When 100 fg of agent DNA was added to Array Card channels the following levels of agent detection (where at least one agent PCR replicate returned a positive result) were observed: Y. pestis 100%, B. mallei & F. tularensis 93%; B. anthracis 71%; B. pseudomallei 43%. For B. mallei & pseudomallei detection the BPM2 PCR, which detects both species, outperformed PCR assays specific to each organism indicating identification of the respective species would not be reproducible at the 100 fg level. Near 100% levels of detection were observed when 100 fg of DNA was added to each PCR in singleplex format with singleplex PCRs also returning sporadic positives at the 10 fg per PCR level. Before evaluating the use of Array Cards for the testing of environmental and clinical sample types, with potential levels of background DNA and PCR inhibitors, users would therefore have to accept a 10-fold reduction in sensitivity of PCR assays on the Array Card format, in order to benefit for the capacity to test multiple samples for multiple agents. A two PCR per agent strategy would allow the testing of 7 samples for the presence of 11 biological agents or 3 samples for 23 biological agents per card (with negative control channels).
Highlights
The Polymerase Chain Reaction (PCR) is commonly used to detect pathogens from various sample types [1,2]
With the total volume of the sample being 100 mL (50 mL DNA extract and 50 mL PCR mastermix) and the channel comprising 4861 mL reaction chambers not all the DNA in the sample would be analysed by a PCR assay
A given PCR chamber will not necessarily contain a PCR assay targeting the agent DNA that has been added to the channel
Summary
The Polymerase Chain Reaction (PCR) is commonly used to detect pathogens from various sample types [1,2]. To act as a screening capability, where one sample is analysed by a panel of PCR assays, PCRs must either be multiplexed where multiple reactions are carried out in a single tube [3,4], or an engineering solution such as microfluidics [5] or robotics [6] be found. Gene expression arrays, based on PCR, have been developed to analyse cDNA generated from an RNA template [7,8]. This cDNA is generally added to the array at the nanogram (ng) scale and individual PCR assays report on the expression of individual genes. Low-level, detection of a bacterial agent, with bacterial genomes known to have a weight at the single figure femtogram (fg) scale, would require detection of genetic material at levels some six orders of magnitude lower than that required for the analysis of gene expression
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