Comparison of electronic microarray to enzyme hybridization assay for multiplex reverse-transcriptase PCR detection of common respiratory viruses in children

Abstract

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Detection of 11 Common Viral and Bacterial Pathogens Causing Community-Acquired Pneumonia or Sepsis in Asymptomatic Patients by Using a Multiplex Reverse Transcription-PCR Assay with Manual (Enzyme Hybridization) or Automated (Electronic Microarray) Detection

“…Understanding this discrepancy between in silico and real-life coverage rates is important for clinical diagnostics and is addressed in one of our ongoing research programs. Both the RT-PCR-EHA and the RT-PCReMA assay were found to have good LODs for all the pathogens, with the LODs of the EHA being equal to (for influenza virus B, RSV B, M. pneumoniae, and C. pneumoniae) or lower than (for influenza virus B, RSV A, S. aureus, S. pneumoniae, L. pneumophila, L. micdadei, and B. pertussis) those of the eMA, consistent with the data previously reported for the respiratory-virus assay and the Hexaplex (17). No inhibition of amplification from clinical specimens spiked with control organisms was observed.…”

“…However, in clinical testing, the EHA detection platform gave 7 false-positive results while the eMA gave 1 false positive out of 3,573 true negatives. The one false-positive eMA result in our testing is the first we have found using this automated detection platform (17). The sample in question was a skin specimen testing as positive for S. aureus by the eMA but as negative in both the initial and subsequent EHA runs, as well as in an Agilent assay run performed with original amplicons from the EHA and eMA and, finally, in the repeat Agilent assay and EHA runs performed with fresh DNA obtained by singleplex S. aureus PCR amplification of the originally extracted NA.…”

“…PCR amplification was carried out with 50-l reaction volumes as described previously (17). PCR mixtures contained 30 l of cDNA input and concentrations of primers, MgCl 2 , and deoxynucleoside triphosphates equivalent to those in the multiplex PCR EHA.…”

“…If the repeat EHA testing resulted in an OD reading of Ն0.4, the specimen was counted as a true positive. Previously described work has demonstrated that the EHA detection method is more sensitive than the eMA (17). If the OD was 0.3 to 0.4 (indeterminate), the specimen was further analyzed as described below.…”

Rapid Multiplex Reverse Transcription-PCR Typing of Influenza A and B Virus, and Subtyping of Influenza A Virus into H1, 2, 3, 5, 7, 9, N1 (Human), N1 (Animal), N2, and N7, Including Typing of Novel Swine Origin Influenza A (H1N1) Virus, during the 2009 Outbreak in Milwaukee, Wisconsin

“…Unfortunately, the NC-400 platform is no longer made or supported. Also, the electronic microarray has been shown to decrease analytical sensitivity compared to that of an EHA (12). The third assay is the resequencing pathogen microarray (RPMFlu), which is designed to detect 86 bacterial and viral agents, including all 16 HA and 9 NA subtypes of avian influenza virus.…”