Abstract
Quantitative PCR diagnostic platforms are moving towards increased sample throughput, with instruments capable of carrying out thousands of reactions at once already in use. The need for a computational tool to reliably assist in the validation of the results is therefore compelling. In the present study, 328 residual clinical samples provided by the Public Health England at Addenbrooke's Hospital (Cambridge, UK) were processed by TaqMan Array Card assay, generating 15 744 reactions from 54 targets. The amplification data were analysed by the conventional cycle-threshold (CT) method and an improvement of the maxRatio (MR) algorithm developed to filter out the reactions with irregular amplification profiles. The reactions were also independently validated by three raters and a consensus was generated from their classification. The inter-rater agreement by Fleiss' kappa was 0.885; the agreement between either CT or MR with the raters gave Fleiss' kappa 0.884 and 0.902, respectively. Based on the consensus classification, the CT and MR methods achieved an assay accuracy of 0.979 and 0.987, respectively. These results suggested that the assumption-free MR algorithm was more reliable than the CT method, with clear advantages for the diagnostic settings.
Highlights
Quantitative polymerase chain reaction has become the standard assay for many diagnostic and research procedures
The clinical samples were obtained between March and May 2015 from residual faecal specimens processed by the Public Health England (PHE) Clinical Microbiology and Public Health Laboratory at Addenbrooke's Hospital (Cambridge, UK)
Quantitative PCR platforms are evolving for higher throughput: for instance, Roche Diagnostic has developed a 1536 well format platform (LightCycler 1536 Instrument) [30] and ThermoFisher Scientific is introducing a 3072 well format for the TaqMan Array Cards (TAC) assay (OpenArray) [31]; a PCR system allocating 300 000 simultaneous reactions has been presented [32]
Summary
Quantitative polymerase chain reaction (qPCR) has become the standard assay for many diagnostic and research procedures. The specific roles of these authors are articulated in the ‘author contributions’ section
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