SPADA Draft Documents

“present” in all targets and “absent” in all non-targets. This traditional definition is problematic 935 due to the various definitions of “presence” and “absence”, i.e.: 1) Edit distance; 2) BLAST 936 alignment score; 3) Melting temperature or delta G; 4) Impact of 3’ terminal primer mismatches. 937 ( hh ) Signature erosion .—Emergence of mutations in the sequences of the assay target 938 regions that may lead to assay failure. This may happen due to the natural course of evolution, 939 especially in viruses, due to genetic drift or shift or deliberate acts. 940 ( ii ) Specificity .—The true negative rate, which is the fraction of actual negative samples that 941 the assay returns a negative result. Assays with high specificity have a low occurrence of false 942 positives. The equation for specificity is given by: 943

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where TN = true negatives and FP = false positives.

( jj ) Target Selection .—Entails identifying suitable “unique regions” in the genome of interest

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for assay design. This may be longer than the actual PCR amplicon.

( kk ) Test Portion .—A quantity of subsample or member of a sample set that is taken for

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analysis by the method.

( ll ) Test and evaluation (T&E) .—A process by which a system or components are tested and 949 results analyzed to provide performance related information. The results provide information to 950 identify risks, empirical data for validation, and assessment of technical performance, 951 specifications, system maturity, and suitability for intended use. 952 ( mm ) Validation .—The establishment of the performance characteristics of a method and 953 provision of objective evidence that the performance requirements for a specified intended use 954 are fulfilled (ISO 16140-1:2016).

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