Closeness of agreement between a quantity value obtained by measurement and the true value of the measured . This is essentially the rate of the assay giving the correct result. Assays with high accuracy have low occurrence of both false positives and false negatives
PCR assay design
An in silico process to select the assay components such as primers and probe.
Refers to the entire process from target identification/selection, assay design, validation, optimization, matrix testing, preparation of the packages
Any improvement (in silico or wet lab testing or both) of existing assays in response to a variety of outcomes from initial testing or field testing. E.g., signature erosion, failure of assays in certain conditions or specific matrices, or in multiplex formats.
In silico (IS) optimization of parameters
Assay optimization (Wet lab)
Refers to wet lab testing of the designs from the in silico process under different conditions of PCR (concentrations of components, temperature, etc)
An overall assessment of the assay including sensitivity, specificity, and LOD of an assay in a particular sample matrix.
Assay stewardship (assay performance monitoring)
In silico validation of assay performance with the availability of new genomic sequences over time.
A comprehensive analysis of the test system (i.e. the sample to answer process including steps such as extraction, testing, and analysis) to define the assay performance characteristics in the intended matrix. The test system is only validated for use in that specific matrix.
A panel of organisms found in the typical matrix of the sample (e.g., human genome or human microbiome for human samples, soil microbes for environmental samples, etc.)
The difference between the expectation of the test result or measurement result and the true value.
The non-target agents, which are potentially cross-reactive, but are not expected to be detected by the method.
A panel of near neighbors that are expected to be negative for the assay. Exceptions (i.e., false positives) are expected and need to be tested.
The strains or isolates or variants of the target agent(s) that the method can detect
A panel of strains of the intended PCR assay target organism to include members that represent the organism’s entire genetic diversity. Ideally, the assay is expected to be positive (i.e. sensitive) for all the panel strains. Exceptions (i.e., false negatives) are expected and need to be tested.
Limit of detection (LOD)
The lowest concentration or mass of analyte in a test sample that can be distinguished from a true blank sample at a specified probability level.
Limit of quantitation (LOQ)
The lowest level of analyte in a test sample that can be reasonably quantified at a specified level of precision.
Totality of components of a material system except the analyte.
Testing the final assay in specific matrices relevant to the end user; e.g., clinically relevant matrices such as blood, sputum, etc. or environmentally relevant matrices such as soil.
Alternate term for background panel.
Organisms and/or substances selected to be either closely related or potentially cross-reactive with the organism and/or substance under test. These are targets that are likely to give a false positive for a given assay.
Probability of detection (POD)
The proportion of positive analytical outcomes for a qualitative method for a given matrix at a given agent level or concentration. POD is concentration dependent.
The true positive rate, which is the fraction of actual positive samples that the assay returns a positive result. This is also called the “probability of detection.” Assays with high sensitivity have a low occurrence of false negatives. Sensitivity should not be confused with the limit of detection. The equation for sensitivity is given by:
where TP = true positives and FN = false negatives.
One or more oligonucleotide sequences (i.e. forward primer and/or reverse primer and/or probe) that experimentally detects (e.g., by PCR or probe-based methods) most or all of the desired target organism sequences (i.e. inclusivity panel) and does not detect most or all non-target sequences (exclusivity panel, near neighbors, and environmental panel). This definition deviates from the traditional definition of a signature as a single oligonucleotide sequence that is “present” in all targets and “absent” in all non-targets. This traditional definition is problematic due to the various definitions of “presence” and “absence” depending on the threshold parameters set for example,
1) Edit distance
2) BLAST alignment score
3) Melting temperature or delta G
4) Impact of 3’ terminal primer mismatches.
Emergence of mutations in the sequences of the signature (primers and probes)that may lead to assay failure. This may happen via the natural course of evolution, especially in viruses, due to genetic drift or shift or deliberate acts.
The true negative rate is the fraction of tests that are true negative divided by the actual number of negative samples (i.e., true negative plus false positive). Assays with high specificity have a low occurrence of false positives. The equation for specificity is given by:
where TN = true negatives and FP = false positives.
Entails identifying suitable “unique regions” in the genome of interest for assay design. These regions may be longer than the actual PCR amplicon.
A quantity of subsample or member of a sample set that is taken for analysis by the method.