title: Validation of assembled nucleic acid-based tests in diagnostic microbiology laboratories.
abstract: Medical microbiology and virology laboratories use nucleic acid tests (NAT) to detect genomic material of infectious organisms in clinical samples. Laboratories choose to perform assembled (or in-house) NAT if commercial assays are not available or if assembled NAT are more economical or accurate. One reason commercial assays are more expensive is because extensive validation is necessary before the kit is marketed, as manufacturers must accept liability for the performance of their assays, assuming their instructions are followed. On the other hand, it is a particular laboratory's responsibility to validate an assembled NAT prior to using it for testing and reporting results on human samples. There are few published guidelines for the validation of assembled NAT. One procedure that laboratories can use to establish a validation process for an assay is detailed in this document. Before validating a method, laboratories must optimise it and then document the protocol. All instruments must be calibrated and maintained throughout the testing process. The validation process involves a series of steps including: (i) testing of dilution series of positive samples to determine the limits of detection of the assay and their linearity over concentrations to be measured in quantitative NAT; (ii) establishing the day-to-day variation of the assay's performance; (iii) evaluating the sensitivity and specificity of the assay as far as practicable, along with the extent of cross-reactivity with other genomic material; and (iv) assuring the quality of assembled assays using quality control procedures that monitor the performance of reagent batches before introducing new lots of reagent for testing.
authors.affiliation: National Serology Reference Laboratory, Fitzroy, Vic., Australia. wayne@nrl.gov.au
authors.forename: Wayne
authors.initials: W
authors.lastname: Dimech
authors.forename: D Scott
authors.initials: DS
authors.lastname: Bowden
authors.forename: Brian
authors.initials: B
authors.lastname: Brestovac
authors.forename: Keith
authors.initials: K
authors.lastname: Byron
authors.forename: Greg
authors.initials: G
authors.lastname: James
authors.forename: Darren
authors.initials: D
authors.lastname: Jardine
authors.forename: Theo
authors.initials: T
authors.lastname: Sloots
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Dax
mesh_terms: D004269:DNA, Bacterial / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis*; D006801:Humans; D007755:Laboratories, Hospital / Q000592:standards*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000592:standards*; D016133:Polymerase Chain Reaction; D017410:Practice Guidelines as Topic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012986:Software Validation
chemical_list: D004269:DNA, Bacterial; D004279:DNA, Viral