Abstract
The University of Louisville Infectious Diseases Laboratory followed the US Food and Drug Administration (FDA) Emergency Use Authorization (EUA) guidance for developing a molecular diagnostic test for SARS CoV-2 to help address the novel coronavirus pandemic. As a Clinical Laboratory Improvement Amendment ‘88 (CLIA) certified, high-complexity clinical laboratory, the Infectious Diseases Laboratory chose to use the Luminex ARIES® platform to evaluate a laboratory developed test. This instrument was already familiar to the Infectious Diseases Laboratory and in use for molecular diagnostic testing for pathogens causing atypical pneumonia and two tick-borne pathogens. The FDA EUA guidance for molecular diagnostic tests recommended limit of detection studies, inclusivity and exclusivity (specificity) analysis, and validation with clinical samples to ensure the performance of the assay was acceptable for use as a molecular diagnostic tool. Data obtained from these experiments demonstrated acceptable performance per FDA guidance, as well as for CLIA requirements. Thus, the real-time Reverse Transcription PCR assay was implemented for diagnostic use on March 27, 2020 and was a great benefit to the local community in responding to the pandemic.
Highlights
Coronaviruses are a large family of single-stranded RNA viruses
Middle East Respiratory Syndrome virus (MERS) and Severe Acute Respiratory Syndrome virus (SARS) are two recent examples of novel coronaviruses that originated in animals and spread to humans.[1]
To meet local testing needs related to the pandemic, the Infectious Diseases Laboratory partnered with Luminex Corporation to validate a SARS-CoV-2 real-time Reverse Transcription PCR assay (RT-PCR) using a slight modification of the CDC primer and probe sets targeting the N1 and N3 nucleocapsid genes, as well as the human RNase P gene, to create a multiplex molecular diagnostic assay
Summary
Coronaviruses are a large family of single-stranded RNA viruses. Middle East Respiratory Syndrome virus (MERS) and Severe Acute Respiratory Syndrome virus (SARS) are two recent examples of novel coronaviruses that originated in animals and spread to humans.[1]. The US Food and Drug Administration’s (FDA) Emergency Use Authorization (EUA) guidance for validation of molecular assays for SARS-CoV-2, published originally on February 29, 2020, allowed highcomplexity, CLIA-certified laboratories to validate molecular assays and submit their data to the FDA for review. As more clinical materials positive for SARS-CoV-2 became available in the course of the pandemic, the FDA updated the EUA guidance (July 2020), requesting that laboratories use true clinical samples rather than contrived samples to supplement the initial data packet. The University of Louisville Infectious Diseases Laboratory is a CLIA-certified, high-complexity laboratory with previous experience using laboratory-developed tests on the Luminex ARIES® system.[3,4,5,6] To meet local testing needs related to the pandemic, the Infectious Diseases Laboratory partnered with Luminex Corporation to validate a SARS-CoV-2 real-time Reverse Transcription PCR assay (RT-PCR) using a slight modification of the CDC primer and probe sets targeting the N1 and N3 nucleocapsid genes, as well as the human RNase P gene, to create a multiplex molecular diagnostic assay. Data was compiled and published, along with that of three other laboratories using the Luminex ARIES® instrument, in a white paper in March 2020.[7]
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