The earliest batch of COVID-19 tests distributed by the U.S. Centers for Disease Control and Prevention (CDC) showed a false positive reactivity of negative controls due to errors in assay design and contamination in one of the assays Components, according to a CDC internal research published this week in the open access journal PLOS ONE.
The full SARS-CoV-2 genome sequence of a patient in Wuhan, China was released on January 12, 2020 and CDC scientists began rapidly developing a real-time RT-PCR diagnostic panel to detect the novel coronavirus. The first RT-PCR panel, distributed to US state health laboratories starting February 5, 2020, was designed to identify three unique spots, or “loci”, on the SARS-CoV-2 genome. Within a few days of the distribution, CDC received reports from several laboratories of false positive reactivity in the negative controls generated by two of the three probes known as N1 and N3. This resulted in changes to the panel, including the removal of the N3 probes.
In the new study, CDC researchers reviewed the design, validation, manufacture and distribution of the diagnostic panel. The team concluded that one source of the negative control’s false positive reactivity was contamination of the N1 component of the RT-PCR kits by a synthetic piece of genetic material. N1 contamination was detected in the initial production batch of kits distributed, resulting in approximately 2% false positive results, but the pre-validation material was not contaminated (0% false positive results). The researchers therefore found that the N1 contamination, which has now been resolved, occurred during quality control after production or while the kits were being packaged.
Meanwhile, the false positive reactivity of the negative control of the N3 probe was due to a design flaw, the scientists report. Two molecules in the N3 probe often bind to each other in the absence of a virus and trigger the fluorescence that signals a positive reaction. This false-positive reactivity of the negative control increased with the age of the RT-PCR test kits, which could explain why early evaluation runs with newly prepared materials did not show high false-positive values.
The team says that this problem with the false positive reactivity of the negative control has since led to improvements in quality control, quality assurance, and assay validation for RT-PCR and other diagnostic tests at the CDC. These changes include additional steps of review and approval by experts independent of the diagnostic panel design team, as well as piloting the diagnostic panels with public health labs to confirm functionality and usability.
For more information, see the CDC website on Laboratory Quality Improvement Activities (https://www.cdc.gov/labs/quality-activities.html).
Lee, JS, et al. (2021) Analysis of the first batch of CDC 2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR diagnostic panel. Plus one. doi.org/10.1371/journal.pone.0260487.