key: cord-0833261-d2lxztgk
authors: Rosebrock, Adam P
title: DNA cross-reactivity of the CDC-specified SARS-COV-2 specimen control leads to potential for false negatives and underreporting of viral infection
date: 2020-11-06
journal: Clin Chem
DOI: 10.1093/clinchem/hvaa284
sha: 8133b6fc8c50398491fcf13998b4364ee5a89b7b
doc_id: 833261
cord_uid: d2lxztgk

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Single-digit copies of genomic DNA are sufficient to generate a strong control signal using the CDC design (data not shown). DNA is copurified by solid phase and liquid-liquid extraction procedures used for isolation of RNA from clinical specimens. qPCR-only (no-RT) reanalysis of RNA samples extracted from COVID-19 case nasopharyngeal swabs yielded strong control signals from all specimens tested (data not shown). More worryingly, DNA cross-reactivity leads to analytical false negatives from true-positive patient samples where RNA has been degraded ( Figure 1 ).

Pooling multiple samples prior to analysis is being used to increase throughput and reduce testing cost (2) (3) (4) . The problems caused by a DNA-reactive control are magnified by pooling:

one RNase-containing sample can render an entire pool virus RNA negative, while a few cells worth of DNA from a single patient are sufficient to generate a specimen and extraction control signal. A DNA-reactive control opens the door to silent assay failures and false-negative reporting of COVID-19 positive individuals from whom virus was successfully collected and whose samples were intact prior to pooling with dominant negative samples.

The absence of viral signal is insufficient for clinical interpretation. Controls must demonstrate that the test worked as intended and would have found virus had it been present. The current goal of testing is not just to find needles in haystacks -it is to conclusively state that individual haystacks contained no needles.

This widely used design has high analytical sensitivity for detecting the SARS-CoV-2 virus, but incorrectly reports "assay success, no virus found" when faced with degraded specimens. A specimen and extraction control that specifically detects human RNA (Figure 1) 

Centers for Disease Control and Prevention. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel -Instructions for Use

Food and Drug Administration. Coronavirus (COVID-19) Update: Facilitating Diagnostic Test Availability for Asymptomatic Testing and Sample Pooling

Pooling of samples for testing for SARS-CoV-2 in asymptomatic people

Evaluation of COVID-19 RT-qPCR test in multi-sample pools