US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2

Xiaoyan Lu; Lijuan Wang; Senthilkumar K. Sakthivel; Brett Whitaker; Janna Murray; Shifaq Kamili; Brian Lynch; Lakshmi Malapati; Stephen A. Burke; Jennifer Harcourt; Azaibi Tamin; Natalie J. Thornburg; Julie M. Villanueva; Stephen Lindstrom

Disclosures

Emerging Infectious Diseases. 2020;26(8):1654-1665. 

In This Article

Abstract and Introduction

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiologic agent associated with coronavirus disease, which emerged in late 2019. In response, we developed a diagnostic panel consisting of 3 real-time reverse transcription PCR assays targeting the nucleocapsid gene and evaluated use of these assays for detecting SARS-CoV-2 infection. All assays demonstrated a linear dynamic range of 8 orders of magnitude and an analytical limit of detection of 5 copies/reaction of quantified RNA transcripts and 1 x 10−1.5 50% tissue culture infectious dose/mL of cell-cultured SARS-CoV-2. All assays performed comparably with nasopharyngeal and oropharyngeal secretions, serum, and fecal specimens spiked with cultured virus. We obtained no false-positive amplifications with other human coronaviruses or common respiratory pathogens. Results from all 3 assays were highly correlated during clinical specimen testing. On February 4, 2020, the Food and Drug Administration issued an Emergency Use Authorization to enable emergency use of this panel.

Introduction

On December 31, 2019, an outbreak of an unexplained acute respiratory disease, later designated coronavirus disease (COVID-19), was reported in Wuhan, China.[1] On January 7, 2020, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), previously known as 2019-nCoV, was identified as the causative agent of the outbreak.[2] On January 10, 2020, a SARS-CoV-2 genome sequence was shared with the global scientific community through an online resource.[3] The virus was genetically most closely related to SARS-CoV and SARS-related bat and civet coronaviruses within the family Betacoronavirus, subgenus Sarbecovirus.[4,5]

To support the potential public health emergency response to COVID-19, the Centers for Disease Control and Prevention (CDC) developed and validated a real-time reverse transcription PCR (rRT-PCR) panel based on this SARS-CoV-2 genome sequence.[3] The panel targeted the nucleocapsid protein (N) gene of SARS-CoV-2. The rRT-PCR panel was validated under the Clinical Laboratory Improvement Amendments (https://www.cms.gov/Regulations-and-Guidance/Legislation/CLIA) for CDC use for diagnosis of SARS-CoV-2 from respiratory clinical specimens. On January 20, 2020, the CDC rRT-PCR panel confirmed an early case of COVID-19 in the United States.[6] The US Food and Drug Administration issued an Emergency Use Authorization to enable emergency use of the CDC rRT-PCR panel as an in vitro diagnostic test for SARS-CoV-2 (https://www.fda.gov/news-events/press-announcements/fda-takes-significant-step-coronavirus-response-efforts-issues-emergency-use-authorization-first). From January 18 through February 27, as part of the COVID-19 response, CDC tested 2,923 specimens from 998 persons for SARS-CoV-2.

As of April 22, ≈2,400,000 confirmed COVID-19 cases and ≈169,000 associated deaths had been identified globally, including ≈770,000 cases and ≈37,000 deaths in the United States.[7] We describe the design and validation of the CDC rRT-PCR panel and present comprehensive data on its performance with multiple specimen types and clinical specimens tested during the early CDC COVID-19 response.

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