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

Abstract

, 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/Regulationsand-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-responseefforts-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

Isothermal Recombinase Polymerase Amplification-lateral Flow Detection of SARS-CoV-2, the Etiological Agent of COVID-19

“…All positive tests had de nitive bands in the lateral ow strips with varying intensities compared with the positive control (11 positive representative tests, Fig 6 1-11), while the test band was absent in the negative samples (11 shown, Fig 6 12-22). The results of the SARS-CoV-2 RPA-LF were 100% concordant the CDC's nucleocapsid rRT-PCR for COVID-19 (10,11) with Ct values ranging from 19-37 (Table1).…”

“…To date, the RPA-LF assays for cutaneous and visceral leishmaniasis (5,6), intestinal protozoa (Cryptosporidium and Giardia) (7), Entamoeba histolytica (8) and Trypanosoma cruzi (9), have shown similar sensitivity and speci city as the respective real-time or conventional PCR assays. nCoV rRT-PCR Panel (10,11). This assay does not require expensive real-time PCR equipment and would allow for testing of suspected COVID19 samples at rural or decentralized laboratories, reducing the time to diagnosis and potential treatment.…”

Clinical sensitivity and interpretation of PCR and serological COVID‐19 diagnostics for patients presenting to the hospital

“…PCR testing using consensus primers has an estimated specificity of >99%. 15 Based on early reports from Wuhan 16 the overall clinical sensitivity is reported around 70%. We found a clinical sensitivity around 95% in the first 5 days after symptom onset and although PCR is an imperfect standard, concurrent IgM/IgA/IgG antibody assessment in the first 5 days post symptom onset does not significantly aid in rendering a current diagnosis; at no point during active infection should serology replace PCR for diagnosis.…”

Overcoming Limitations in the Availability of Swabs Systems Used for SARS-CoV-2 Laboratory Diagnostics

“…Once collected, respiratory specimens are most commonly analyzed in diagnostic laboratories by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA 8,9 . Some RT-PCR tests are developed in-house, often following recommendations of reference laboratories and o cial institutions [10][11][12] . Other tests are commercially available and are in research use only (RUO) status or certi ed as invitro diagnostics (IVD) [13][14][15][16] .…”