Intended Use

The CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel is a real-time RT-PCR test intended for the qualitative detection of nucleic acid from the 2019-nCoV in upper and lower respiratory specimens (such as nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage, and nasopharyngeal wash/aspirate or nasal aspirate) collected from individuals who meet 2019-nCoV clinical and/or epidemiological criteria.

Results are for the identification of 2019-nCoV RNA. The 2019-nCoV RNA is generally detectable in upper and lower respiratory specimens during infection.

Positive results are indicative of active infection with 2019-nCoV but do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. Laboratories within the United States and its territories are required to report all positive results to the appropriate public health authorities.

Negative results do not preclude 2019-nCoV infection and should not be used as the sole basis for treatment or other patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.

Testing with the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel is intended for use by trained laboratory personnel who are proficient in performing real-time RT-PCR assays. The CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel is only for use under a Food and Drug Administration’s Emergency Use Authorization.

Milestones

• Available for use under the Emergency Use Authorization (EUA) as per FDA
• Detection of viral RNA not only aids in the diagnosis of illness put also provides epidemiological and surveillance information

Principle of the Procedure

The oligonucleotide primers and probes for detection of 2019-nCoV were selected from regions of the virus nucleocapsid (N) gene. The panel is designed for specific detection of the 2019-nCoV (two primer/probe sets). An additional primer/probe set to detect the human RNase P gene (RP) in control samples and clinical specimens is also included in the panel.

RNA isolated and purified from upper and lower respiratory specimens is reverse transcribed to cDNA and subsequently amplified in the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with SDS version 1.4 software. In the process, the probe anneals to a specific target sequence located between the forward and reverse primers. During the extension phase of the PCR cycle, the 5’ nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored at each PCR cycle by Applied Biosystems 7500 Fast Dx Real-Time PCR System with SDS version 1.4 software.

Detection of viral RNA not only aids in the diagnosis of illness but also provides epidemiological and
surveillance information.

Reagent and Controls Preparation

Primer and Probe Preparation
1) Upon receipt, store dried primers and probes at 2-8°C.
2) Precautions: These reagents should only be handled in a clean area and stored at appropriate temperatures (see below) in the dark. Freeze-thaw cycles should be avoided. Maintain cold when thawed.
3) Using aseptic technique, suspend dried reagents in 1.5 mL of nuclease-free water (50X working concentration) and allow to rehydrate for 15 min at room temperature in the dark.
4) Mix gently and aliquot primers/probe in 300 μL volumes into 5 pre-labeled tubes. Store a single aliquot of primers/probe at 2-8oC in the dark. Do not refreeze (stable for up to 4 months). Store remaining aliquots at ≤ -20oC in a non-frost-free freezer.

2019-nCoV Positive Control (nCoVPC) Preparation
1) Precautions: This reagent should be handled with caution in a dedicated nucleic acid handling area to prevent possible contamination. Freeze-thaw cycles should be avoided. Maintain on ice when thawed.
2) Resuspend dried reagent in each tube in 1 mL of nuclease-free water to achieve the proper concentration. Make single use aliquots (approximately 30 μL) and store at ≤ -70oC.
3) Thaw a single aliquot of diluted positive control for each experiment and hold on ice until adding to plate. Discard any unused portion of the aliquot.

Human Specimen Control (HSC) (not provided)
1) Human Specimen Control (HSC) or one of the listed acceptable alternative extraction controls must be extracted and processed with each specimen extraction run.
2) Refer to the Human Specimen Control (HSC) package insert for instructions for use.

No Template Control (NTC) (not provided)
1) Sterile, nuclease-free water
2) Aliquot in small volumes
3) Used to check for contamination during specimen extraction and/or plate set-up

Clinical Performance

As of February 22, 2020, CDC has tested 2071 respiratory specimens from persons under investigation (PUI) in the U.S. using the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. Specimen types include bronchial fluid/wash, buccal swab, nasal wash/aspirate, nasopharyngeal swab, nasopharyngeal/throat swab, oral swab, sputum, oropharyngeal (throat) swab, swab (unspecified), and throat swab.

Two thousand twenty-one (2021) respiratory specimens of the 2071 respiratory specimens tested negative by the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. Forty-nine (49) of the 2071 respiratory specimens tested positive by the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. Only one specimen (oropharyngeal (throat) swab) was invalid. Of the 49 respiratory specimens that tested positive by the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel, seventeen (17) were confirmed by genetic sequencing and/or virus culture (positive percent agreement = 17/17, 95% CI: 81.6%-100%)

During the early phase of the testing, a total of 117 respiratory specimens collected from 46 PUI subjects were also tested with two analytically validated real-time RT-PCR assays that target separate and independent regions of the nucleocapsid protein gene of the 2019-nCoV, N4 and N5 assays. The nucleocapsid protein gene targets for the N4 and N5 assays are different and independent from the nucleocapsid protein gene targets for the two RT-PCR assays included in the CDC 2019-nCoV Real-Time RT

PCR Diagnostic Panel, N1 and N2. Any positive result from the N4 and/or the N5 assay was further investigated by genetic sequencing.

Performance of the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel testing these 117 respiratory specimens was estimated against a composite comparator. A specimen was considered comparator negative if both the N4 and the N5 assays were negative. A specimen was considered comparator positive when the N4 and/or the N5 assay generated a positive result, and the comparator positive result(s) were further investigated and confirmed to be 2019-nCoV RNA positive by genetic sequencing.

Performance Characteristics

Limit of Detection (LoD)

LoD studies determine the lowest detectable concentration of 2019-nCoV at which approximately 95% of all (true positive) replicates test positive. The LoD was determined by limiting dilution studies using characterized samples.

The analytical sensitivity of the rRT-PCR assays contained in the CDC 2019 Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel were determined in Limit of Detection studies. Since no quantified virus isolates of the 2019-nCoV are currently available, assays designed for detection of the 2019-nCoV RNA were tested with characterized stocks of in vitro transcribed full length RNA (N gene; GenBank accession: MN908947.2) of known titer (RNA copies/µL) spiked into a diluent consisting of a suspension of human A549 cells and viral transport medium (VTM) to mimic clinical specimen. Samples were extracted using the QIAGEN EZ1 Advanced XL instrument and EZ1 DSP Virus Kit (Cat# 62724) and manually with the QIAGEN DSP Viral RNA Mini Kit (Cat# 61904). Real-Time RT-PCR assays were performed using the ThemoFisher Scientific TaqPath™ 1-Step RT-qPCR Master Mix, CG (Cat# A15299) on the Applied Biosystems™ 7500 Fast Dx RealTime PCR Instrument according to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel instructions for use.

A preliminary LoD for each assay was determined testing triplicate samples of RNA purified using each extraction method. The approximate LoD was identified by extracting and testing 10-fold serial dilutions of characterized stocks of in vitro transcribed full-length RNA. A confirmation of the LoD was determined using 3-fold serial dilution RNA samples with 20 extracted replicates. The LoD was determined as the lowest concentration where ≥ 95% (19/20) of the replicates were positive.