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Writer affiliations: Facilities for Illness Management and Prevention, Atlanta, Georgia, USA (B. Shu, M.Okay. Kirby, W.G. Davis, C. Warnes, J. Liddell, J. Liu, Okay.-H. Wu, N. Hassell, A.J. Benitez, M.M. Wilson, M.W. Keller, B.L. Rambo-Martin, Y. Camara, J. Winter, R.J. Kondor, B. Zhou, S. Spies, L.E. Rose, J.M. Winchell, B.M. Limbago, D.E. Wentworth, J.R. Barnes); Battelle Memorial Institute, Atlanta (W.G. Davis, J. Liddell); Leidos Inc, Atlanta (Y. Camara)
An outbreak of pneumonia of unknown etiology in Wuhan, China, was reported to the World Well being Group on December 31, 2019 (1). Researchers decided that the sickness, later referred to as coronavirus illness (COVID-19), was attributable to a beforehand unidentified betacoronavirus, extreme acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (2). SARS-CoV-2 quickly unfold around the globe, and on March 11, 2020, the World Well being Group declared a pandemic (3). By January 2021, SARS-CoV-2 had contaminated >96 million individuals and prompted >2 million deaths worldwide (4).
The excessive demand for molecular testing for SARS-CoV-2 has contributed to international shortages of diagnostic sources, together with reagents, enzymes utilized in reverse transcription PCR (RT-PCR), plastic consumables, and workers availability (5,6). Environment friendly diagnostic assessments can cut back pressure on the testing system and reduce turnaround time. To enhance testing effectivity, we developed the Facilities for Illness Management and Prevention (CDC) Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay, which is selective for influenza A and B viruses and SARS-CoV-2. This quadruplex real-time RT-PCR (rRT-PCR) concurrently detects and distinguishes RNA of influenza A virus, influenza B virus, and SARS-CoV-2 in higher and decrease respiratory specimens. To observe specimen high quality management, the assay additionally detects the Homo sapiens (human) RNase P (RP) gene. As a result of the Flu SC2 Multiplex Assay can take a look at 93 samples in a 96-well plate, this know-how improves the throughput of SARS-CoV-2 testing by 3-fold in contrast with the CDC 2019-nCoV Actual-Time RT-PCR Diagnostic Panel (7). The Flu SC2 Multiplex Assay additionally concurrently detects influenza A and B viruses, thereby decreasing the general pressure on testing services, particularly throughout influenza season. Continued testing and surveillance of influenza viruses in the course of the COVID-19 pandemic present essential steerage on collection of candidate vaccine strains; these processes additionally determine antiviral resistance genes and novel influenza viruses which have pandemic potential (8).
We evaluated present and novel SARS-CoV-2 primers and probes to determine the optimum SC2 assay parts for this quadruplex rRT-PCR (Appendix Table 1). The SC2 assay parts are selective for the three′ area of the SARS-CoV-2 genome from the carboxy terminus of the nucleocapsid (N) gene into the three′ untranslated area (UTR). The primer and probe sequences for the influenza A (InfA), influenza B (InfB), and RP targets are similar to these used within the singleplex assays of the US Meals and Drug Administration (FDA)–accredited CDC Human Influenza Virus Actual-Time RT-PCR Detection and Characterization Panel [510(k) no. K200370] (9). The Flu SC2 Multiplex Assay is selective for the matrix (M) gene section of the influenza A virus, the nonstructural (NS) gene section of the influenza B virus, and the human ribonuclease P/MRP subunit P30 gene; the InfA assay is designed for common detection of all influenza A viruses and InfB assay is designed for common detection of all influenza B viruses (10–14). The InfA assay was not too long ago up to date to handle evolutionary modifications and reactivity challenges; the up to date CDC Human Influenza Virus Actual-Time RT-PCR Diagnostic Panel was cleared by FDA in 2020 (9). On July 2, 2020, FDA granted an emergency use authorization (EUA) for in vitro diagnostic use of the Flu SC2 Multiplex Assay (15).
Multiplex detection of RNA from influenza A virus, influenza B virus, and SARS-CoV-2 can enhance testing capability and cut back use of reagents. The elevated throughput can protect workers sources and cut back turnaround time. The Flu SC2 Multiplex Assay and related panels determine co-infections or various causes of influenza-like and COVID-19–like diseases. The Flu SC2 Multiplex Assay can allow assortment of essential information on influenza A and B viruses and SARS-CoV-2, in addition to the prevalence of co-infection amongst these respiratory viruses.
Influenza Viruses and SARS-CoV-2
Influenza viruses had been grown to excessive titer in Madin-Darby Canine Kidney cells or embryonated hen eggs. Infectious virus titer within the cell tradition supernatant or allantoic fluid was measured through the use of 50% tissue tradition infectious dose (TCID50) or 50% egg infectious dose (EID50) (16). The SARS-CoV-2 virus (2019-nCoV/USA-WA1/2020; GenBank accession no. MT576563) was grown to excessive titer in Vero cells; the infectious virus titer within the cell tradition supernatant was measured through the use of TCID50 (16). Whole nucleic acids had been extracted through the use of the EZ1 DSP Virus Equipment on the EZ1 Superior XL automated extractor (QIAGEN, https://www.qiagen.com).
Primers and Probes
Primers and probes had been chosen from extremely conserved areas of the SARS-CoV-2 genome based mostly on ≈4,000 sequences accessible in GISAID (https://www.gisaid.org) in March 2020 (Table 1). Primer Specific 3.0.1 software program (Thermo Fisher Scientific, https://www.thermofisher.com) was used to design primers that had annealing temperatures of ≈60°C and probes that had annealing temperatures of ≈68°C.
The multiplex assay probes had been synthesized through the use of ZEN or TAO Double-Quenched Probes labeled on the 5′ finish utilizing reporter 6-carboxyfluorescein (FAM) for InfA, Yakima Yellow for InfB, Texas Purple-XN for SARS-CoV-2, and Cyanine 5 (Cy 5) for RP targets (Built-in DNA Applied sciences, Inc., https://www.idtdna.com). The InfA and InfB probes had been quenched with ZEN between nucleotides 9 and 10 and with Iowa Black FQ on the 3′ finish; the SARS-CoV-2 and RP probes had been quenched with TAO between nucleotides 9 and 10 and with Iowa Black RQ on the 3′ finish (Built-in DNA Applied sciences, Inc.). Primers and Taqman hydrolysis probes had been synthesized by Built-in DNA Applied sciences and the CDC Biotechnology Core Facility Department (Division of Scientific Assets, Nationwide Heart for Rising and Zoonotic Infectious Ailments; Atlanta, Georgia, USA).
rRT-PCR Response Circumstances
The rRT-PCR reactions of the Flu SC2 Multiplex Assay had been optimized and carried out through the use of the TaqPath 1-Step Multiplex Grasp Combine (No Rox) (Thermo Fisher Scientific) and the 7500 Quick Dx Actual-Time PCR Instrument (Thermo Fisher Scientific). The ultimate quantity of 25 μL included 6.25 μL of TaqPath 1-Step Multiplex Grasp Combine (No Rox) and 5 μL RNA. We used last concentrations of 400 nmol for the InfA F1 and F2 primers, 600 nmol for the InfA R1 primer, and 200 nmol for the InfA R2 primer; all different primers had last concentrations of 800 nmol. Probes had a last focus of 200 nmol. Response circumstances for the multiplex rRT-PCR had been based mostly on circumstances for the CDC rRT-PCR Flu Panel, however we decreased the reverse transcription step from 30 min to fifteen min (9,17). We used the next thermocycling circumstances for rRT-PCR: 25°C for two min, 50°C for 15 min, Taq activation at 95°C for two min, 45 cycles at 95°C for 15 sec, and 55°C for 30 sec. We carried out comparator reactions utilizing influenza singleplex rRT-PCR and the CDC 2019-nCoV Actual-Time RT-PCR Diagnostic Panel, as described beforehand (7,10,17).
Analytical Sensitivity and Specificity
A quantified artificial RNA materials (Armored RNA Quant CDC-9; Asuragen, Inc., https://asuragen.com) was used to check analytical sensitivity. The artificial RNA included primer–probe area sequences derived from the M gene of A/Brisbane/02/2018_(H1N1)pdm09 (GISAID accession no. EPI1799928), for the InfA goal, the NS gene of B/Colorado/06/2017_Victoria (GISAID accession no. EPI1056634) for the InfB goal, and the Homo sapiens (human) ribonuclease P/MRP subunit P30 gene for the RP goal. We used RNA extracted from propagated, A/Illinois/20/2018_(H1N1)pdm09 (GISAID accession no. EPI1220313; GenBank accession no. MH359945), and B/Colorado/06/2017_Victoria viruses to check the analytical sensitivity of the InfA and InfB targets. We used Twist Artificial SARS-CoV-2 RNA Management 2 (Twist Bioscience, https://www.twistbioscience.com) and RNA extracted from propagated SARS-CoV-2 virus (2019-nCoV/USA-WA1/2020) to evaluate analytical sensitivity of the SARS-CoV-2 goal.
We evaluated assay specificity through the use of a panel of influenza A virus, influenza B virus, and SARS-CoV-2. This panel included influenza A(H1/H3) variant viruses that normally flow into amongst swine and have prompted outbreaks and pandemics in human populations (18–22).
We used a group of influenza C viruses, coronaviruses, and human noninfluenza respiratory pathogens to check the analytical specificity of the Flu SC2 Multiplex Assay. We additionally examined the specificity of the SARS-CoV-2 goal with an RNA transcript generated from a clone representing nt 27768–29738 of the extreme acute respiratory syndrome coronavirus (SARS-CoV)/Urbani genome, which accommodates the complete N gene by way of the three′-terminus, and a full SARS-CoV viral genome.
To check sensitivity to co-infection, we created a serial dilution with nucleic acids extracted from A/Illinois/20/2018_(H1N1)pdm09, B/Colorado/06/2017_Victoria, 2019-nCoV/USA-WA1/2020, and adenocarcinomic human alveolar basal epithelial cells (A549). We examined the dilution by the Flu SC2 Multiplex Assay, influenza A and influenza B singleplex rRT-PCR from the CDC rRT-PCR Flu Dx Panel Influenza A/B Typing Equipment, and the N1 part of the CDC 2019-nCoV Actual-Time RT-PCR Diagnostic Panel.
In Silico Evaluation
We examined the specificity and sensitivity of every primer and probe oligonucleotide sequence for the SARS-CoV-2 goal of the Flu SC2 Multiplex Assay by BLAST evaluation (https://blast.ncbi.nlm.nih.gov/blast.cgi) in opposition to the nr/nt database and the Nationwide Heart for Biotechnology Iinformation and GISAID β Coronaviridae nucleotide database. We analyzed outcomes and assessed for potential non–SARS-CoV-2 matches (Appendix). We in contrast the primer and probe sequences with SARS-CoV-2 variant sequences accessible in GISAID on January 19, 2021, together with 501Y.V1, a B.1.1.7 variant from the UK; 501Y.V2, a B.1.351 variant from South Africa; and 501Y.V3, a P.1 variant from Brazil.
Assay Efficiency with Scientific Specimens
We evaluated the scientific efficiency of the Flu SC2 Multiplex Assay utilizing 104 higher and decrease respiratory specimens, together with oral swab, throat swab, nasopharyngeal swab, oropharyngeal swab, and sputum samples. Whole nucleic acids had been extracted from 120 μL of every scientific specimen through the use of the EZ1 DSP Virus Equipment on the EZ1 Superior XL automated extractor (QIAGEN). The extracted materials was eluted in 120 μL elution buffer. Specimens had been examined with the Flu SC2 Multiplex Assay, the CDC Human Influenza Actual-Time RT-PCR Diagnostic Panel: Influenza A/B Typing Equipment model 2 [510 (k) no. K200370], or the CDC 2019-nCoV Actual-Time RT-PCR Diagnostic Panel, as described beforehand (7,9).
Growing the SARS-CoV-2 Goal
We recognized candidate SARS-CoV-2 targets and evaluated them by an in silico screening course of. This course of recognized targets with only a few mismatches throughout the accessible SARS-CoV-2 genomes and accounted for RNA structural parts identified to be important for associated betacoronaviruses. In whole, we examined 17 SARS-CoV-2 assay designs in singleplex format; we subsequently examined a subset of those candidates utilizing the multiplex format, together with revealed targets within the RNA-dependent RNA polymerase and E gene areas (Appendix Desk 1) (23). We chosen for the assay the SARS-CoV-2 goal with the very best ranges of sensitivity and specificity and that precisely recognized residual scientific respiratory specimens.
The SARS-CoV-2 assay is selective for the three′ area of the SARS-CoV-2 genome from the carboxy terminus of the of the N gene into the three′-UTR (Appendix Determine). This area is expressed at excessive ranges in contaminated cells and is very conserved as a result of it encodes a cis-acting RNA pseudoknot important for the transcription and replication of intently associated betacoronaviruses (24).
Analytical Sensitivity of Flu SC2 Multiplex Assay
We decided the analytical sensitivity of the Flu SC2 Multiplex Assay by calculating the bounds of detection utilizing extracted RNA from influenza A virus, influenza B virus, and SARS-CoV-2. We used serial 10-fold dilutions of extracted RNA to determine an endpoint for detection with every primer and probe set included within the multiplex assay (information not proven). After a detection vary was established, we examined serial 5-fold dilutions of extracted RNA from every virus at titers close to the restrict of detection (LOD) with the Flu SC2 Multiplex Assay, the CDC 2019-nCoV Actual-Time RT-PCR Diagnostic Panel, or the CDC rRT-PCR Flu Dx Panel: Influenza A/B Typing Equipment model 2 [510 (k) no. K200370] (Table 2). We decided the bounds of detection to be 102.0 TCID50 for influenza A, 102.2 EID50 for influenza B, and 100.3 TCID50 for SARS-CoV-2 (Table 2). These values correspond to 10–0.3 TCID50 for every influenza A response, 10–0.1 EID50 for influenza B, and 10–2.0 TCID50 for SARS-CoV-2 (i.e., 5 µl RNA/response). We confirmed the LOD by way of additional testing of 20 replicate viral isolates combined with A549 cells on the established LOD and at a 5-fold dilution step above the established LOD; this course of demonstrated that the multiplex assay can detect >95% of samples on the lowest detectable concentrations (Table 3; Appendix Desk 2). The SD throughout the 20-replicate experiment was very low, demonstrating the consistency of the multiplex outcomes even on the LOD (Table 3).
We used an engineered RNA assemble (Armored RNA Quant CDC-9; Asuragen, Inc.) containing the goal sequences for the InfA, InfB, and RP assays to check the copy quantity sensitivity of the multiplex assay by way of serial dilutions. We assessed copy quantity sensitivity of the SARS-CoV-2 assay through the use of a serial dilution of an artificial SARS-CoV-2 genome (GenBank accession no. MN908947.3; Twist Bioscience). All targets within the assay may detect as few as 5 RNA copies per response (Table 4).
Analytical Specificity of Flu SC2 Multiplex Assay
Initially, the Flu SC2 Multiplex Assay was screened utilizing no template management reactions; we discovered no intramolecular or intermolecular nonspecific interactions that resulted in any merchandise (information not proven). The specificity of the primers and probes was evaluated with viral RNA from 13 influenza A, 2 influenza B, and 1 SARS-CoV-2 isolate. The viral RNAs had been examined at excessive and low titers; every assay precisely detected the corresponding viral goal (Table 5). We noticed no cross-reactivity among the many 4 targets throughout the assays, nor did we observe any bleed-through fluorescence imaging from neighboring channels when testing the person assays (Table 5).
To verify that the SARS-CoV-2 assay was particular to that virus, we examined 6 identified human coronaviruses, 2 alphacoronaviruses, 2 group A betacoronaviruses, and a couple of group B betacoronavirus (i.e., SARS-CoV and Center East respiratory syndrome coronavirus [MERS-CoV]), in addition to an RNA transcript together with the complete SARS-CoV N gene area by way of the three′ UTR. No cross-reactivity was noticed, demonstrating the excessive specificity of the assay (Appendix Desk 3).
To additional consider the specificity of the multiplex assay, we additionally examined widespread respiratory pathogens and genetic close to neighbors of viruses chosen for by the assay. Nucleic acids from excessive titer viral preparations had been extracted and examined with the Flu SC2 Multiplex Assay; no cross-reactivity was noticed (Appendix Desk 4).
An intensive in silico BLAST evaluation of the primer and probe sequences for the SARS-CoV-2 goal confirmed that the assay is restricted to SARS-CoV-2; no proof of non–SARS-CoV-2 goal matches was discovered (Figure; Appendix Desk 5). These outcomes display that Flu SC2 Multiplex Assay is restricted to influenza A viruses, influenza B viruses, and SARS-CoV-2; it doesn’t detect different respiratory pathogens or shut kinfolk, together with SARS-CoV.
An in silico evaluation in contrast the genomes of the rising SARS-CoV-2 variants B.1.1.7, B.1.351, and P.1 with the sequence of the SARS-CoV-2 goal. This evaluation demonstrated that in January 2021, most (>99.5%) of the variant virus sequencing information was similar to the SARS-CoV-2 goal sequence; of the genomes that had <100% match, none besides 2 sequences displayed >1 mismatch for any area of the assay (Appendix Desk 6). Subsequently, the Flu SC2 Multiplex Assay ought to precisely detect the B.1.1.7, B.1.351, and P.1 SARS-CoV-2 variants.
Co-An infection Sensitivity of Flu SC2 Multiplex Assay
We evaluated the analytical sensitivity of the multiplex assay within the context of a mock co-infection state of affairs by testing a mix of nucleic acids extracted from influenza A, influenza B, SARS-CoV-2, and A549 cells with the Flu SC2 Multiplex Assay and the InfA, InfB, and N1 singleplex assays (7,9). The outcomes demonstrated that the multiplex assay can detect all 4 targets concurrently at comparable or greater sensitivity ranges than every singleplex comparator (Appendix Desk 7).
Efficiency on Scientific Specimens
We evaluated the scientific efficiency of the multiplex assay through the use of residual scientific respiratory specimens. Nucleic acids had been extracted from 104 potential and retrospective scientific specimens, together with 33 SARS-CoV-2–constructive, 30 influenza A–constructive, 30 influenza B–constructive, and 11 adverse residual scientific samples. The samples had been examined with the Flu SC2 Multiplex Assay; the outcomes had been in 100% settlement with the anticipated worth for every specimen (Table 6; Appendix Tables 8, 9).
SARS-CoV-2 emerged in December 2019 and shortly unfold, inflicting the COVID-19 pandemic. Because the SARS-CoV-2 an infection price elevated, the demand for viral diagnostic testing additionally elevated. The Flu SC2 Multiplex Assay will increase throughput and makes use of much less reagent than the CDC 2019-nCoV Actual-Time RT-PCR Diagnostic Panel, thus enhancing SARS-CoV-2 testing effectivity. The multiplex assay permits laboratories to concurrently take a look at for influenza viruses and SARS-CoV-2, an utility that’s particularly helpful as a result of influenza virus and SARS-CoV-2 infections trigger related indicators and signs (25,26). Though not described on this article, extra enzyme grasp combine combos, nucleic acid extraction platforms, and an alternate producer had been added to the assay EUA, additional enhancing its utility (15,27). CDC granted the appropriate of reference to all information submitted to the FDA for EUA authorization of the Flu SC2 Multiplex Assay. A number of industrial suppliers have leveraged the information to provide multiplex kits, together with the BioSearch Valuepanel (LGC BioSearch Applied sciences, https://www.biosearchtech.com), PrimeTime SARS-CoV-2/Flu Take a look at Built-in DNA Applied sciences, Inc., Accuplex (consists of assay for human respiratory syncytial virus) (SeraCare Life Sciences, Inc., https://www.seracare.com), BioRad Reliance (Bio-Rad Laboratories, Inc., https://www.bio-rad.com), and FLU SC2 RT-PCR (InGenuityD Diagnostics, https://ingenuityd.com).
The analytical sensitivity of the Flu SC2 Multiplex Assay was evaluated; every part was akin to the singleplex variations of every assay. The assay detects titers as little as 102.2–100.3 TCID50 or EID50 (or 10–2.0–10−0.1 TCID50 or EID50/response) of influenza A viruses, influenza B viruses, and SARS-CoV-2, or as few as 5 RNA copies/response. We noticed no cross-reactivity among the many targets, even at excessive viral titers; none with the opposite 6 identified human coronaviruses, together with SARS-CoV and MERS-CoV; and none with influenza C cultured viruses or different widespread noninfluenza respiratory pathogens (28). The Flu SC2 assays manufactured by CDC are evaluated to make sure that the LOD of every lot is comparable with the LOD established within the EUA. High quality assessments guarantee restricted variability: heaps which have a variance of >2 cycle thresholds from the EUA submission information in opposition to customary high quality management virus dilution collection are deemed unacceptable for distribution (information not proven). These requirements be certain that sensitivity and specificity are maintained by way of the manufacturing course of.
The SARS-CoV-2 goal utilized by the multiplex panel was chosen from a conserved and important area of the N gene (29). Analytical analysis and in silico evaluation demonstrated the goal is delicate and particular to SARS-CoV-2 and won’t detect different human coronaviruses, together with SARS-CoV and MERS-CoV. The in silico evaluation of 376,469 SARS-CoV-2 sequences accessible in GISAID in January 2021 indicated that >99.9% of the viruses have <1 mismatch inside a single primer or probe of the SARS-CoV-2 assay (Appendix Desk 5). An in silico evaluation of genomes from the rising SARS-CoV-2 B.1.1.7, B.1.351, and P.1 variants demonstrated that the goal is similar to the genome sequence for >99.5% of those variant genomes (Appendix Desk 6). The Flu SC2 Multiplex Assay ought to detect these rising variants as a result of the mutations related to these variants are positioned inside a special area of the genome than the goal.
The Flu SC2 Multiplex Assay was evaluated utilizing a reference panel developed by the FDA for assessing diagnostic nucleic acid amplification assessments for SARS-CoV-2 (30,31). The panel consisted of reference SARS-CoV-2 materials, blinded samples, and a protocol offered by the FDA. The analysis included vary discovering and confirmatory research for LOD, in addition to blinded pattern testing to determine specificity and additional affirmation of the LODs. The LOD of the Flu SC2 Multiplex Assay utilizing the FDA panel was 5.7 × 103 nucleic acid amplification take a look at detectable models/mL, with no observable cross-reactivity with MERS-CoV (32).
In abstract, the Flu SC2 Multiplex Assay demonstrates a excessive degree of specificity and sensitivity. In a single response, it may possibly detect and distinguish 3 main respiratory viruses in addition to the human high quality management goal, thereby growing the testing throughput. Further benefits of the Flu SC2 Multiplex Assay embrace fewer freeze-thaw cycles, decreased potential for contamination by way of a discount within the variety of reactions, and fewer alternatives for pipetting errors. With this multiplex assay, customers can quickly take a look at massive quantities of samples. Though the influenza season for 2020–21 had traditionally few circumstances, this assay will probably be helpful in upcoming influenza seasons when influenza may co-circulate with SARS-CoV-2.
Drs. Shu and Kirby are biologists with the Genomics and Diagnostics Crew within the Influenza Division, Nationwide Heart for Immunization and Respiratory Ailments, Facilities for Illness Management and Prevention, Atlanta, Georgia, USA. Their analysis pursuits embrace detection of influenza viruses and extreme acute respiratory syndrome coronavirus 2.
We acknowledge and thank the Laboratory and Testing Process Pressure group, together with Kevin Karem, Wendi Kuhnert-Tallman, Jeff Johnson, Michele Owen, Christopher Elkins, Victoria Olson, Alison Laufer Halpin, and Sean Courtney, for steerage, sources, and suggestions in the course of the improvement of the Flu SC2 Multiplex Assay. We additionally acknowledge and thank the CDC Division of Scientific Assets, Nationwide Heart for Rising and Zoonotic Infectious Ailments, for his or her help with primer and probe synthesis in the course of the improvement part of the assay. Particular person exterior consultants had been consulted to evaluate the design and strategy of the Flu SC2 Multiplex Assay; we acknowledge and thank Tom Slezak, Timothy Minogue, and Pardis Sabeti for his or her time and suggestions. We want to acknowledge and thank the general public well being laboratories that carried out preliminary testing and suggestions of the Flu SC2 Multiplex Assay, together with Matthew Charles, Leslie Chapman, Christopher Vogt, and Jamie Pierce of the Illinois Division of Public Well being Division of Laboratories; Peter Shult and Erik Reisdorf of the Wisconsin State Laboratory of Hygiene; Carol Loring of the New Hampshire Public Well being Laboratories; and Rene C. Hull and Kirsten St. George of the Wadsworth Heart, New York State Division of Well being. Lastly, we thank and acknowledge the authors, in addition to the originating and submitting laboratories, who submitted sequences to the GISAID database (https://www.gisaid.org).
Steered quotation for this text: Shu B, Kirby MKK, Davis WG, Warnes C, Liddell J, Liu J, et al. Multiplex real-time reverse transcription PCR for influenza A virus, influenza B virus, and extreme acute respiratory syndrome coronavirus 2. Emerg Infect Dis. 2021 July [date cited]. https://doi.org/10.3201/eid2707.210462
The conclusions, findings, and opinions expressed by authors contributing to this journal don’t essentially mirror the official place of the U.S. Division of Well being and Human Providers, the Public Well being Service, the Facilities for Illness Management and Prevention, or the authors’ affiliated establishments. Use of commerce names is for identification solely and doesn’t suggest endorsement by any of the teams named above.