key: cord-269488-7fy6exsd authors: Zhen, Wei; Berry, Gregory J. title: Development of a New Multiplex Real Time RT-PCR Assay for SARS-CoV-2 Detection date: 2020-09-19 journal: J Mol Diagn DOI: 10.1016/j.jmoldx.2020.09.004 sha: doc_id: 269488 cord_uid: 7fy6exsd We describe the development of a new multiplex real time reverse transcription (RT)-PCR test for detection of SARS-CoV-2, with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) and a hydrolysis Taqman probe designed to specifically detect SARS-CoV-2. We then evaluated the limit of detection (LOD) and clinical performance of this new assay. A LOD study with inactivated virus exhibited equal performance to the modified CDC assay with a final LOD of 1,301 ± 13 genome equivalents/ml for the Northwell Health Laboratories laboratory developed test (NWHL LDT) vs. 1,249 ± 14 genome equivalents/ml for the modified CDC assay. Additionally, a clinical evaluation with 270 nasopharyngeal (NP) swab specimens exhibited 98.5% positive percent agreement and 99.3% negative percent agreement compared to the modified CDC assay. The NWHL LDT multiplex design allows testing of 91 patients per plate, versus a maximum of 29 patients per plate on the modified CDC assay, providing the benefit of testing significantly more patients per run and saving reagents, during a time when both of these parameters are critical. The results demonstrate that the NWHL LDT multiplex assay performs as well as the modified CDC assay, but is more efficient and cost effective and can be used as a diagnostic assay and for epidemiological surveillance and clinical management of SARS-CoV-2. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered as etiologic agent of Coronavirus Disease 2019 in the city of Wuhan, Hubei providence, China by the end of December of 2019 (1) , and is the seventh coronavirus known to infect humans and also to be transmitted from human to human. The four seasonal coronaviruses (HKU1, NL63, OC43 and 229E) are associated with mild symptoms, whereas SARS-CoV, MERS-CoV and SARS-CoV-2 can cause severe acute respiratory disease (2, 3) . SARS-CoV-2 belongs to the betacoronavirus genus and is an enveloped, singlestrand RNA virus with a ~29.8 kb genome, which can cause a wide range of clinical presentations from asymptomatic or mild illness to fatal outcomes (4, 5 J o u r n a l P r e -p r o o f Primers and probe design: Available whole genome sequence of SARS-CoV-2 (as of February 27, 2020) retrieved from the NCBI GenBank database and the Global Initiative on Sharing All Influenza Database (GISAID) were aligned using Clustal Omega software from EMBL-EBI. The designed primers and probe showed 100% similarity with targeted region of S gene of all SARS-CoV-2 genome sequences only ( Table 1 ). The primers and probe were designed using Primer Express 3.0 software in the S gene of SARS-CoV-2 and were synthesized by Integrated DNA Technologies, Inc. (IDT; Coralville, IA). In addition, the primers and probe of human RNase P gene used for the assay internal control were also synthesized by IDT and were the same sequences used in the CDC assay (v3) for this gene target (https://www.cdc.gov/coronavirus/2019-nCoV/lab/index.html. Accessed 13 July 2020). Here, the 5' base of probe of RNase P gene was modified and labeled with Cy5, and the 3' base of probe was labeled with Black Hole Quencher 2(BHQ2) to allow multiplexing of the assay. In order to monitor the extraction process, a negative extraction control was included in each extraction run, the RNase P target must be detected from this control. In this one-step, qualitative real time RT-PCR assay, TaqPath 1-step RT-qPCR kit (Catalog no. A15299, Thermo Fisher Scientific) was used to perform cDNA synthesis and PCR amplification on the 96well plate at a 20 µL final reaction volume. After PCR condition was optimized, the PCR reaction mix Results: Using a primer and probe design tool, the assay primers and probe specifically targeting S gene of SARS-CoV-2 (Table 1) Analytical sensitivity: The LOD of the NWHL LDT was 1301±13 GE/ml for the S gene target. For the modified CDC assay, the LOD was 1249±14 GE/mL for the N1 target and 946±11 GE/ml for the N2 target ( Table 2 ). The final LOD of modified CDC assay was 1249±14 GE/mL in accordance with the result interpretation algorithm. Analytical specificity: In silico analysis and Blastn analysis were performed against the standard and betacoronavirus database of National Center for Biotechnology Information (NCBI) and results showed no cross-reactivity with other respiratory pathogens; Clinical specimens positive respiratory pathogens were also tested to further evaluate the specificity of the NWHL LDT. According to the results shown in the modified CDC assay and the NWHL LDT assay were positive. In the present study, a multiplex real time RT-PCR assay was developed and validated for SARS-CoV-2 specific detection in NP specimens on the 7500 Fast Dx real time PCR instrument. The findings demonstrate that the NWHL LDT has comparable clinical performance for the specific detection of SARS-CoV-2 RNA in NP specimens and is more efficient and cost effective in comparison to the modified CDC assay. Since the modified CDC assay has been compared to multiple other commercially-available diagnostic assays and has equal or better performance, the performance of the NWHL LDT would also show equivalent performance (6) . The design also showed significant advantages over the modified CDC assay, since only one set of primer and probe Master Mix is required to prepare and dispense per specimen, in contrast to three sets of Master Mix preparation and the use of three wells for each patient specimen by the modified CDC assay. The multiplex design of the NWHL LDT allows testing of 91 patients per plate, versus a maximum of 29 patients per plate on the modified CDC assay. Overall, this allows laboratories to run more than 3 times as many patients per run and also adds to the ease of setting up each run. Additionally, the saving of hands-on time, reagents, and consumables is another advantage at a stage where there are currently global shortages of reagents and major assay supply chain issues. The design of the primers and probe for the NWHL LDT is based on multiple sequence alignments of all SARS-CoV-2 genome sequences that were available between January 11 and February 27 of 2020. Since RNA viruses are well known for their high mutation and recombination rates (9) Limitations of the current study include that the NWHL LDT is a single site evaluation at Northwell Health Laboratories and only a single target gene for SARS-CoV-2 detection was used. While there has been a trend toward dual-target design in commercial assays for the detection of this highly contagious pathogen (6, 10, 11) , occasional monitoring of SARS-CoV-2 sequences to verify that mutations have not developed in the region targeted by the NWHL LDT primers and probe is an adequate quality monitor to ensure continued consistent analytical performance. In addition, other targets could potentially be added to this assay in the future to further increase sensitivity and specificity. As an additional future plan, we could also potentially further improve the analytical sensitivity of the NWHL LDT by increasing the input volume and decreasing the elution volume during the nucleic acid extraction step. In summary, The NWHL LDT has comparable analytical sensitivity and accuracy for specific detection of SARS-CoV-2 RNA and also showed superior efficiency and cost-effectiveness when compared to the modified CDC assay. This assay can also be somewhat easily-established by other laboratories for diagnostic use. J o u r n a l P r e -p r o o f A pneumonia outbreak associated with a new coronavirus of probable bat origin Hosts and sources of endemic human coronavirus Covid-19 -Navigating the Uncharted Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China SARS-CoV-2 and COVID-19: The most important research questions Comparison of Four Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2 in Nasopharyngeal Specimens The measurement of observer agreement for categorical data Interrater reliability: the kappa statistic RNA virus mutations and fitness for survival Clinical Evaluation of Three Sample-To-Answer Platforms for the Detection of SARS-CoV-2 Comparing the analytical performance of three SARS-CoV-2 molecular diagnostic assays We would like to thanks Dr. Kirsten St. George and Daryl Lamson of the New York State Department of Health for providing the non-infectious human HEL cell line.