key: cord-0929067-cb7cyxlo authors: Freire-Paspuel, Byron; Garcia-Bereguiain, Miguel Angel title: Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2 and RP targets for SARS-CoV-2 diagnosis date: 2020-10-25 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2020.10.047 sha: 20d58c959bf5a96264af0a91f65cc5b033fe7c06 doc_id: 929067 cord_uid: cb7cyxlo BACKGROUND: Several RT-qPCR kits are available for SARS-CoV-2 diagnosis, some of them with Emergency Use Authorization (EUA) by FDA like de nCoV19 CDC kit that includes 2 targets for detection of SARS-CoV-2 (N1, N2) and one target for RNA extraction quality control (RP), all them labelled with FAM so 3 PCR reactions are required per sample. METHODS: We designed a triplex RT-qPCR assay based on nCoV19 primers and probes where N1, N2 and RP are labelled with FAM, HEX and Cy5, respectively, so a single PCR reaction is required for each sample for SARS-CoV-2 diagnosis. RESULTS: 172 samples where analyzed with both the singleplex and triplex assays; 86 samples tested SARS-CoV-2 negative for both assays, so the triplex assay specificity is 100%; 86 samples tested SARS-Co-V 2 positive for the singleplex assay and 84 tested positive for the triplex assay, so the sensitivity was 97.7%. The limit of detection (LOD) for the triplex assay was found to be 1000 copies/mL. CONCLUSIONS: This new triplex RT-qPCR assay based on primers and probes from the CDC protocol is a highly reliable tool for SARS-CoV-2 diagnosis that could speed up detection and save reagents during current SARS-CoV-2 testing supplies shortage. was found to be 1000 copies/mL. Conclusions. This new triplex RT-qPCR assay based on primers and probes from the CDC protocol is a highly reliable tool for SARS-CoV-2 diagnosis that could speed up detection and save reagents during current SARS-CoV-2 testing supplies shortage. Keywords: SARS-CoV2, triplex, CDC, RT-qPCR The COVID19 pandemia has challenged public health systems worldwide, mainly for patient care or surveillance and control, but also to guarantee the supplies and the quality of SARS-CoV-2 However, the main limitation for the CDC protocol relies on the need to run 3 PCR reactions per sample, as all the probes are labelled with the dye FAM (1, 2) . This singleplex PCR protocol uses large amounts of reagents and reduce laboratory testing capacity, specially at small scale facilities, both of which have become crucial during the ongoing coronavirus disease pandemic, particularly at developing countries. Moreover, although several multiplex assays are commercially available, they usually depend on specific platforms or providers, and also are substantially more expensive than the CDC protocol. We herein present the development of a triplex assay to detect SARS-CoV-2 RNA from nasopharyngeal swabs based on the CDC designed probes and primers N1, N2 and RP, and the evaluation of its clinical performance and analytical sensitivity using singleplex 2019-nCoV CDC EUA kit as a gold standard. J o u r n a l P r e -p r o o f Study design. 172 clinical specimens (nasopharyngeal swabs collected on 0.5mL TE pH 8 buffer) were included on this study, coming from individuals selected for SARS-CoV-2 surveillance at "Universidad de Las Américas" SARS-CoV-2 diagnosis lab in Quito (Ecuador). Also, eight negative controls (TE pH 8 buffer) were included as control for carryover contamination, one for each set of RNA extractions. RT-qPCR for SARS-CoV-2 diagnosis using the N1, N2 and RP triplex assay. Same RNA extractions from all the samples included on the study were tested using our triplex RT-qPCR assay. N1 probe was labelled with FAM dye and BHQ1 quencher, N2 probe was labelled with HEX dye and BHQ1 quencher, and RP probe was labelled with Cy5 dye and TAO and Iowa Black RQ. All primers and probes used for the triplex assay were purchased from IDT (USA). Triplex reactions contained N1 and N2 primers and probes at final concentration of 0.5 and 0.13 µM, respectively. For the RP primers and probe, the final concentration was 0.3 and 0.075 µM, respectively. The triplex assay was also performed at a CFX96 Thermal cycler (BioRad). The RT-PCR conditions were: a) retrotrancrisption: 5min at 50C; b) initial denaturalization: 1min at 95C; c) cyclic amplification: 42 cycles of 15 sec at 95C5 + 1min at 55C. Analytical Sensitivity. Limit of detection (LoD) was performed using commercial positive control 2019-nCoV N positive control (IDT, USA), provided at 200.000 genome equivalents/mL. Ethics statement. All samples have been submitted for routine patient care and diagnostics. This study is part of a project that obtained "Comité de ética para investigación en seres humanos de la Universidad de Las Américas" approval. No extra specimens were specifically collected for this validation study. All data used in the current study was anonymized prior to being obtained by the authors. Clinical performance of triplex assay compared to the CDC gold standard protocol. (Table 1 and Supplementary Table 1) . All the 86 samples tested negative for 2019-nCoV CDC kit were also SARS-CoV-2 negative for the triplex assay, so the specificity obtained in our study was 100%. 84 samples out of 86 SARS-CoV-2 positive ones were also positive for the triplex assay, so the sensitivity for the triplex assay compared to 2019-nCoV CDC EUA was 97.7% (Table 1 The viral loads detailed on Supplementary Table 1 were calculated running a calibration curve with 2019-nCoV N positive control (IDT, USA). As the LoD is defined as the lowest viral load in which all replicates are detected (100% sensitivity), our data indicates that the LoD for N1 and N2 probes was 1000 viral RNA copies/mL of sample (in our conditions, doing RNA extractions from 0.2mL of viral transport media and eluting in 40uL at the end of the extraction, that means 5 RNA copies/uL of RNA extraction solution), as detailed on Table 2 . The viral loads for the only two samples that were singleplex positive but triplex assay negative were 1.56 copies/uL (sample 85, Table 2 ) and 2.41 copies/uL (sample 84, Supplementary Table 1 ). We herein describe the development of a triplex assay for SARS-CoV-2 RT-qPCR for the same targets included on the EUA FDA approved CDC singleplex assay. Primers and probes for N1, N2 and RP for our triplex assay were purchased from IDT (USA) which is among the few companies endorsed by CDC to purchase its CDC designed SARS-CoV-2 singleplex kit. Although the main limitation of our study is the sample size (172 specimens), our results support that the triplex J o u r n a l P r e -p r o o f assay described here for N1, N2 and RP have a great performance in terms on sensitivity and specificity compared to 2019-nCoV CDC EUA, with values of 97.7% and 100%, respectively. Moreover, as we have described on the results, the LoD for the triplex assay was set at 1000 viral RNA copies/mL of sample, that according to our experimental procedure is equivalent to 5 viral RNA copies/uL of RNA extraction solution. Actually, the only 2 SARS-CoV-2 positive samples that failed for the triplex assay had viral loads below the calculated LoD. It is important to notice that this triplex assay for SARS-CoV-2 RT-qPCR has been validated only for the instruments (CFX96 from Biorad) and chemistries described here and could need extra validation before implementation for others. Although other triplex assays protocols have been recently published using some of the CDC designed primers and probes (8, 9) , our triplex assay protocol is the first one using the exactly same set of primers and probes for N1, N2 and RP gene targets than the CDC FDA EUA singleplex protocol. This N1, N2 and RP triplex RT-qPCR represents an affordable alternative to other commercial triplex assays. For instance, in the context of Ecuador, while the cost per reaction for the triplex assay is below 2 USD, triplex assays for other commercial brands are usually above 15 USD per reaction. For any laboratory worldwide currently using the CDC protocol, this triplex assay would be easy to improve and would also speed up diagnosis while saving reagents, both necessary to improve testing capacity for SARS-CoV-2. This study was funded by Universidad de Las Americas (Quito, Ecuador). All authors contributed to study conceptualization, experimental procedures and revision and approval of final version of the manuscript. Byron Freire-Paspuel and Miguel Angel Garcia-Bereguiain analyzed the data and wrote the manuscript. All authors have no conflict of interest to declare. US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2. Emerging Infectious Diseases Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons for Coronavirus Disease 2019 (COVID-19). Center for Diseases Control and Prevention, USA Comparison of Abbott ID Now, Diasorin Simplexa, and CDC FDA EUA methods for the detection of SARS-CoV-2 from nasopharyngeal and nasal swabs from individuals diagnosed with COVID-19. Accepted Manuscript Posted Online 17 Comparative Performance of SARS-CoV-2 Detection Assays using Seven Different Primer/Probe Sets and One Assay Kit. JCM Accepted Manuscript Posted Online 8 Evaluation of nCoV-QS (MiCo BioMed) for RT-qPCR Detection of SARS-CoV-2 From Nasopharyngeal Samples Using CDC FDA EUA qPCR Kit as a Gold Standard: An Example of the Need of Validation Studies Cotton-Tipped Plastic Swabs for SARS-CoV-2 RT-qPCR Diagnosis to Prevent Supply Shortages. Front Cell Infect Microbiol Miguel Angel Garcia-Bereguiain. Sample pooling of RNA extracts to speed up SARS-CoV-2 diagnosis using CDC FDA Triplex Real-Time RT-PCR for Severe Acute Respiratory Syndrome Coronavirus 2. Emerging Infectious Diseases Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR J o u r n a l P r e -p r o o f We specially Oscar Espinosa and Dr Tannya Lozada from "Dirección General de Investigación de la Universidad de Las Américas", and also the authorities from Universidad de Las Américas, for logistic support to make SARS-CoV-2 diagnosis possible in our lab.