key: cord-0914831-bm545scp authors: Chang, Chih-Kai; Jian, Ming-Jr; Chung, Hsing-Yi; Lin, Jung-Chung; Hsieh, Shan-Shan; Tang, Sheng‐Hui; Perng, Cherng-Lih; Chen, Chien-Wen; Hung, Kuo-Sheng; Chang, Feng-Yee; Shang, Hung-Sheng title: Clinical Comparative Evaluation of the LabTurbo(TM) AIO(®) Reverse Transcription-Polymerase Chain Reaction and World Health Organization-Recommended Assays for the Detection of Emerging SARS-CoV-2 Variants of Concern date: 2022-02-22 journal: Infect Drug Resist DOI: 10.2147/idr.s349669 sha: 0a8f78c670cb3e139c16f9ad2975b5c61ee62292 doc_id: 914831 cord_uid: bm545scp PURPOSE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent behind coronavirus disease-2019 (COVID-19). Single-plex reverse transcription-polymerase chain reaction (RT-PCR)-based assays are widely used for COVID-19 detection but exhibit decreased sensitivity and specificity in detecting the rapidly spreading SARS-CoV-2 variants; in contrast, multiplex RT-PCR reportedly yields better results. Here, we aimed at comparatively analyzing the clinical performance of the LabTurbo(TM) AIO COVID-19 RNA testing kit, a multiplex quantitative RT-PCR kit, including a three-target (E, N1, and RNase P), single-reaction, triplex assay used for SARS-CoV-2 detection, with that of the WHO-recommended RT-PCR assay. MATERIALS AND METHODS: Residual, natural, nasopharyngeal swabs obtained from universal transport medium specimens at SARS-CoV-2 testing centers (n = 414) were collected from May to October 2021. For SARS-CoV-2 qRT-PCR, total viral nucleic acid was extracted. The limit of detection (LOD) and the comparative clinical performances of the LabTurbo(TM) AIO COVID-19 RNA kit and the WHO-recommended RT-PCR assay were assessed. Statistical analysis of the correlation was performed and results with R(2) values >0.9 were considered to be highly correlated. RESULTS: The LOD of the LabTurbo(TM) AIO COVID-19 RNA kit was 9.4 copies/reaction for the target genes N1 and E. The results obtained from 102 SARS-CoV-2-positive and 312 SARS-CoV-2-negative samples showed 100% correlation with previous WHO-recommended RT-PCR assay results. CONCLUSION: Multiplex qRT-PCR is a critical tool for detecting unknown pathogens and employs multiple target genes. The LabTurbo(TM) AIO COVID-19 RNA testing kit provides an effective and efficient assay for SARS-CoV-2 detection and is highly compatible with SARS-CoV-2 variants. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a member of the beta-coronavirus lineage B (B-βCoV), is spreading rapidly worldwide. This virus causes an infectious respiratory disease known as coronavirus disease-2019 (COVID- 19) . 1, 2 On January 30, 2020, the World Health Organization (WHO) declared this disease a "Public Health Emergency of International Concern", given the serious global health crisis caused by SARS-CoV-2. 3 The clinical symptoms of COVID- 19 are similar to that of infections caused by influenza viruses or other common respiratory viruses. 4 The ongoing global pandemic caused by multiple SARS-CoV-2 variants emerged in the Fall of 2020. These variants are more transmissible and infectious than the original strain, leading to their rapid spread. 5 The SARS-CoV-2 B.1.1.7 (Alpha) variant was first detected in the United Kingdom in September 2020; its prevalence has increased in numerous counTries, including the United States and Europe. 6, 7 Additionally, the B.1.351 (Beta) and B.1.617 (Delta) variants were first identified in South Africa and India in December 2020, respectively. Furthermore, these variants have been associated with high mortality rates. 8 SARS-CoV-2 nucleic acid testing using reverse transcription-polymerase chain reaction (RT-PCR), the gold standard for detecting acute infections [9] [10] [11] was developed by Corman et al 12 as recommended by the WHO and the Centers for Disease Control (CDC) 13 and is used worldwide. However, some studies have reported that regular single-plex RT-PCR cannot detect SARS-CoV-2 variants, leading to decreased sensitivity and specificity of SARS-CoV-2 detection. Moreover, N, a SARS-CoV-2 target gene, was reported to show dropout or cycle threshold (Ct) value shifts in the evaluation of B.1.1.7 variants. 14, 15 The primer/probe sets recommended by the WHO and the CDC use identical fluorescent reporters. These require each target gene to be assayed separately, leading to high reagent consumption and low production capacity when evaluating samples in a 96-well plate. Although commercially available systems, including the Cepheid Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA), DiaSorin Simplexa (Saluggia, Italy), and Cobas 6800 (Roche Diagnostics, Indianapolis, IN), show high sensitivity and require short testing times, these require unique instruments. Therefore, more accessible assays are needed to confirm or rule out SARS-CoV-2 infection. Multiplex quantitative (q)RT-PCR assays can yield multiple results in a single test run effectively and efficiently, with decreased reagent consumption and shorter test duration. The LabTurbo TM AIO COVID-19 RNA testing kit (Cat. No. Acov11240; Taigen Bioscience Corp., Taipei, Taiwan) is a commercially available, multiplex, quantitative RT-PCR kit for a three-target (E, N1, and RNase P), single-reaction, triplex assay for SARS-CoV-2 detection and is highly compatible with many other real-time PCR platforms. Here, we evaluated the clinical performance of the LabTurbo TM AIO COVID-19 RNA testing kit and determined its limit of detection (LOD) compared with current WHO-recommended qRT-PCR assays. Between May and October 2021, 414 residual, natural, nasopharyngeal swabs were obtained from universal transport medium specimens at SARS-CoV-2 testing centers and frozen at −80°C. The Taiwan CDC Central Laboratory confirmed that 102 original specimens tested positive for SARS-CoV-2 using the WHO-recommended RT-PCR assay, 12 while the remaining 312 specimens were negative. All procedures performed in this study were approved by the Institutional Review Board of the Tri-Service General Hospital (TSGHIRB No. C202005041) registered in March 2021. Written informed consent was obtained from the patients. Total viral nucleic acid was extracted from 300 µL of each nasopharyngeal swab in a universal transport medium and eluted in a final volume of 60 µL using the LabTurbo Virus Mini Kit (Cat. No. LVN480-300) and an automated LabTurbo 48 Compact extraction system (Taigen Bioscience Corp., Taipei, Taiwan) according to the manufacturer's instructions. Each extracted viral RNA was stored at −80°C until use. The five different primer/probe sets and sequences used for the SARS-CoV-2 detection assays are listed in Table 1 . For single-plex qRT-PCR, E and N primer/6-carboxyfluorescein (FAM)-labeled probe sets were designed for the non-coding https://doi.org/10.2147/IDR.S349669 Infection and Drug Resistance 2022:15 596 region, as described previously 12 for SARS-CoV-2 qRT-PCR. The LabTurbo TM AIO COVID-19 RNA testing kit for SARS-CoV-2 multiplex qRT-PCR, containing reverse transcriptase, primer/probe mixture, and 2× PCR master mix, was utilized according to the manufacturer's instructions. In this kit, probes for E and N1 were labeled with Hexachlorofluorescein (HEX) and FAM fluorescent reporters, respectively. RNase P primer/Cy5-labeled probe sets (designed as quality control for RNA extraction to detect human nucleic acids) were developed by the CDC using fluorescent reporter modification and are published on the CDC website. 13, 16 Each 25 µL reaction mixture contained 12.5 µL of 2× PCR master mix, 2.5 µL of primer/probe mixture, 1.25 µL of reverse transcriptase, 2.75 µL of RNase-free water, and 6 µL of extracted RNA template. SARS-CoV-2 qRT-PCR was performed on a Rotor-Gene-Q real-time PCR instrument (Qiagen, Hilden, Germany) and a Roche LightCycler ® 96 instrument (Roche Diagnostics, Indianapolis, IN) under the following thermocycling conditions: Reverse transcription at 55°C for 10 min and pre-denaturation at 95°C for 1 min, followed by 45 cycles of 95°C for 10s and 60°C for 15s. A Ct value of < 36 was considered a true positive while a Ct value of > 36 indicated re-testing. Briefly, the VirSNiP SARS-CoV-2 assay 17 (TIB Molbiol, Berlin, Germany) included real-time RT-PCR post-melting curve analysis to detect six spike gene mutations as follows: N501Y, H69/V70, K417N, E484K, P681R, and L452R in SARS-CoV-2 VOC. Distinguishment of SARS-CoV-2 VOCs was based on spike gene mutations detected as follows: N507Y and del H69/V70 for B. Dilutions of SARS-CoV-2-positive clinical samples were to preliminarily assess analytical sensitivity. To determine the LOD of the LabTurbo TM AIO COVID-19 RNA testing kit for both E and N1, two-fold serial dilutions of an AMPLIRUN SARS-CoV-2 RNA control (Vircell, Granada, Spain) from 300 to 9.4 copies/reaction were used (n = 20). The LOD was defined as a 95% probability of 20 replicates testing positive. The specificity of the LabTurbo TM AIO COVID-19 RNA testing kit was evaluated using three common upper respiratory tract viruses, as described in our previous report. 18 Samples positive for influenza A, influenza B, and respiratory syncytial virus were obtained from viral cultures at the Taiwan CDC Viral Infection Contract Laboratory. Analytical sensitivity (LOD) was preliminarily assessed at concentrations around the detection endpoint using serial dilutions. To accurately determine the analytical sensitivity, 20 replicate reactions were performed, yielding 9.4 copies per reaction for both N1 and E using the LabTurbo TM AIO COVID-19 RNA testing kit ( Table 2) . Clinical samples obtained from respiratory viruses, including those positive for influenza A, influenza B, respiratory syncytial virus, rhinovirus, parainfluenza virus, and adenovirus, as well as cell culture supernatants, were used to assess the kit's analytical specificity. The E and N1 primer/probe sets from the LabTurbo TM AIO COVID-19 RNA testing kit were found to be highly specific for SARS-CoV-2. Furthermore, they did not show cross-reactivity with the tested viruses or the cell culture supernatants ( Table 3) . The RNase P target gene was not included in this comparison as it was used as quality control for RNA extraction. There were 102 SARS-CoV-2-positive samples and 312 SARS-CoV-2-negative samples among the 414 clinical samples analyzed using the LabTurbo TM AIO COVID-19 RNA testing kit, demonstrating 100% correlation with the results obtained using the WHO-recommended RT-PCR assay. The Ct values for these two assays were highly correlated when the Rotor-Gene-Q real-time PCR instrument (LabTurbo TM -N1 versus WHO-RdRp target, R 2 = 0.9407; LabTurbo TM -E versus WHO-E target, R 2 = 0.9778) ( Figure 1A and B; Table S1), and the Roche LightCycler ® 96 instrument (LabTurbo TM -N1 versus WHO-RdRp target, R 2 = 0.9401; LabTurbo TM -E versus WHO-E target, R 2 = 0.9725) ( Figure 1C and D; Table S2 ) were used. Tables S1 and S2 ). As of October 24, 2021, over 243 million cases of COVID-19 have been confirmed worldwide, with a death toll surpassing 4 million people. 19 Various detection assays for SARS-CoV-2 have been developed since the pandemic began. RT-qPCR is currently being used as a reliable method for detecting symptomatic and asymptomatic patients infected with SARS-CoV-2. Given the rapid spread of SARS-CoV-2 VOC worldwide, reliable, early, and accurate diagnosis is crucial to detect these variants. Although whole-genome sequencing is a standard method to detect the variants, it has limited accessibility and requires sophisticated infrastructure and trained personnel. 20 Currently, the rapid spread of SARS-CoV-2 variants poses a significant public health challenge. A rapid molecular method to screen for SARS-CoV-2 VOC is necessary. A SARS-CoV-2 RT-PCR with additional VirSNiP probe-based melting curve assay for SARS-CoV-2 variants is a feasible, rapid, and efficient screening strategy that can be implemented in laboratories to perform VOC testing on SARS-CoV-2positive samples. We observed an increase in the proportion of UK B. Some studies have reported that regular single-plex RT-PCR cannot reliably detect SARS-CoV-2 variants, resulting in decreased sensitivity and the specificity of SARS-CoV-2 detection. Moreover, instances of N dropout or Ct value shift have been reported in association with the assay, especially with the B.1.1.7 lineage. 14 Multiplex PCR can increase the sensitivity and the specificity of SARS-CoV-2 variant detection and its dual or triple target approach can mitigate the risk of loss of sensitivity. During the earlier stages of the COVID-19 outbreak, the sets of RT-qPCR primers and probes were publicly available and were used by several laboratories worldwide (Table 1) . Assays performed using these sets used identical fluorescent reporters as probes. They also required each target gene to be assayed separately, leading to high reagent consumption and low production capacity when analyzing samples using 96-well plates. The LabTurbo TM AIO COVID-19 RNA testing kit used in this study employs a single-reaction triplex assay for SARS-CoV-2 detection. The advantages of using a multiplex qRT-PCR assay are its high capacity in a single test run, effective and efficient detection, decreased reagent Simplexa (Saluggia, Italy), and Cobas 6800 (Roche Diagnostics, Indianapolis, IN), with high sensitivity and short testing times. However, all of these assays require unique instruments. Multiplex qRT-PCR is a critical tool for detecting unknown pathogens and allows multiple target gene detection. The LabTurbo TM AIO COVID-19 RNA testing kit provides an effective and efficient method for SARS-CoV-2 detection and is highly compatible with SARS-CoV-2 variants. COVID-19, coronavirus disease 2019; LOD, the limit of detection; RT-PCR, reverse transcriptase-polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; VOC, variant of concern; WHO, World Health Organization; CDC, Centers for Disease Control. 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Coronavirus disease (COVID-19) epidemiological update SARS-CoV-2 variants detection using TaqMan SARS-CoV-2 mutation panel molecular genotyping assays Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia Infection and Drug Resistance Infection and Drug Resistance is an international, peer-reviewed open-access journal that focuses on the optimal treatment of infection (bacterial, fungal and viral) and the development and institution of preventive strategies to minimize the development and spread of resistance. The journal is specifically concerned with the epidemiology of antibiotic resistance and the mechanisms of resistance development and diffusion in both hospitals and the community. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. This study was supported by the Tri-Service General Hospital, Taipei, Taiwan, ROC, (grant number TSGH-D-111086). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. The authors declare no conflict of interest.