The Microlab Nimbus (Hamilton Italia Srl, Agrate Brianza, Italy) 72 batch sample loading system (which is a compact, automated, multi-channel, fast, and flexible, instrument) was used to perform molecular tests for the identification of SARS-CoV-2 with nasopharyngeal swabs. This was achieved by automatically extracting RNA and preparing RT-PCR reaction plates (Allplex™ 2019-nCoV Assay (Seegene Inc.; Seoul, Republic of Korea). Amplification was performed with a Real Time-PCR amplifier on CFX96 ™ Dx (Bio-Rad Laboratories, Inc., Segrate, Milano, Italy) and an analysis of the results was carried out on dedicated Seegene Viewer software. The method foresees that the detection of the target gene takes place when the Cycle Threshold (Ct) is lower than 40. The test detects the E, N, RdRP/S genes. The analytical sensitivity declared by the kit is equal to 4.167 copies/μL for gene E, 1250 copies/μL for the RdRP gene, and 4167 copies/μL for the N gene, respectively. Regarding the analytical specificity, the analysis performed in silico did not show cross-reactivity with the 65 potential respiratory pathogens tested (Allplex™ 2019-nCoV Assay-Seegene). All the samples sequenced were selected and identified exclusively in accordance with these criteria.
Allplex 2019 ncov assay
Manufactured by Seegene
Sourced in Cameroon, Spain, Italy, Germany, United States
The Allplex™ 2019-nCoV Assay is a real-time RT-PCR test designed for the qualitative detection of SARS-CoV-2 nucleic acid. The test targets three viral genes: E gene, RdRp gene, and N gene.
Automatically generated - may contain errors
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141 protocols using allplex 2019 ncov assay
1
Automated SARS-CoV-2 Detection Using Microlab Nimbus
2
SARS-CoV-2 Detection by Multiplex RT-PCR
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Nasopharyngeal swabs were tested for SARS-CoV-2 infection with Seegene Allplex™2019-nCoV assay (Seegene, Seoul, South Korea), according to the manufacturer’s protocol. Automated RNA extraction and PCR setup were carried out using Seegene NIMBUS, an automatic liquid handling workstation. rRT-PCR was run on a CFX96TMDx platform (Bio-Rad Laboratories, Inc., CA, USA) and subsequently interpreted by Seegene’s Viewer Software. The Seegene Allplex™2019-nCoV assay identifies the virus by multiplex real-time PCR targeting three viral genes (E, RdRP and N), thus complying with international validated testing protocols [10] .
3
Retrospective Study of Cancer Patients with COVID-19
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This study retrospectively describes a cohort of cancer patients over 18 years old consecutively admitted to CHEDV during the year 2020 who tested positive for SARS-CoV-2. Cases were considered if had a positive real-time polymerase chain reaction (RT-PCR) test from a nasal or throat swab and, when available, a radiological or clinical diagnosis of COVID-19. From March 17 to 31 December of 2020 a total of 2755 CHEDV patients tested positive for SARS-CoV-2, of which 127 (4.7%) were cancer patients.
SARS-CoV-2 laboratory diagnosis was performed using Seegene® Allplex 2019-nCoV Assay (Seegene Inc., Seoul, Korea). This assay targets four viral genes (E, N, and S/RdRp), and results were analyzed using Seegene Viewer software (Seegene Inc., Seoul, Korea).
SARS-CoV-2 laboratory diagnosis was performed using Seegene® Allplex 2019-nCoV Assay (Seegene Inc., Seoul, Korea). This assay targets four viral genes (E, N, and S/RdRp), and results were analyzed using Seegene Viewer software (Seegene Inc., Seoul, Korea).
4
COVID-19 Mechanically Ventilated Patients
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This study was approved by the University Health Network and Sinai Health System research ethics boards (20-5378.1 and 20-0115-C) and follows the Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis (TRIPOD) guidelines.19 (link) Written informed consent was waived given the retrospective design and the use of de-identified data. The records of patients admitted to intensive care units (ICUs) at Sinai Health System, Toronto Western Hospital and Toronto General Hospital were reviewed from 1 March 2020 to 15 December 2020. Adult patients (≥ 18 yr) with polymerase chain reaction-confirmed SARS-CoV2 infection (Seegene Allplex™ 2019 n-CoV assay; Seegene Inc., Seoul, South Korea) receiving invasive mechanical ventilation at any point during their ICU stay were eligible for inclusion. Patients prescribed high-flow nasal cannula or non-invasive ventilation (e.g., continuous positive airway pressure or bilevel positive airway pressure) were not eligible. A team of experienced clinical data abstractors assessed electronic and paper charts. Data were collected and stored on a centralized electronic database.
5
Placental SARS-CoV-2 Infection Analysis
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A total of 198 placentas delivered from SARS-CoV-2-positive pregnant women were submitted for conventional pathological analysis. Sampling was performed according to recommendations of the Amsterdam Consensus for placental sampling and report statements [10 (link)]. All placentas were immunohistochemically investigated for the presence of SARS-CoV-2 with the anti-SARS-CoV-2 nucleoprotein (Sino Biological, Wayne, PA) according to manufacturer's instructions. Immunohistochemically positive cases were further analyzed by in situ hybridization (ISH) with the RNAscope-ProbeV-nCoV2019-S (Advanced Cell Diagnostics, Hayward, CA). Thirty-three placentas were investigated for SARS-CoV-2 infection through retrotranscriptase PCR (RT-PCR). Fresh tissue samples and/or swabs were investigated by RT-PCR for SARS-CoV-2 with either the Allplex 2019-nCoV Assay (Seegene, Seoul, South Korea) or the Taqman 2019-nCoV Assay kit v.2 (ThermoFisher Scientific, Waltham, MA, US). For formaldehyde-fixed, paraffin embedded samples, RNA was obtained by High Pure FFPET extraction kit (Roche Diagnostics Gmbh) and further analyzed with CLART®COVID-19 (Genomica SAU) following the manufacturer's instructions. Clinical data were retrieved from the institutional records. Significance of association between qualitative variables was tested by Fisher's exact test with an α-risk of 0.05.
6
COVID-19 Respiratory Specimens Analysis
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This study included 152 respiratory specimens (66 from bronchoalveolar lavage fluid and 86 from sputum) collected from patients admitted to Tor Vergata University Hospital due to respiratory failure between March 2020 and June 2020. They were 93 males and 59 females (average age 65 years) who were admitted to our hospital due to moderate/severe acute respiratory syndrome, and 23 of them required assisted ventilation in the ICU (10 females and 13 males with average ages of 73.4 and 75,5, respectively). Due to the COVID-19 pandemic, nasopharyngeal swabs were also collected from all patients for SARS-CoV-2 assay. The samples were collected by well-trained staff using a nasopharyngeal Eswab™ (Copan, Brescia-Italy) and were analyzed using the Allplex™ 2019-nCov Assay (Seegene).
7
SARS-CoV-2 Detection in Ocular Samples
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Total RNA was isolated from conjunctival swab and corneal epithelium recovered from post-mortem patients using a modified Chomczyńsky and Sacchi’s protocol adapted for poorly cellular samples [15 (link)]. RNA integrity was measured for RNAs extracted from the 23 samples using the NanoDrop 2000 (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s instructions. Real-time reverse transcriptase (RT)-PCR for SARS-CoV-2 determination was carried out manually by using Allplex™ 2019n-CoV assay designed for the qualitative detection of the novel coronavirus in respiratory samples (Seegene, Seoul, South Korea). In the case of nasopharyngeal swabs, RNA extraction and PCR set-up were carried out on NIMBUS (Seegene), an automated liquid handling workstation.
Regarding NPS, RT-PCR was performed on a CFX96TMDx platform (Bio-Rad Laboratories, Inc., Irvine, CA, USA) followed by interpretation by Seegene’s Viewer Software. The Allplex™ 2019n-CoV assay is a multiplex real-time PCR targeting the common envelope (E) gene, and the specific nucleocapsid (N) and RNA-dependent-RNA-polymerase (RdRp) genes, complying with the international validated protocols [16 (link)].
Positive and negative controls were included in both cases.
Regarding NPS, RT-PCR was performed on a CFX96TMDx platform (Bio-Rad Laboratories, Inc., Irvine, CA, USA) followed by interpretation by Seegene’s Viewer Software. The Allplex™ 2019n-CoV assay is a multiplex real-time PCR targeting the common envelope (E) gene, and the specific nucleocapsid (N) and RNA-dependent-RNA-polymerase (RdRp) genes, complying with the international validated protocols [16 (link)].
Positive and negative controls were included in both cases.
8
SARS-CoV-2 Detection Protocol in Nasopharyngeal Swabs
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Nasopharyngeal swabs were taken and examined for SARS-CoV-2 by real-time RT-PCR performed with internal positive and negative controls, according to World Health Organization guidelines. The Allplex 2019-nCoV assay (Seegene, Seoul, Korea) was used until 10 March 2020, after which time the COBAS SARS-Cov-2 6800/8800 assay (Roche Pharmaceuticals, Basel, Switzerland) was employed.
The study protocol was approved by the statutory research committee in LHS and the Shamir Medical Center Institutional Review Board on human research.
The study protocol was approved by the statutory research committee in LHS and the Shamir Medical Center Institutional Review Board on human research.
9
SARS-CoV-2 and Dengue Diagnostic Protocols
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SARS-CoV-2 infections were diagnosed with nasopharyngeal swabs using the CDC 2019-nCoV RT-PCR Diagnostic Panel protocol (CDC, Atlanta, GA), VIASURE SARS-CoV-2 RT-PCR Detection Kit (Certest Biotec S.L., Zaragoza, Spain), Allplex 2019-nCoV Assay (Seegene Inc, Seoul, South Korea), or AccuPower SARS-CoV-2 Multiplex RT-PCR Kit (Bioneer Corporation, Daedeok-gu, South Korea). Dengue cases were diagnosed through blood samples using the SD BIOLINE Dengue DUO NS1 Ag + Ab Combo test (Alere Inc., Waltham, MA). None of the participants classified as having dengue had confirmation of the virus by molecular detection because of the unavailability of the test in Colombia in routine clinical practice.
10
RT-PCR for SARS-CoV-2 Detection and Quantification
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The RT-PCR test for SARS-CoV-2 was performed using the Allplex™ 2019-n-CoV-assay (Seegene®, Seoul, ROK), and aimed to detect the envelope gene, RNA-dependent RNA polymerase gene, and nucleocapsid protein gene. The cycle threshold (Ct) value means the cycle in which virus is detected through virus amplification, and the lower the Ct value, the higher the amount of viral load. The Ct value of all genes was detected less than 40 cycles was defined as positive.9 (link)
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