A total of 22 respiratory pathogens were used in our initial validation analysis of the specificity of the study primers. The strain names of these 22 pathogens are listed in Table 1 . The Middle East respiratory syndrome coronavirus (MERS-CoV) EMC strain was kindly provided by Dr. Ron A. M. Fouchier, Erasmus Medical Center, Rotterdam, the Netherlands. The SARS-CoV Frankfurt 1 strain was kindly provided by J. Ziebuhr, University of Würzburg, Germany. The clinical isolates of human coronaviruses (HCoV)-HKU1, -OC43, -NL63, and -229E were described by previous studies.31 (link)–33 (link)In vitro-transcribed RNA (GenBank accession number MN908947), synthesized using a ScriptMax Thermo T7 Transcription Kit (TOYOBO, Osaka, Japan), was used to determine the detection limit of the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan).
Loopamp sars cov 2 detection kit
Manufactured by Eiken Chemical
Sourced in Japan
The Loopamp SARS-CoV-2 Detection kit is a laboratory equipment product designed for the detection of SARS-CoV-2 genetic material. The kit utilizes the loop-mediated isothermal amplification (LAMP) technique to amplify and detect specific regions of the SARS-CoV-2 genome.
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Lab products found in correlation
5 protocols using loopamp sars cov 2 detection kit
1
Validation of SARS-CoV-2 Detection Assay
2
SARS-CoV-2 Detection via RT-PCR and LAMP
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Nasal, pharynx, and rectal swab samples were collected in 1 mL DMEM supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin (Nacarai Tesque). RNA was extracted from swab samples using a QiaAmp Viral RNA kit (Qiagen) according to the manufacturer’s instructions. For the detection of viral RNA, 5 μL of extracted RNA was subjected to one-step real-time RT-PCR using a QunatiTect Probe RT-PCR (Qiagen) kit on a Light-Cycler 480 II (Roche Diagnostics). The following primer and probe sets were used: forward primer 5'-AAATTTTGGGGACCAGGAAC-3' and reverse primer 5'-TGGCAGCTGTGTAGGTCAAC-3' with probe FAM-5'-ATGTCGCGCATTGGCATGGA-3'-BHQ1. The reaction conditions of RT-PCR were 50 °C for 30 min (reverse transcription) and 95 °C for 15 min (activation of the polymerase), 45 cycles of 15 s at 95 °C (denaturation) followed by 60 s at 60 °C (annealing and extension). LAMP assay was tested using 10 μL of extracted RNA form swab samples by Loopamp SARS-CoV-2 detection kit (Eiken Chemical Co.) according to the manufacturer’s instructions.
3
SARS-CoV-2 Antigen Testing in Healthcare Setting
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Case definition included all healthcare workers and all patients associated to the ward from January 27 to January 31, 2022 with a positive SARS-COV-2 antigen test using the Lumipulse G SARS-CoV-2 Ag assay (Fujirebio, Tokyo, Japan), which detects the SARS-CoV-2 nucleocapsid (N) protein [9 (link)]. Non-case definition included all healthcare workers and all patients associated to the ward from January 27 to January 31, 2022 with a negative SARS-CoV-2 antigen test. Cases with judgment-pending results in Lumipulse G were confirmed using the Loopamp™ SARS-CoV-2 Detection Kit (Eiken Chemical, Tokyo, Japan) [10 (link)], which targets genes encoding the nucleocapsid (N) and the RNA-dependent RNA polymerase of SARS-CoV-2, or the ID NOW COVID-19 assay (Abbott Rapid Diagnostic, Scarborough, ME, USA), which uses isothermal nucleic acid amplification of RNA-dependent RNA polymerase viral targets [11 (link),12 (link)]. Saliva or nasopharyngeal swab specimens were obtained from all members of the ward (including all healthcare workers and patients). The type of variant strain was not evaluated by laboratory methods.
4
SARS-CoV-2 Detection using RT-LAMP
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Assays were performed using a Loopamp™ SARS-CoV-2 Detection Kit (Eiken Chemical, Tokyo, Japan), targeting genes encoding the nucleocapsid (N) and RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, in accordance with the manufacturer’s instructions. The reaction mixture consisted of RNA sample (10 μL) and the provided master mix including a set of primers (15 μL). The mixture was incubated at 62.5 °C for 35 min, with turbidity measured every 6 s using a real-time Loopamp EXIA turbidity meter (Eiken Chemical). Assays were scored as positive when the differential value approached 0.05, at which point the threshold time (Tt) was recorded. In addition, LAMP positive reactions can also be detected visually, through fluorescence, using calcein staining; therefore, we reassessed RT-LAMP reactions using this visual endpoint detection [9 (link)].
5
RT-LAMP for SARS-CoV-2 Detection
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According to the manufacturer’s instructions, RT-LAMP was performed using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical Co., Ltd., Tokyo, Japan). The reaction volume of 25 μL contained 10 μL of purified RNA and 15 μL of reaction mix containing 172.3 ng/μL of SARS-CoV-2 specific primer sets. The mixture was incubated for 35 min at 62.5 °C, and the process was monitored using a Loopamp Real-time Turbidimeter (LA-200; Eiken Chemical). For visual evaluation of fluorescence, the reaction tube was illuminated with ultraviolet light using an ultraviolet illumination system (WSE-5300; ATTO, Tokyo, Japan) and observed by the naked eye.
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