The TaqPath RT-PCR COVID-19 Combo Kit Assay is US Food and Drug Administration approved under Emergency Use Authorization. Multiplex RT-qPCR was performed according to the manufacturer’s instructions (Thermo Fisher Scientific; catalog no. A47814). Viral nucleic acids were detected by using primers and probes targeting the N, S, and Orf1ab genes. A pair of primers against the extraction controls (MS2) was also included in the same reaction. RT-qPCR reactions were performed on either an ABI7500 FAST DX Real-Time PCR instrument (Thermo Fisher Scientific; catalog no. 4406985) or a QuantStudio 5 Real-Time PCR instrument (Thermo Fisher Scientific; catalog no. A34322). Positive samples were identified by using the Applied Biosystems COVID-19 Interpretive Software version 1.3 (for ABI7500 FAST DX; Foster City, CA) or Applied Biosystems COVID-19 Interpretive Software version 2.3 (for QuantStudio 5).
Abi7500 fast dx real time pcr instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI7500 FAST DX Real-Time PCR instrument is a laboratory equipment designed for performing real-time polymerase chain reaction (PCR) analysis. It is capable of detecting and quantifying specific nucleic acid sequences in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 5 real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
Covid 19 interpretive software version 2, by Thermo Fisher Scientific (1 mentions)
Abi 7500 fast dx, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Cfx96 touch real time pcr system, by Bio-Rad (1 mentions)
Omni plant rna kit dnase 1, by CoWin Biotech (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Xn 2000, by Sysmex (1 mentions)
Cobas 6000, by Roche (1 mentions)
5 protocols using abi7500 fast dx real time pcr instrument
1
TaqPath RT-PCR COVID-19 Combo Kit Assay
2
Quantitative Real-Time PCR Methodology
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The total RNA extraction from tissues and embryos was performed using TRIzol reagent (Invitrogen, Waltham, MA, USA), followed by reverse transcription using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). The synthesized cDNA was used for real-time quantitative PCR (RT-qPCR) with SsoAdvanced Universal Supermixes (Bio-Rad Laboratories, Hercules, CA, USA), GoTaq qPCR Master Mix (Promega, Madison, WI, USA) in CFX96 Touch Real-Time PCR System (Bio-Rad Laboratories), and ABI 7500 Fast Dx Real-Time PCR Instrument (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Genes of interest were amplified by 40 cycles using a standard three-step protocol (95 °C, 20 s; 65 °C, 15 s; 72 °C, 30 s). All primers used in RT-qPCR are listed in Table S1 .
3
qRT-PCR Analysis of RhUBI2 and Actin
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Total RNA was extracted using the OmniPlant RNA Kit (DNase I) (CoWin Biosciences, Taizhou, China). The first strand of cDNA was synthesized from 15 μL of total RNA by using MonScript™ RTIII All-in-One Mix with dsDNase (Monad Biotechnology Co., Ltd., Wuhan, China). RhUBI2 (JK618216) and Actin were used as internal controls [40 (link)]. Primers were designed using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) and synthesized by Sangon Biotech (Shanghai, China) (Table S2 ). qRT-PCR was performed on a ABI 7500 FAST DX Real-Time PCR instrument (Thermo Fisher Scientific, Inc., Waltham, USA). Each reaction was conducted in a 20 μL mixture containing 10 μL of 2× Universal Blue SYBR Green qPCR Master Mix (Wuhan Servicebio Biotechnology Co., Ltd., Wuhan, China), 7.4 μL of RNase-free H2O, 1 μL of cDNA, 0.8 μL of forward primer, and 0.8 μL of reverse primer. The PCR machine was programmed as follows: PCR initial activation step for 2 min at 95 °C, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. The relative gene expression was calculated using the 2−ΔΔCT method [41 (link)].
4
Influenza Virus Molecular Detection
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Viral RNA was extracted from 200 μl of the specimen' aliquot using the QIAamp viral mini RNA kit (Qiagen, Germany) following the manufacturer' instructions. RNA was eluted in 100 μl of RNase/DNase-free elution buffer provided with the kit. Extracted RNA was analysed for the presence of influenza viruses by real-time reverse transcription-polymerase chain reaction (RT-PCR) on the ABI 7500 Fast Dx Real-time PCR instrument (Applied Biosystems, US). The reagents, primers and probes used for influenza viruses' detection and subtyping were from the US Centers for Disease Control and Prevention (US CDC). To assess the quality of each sample, the human ribonuclease P gene was amplified from all samples tested. All real-time RT-PCR data were analysed using the ABI sequence detection software version 1.4 (Applied Biosystems, US).
5
Comprehensive Biochemical Analysis Protocol
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All biochemical parameters were determined using the standard manufacturer’s procedures. Blood hemoglobin level was evaluated on XN-2000 (Sysmex Corporation, Kobe, Japan). Urinary proteins, hemoglobin, and glucose were quantified using the semi-quantitative automated reagent strip test Iris iRICELL2000 (Beckman Coulter, Brea, CA, USA). The dipstick urine test detects hemoglobin at four levels: 0.03, 0.10, 0.50, and ≥1.0 mg/dL; proteins at three levels: 10, 50, and 100 mg/dL; and glucose at three levels: 50, 100, and 200 mg/dL. Detection and quantification of the BK virus (BKV) and Cytomegalovirus (CMV) in the plasma were assessed with the real-time PCR method, using the diagnostic test R-gene Kit (bioMérieux, Marcy l’Etoile, France). The number of virus copies was automatically calculated by the ABI 7500 Fast Dx Real-Time PCR Instrument (Applied Biosystems, Foster City, CA, USA) relative to the standard curve. The creatinine concentration in serum was measured on a Cobas6000 (Roche, Risch-Rotkreuz, Switzerland). Next, eGFR was estimated from creatinine concentration, age, and sex of the subject using the chronic kidney disease epidemiology collaboration (CKD-EPI) equation according to the current 2012 Kidney Disease: Improving Global Outcomes (KDIGO) guidelines [18 ].
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