7500 fast dx real time pcr instrument

We conducted a cross-sectional study to determine the diagnostic performance of the rapid antigen test compared to RT-PCR. Two swabs were taken from each individual, one nasal swab for the antigen test and one nasopharyngeal swab for the RT-PCR. For rapid antigen test, Abbott panbio COVID-19 antigen rapid test device (Abbott Rapid Diagnostic Jena GmbH, Jena, Germany) to detect SARS-CoV-2 nucleocapsid protein was used. The contained membrane strip is pre-coated with immobilized anti-SARS-CoV-2 antibody on the test line and mouse monoclonal anti-chicken IgY on the control line (13 ). The nasopharyngeal samples for RT-PCR were transferred to a viral transport media immediately after collection and transported to a COVID-19 laboratory for testing. The RT-PCR test was conducted using Thermo Fisher Scientific (Waltham, MA) TaqPath 1-Step RT-qPCR Master Mix, CG on the Applied Biosystems (Foster City, CA) 7500 Fast Dx RealTime PCR Instrument. The assay used followed the WHO protocol and targeted the E gene. If the E gene was detected, the sample was then confirmed by RdRP and N genes (14 ). The E gene Ct value was reported and used in this study. Ct values >40 were considered negative. Positive (virus-like particles of SARS-CoV-2 and RNase P) and negative (RNase-free Water) controls were included for quality control purposes.

Real-time PCR validation of species-specific primers was performed using a 7500 Fast Dx real-time PCR instrument with SDS software (Applied Biosystems, USA). The reaction mix contained 12.5 µl of Maxima SYBR Green/ROX qPCR Master Mix (2×) (Thermo Scientific, USA) 400 nM of each primer, 20 ng of DNA template of each mildew isolate and nuclease-free water to reach a final volume of 25 µl. No template controls (NTC) were systematically included in triplicate. The thermal cycling conditions comprised an initial denaturation step of 7 min at 95 °C for 30 s and annealing at 60 °C for the 30 s. A melt curve analysis using a temperature gradient from 60 °C to 100 °C and a ramp speed of 0.5 °C/s and continuous fluorescent measurement was performed after the last cycle.
Species-specific qPCR assay performed, DNA isolated from pure cultures of both the mildew isolates CsKP07 and CsKD11, used as the positive control. Reactions containing DNA from healthy cucurbit plants and NTC reactions were used as negative controls. All qPCR assays were performed in triplicate. The expected size of both the mildew pathogens was confirmed by gel electrophoresis, as described above.

RNA extraction was conducted using the NucleoSpin RNA Plus RNA purification kit (Macherey-Nagel, Düren, Germany). RNA samples were stored at −80 °C, and RNA integrity was tested with bleach agarose gel [111 (link)]. cDNA was synthesized using the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio USA, San Jose, CA, USA). Real-time quantitative PCRs (qPCRs) were performed in duplicates using 5 ng of cDNA per reaction amplified by SYBR FAST qPCR Master Mix (KK4602, Kapa Biosystems, Wilmington, MA, USA) in a StepOne™ Real-Time PCR System (Applied Biosystems™, Warrington, UK) and a 7500 Fast Dx Real-Time PCR Instrument (Applied Biosystems™, Warrington, UK). CYLD qPCR primers recognize a region from exon 2 to exon 3 (spanning from 645 to 753, transcript NM_173369.3). The complete list of primer sequences can be found in Table S1.