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1、VIROLOGICASINICA2016,31(1):85–88DOI:10.1007/s12250-015-3653-9LETTERTwo-tubemultiplexreal-timereversetranscriptionPCRtodetectsixhumancoronavirusesDearEditor,lection Database (National Center for Biotechnology In-formation, Bethesda, MD, USA) for our study usingCoronaviruses are enveloped positive-str
2、and RNA vir-Primer Premier 5.0. Different primer and probe combina-uses with 27–33 kb genomes. These viruses are classi-tions were evaluated in preliminary experiments. Basedfied into four genera, namely Alphacoronavirus, Betacor-on these experiments, primers for NL63, 229E, andonavirus,Gammacoronav
3、irus, and DeltacoronavirusSARS were grouped into one triplex reaction, while(Adams and Carstens, 2012). The Middle East respirat-those for MERS, OC43, and HKU1 were grouped intoory syndrome coronavirus (MERS-CoV), which was firstanother (Table 1). Viral targets were amplified on aand only recently i
4、dentified in the Middle East, belongsCFX96 real-time PCR system (Bio-Rad, USA) usingto the genus Betacoronavirus (Zaki et al., 2012). The hu-One Step RT-PCR Enzyme Mix (TaKaRa, Japan) in 25man coronaviruses HCoV-NL63, HCoV-229E, SARS-μL reactions containing 12.5 µL 2 × PCR buffer, 0.5 µLCoV, HCoV-OC
5、43, MERS-CoV, and HCoV-HKU1 areRT enzyme mix, 0.5 µL Taq enzyme mix, 2 µL templateassociated with high-morbidity respiratory distress, in-DNA, as well as primers and probes added from 10 ×cluding acute respiratory tract infection, pneumonia, andmixtures. Final concentrations are listed Table 1. Reac
6、-bronchiolitis (Gaunt et al., 2010; Zaki et al., 2012; Lu ettions consisted of 5 min reverse transcription at 42 °C,al., 2014). Of these, HCoV-NL63, HCoV-229E, HCoV-10 s denaturation at 95 °C, and 40 cycles at 95 °C for 10OC43, and HCoV-HKU1 are frequently isolated froms and 62 °C for 45 s. Human RN
7、ase P gene was ampli-patients, and are globally distributed, although preval-fied as internal control. Data were analyzed by univari-ence varies with time and geographical region (Geng andate statisti
