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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
