H9亚型禽流感病毒荧光RT-PCR检测方法

ICS11.220B41GB/T19438.4—2004H9RT-PCRMethodofthereal-timeRT-PCRforthedetectionofAvianInfluenzavirussubtypeH92004-2-152004-2-15食品伙伴网—2004IGB/T1.1—20001食品伙伴网—20041H9RT-PCR1RT-PCRH9H92GB/T19438.1-2004RT-PCR3RT-PCR-CtRNADEPCPBSATaqTaqDNA4H9RT-PCRTaqManH95’3’5’FAMR3’TAMRA5’QPCRRQRPCRTaqTaq5’→3’RRQTaqRQR55.1RNADEPC食品伙伴网—20042-20oCPBS121215min10000U/mL75%:RNAGB668292-20oCH9RT-PCR1:A5.2RT-PCR12000r/min2000r/min(2oC8oC-20oC)10µL100µL1000µLPCREppendorf1.5mL66.11212oC15min1.5mLEppendorf6.26.2.1351.0mLPBS1.5mLEppendorf6.2.26.2.3Eppendorf6.36.46.4.130min1.5mLEppendorf6.4.22.0g10mLPBS4oC3000r/min15min1.5mLEppendorf6.51食品伙伴网—200432oC8oC24h-70oC377.1GB/T19438.1-2004C7.27.2.1n1.5mLEppendorfn7.2.2600µL200µL200µL5s4oC12000r/min15min7.2.36.2.11.5mLEppendorf500µL-20oC6.2.2500µL7.2.44oC12000r/min15minEppendorf()600µL757.2.54oC12000r/min10minEppendorf7.2.64000g10sEppendorf3minRNA7.2.711µLDEPCRNA2000r/min5sRNA2hPCR-70oC7.37.3.1RT-PCRTaq2000r/min5sRT-PCRnnRT-PCRTaq15µL0.25µL41RT-PCRRT-PCR15µL7.3.2RT-PCR7.1.7RNA10µL500r/min30s7.3.3RT-PCR食品伙伴网—200447.2.2PCRRT-PCR42oC/30min92oC/3min92oC/10s45oC/30s72oC/1min592oC/10s60oC/30s40Ct88.18.28.2.1Ct8.2.2Ct28.08.38.3.1CtH98.3.2Ct≤30H98.3.3Ct30食品伙伴网—20045AH9RT-PCRA.14830mL1DEPC1mL1H9RT-PCR750µL1RT-PCR1/12Taq12µL11mL1RNA1mL1A.2A.2.1RNA4A.2.2DEPC1%DEPCRNAA.2.3H9RT-PCRH9A.3A.3.1A.3.2A.3.3RT-PCR食品伙伴网