melt curve_melt curve测定原理_amplification plot(3)

>=Objective To establish a rapid assay for detection of RSV A and B subtypes by. real-time reverse transcription PCR. Methods According to RSV G protein sequence, three primers which contained one primer for both subtypes and two for either A or B subtype were designed. Detection of RSV A and B subtypes were based on determination of specific melting temperatures(Tm) by real-time PCR and melting curve ysis using SYBR Green I fluorescent dye. Results Tm value of RSV Long strains (A subtype) and...

>=Objective To establish a rapid assay for detection of RSV A and B subtypes by. real-time reverse transcription PCR. Methods According to RSV G protein sequence, three primers which contained one primer for both subtypes and two for either A or B subtype were designed. Detection of RSV A and B subtypes were based on determination of specific melting temperatures(Tm) by real-time PCR and melting curve ysis using SYBR Green I fluorescent dye. Results Tm value of RSV Long strains (A subtype) and CH18375 strains(B subtype) by using this method was 84.06± 0.38℃ and 89.06±0.32℃,respectively; and no cross-reactivity was observed with the other respiratory viruses. Of the 96 samples, 58 were positive for RSV, and 42(72.4%) of them were A subtype, 16(27.6%) of them were B subtype. Conclusion Detection RSV subtype by real-time RT-PCR and melting curve ysis using SYBR Green I is more rapid, and specific.

目的建立SYBR Green Ⅰ实时逆转录聚合酶链反应(RT-PCR)快速检测呼吸道合胞病毒(respiratory syncytial virus,RSV)A、B亚型的方法．方法根据RSV G蛋白编码基因的核苷酸序列设计三条引物,其中P1为RSV通用引物,P2、P3分别为A、B亚型特异性引物．应用SYBR Green Ⅰ荧光染料结合熔解曲线分析,测定PCR产物特征性熔解温度(melting temperatures,Tm)鉴别RSV A、B亚型．结果本法对RSV Long株(A亚型)和CH18375株 (B亚型)进行检测,其Tm均值分别为84．06±0．38℃和89．06±0．32℃;与常见呼吸道病毒间无交叉反应;96例临床标本中检出RSV阳性58例,其中A亚型占72．4％(42/58),B亚型占 27．6％(16/58)．结论 SYBR Green Ⅰ实时逆转录PCR结合熔解曲线分析检测RSV及亚型具有快速、简便、特异等特点,可对临床标本直接分型．

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