Two kinds of methods RT-PCR and IC-PCR were used to detect pathogen of virus disease of pumpkin and the sensitivity of the two methods was compared. The results showed that PRSV-W and CMV were detected in diseased sam...Two kinds of methods RT-PCR and IC-PCR were used to detect pathogen of virus disease of pumpkin and the sensitivity of the two methods was compared. The results showed that PRSV-W and CMV were detected in diseased samples gathered in Yunnan Province, while WMV and CMV were detected in diseased samples gathered in Heilongjiang Province. The sensitivity of RT-PCR is higher than that of IC-PCR, but the effect of IC-PCR in the specialization of bonding reaction and requisition for experiment material is better than that of RT-PCR.展开更多
采用传统生物学方法、显色培养基方法、自制三抗体夹心ELISA试剂盒(TAS-ELISA)、基因组直接PCR(DNA Direct-PCR)、菌裂解直接PCR(Bacterium Direct-PCR)、免疫捕捉PCR(IC-PCR)、生物PCR(Bio-PCR)、SYBR Green染料实时荧光PCR(SYBR Green...采用传统生物学方法、显色培养基方法、自制三抗体夹心ELISA试剂盒(TAS-ELISA)、基因组直接PCR(DNA Direct-PCR)、菌裂解直接PCR(Bacterium Direct-PCR)、免疫捕捉PCR(IC-PCR)、生物PCR(Bio-PCR)、SYBR Green染料实时荧光PCR(SYBR Green Real time-PCR)、探针实时荧光PCR(TaqMan Real time-PCR)检测纯菌液及模拟带菌食品中的单核细胞增生性李斯特菌(Listeria monocytogenes,LM)。结果表明:传统生物学培养方法出现假阳性;TAS-ELISA、DNADirect-PCR、Bacterium Direct-PCR、IC-PCR最低检测限分别为106、102、105、104CFU/ml;显色培养基、SYBR Green Real time-PCR灵敏度达102CFU/ml;Bio-PCR、TaqMan Real time-PCR检测灵敏度最高,均为101CFU/ml。显色培养基、TAS-ELISA操作简单,适合大量样品的初检;IC-PCR具有灵敏、特异、快速、经济的优点,特别适合大体积样品中微量病原的检测;Bio-PCR、TaqMan Real time-PCR灵敏度高、特异性好,适合阳性样品的复检以及科研使用。展开更多
【研究目的】黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的快速鉴定检测是控制该潜在危险性有害生物蔓延的有效途径。【方法】通过汁液摩擦接种、透射电镜观察、双抗夹心酶联免疫吸附测定法(DAS-ELISA)和RT-PCR方...【研究目的】黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的快速鉴定检测是控制该潜在危险性有害生物蔓延的有效途径。【方法】通过汁液摩擦接种、透射电镜观察、双抗夹心酶联免疫吸附测定法(DAS-ELISA)和RT-PCR方法对该病毒进行鉴定;同时对RT-PCR反应体系及扩增程序进行优化,建立CGMMV免疫捕获RT-PCR(IC-RT-PCR)检测方法。【结果】血清学、生物学、电镜观察和分子生物学方法证明供试样本为黄瓜绿斑驳花叶病毒引致。IC-RT-PCR方法可以简化RNA的提取,降低对试验材料要求。【结论】IC-RT-PCR的建立为CGMMV的检测提供了操作更为简便、特异性更强的快速、准确的检测方法。展开更多
By Using the modified method to extract Rice dwarf virus virions from infected rice leaves,a concentration 3.854mg/mL of purified virus virions reached,and the Rabbits was immunized with purified virions,yielding high...By Using the modified method to extract Rice dwarf virus virions from infected rice leaves,a concentration 3.854mg/mL of purified virus virions reached,and the Rabbits was immunized with purified virions,yielding high titer antiserum;1:20480 by the indirect enzyme-linked immunosorbent assay(I-ELISA)test.The antiserum successfully detected three rice samples from Kunming,Yuxi and Luliang by I-ELISA.Immunocapture polymerase chain reaction(IC-PCR),respectively was the utilized to show that serologic and molecular methods were successfully used for detection of Rice dwarf virus.展开更多
基金The Open Fundation Item of Agricultural Biotechnological Key Laboratory of Yunnan Province (2001B- 3)
文摘Two kinds of methods RT-PCR and IC-PCR were used to detect pathogen of virus disease of pumpkin and the sensitivity of the two methods was compared. The results showed that PRSV-W and CMV were detected in diseased samples gathered in Yunnan Province, while WMV and CMV were detected in diseased samples gathered in Heilongjiang Province. The sensitivity of RT-PCR is higher than that of IC-PCR, but the effect of IC-PCR in the specialization of bonding reaction and requisition for experiment material is better than that of RT-PCR.
文摘采用传统生物学方法、显色培养基方法、自制三抗体夹心ELISA试剂盒(TAS-ELISA)、基因组直接PCR(DNA Direct-PCR)、菌裂解直接PCR(Bacterium Direct-PCR)、免疫捕捉PCR(IC-PCR)、生物PCR(Bio-PCR)、SYBR Green染料实时荧光PCR(SYBR Green Real time-PCR)、探针实时荧光PCR(TaqMan Real time-PCR)检测纯菌液及模拟带菌食品中的单核细胞增生性李斯特菌(Listeria monocytogenes,LM)。结果表明:传统生物学培养方法出现假阳性;TAS-ELISA、DNADirect-PCR、Bacterium Direct-PCR、IC-PCR最低检测限分别为106、102、105、104CFU/ml;显色培养基、SYBR Green Real time-PCR灵敏度达102CFU/ml;Bio-PCR、TaqMan Real time-PCR检测灵敏度最高,均为101CFU/ml。显色培养基、TAS-ELISA操作简单,适合大量样品的初检;IC-PCR具有灵敏、特异、快速、经济的优点,特别适合大体积样品中微量病原的检测;Bio-PCR、TaqMan Real time-PCR灵敏度高、特异性好,适合阳性样品的复检以及科研使用。
文摘【研究目的】黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的快速鉴定检测是控制该潜在危险性有害生物蔓延的有效途径。【方法】通过汁液摩擦接种、透射电镜观察、双抗夹心酶联免疫吸附测定法(DAS-ELISA)和RT-PCR方法对该病毒进行鉴定;同时对RT-PCR反应体系及扩增程序进行优化,建立CGMMV免疫捕获RT-PCR(IC-RT-PCR)检测方法。【结果】血清学、生物学、电镜观察和分子生物学方法证明供试样本为黄瓜绿斑驳花叶病毒引致。IC-RT-PCR方法可以简化RNA的提取,降低对试验材料要求。【结论】IC-RT-PCR的建立为CGMMV的检测提供了操作更为简便、特异性更强的快速、准确的检测方法。
文摘By Using the modified method to extract Rice dwarf virus virions from infected rice leaves,a concentration 3.854mg/mL of purified virus virions reached,and the Rabbits was immunized with purified virions,yielding high titer antiserum;1:20480 by the indirect enzyme-linked immunosorbent assay(I-ELISA)test.The antiserum successfully detected three rice samples from Kunming,Yuxi and Luliang by I-ELISA.Immunocapture polymerase chain reaction(IC-PCR),respectively was the utilized to show that serologic and molecular methods were successfully used for detection of Rice dwarf virus.
