目的探讨巨细胞病毒(C M V)D N A检测在肾移植术后C M V病预防中的作用。方法采用R ealtim e-PC R定量检测肾移植受者外周血白细胞中C M V-D N A,术后4月内每周1次。结果95例患者中有55例检出C M V-D N A阳性(57.9%),初次检出C M V-D N ...目的探讨巨细胞病毒(C M V)D N A检测在肾移植术后C M V病预防中的作用。方法采用R ealtim e-PC R定量检测肾移植受者外周血白细胞中C M V-D N A,术后4月内每周1次。结果95例患者中有55例检出C M V-D N A阳性(57.9%),初次检出C M V-D N A的平均时间为(25.6±16.3)d,C M V-D N A的平均拷贝数为(1396.4±445.2)/105白细胞。在随访过程中,18例患者的C M V-D N A水平超过警戒线—800copy/105白细胞,经调整免疫抑制剂后,均降至警戒线以下,所需平均时间为(16.3±8.5)d。所有患者均未出现C M V病。结论C M V-D V A检测可用于预防肾移植术后C M V病的发生。展开更多
马铃薯A病毒(Potato virus A,PVA)是我国重要的检疫性有害生物。本研究根据PVA中CP基因(coat protein gene)的保守序列,设计了两套PCR引物和TaqMan探针,建立了双引物探针RT-RealtimePCR检测PVA的方法。该方法采用实时荧光PCR技术,有效...马铃薯A病毒(Potato virus A,PVA)是我国重要的检疫性有害生物。本研究根据PVA中CP基因(coat protein gene)的保守序列,设计了两套PCR引物和TaqMan探针,建立了双引物探针RT-RealtimePCR检测PVA的方法。该方法采用实时荧光PCR技术,有效地提高了检测的灵敏度;同时两套引物探针相互验证,有效提高了结果的准确性。实验结果表明,本方法准确、灵敏、简便、快速,检出低限可达0.5fg/μL植物总RNA。展开更多
目的:为建立通过测定转换样品T4而检验样品大肠杆菌的新方法,探讨快速测定转换样品中T4的PCR技术。方法:以T4 DNA纯化样品水稀释液和转换样品沸水浴处理产物分别为模板样液,经过两对引物PCR及real time PCR的试用和比较,优选定量检测转...目的:为建立通过测定转换样品T4而检验样品大肠杆菌的新方法,探讨快速测定转换样品中T4的PCR技术。方法:以T4 DNA纯化样品水稀释液和转换样品沸水浴处理产物分别为模板样液,经过两对引物PCR及real time PCR的试用和比较,优选定量检测转换样品中T4的适宜方法和条件。结果:源自T4 ligase的一对引物具有较高的灵敏度和特异性;针对纯化T4DNA配制的模板样液,优化的PCR和real time PCR至少可分别精确检出39.25和3.925 pg/ml的T4DNA;针对沸水浴处理转换样品制备的模板样液,PCR和real time PCR可清楚区别不同浓度的转换样品,PCR的检出极限为500 PFU/ml T4的转换样品,real time PCR的检测极限为35 PFU/ml T4的转换样品。结论:采用PCR和real timePCR可快速定量测定转换样品中的T4含量,real time PCR比PCR的灵敏度高约一个数量级。展开更多
李痘病毒(Plum pox virus,PPV)是我国重要的植物检疫性有害生物。本研究根据PPV中CP基因(coat protein gene)的保守序列,设计了3条PCR引物和1条TaqMan探针,建立了半巢式-RT-RealtimePCR检测PPV的方法。该方法有机地结合了巢式PCR和实时...李痘病毒(Plum pox virus,PPV)是我国重要的植物检疫性有害生物。本研究根据PPV中CP基因(coat protein gene)的保守序列,设计了3条PCR引物和1条TaqMan探针,建立了半巢式-RT-RealtimePCR检测PPV的方法。该方法有机地结合了巢式PCR和实时荧光PCR技术;3条引物形成的2套PCR体系相互验证,有效提高了结果的准确性;荧光探针有效提高了检测的灵敏度。实验结果表明,本方法准确、灵敏、简便、快速,检出低限可达37fg/μL植物总RNA。展开更多
马铃薯黑环斑病毒(Potato black ringspot virus,PBRSV)是马铃薯重要病毒病害之一。本研究根据PBRSV基因组中RNA依赖的RNA聚合酶(RDRP)保守序列,设计合成了两对巢式PCR引物和1条Taq-MAN荧光探针,建立了巢式-多重RT-Realtime PCR检测PBRS...马铃薯黑环斑病毒(Potato black ringspot virus,PBRSV)是马铃薯重要病毒病害之一。本研究根据PBRSV基因组中RNA依赖的RNA聚合酶(RDRP)保守序列,设计合成了两对巢式PCR引物和1条Taq-MAN荧光探针,建立了巢式-多重RT-Realtime PCR检测PBRSV的新方法。该方法采用E.Z.N.ATM试剂盒快速提取植物总RNA,并有机地结合了巢式PCR、多重PCR和探针检测技术。实验结果表明,该方法检测灵敏度可达450fg/μL植物总RNA。利用4对引物和1条TaqMAN探针对PCR阳性产物进行确认,本方法检测的准确性、灵敏度比巢式PCR、单重Realtime PCR等方法高。展开更多
Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without val...Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.展开更多
文摘目的探讨巨细胞病毒(C M V)D N A检测在肾移植术后C M V病预防中的作用。方法采用R ealtim e-PC R定量检测肾移植受者外周血白细胞中C M V-D N A,术后4月内每周1次。结果95例患者中有55例检出C M V-D N A阳性(57.9%),初次检出C M V-D N A的平均时间为(25.6±16.3)d,C M V-D N A的平均拷贝数为(1396.4±445.2)/105白细胞。在随访过程中,18例患者的C M V-D N A水平超过警戒线—800copy/105白细胞,经调整免疫抑制剂后,均降至警戒线以下,所需平均时间为(16.3±8.5)d。所有患者均未出现C M V病。结论C M V-D V A检测可用于预防肾移植术后C M V病的发生。
文摘马铃薯A病毒(Potato virus A,PVA)是我国重要的检疫性有害生物。本研究根据PVA中CP基因(coat protein gene)的保守序列,设计了两套PCR引物和TaqMan探针,建立了双引物探针RT-RealtimePCR检测PVA的方法。该方法采用实时荧光PCR技术,有效地提高了检测的灵敏度;同时两套引物探针相互验证,有效提高了结果的准确性。实验结果表明,本方法准确、灵敏、简便、快速,检出低限可达0.5fg/μL植物总RNA。
文摘目的:为建立通过测定转换样品T4而检验样品大肠杆菌的新方法,探讨快速测定转换样品中T4的PCR技术。方法:以T4 DNA纯化样品水稀释液和转换样品沸水浴处理产物分别为模板样液,经过两对引物PCR及real time PCR的试用和比较,优选定量检测转换样品中T4的适宜方法和条件。结果:源自T4 ligase的一对引物具有较高的灵敏度和特异性;针对纯化T4DNA配制的模板样液,优化的PCR和real time PCR至少可分别精确检出39.25和3.925 pg/ml的T4DNA;针对沸水浴处理转换样品制备的模板样液,PCR和real time PCR可清楚区别不同浓度的转换样品,PCR的检出极限为500 PFU/ml T4的转换样品,real time PCR的检测极限为35 PFU/ml T4的转换样品。结论:采用PCR和real timePCR可快速定量测定转换样品中的T4含量,real time PCR比PCR的灵敏度高约一个数量级。
文摘李痘病毒(Plum pox virus,PPV)是我国重要的植物检疫性有害生物。本研究根据PPV中CP基因(coat protein gene)的保守序列,设计了3条PCR引物和1条TaqMan探针,建立了半巢式-RT-RealtimePCR检测PPV的方法。该方法有机地结合了巢式PCR和实时荧光PCR技术;3条引物形成的2套PCR体系相互验证,有效提高了结果的准确性;荧光探针有效提高了检测的灵敏度。实验结果表明,本方法准确、灵敏、简便、快速,检出低限可达37fg/μL植物总RNA。
基金Supported by the Knowledge Innovation Program of Chinese Academy of Sciences(No.KSCX2-EW-G-12B)the Knowledge Innovation Program of the Chinese Academy of Sciences(No.KZCX2-EW-Q213)the National High Technology Research and Development Program of China (863 Program)(No.2012AA10A412)
文摘Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.
