菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)是我国进境植物检疫性有害生物。为解决普通RT-PCR不能直接检测大豆病种子中该病毒的问题,将简便快速的胶体金免疫层析技术(GICA)和高灵敏度的普通RT-PCR检测技术有机地结合起来,建立了GICA-...菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)是我国进境植物检疫性有害生物。为解决普通RT-PCR不能直接检测大豆病种子中该病毒的问题,将简便快速的胶体金免疫层析技术(GICA)和高灵敏度的普通RT-PCR检测技术有机地结合起来,建立了GICA-RT-PCR检测新方法。即先用GICA捕获病毒,将捕获的病毒直接进行RT-PCR扩增,简化了检测的步骤,提高了检测的灵敏度。结果表明:用该方法检测提纯病毒灵敏度达到0.01μg.mL-1、检测大豆病叶和病大豆种皮,灵敏度达到10-4,分别比GICA检测提高了20倍、10倍和100倍。GICA-RT-PCR可进一步确认GICA的结果。展开更多
采用RT-PCR、RT-半巢式PCR(seminested PCR)和RT-环介导等温扩增(LAMP)三种分子生物学方法分别检测了牡蛎中经粪便污染的诺如病毒。三种方法所采用的特异性引物均针对诺如病毒高度保守的N/S结构域。结果显示:一步法RT-PCR较两步法结果...采用RT-PCR、RT-半巢式PCR(seminested PCR)和RT-环介导等温扩增(LAMP)三种分子生物学方法分别检测了牡蛎中经粪便污染的诺如病毒。三种方法所采用的特异性引物均针对诺如病毒高度保守的N/S结构域。结果显示:一步法RT-PCR较两步法结果理想但仍不能有效去除食品中的PCR反应抑制物。RT-半巢式PCR和RT-LAMP的特异性和敏感性都远远优于RT-PCR,但是RT-半巢式PCR操作繁琐费时。RT-LAMP扩增程序简单、反应时间短,且不需要精密的温度循环装置,在产物中加入SYBR Green Ⅰ染料后可用肉眼直接判断反应结果。因此,RT-LAMP有望发展成为快速检测牡蛎中诺如病毒的有效手段。展开更多
[Objective] This study aimed to clone and analyze the sequence of CHS gene from Acer truncatum leaves. [Method] Using A. truncatum cultivars No.1-6 as experimental materials, total RNA was extracted from A. truncatum ...[Objective] This study aimed to clone and analyze the sequence of CHS gene from Acer truncatum leaves. [Method] Using A. truncatum cultivars No.1-6 as experimental materials, total RNA was extracted from A. truncatum leaves with the modified CTAB method. CHS gene sequences were downloaded from the NCBI and aligned by BLAST. Degenerate primers were designed by DNAMAN and Primer- premier5 to amplify the target band. CHS gene fragment was amplified by RT-PCR and ligated to pMD18-T vector. The identified positive colonies were sequenced. [Result] A 1 365 bp fragment was amplified. Sequence analysis suggested that the obtained fragment encoded 365 amino acids and shared above 90% homology to nucleotide sequence of CHS gene from A. palmatum and A. [Conclusion] In this study, CHS gene was successfully cloned from A. truncatum for the first time, which laid the foundation for efficient utilization of CHS gene.展开更多
Objective: To investigate the differen t expression of three isozymes of nitric oxide synthase (NOS) in diabetic rat colo ns and the contribution to the colonic dysfunction. Methods: Sprague-Dawley (SD) rats were us...Objective: To investigate the differen t expression of three isozymes of nitric oxide synthase (NOS) in diabetic rat colo ns and the contribution to the colonic dysfunction. Methods: Sprague-Dawley (SD) rats were used in this experiment and diabetes were induc ed by streptozotocin (65 mg/kg, i.v.). Three isozymes of NOS (nNOS, iNOS and eNO S) expression in proximal and distal colon were measured in two weeks after diab etes induction using the methods of immunocytochemistry and semi-quantitative r everse transcription-polymerase chain reaction (RT-PCR). Results: Positive immunoreactivity for nNOS was found in intermuscular and submucous plexus neuronal cells, neither eNOS nor iNOS had been found in any layers of col on in the two groups. The expression of nNOS mRNA was significantly increased in diabetic colon than that in control rats as determined by RT-PCR. The eNOS mRN A level of diabetic colon was lower compared to thecontrol rats, while no expres sion of iNOS mRNA was found in the normal or diabetic rats. Conclusion: This report has demonstrated that nNOS increased and eNOS decreased in rat colon in the early stages of diabetes. NO production by the nNOS might play a key role in colonic dysfunction, as supported by raised nNOS mRNA and enzyme e xpression in the diabetic colon. Reduced eNOS activity might also contribute to colonic dysfunction in experimental diabetes.展开更多
Objective To investigate the dynamic change of coxsackievirus B3 (CVB3) in murine model ofchronic myocarditis and revel the molecular mechanism of the persistent infection of the tirus.Methods Strand - specific RT- PC...Objective To investigate the dynamic change of coxsackievirus B3 (CVB3) in murine model ofchronic myocarditis and revel the molecular mechanism of the persistent infection of the tirus.Methods Strand - specific RT- PCR(ssRT- PCR), quantitative RT- PCR (qRT - PCR) and multiplexRT- PCR(mRT- PCR). Results The positive strand of CVB3 RNA existed in heart tissue up to 3 monthsalthough its amount decreased by 103～4 folds from acute to chronic phase. The negative strand RNA for virusreplication kept its amount on la moleculars per gram heart tissue. Some conserved areas of virus RNA 5’NTRand 3’NTR were lost in chronic phase. Conclusion The virus kept replication during the whole phase ofmyocarditis and speeded down on chronic period in the status of persistent infection. That may be due to theterminal lose of CVB RNA.展开更多
文摘菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)是我国进境植物检疫性有害生物。为解决普通RT-PCR不能直接检测大豆病种子中该病毒的问题,将简便快速的胶体金免疫层析技术(GICA)和高灵敏度的普通RT-PCR检测技术有机地结合起来,建立了GICA-RT-PCR检测新方法。即先用GICA捕获病毒,将捕获的病毒直接进行RT-PCR扩增,简化了检测的步骤,提高了检测的灵敏度。结果表明:用该方法检测提纯病毒灵敏度达到0.01μg.mL-1、检测大豆病叶和病大豆种皮,灵敏度达到10-4,分别比GICA检测提高了20倍、10倍和100倍。GICA-RT-PCR可进一步确认GICA的结果。
文摘采用RT-PCR、RT-半巢式PCR(seminested PCR)和RT-环介导等温扩增(LAMP)三种分子生物学方法分别检测了牡蛎中经粪便污染的诺如病毒。三种方法所采用的特异性引物均针对诺如病毒高度保守的N/S结构域。结果显示:一步法RT-PCR较两步法结果理想但仍不能有效去除食品中的PCR反应抑制物。RT-半巢式PCR和RT-LAMP的特异性和敏感性都远远优于RT-PCR,但是RT-半巢式PCR操作繁琐费时。RT-LAMP扩增程序简单、反应时间短,且不需要精密的温度循环装置,在产物中加入SYBR Green Ⅰ染料后可用肉眼直接判断反应结果。因此,RT-LAMP有望发展成为快速检测牡蛎中诺如病毒的有效手段。
基金Supported by Agricultural Improved Variety Project of Shandong Province(LKZ[2014]No.96)
文摘[Objective] This study aimed to clone and analyze the sequence of CHS gene from Acer truncatum leaves. [Method] Using A. truncatum cultivars No.1-6 as experimental materials, total RNA was extracted from A. truncatum leaves with the modified CTAB method. CHS gene sequences were downloaded from the NCBI and aligned by BLAST. Degenerate primers were designed by DNAMAN and Primer- premier5 to amplify the target band. CHS gene fragment was amplified by RT-PCR and ligated to pMD18-T vector. The identified positive colonies were sequenced. [Result] A 1 365 bp fragment was amplified. Sequence analysis suggested that the obtained fragment encoded 365 amino acids and shared above 90% homology to nucleotide sequence of CHS gene from A. palmatum and A. [Conclusion] In this study, CHS gene was successfully cloned from A. truncatum for the first time, which laid the foundation for efficient utilization of CHS gene.
文摘Objective: To investigate the differen t expression of three isozymes of nitric oxide synthase (NOS) in diabetic rat colo ns and the contribution to the colonic dysfunction. Methods: Sprague-Dawley (SD) rats were used in this experiment and diabetes were induc ed by streptozotocin (65 mg/kg, i.v.). Three isozymes of NOS (nNOS, iNOS and eNO S) expression in proximal and distal colon were measured in two weeks after diab etes induction using the methods of immunocytochemistry and semi-quantitative r everse transcription-polymerase chain reaction (RT-PCR). Results: Positive immunoreactivity for nNOS was found in intermuscular and submucous plexus neuronal cells, neither eNOS nor iNOS had been found in any layers of col on in the two groups. The expression of nNOS mRNA was significantly increased in diabetic colon than that in control rats as determined by RT-PCR. The eNOS mRN A level of diabetic colon was lower compared to thecontrol rats, while no expres sion of iNOS mRNA was found in the normal or diabetic rats. Conclusion: This report has demonstrated that nNOS increased and eNOS decreased in rat colon in the early stages of diabetes. NO production by the nNOS might play a key role in colonic dysfunction, as supported by raised nNOS mRNA and enzyme e xpression in the diabetic colon. Reduced eNOS activity might also contribute to colonic dysfunction in experimental diabetes.
文摘Objective To investigate the dynamic change of coxsackievirus B3 (CVB3) in murine model ofchronic myocarditis and revel the molecular mechanism of the persistent infection of the tirus.Methods Strand - specific RT- PCR(ssRT- PCR), quantitative RT- PCR (qRT - PCR) and multiplexRT- PCR(mRT- PCR). Results The positive strand of CVB3 RNA existed in heart tissue up to 3 monthsalthough its amount decreased by 103～4 folds from acute to chronic phase. The negative strand RNA for virusreplication kept its amount on la moleculars per gram heart tissue. Some conserved areas of virus RNA 5’NTRand 3’NTR were lost in chronic phase. Conclusion The virus kept replication during the whole phase ofmyocarditis and speeded down on chronic period in the status of persistent infection. That may be due to theterminal lose of CVB RNA.
