本研究建立了检测牛副流感病毒3型的SYBR Green Ⅰ Q-PCR方法。根据Gen Bank发表的BPIV3序列进行对比分析,选取P基因上保守区域设计特异性引物。优化反应体系,建立Q-PCR检测方法,并对该方法的特异性、敏感性和重复性进行验证。结果显示...本研究建立了检测牛副流感病毒3型的SYBR Green Ⅰ Q-PCR方法。根据Gen Bank发表的BPIV3序列进行对比分析,选取P基因上保守区域设计特异性引物。优化反应体系,建立Q-PCR检测方法,并对该方法的特异性、敏感性和重复性进行验证。结果显示,构建的标准曲线在10~4~10~8copies.μL^(-1)内具有较好的线性关系,相关系数达到0.998,斜率为-3.370,Q-PCR扩增效率为98%。特异性试验中,利用该方法对BPIV3a及BPIV3c进行检测为阳性,对牛疱疹病毒I型、牛病毒性腹泻病毒进行检测,结果呈阴性。敏感性试验中,该方法对标准品的最小检出量为1.0×10~3 copies.μL^(-1)。重复性试验中,Ct值变异系数均小于1.0%。表明SYBR Green Ⅰ Q-PCR法能够快速地对病原进行诊断,该方法具有较强的特异性、良好的敏感性及重复性,为实验室诊断及定量分析提供了更快速、稳定、可靠的方法。展开更多
采用传统纯培养方法对内蒙古不同地区的12份绵羊奶油样品中的乳酸菌进行分离纯化,运用16S r DNA序列分析方法进行属种鉴定,同时采用实时荧光定量聚合酶链反应(q-PCR)技术对包头地区样品中优势菌群数量进行了定量研究。分离鉴定结果表明...采用传统纯培养方法对内蒙古不同地区的12份绵羊奶油样品中的乳酸菌进行分离纯化,运用16S r DNA序列分析方法进行属种鉴定,同时采用实时荧光定量聚合酶链反应(q-PCR)技术对包头地区样品中优势菌群数量进行了定量研究。分离鉴定结果表明三个地区绵羊发酵奶油中分离鉴定的44株乳酸菌,其中Lactococcus lactis subsp.Lactis为优势菌群。q-PCR定量结果表明3种优势菌属的数量关系为Lac.Lactis.subsp.lactis>L.plantarum>Leu.Mesenteroides。展开更多
Rhizoctonia solani is a soil-borne pathogenic fungus with several distinct isolates that have been classified based on their anastomosis groups （AG＇s）. Many isolates of these fungi contain double-stranded viral RNA...Rhizoctonia solani is a soil-borne pathogenic fungus with several distinct isolates that have been classified based on their anastomosis groups （AG＇s）. Many isolates of these fungi contain double-stranded viral RNA （dsRNA） that are cytoplasmic and viral in origin. Research in our laboratory has studied the epidemiology and molecular biology of viral RNA in R. solani, making it a useful biological model in the development of protocols for the rapid identification of biological agents. In the present study the dsRNA from the isolate EGR-4 which is characteristically large at 3.301 Kb was purified. Attempts to clone middle （M）-size dsRNA fragments from R, solani have been very difficult primarily due to artifacts that co-purify including large （L）-size dsRNA in the fungus. Various MgC12 concentrations were tested to optimize full length dsRNA PCR product. Magnesium is required for DNA polymerase, and EGR-4 requires a specific concentration; thus, several MgC1z concentrations were tested. The dsRNA was analyzed by gel electrophoresis. The gel-purified, nuclease-treated dsRNA was reverse transcribed into cDNA and ligated into the p-jet cloning vector and transformed using E. coli. All such clones were sequenced and forward and reverse primers were generated using BLAST sequence via Biosearch Technology. The plasmids were purified from transformed cultures and amplified using real-time PCR （RTqPCR） with the primers （reverse CCACCGGAAGAGGGAAATCC, forward AGCGCTGACCTTGCTATCGA ATC） and probe （5＇ Fam-AGTGCCGATCAGCCCTCCACCG-BHQ 1 3＇）. The ideal primer/probe concentration was determined through optimization by comparing the lowest threshold concentration （Ct） values using the plasmid cDNA as a template.展开更多
氨氧化反应是硝化作用的关键步骤,参与这一反应的微生物是氨氧化细菌(AOB)和氨氧化古菌(AOA).对新疆艾比湖湿地盐节木根际和非根际土壤的氨氧化微生物进行群落结构和丰度分析,并探究其与土壤理化因子的相关性.同时,以氨单加氧酶基因(amo...氨氧化反应是硝化作用的关键步骤,参与这一反应的微生物是氨氧化细菌(AOB)和氨氧化古菌(AOA).对新疆艾比湖湿地盐节木根际和非根际土壤的氨氧化微生物进行群落结构和丰度分析,并探究其与土壤理化因子的相关性.同时,以氨单加氧酶基因(amo A)为分子标记,构建克隆文库和测序并与q-PCR法结合研究AOA、AOB的群落结构和丰度,利用Pearson相关分析法探究其与环境因子的相关性.结果表明,根际土壤中AOB的多样性高于AOA,amo A基因序列多属于土壤/水体沉积物分支,AOB克隆文库中的所有序列均属于亚硝化单胞菌属(Nitrosomonas).根际土壤中AOA amo A和AOB amo A的数量分别为2.09×104和2.91×105copies·g^(-1),AOB/AOA的比值为13.9;非根际土壤中AOA amo A和AOB amo A的数量分别为3.85×104和4.76×105copies·g^(-1),AOB/AOA的比值为12.36.相关分析显示,氨氧化微生物的群落结构和丰度与电导率(EC)、有机质(OM)、速效氮(AN)、氨氮(NH_4^+-N)和总氮(TN)等环境因子显著相关.这些结果表明,根际土壤中AOB的群落多样性高于AOA,根际和非根际土壤中AOB的丰度均高于AOA,说明在艾比湖湿地AOB是氨氧化微生物的优势种群,且EC、OM、AN、NH_4^+-N和TN可能会影响氨氧化微生物的群落结构和丰度.展开更多
文摘本研究建立了检测牛副流感病毒3型的SYBR Green Ⅰ Q-PCR方法。根据Gen Bank发表的BPIV3序列进行对比分析,选取P基因上保守区域设计特异性引物。优化反应体系,建立Q-PCR检测方法,并对该方法的特异性、敏感性和重复性进行验证。结果显示,构建的标准曲线在10~4~10~8copies.μL^(-1)内具有较好的线性关系,相关系数达到0.998,斜率为-3.370,Q-PCR扩增效率为98%。特异性试验中,利用该方法对BPIV3a及BPIV3c进行检测为阳性,对牛疱疹病毒I型、牛病毒性腹泻病毒进行检测,结果呈阴性。敏感性试验中,该方法对标准品的最小检出量为1.0×10~3 copies.μL^(-1)。重复性试验中,Ct值变异系数均小于1.0%。表明SYBR Green Ⅰ Q-PCR法能够快速地对病原进行诊断,该方法具有较强的特异性、良好的敏感性及重复性,为实验室诊断及定量分析提供了更快速、稳定、可靠的方法。
文摘采用传统纯培养方法对内蒙古不同地区的12份绵羊奶油样品中的乳酸菌进行分离纯化,运用16S r DNA序列分析方法进行属种鉴定,同时采用实时荧光定量聚合酶链反应(q-PCR)技术对包头地区样品中优势菌群数量进行了定量研究。分离鉴定结果表明三个地区绵羊发酵奶油中分离鉴定的44株乳酸菌,其中Lactococcus lactis subsp.Lactis为优势菌群。q-PCR定量结果表明3种优势菌属的数量关系为Lac.Lactis.subsp.lactis>L.plantarum>Leu.Mesenteroides。
文摘Rhizoctonia solani is a soil-borne pathogenic fungus with several distinct isolates that have been classified based on their anastomosis groups （AG＇s）. Many isolates of these fungi contain double-stranded viral RNA （dsRNA） that are cytoplasmic and viral in origin. Research in our laboratory has studied the epidemiology and molecular biology of viral RNA in R. solani, making it a useful biological model in the development of protocols for the rapid identification of biological agents. In the present study the dsRNA from the isolate EGR-4 which is characteristically large at 3.301 Kb was purified. Attempts to clone middle （M）-size dsRNA fragments from R, solani have been very difficult primarily due to artifacts that co-purify including large （L）-size dsRNA in the fungus. Various MgC12 concentrations were tested to optimize full length dsRNA PCR product. Magnesium is required for DNA polymerase, and EGR-4 requires a specific concentration; thus, several MgC1z concentrations were tested. The dsRNA was analyzed by gel electrophoresis. The gel-purified, nuclease-treated dsRNA was reverse transcribed into cDNA and ligated into the p-jet cloning vector and transformed using E. coli. All such clones were sequenced and forward and reverse primers were generated using BLAST sequence via Biosearch Technology. The plasmids were purified from transformed cultures and amplified using real-time PCR （RTqPCR） with the primers （reverse CCACCGGAAGAGGGAAATCC, forward AGCGCTGACCTTGCTATCGA ATC） and probe （5＇ Fam-AGTGCCGATCAGCCCTCCACCG-BHQ 1 3＇）. The ideal primer/probe concentration was determined through optimization by comparing the lowest threshold concentration （Ct） values using the plasmid cDNA as a template.
文摘氨氧化反应是硝化作用的关键步骤,参与这一反应的微生物是氨氧化细菌(AOB)和氨氧化古菌(AOA).对新疆艾比湖湿地盐节木根际和非根际土壤的氨氧化微生物进行群落结构和丰度分析,并探究其与土壤理化因子的相关性.同时,以氨单加氧酶基因(amo A)为分子标记,构建克隆文库和测序并与q-PCR法结合研究AOA、AOB的群落结构和丰度,利用Pearson相关分析法探究其与环境因子的相关性.结果表明,根际土壤中AOB的多样性高于AOA,amo A基因序列多属于土壤/水体沉积物分支,AOB克隆文库中的所有序列均属于亚硝化单胞菌属(Nitrosomonas).根际土壤中AOA amo A和AOB amo A的数量分别为2.09×104和2.91×105copies·g^(-1),AOB/AOA的比值为13.9;非根际土壤中AOA amo A和AOB amo A的数量分别为3.85×104和4.76×105copies·g^(-1),AOB/AOA的比值为12.36.相关分析显示,氨氧化微生物的群落结构和丰度与电导率(EC)、有机质(OM)、速效氮(AN)、氨氮(NH_4^+-N)和总氮(TN)等环境因子显著相关.这些结果表明,根际土壤中AOB的群落多样性高于AOA,根际和非根际土壤中AOB的丰度均高于AOA,说明在艾比湖湿地AOB是氨氧化微生物的优势种群,且EC、OM、AN、NH_4^+-N和TN可能会影响氨氧化微生物的群落结构和丰度.
