目的建立结核分枝杆菌IS6110和IS1081微滴数字PCR（droplet digital PCR，ddPCR）检测体系，并用于不同类型临床样本的检测。方法常规提取13株结核分枝杆菌和14株非结核分枝杆菌临床分离株DNA，9例结核分枝杆菌阳性肺结核患者和4例其他...目的建立结核分枝杆菌IS6110和IS1081微滴数字PCR（droplet digital PCR，ddPCR）检测体系，并用于不同类型临床样本的检测。方法常规提取13株结核分枝杆菌和14株非结核分枝杆菌临床分离株DNA，9例结核分枝杆菌阳性肺结核患者和4例其他肺部疾病患者痰DNA，12例结核分枝杆菌阳性肺结核患者和7例健康者血浆DNA。设计合成IS6110和IS1081扩增引物和检测探针，建立ddPCR检测体系。应用该体系检测分枝杆菌、痰和血浆3种DNA样本中靶标拷贝数，进行统计学分析。结果建立了能同时检测IS6110和IS1081的ddPCR体系，该体系检测2个靶标的线性范围分别为0．5～8733和0．2～2893拷贝／μl反应体系。该体系具有较好的重复性（r〉0．95），以非结核分枝杆菌为对照检测结核分枝杆菌的灵敏度和特异度均为100％。在痰和血浆DNA样本检测中，2个靶标拷贝数在肺结核组均显著高于对照组。结论结核分枝杆菌特异性核酸的ddPCR体系，可用于肺结核患者临床分离株、痰和血浆样本的微量核酸检测，对提高结核病病原学诊断能力有重要意义。展开更多
DNA extracted directly from the living nodules of Casuarina cunninghamiana, C.collina, C.glauca, Alnus cremastogyne, A.trabeculosa and Myrica rubra and also from 21 Frankia strains isolated from the root nodules of th...DNA extracted directly from the living nodules of Casuarina cunninghamiana, C.collina, C.glauca, Alnus cremastogyne, A.trabeculosa and Myrica rubra and also from 21 Frankia strains isolated from the root nodules of the actinorhizal plants in Fujian, including C. cunninghamiana, C.equisetifolia, C.glauca, A.cremastogyne and M.rubra. PCR amplification was conducted with the primers targeting the 3’ end of the 16S rDNA, the IGS, and the 5’ part of the 23S rDNA (i.e.,rrn region). PCR products were then analyzed by using a set of restriction endonucleases. Two distinct genetic groups were recognized on the basis of these restriction patterns. All Frankia strains associated with the host species of Casuarina were assigned to the same group. Frankia living in the nodules of Myrica and Alnus belonged to the other group. In Myrica-Alnus group, there was two sub-group which one included A.cremastogyme and the other contained A.trabeculosa and M.rubra. The results of RFLP analysis showed that the genetic diversity of Frankia associated with Casuarina could be lower, but Frankia existed in the soils of Fujian Province would have more richness in genetic diversity. The results also reflected that host plant has an ability to choose the strains to form a symbiont.展开更多
Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different...Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces （Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi） were tested by direct tissue blot immuno-assay （DTBIA）, and 158 of them were tested positively, which therefore were subjected to coat protein gene （CPG）/Hinf Ⅰ restriction fragment length polymorphism （RFLP） analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.展开更多
文摘目的建立结核分枝杆菌IS6110和IS1081微滴数字PCR（droplet digital PCR，ddPCR）检测体系，并用于不同类型临床样本的检测。方法常规提取13株结核分枝杆菌和14株非结核分枝杆菌临床分离株DNA，9例结核分枝杆菌阳性肺结核患者和4例其他肺部疾病患者痰DNA，12例结核分枝杆菌阳性肺结核患者和7例健康者血浆DNA。设计合成IS6110和IS1081扩增引物和检测探针，建立ddPCR检测体系。应用该体系检测分枝杆菌、痰和血浆3种DNA样本中靶标拷贝数，进行统计学分析。结果建立了能同时检测IS6110和IS1081的ddPCR体系，该体系检测2个靶标的线性范围分别为0．5～8733和0．2～2893拷贝／μl反应体系。该体系具有较好的重复性（r〉0．95），以非结核分枝杆菌为对照检测结核分枝杆菌的灵敏度和特异度均为100％。在痰和血浆DNA样本检测中，2个靶标拷贝数在肺结核组均显著高于对照组。结论结核分枝杆菌特异性核酸的ddPCR体系，可用于肺结核患者临床分离株、痰和血浆样本的微量核酸检测，对提高结核病病原学诊断能力有重要意义。
文摘DNA extracted directly from the living nodules of Casuarina cunninghamiana, C.collina, C.glauca, Alnus cremastogyne, A.trabeculosa and Myrica rubra and also from 21 Frankia strains isolated from the root nodules of the actinorhizal plants in Fujian, including C. cunninghamiana, C.equisetifolia, C.glauca, A.cremastogyne and M.rubra. PCR amplification was conducted with the primers targeting the 3’ end of the 16S rDNA, the IGS, and the 5’ part of the 23S rDNA (i.e.,rrn region). PCR products were then analyzed by using a set of restriction endonucleases. Two distinct genetic groups were recognized on the basis of these restriction patterns. All Frankia strains associated with the host species of Casuarina were assigned to the same group. Frankia living in the nodules of Myrica and Alnus belonged to the other group. In Myrica-Alnus group, there was two sub-group which one included A.cremastogyme and the other contained A.trabeculosa and M.rubra. The results of RFLP analysis showed that the genetic diversity of Frankia associated with Casuarina could be lower, but Frankia existed in the soils of Fujian Province would have more richness in genetic diversity. The results also reflected that host plant has an ability to choose the strains to form a symbiont.
基金supported by the National Natural Science Foundation of China(30471205)under the program"Comparison Research on Strains of Citrus tristeza virus."
文摘Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces （Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi） were tested by direct tissue blot immuno-assay （DTBIA）, and 158 of them were tested positively, which therefore were subjected to coat protein gene （CPG）/Hinf Ⅰ restriction fragment length polymorphism （RFLP） analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.
