Plague caused by Yersinia pestis is one of the infectious diseases subject to the International Health Regulations (IHR). Permanent monitoring of the focal plague areas is mandatory in order to enable prompt control m...Plague caused by Yersinia pestis is one of the infectious diseases subject to the International Health Regulations (IHR). Permanent monitoring of the focal plague areas is mandatory in order to enable prompt control measures to prevent the spread of the disease. Therefore, the availability of efficient diagnosis tests is of paramount importance. Here, we describe a loop-mediated isothermal amplification (LAMP)-based procedure for rapid Y. pestis detection. We constructed a set of LAMP primers, which were used in assays to establish the reaction conditions that would lead to the quick visualization of the results by evaluating the test tube with the naked eye. The primers were specifically designed to target the caf1 gene located on pFra/Tox (pMT), a prototypical plasmid of Y. pestis. The LAMP procedure was performed at 65°C for 45 min in a water bath and allowed for the detection of at least 10 pg of bacterial DNA. Due to its simplicity, specificity, sensitivity and rapidity, the LAMP technique is an additional tool that may be implemented in routine plague diagnoses, especially in emergencies.展开更多
A loop-mediated isothermal amplification （LAMP） assay was designed and evaluated for rapid de- tection of the toxic microalgae Alexandrium catenella and A. minutum, which can produce paralytic shellfish poisoning （...A loop-mediated isothermal amplification （LAMP） assay was designed and evaluated for rapid de- tection of the toxic microalgae Alexandrium catenella and A. minutum, which can produce paralytic shellfish poisoning （PSP）. Two sets of four specific primers targeting these two species were derived from the sequence of internal transcribed spacer （ITS） of ribosomal DNA. The method worked well in less than an hour under isothermal conditions of 65℃. LAMP specificity was validated in closely related algae as a comparison, suggesting the strict specificity of the LAMP primers. Two visual inspection approaches were feasible to interpret the positive or negative results. The detection lim- its of A. catenella and A. minutum samples using the LAMP assay were found to be 5.6 and 4.5 pg DNA, respectively. The sensitivity of this LAMP assay was 10 or 100-fold higher than Polymerase Chain Reaction （PCR） method in detecting the two microalgae. These characteristics of species specificity, sensitivity, and rapidity suggest that this method has the potentiality in the monitoring of red tide caused by A. catenella and A. minutum.展开更多
Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting ...Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in nonhuman primate laboratory animals. Materials and Methods A total of 205 fecal samples of rhesus monkeys were detected in this LAMP assay. The specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed,and real-time polymerase chain reaction( REAL-TIME PCR) assay was employed as control. Results The LAMP method established here needed only 45 min to complete the reaction at 63℃. Its detection limit was 10 pg / μL and with a high specificity. The positive rate of Shigella and Salmonella was 1. 5% and 6. 3%,respectively. Conclusions Here we have established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for rapid detection of bacterial disease in macaques. The development of this rapid detection kit is underway,and it will be helpful to the diarrhea detection.展开更多
目的为快速检测微小隐孢子虫,建立并优化了以SYBR Green I显色的环介导等温扩增快速检测隐孢子虫的方法。方法根据微小隐孢子虫18SrRNA基因的4条特异LAMP引物,设计2条环引物,建立了微小隐孢子虫LAMP检测方法。结果该方法省去95℃热变性...目的为快速检测微小隐孢子虫,建立并优化了以SYBR Green I显色的环介导等温扩增快速检测隐孢子虫的方法。方法根据微小隐孢子虫18SrRNA基因的4条特异LAMP引物,设计2条环引物,建立了微小隐孢子虫LAMP检测方法。结果该方法省去95℃热变性和80℃酶失活过程,其最佳反应温度和时间是63℃和60min,Betaine、MgSO4、dNTP Mixture、Bst DNA聚合酶大片段的最佳浓度分别是0.8mol/L、8mmol/L、0.8mmol/L、8U/25μL,内外引物浓度分别为1.2μmol/L、0.2μmol/L,添加环引物后体系反应时间缩短为20min。对贾第虫、肠阿米巴、柔嫩艾美耳球虫、类圆线虫检测为阴性。该方法简便、快速,无需特殊设备。结论和常规PCR方法比较,LAMP检测隐孢子虫的灵敏度提高了100倍。该方法的建立为野外和临床隐孢子虫的快速检测提供技术手段。展开更多
文摘Plague caused by Yersinia pestis is one of the infectious diseases subject to the International Health Regulations (IHR). Permanent monitoring of the focal plague areas is mandatory in order to enable prompt control measures to prevent the spread of the disease. Therefore, the availability of efficient diagnosis tests is of paramount importance. Here, we describe a loop-mediated isothermal amplification (LAMP)-based procedure for rapid Y. pestis detection. We constructed a set of LAMP primers, which were used in assays to establish the reaction conditions that would lead to the quick visualization of the results by evaluating the test tube with the naked eye. The primers were specifically designed to target the caf1 gene located on pFra/Tox (pMT), a prototypical plasmid of Y. pestis. The LAMP procedure was performed at 65°C for 45 min in a water bath and allowed for the detection of at least 10 pg of bacterial DNA. Due to its simplicity, specificity, sensitivity and rapidity, the LAMP technique is an additional tool that may be implemented in routine plague diagnoses, especially in emergencies.
基金The Science and Technology Commission of Shanghai Municipality under contract Nos 06235810108DZ1980802 and 10JC1418600a special research fund for the national non-profit institutes (East China Sea Fisheries Research Institute) under contract Nos 2007M22 and 2007Z01
文摘A loop-mediated isothermal amplification （LAMP） assay was designed and evaluated for rapid de- tection of the toxic microalgae Alexandrium catenella and A. minutum, which can produce paralytic shellfish poisoning （PSP）. Two sets of four specific primers targeting these two species were derived from the sequence of internal transcribed spacer （ITS） of ribosomal DNA. The method worked well in less than an hour under isothermal conditions of 65℃. LAMP specificity was validated in closely related algae as a comparison, suggesting the strict specificity of the LAMP primers. Two visual inspection approaches were feasible to interpret the positive or negative results. The detection lim- its of A. catenella and A. minutum samples using the LAMP assay were found to be 5.6 and 4.5 pg DNA, respectively. The sensitivity of this LAMP assay was 10 or 100-fold higher than Polymerase Chain Reaction （PCR） method in detecting the two microalgae. These characteristics of species specificity, sensitivity, and rapidity suggest that this method has the potentiality in the monitoring of red tide caused by A. catenella and A. minutum.
文摘Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in nonhuman primate laboratory animals. Materials and Methods A total of 205 fecal samples of rhesus monkeys were detected in this LAMP assay. The specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed,and real-time polymerase chain reaction( REAL-TIME PCR) assay was employed as control. Results The LAMP method established here needed only 45 min to complete the reaction at 63℃. Its detection limit was 10 pg / μL and with a high specificity. The positive rate of Shigella and Salmonella was 1. 5% and 6. 3%,respectively. Conclusions Here we have established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for rapid detection of bacterial disease in macaques. The development of this rapid detection kit is underway,and it will be helpful to the diarrhea detection.
文摘目的为快速检测微小隐孢子虫,建立并优化了以SYBR Green I显色的环介导等温扩增快速检测隐孢子虫的方法。方法根据微小隐孢子虫18SrRNA基因的4条特异LAMP引物,设计2条环引物,建立了微小隐孢子虫LAMP检测方法。结果该方法省去95℃热变性和80℃酶失活过程,其最佳反应温度和时间是63℃和60min,Betaine、MgSO4、dNTP Mixture、Bst DNA聚合酶大片段的最佳浓度分别是0.8mol/L、8mmol/L、0.8mmol/L、8U/25μL,内外引物浓度分别为1.2μmol/L、0.2μmol/L,添加环引物后体系反应时间缩短为20min。对贾第虫、肠阿米巴、柔嫩艾美耳球虫、类圆线虫检测为阴性。该方法简便、快速,无需特殊设备。结论和常规PCR方法比较,LAMP检测隐孢子虫的灵敏度提高了100倍。该方法的建立为野外和临床隐孢子虫的快速检测提供技术手段。
