When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment(e.g.heating)is enough to obtain rather sensitive responses....When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment(e.g.heating)is enough to obtain rather sensitive responses.Even test samples without any pretreatment can be used as template.This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies.In this study,using Stx1 gene from E.coli as model,we verified that viable cells,dead cells and extracellular DNA could function as template in the LAMP assay.In the incubation at 63℃,viable bacteria in the LAMP reaction mixture lysed completely within 2 min,providing DNA template for nucleic acid amplification.The Stx1 gene in diluted culture medium,spiked tap water,spiked seawater and real seawater all could be detected,with or without the step of DNA extraction.We found that the complex substances in real sample(e.g.natural seawater)exhibited considerable inhibitory effect on the sensitivity of the LAMP assay.These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring,bio-resource surveys,food safety,etc.in particular those based on environmental DNA.展开更多
Rice bacterial leaf brown spot disease caused by Pseudomonas syringae pv.syringae(Pss)is a major disease on rice.In recent years,Pss has emerged worldwide,seriously affecting rice production.It is very important to es...Rice bacterial leaf brown spot disease caused by Pseudomonas syringae pv.syringae(Pss)is a major disease on rice.In recent years,Pss has emerged worldwide,seriously affecting rice production.It is very important to establish a rapid detection method of Pss for the diagnosis and prevention of this disease.In order to robust and accurately diagnose the rice bacterial leaf brown spot disease in the field and laboratory,an assay system for the Pss was developed in this study,and the specific sequence of hrcN was used as the target,based on loop-mediated isothermal amplification(LAMP).The best detection system was MgSO 48 mmol·L^(-1),Bst DNA polymerase 8 U,dNTP 1.4 mmol·L^(-1),the ratio of internal and outer primers was 2:1,the reaction temperature was 63℃,the reaction time was 45 min,and the lowest sensitivity was 104 CFU·mL^(-1).This results provided an accurate and robust method for laboratory and field diagnosis of bacterial leaf brown spot disease of rice.展开更多
【目的】开发外来入侵生物三裂叶豚草和豚草不同生育期、不同部位的环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,以达到田间快速、准确和高效识别的目的。【方法】以SYBR Green Ⅰ为指示剂,分别针对三裂叶豚草和...【目的】开发外来入侵生物三裂叶豚草和豚草不同生育期、不同部位的环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,以达到田间快速、准确和高效识别的目的。【方法】以SYBR Green Ⅰ为指示剂,分别针对三裂叶豚草和豚草不同发育阶段(幼苗期、生长期、种子期)开展LAMP技术开发。【结果】特异性验证结果显示,所检测杂草的LAMP产物均呈阳性(产生白色沉淀),而与其对照的其他2种杂草的LAMP产物均为阴性(无白色沉淀)。灵敏度检测结果显示,该体系的DNA最低检测限为10^(-10) ng·μL-1,比常规聚合酶链式反应灵敏度高。【结论】本研究建立的LAMP检测体系能有效应用于三裂叶豚草和豚草样本的快速检测,为其快速、高效识别提供技术支撑。展开更多
The purpose of this study was to establish a method for the rapid detection of infectious pancreatic necrosis virus(IPNV,Jasper serotype)using reverse transcription loop-mediated isothermal amplification(RT-LAMP).Four...The purpose of this study was to establish a method for the rapid detection of infectious pancreatic necrosis virus(IPNV,Jasper serotype)using reverse transcription loop-mediated isothermal amplification(RT-LAMP).Four groups of specific primers were designed,according to the genome sequence of a Chinese IPNV isolate ChRtm213.The results showed that primer set B2 had the best amplification effect.When the final concentration of Mg2+was 6 mmol·L-1,dNTPs were 1 mmol·L-1 and betaine was 0.4 mol·L-1,the reaction could be completed in a 63℃water bath within 60 min.This RT-LAMP assay for the detection of IPNV had no cross-reactivity with infectious hematopoietic necrosis virus,viral hemorrhagic septicemia virus,grass carp reovirus and spring viremia of carp virus.The detection limit was 3.2×10-12 ng·μL-1.The sensitivity of this method was 10-fold higher than that of a previously published RT-LAMP assay for detecting the Spajarup(Sp)serotype of IPNV.This method,aimed at detecting IPNV isolates that were currently prevalent in China,possessed the characteristics of strong specificity,high sensitivity and direct interpretation by the naked eyes.The IPNV RT-LAMP was successfully applied to determine the clinical samples,which indicated the IPNV RT-LAMP assay was suitable for the rapid and large-scale detections of IPNV in China.展开更多
文摘When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment(e.g.heating)is enough to obtain rather sensitive responses.Even test samples without any pretreatment can be used as template.This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies.In this study,using Stx1 gene from E.coli as model,we verified that viable cells,dead cells and extracellular DNA could function as template in the LAMP assay.In the incubation at 63℃,viable bacteria in the LAMP reaction mixture lysed completely within 2 min,providing DNA template for nucleic acid amplification.The Stx1 gene in diluted culture medium,spiked tap water,spiked seawater and real seawater all could be detected,with or without the step of DNA extraction.We found that the complex substances in real sample(e.g.natural seawater)exhibited considerable inhibitory effect on the sensitivity of the LAMP assay.These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring,bio-resource surveys,food safety,etc.in particular those based on environmental DNA.
基金Supported by the Natural Science Foundation of Heilongjiang Province(Topic C2017032)Heilongjiang Province Applied Technology Research and Development Program(Topic GA19B104)the National Key Research and Development Program(Topic 2018YFD0300105)。
文摘Rice bacterial leaf brown spot disease caused by Pseudomonas syringae pv.syringae(Pss)is a major disease on rice.In recent years,Pss has emerged worldwide,seriously affecting rice production.It is very important to establish a rapid detection method of Pss for the diagnosis and prevention of this disease.In order to robust and accurately diagnose the rice bacterial leaf brown spot disease in the field and laboratory,an assay system for the Pss was developed in this study,and the specific sequence of hrcN was used as the target,based on loop-mediated isothermal amplification(LAMP).The best detection system was MgSO 48 mmol·L^(-1),Bst DNA polymerase 8 U,dNTP 1.4 mmol·L^(-1),the ratio of internal and outer primers was 2:1,the reaction temperature was 63℃,the reaction time was 45 min,and the lowest sensitivity was 104 CFU·mL^(-1).This results provided an accurate and robust method for laboratory and field diagnosis of bacterial leaf brown spot disease of rice.
文摘桑源阴沟肠杆菌是引起桑树发生枯萎症状的重要病原之一。对桑源阴沟肠杆菌全基因组序列进行分析,得到桑源阴沟肠杆菌特异基因序列,并以该特异基因序列为靶序列,设计筛选合适的环介导等温扩增(loop-mediated isothermal amplification,LAMP)引物组,通过荧光扩增曲线法、PCR产物琼脂糖凝胶电泳法及PCR产物SYBR Green Ⅰ染色法3种判定方法优化反应体系,进而建立桑源阴沟肠杆菌LAMP检测技术和产品。采用Yt4引物组,在60℃、外内引物浓度比为1∶10的最优反应条件下,桑源阴沟肠杆菌阳性质粒检测灵敏度为25×10^6拷贝/μL。在特异性实验中,桑源阴沟肠杆菌菌株的阳性反应能产生S型荧光扩增曲线,电泳法可得到梯形条带,染色法反应液变为绿色,而在其他供试菌株及阴性对照中均未发生这些现象。应用建立的LAMP检测法和普通PCR方法分别对来自4个不同地区的11个桑树枯萎症状样品进行同步平行检测,2种方法的检测结果一致,但LAMP检测法更简单、快捷和灵敏。建立的桑源阴沟肠杆菌LAMP检测方法,为桑源阴沟肠杆菌的快速检测及桑树枯萎病的诊断提供了新技术。
基金Supported by the National Natural Science Foundation of China(31802345)China Postdoctoral Science Foundation(2018M630893)Heilongjiang Province Postdoctoral Science Foundation(LBH-Z18275)。
文摘The purpose of this study was to establish a method for the rapid detection of infectious pancreatic necrosis virus(IPNV,Jasper serotype)using reverse transcription loop-mediated isothermal amplification(RT-LAMP).Four groups of specific primers were designed,according to the genome sequence of a Chinese IPNV isolate ChRtm213.The results showed that primer set B2 had the best amplification effect.When the final concentration of Mg2+was 6 mmol·L-1,dNTPs were 1 mmol·L-1 and betaine was 0.4 mol·L-1,the reaction could be completed in a 63℃water bath within 60 min.This RT-LAMP assay for the detection of IPNV had no cross-reactivity with infectious hematopoietic necrosis virus,viral hemorrhagic septicemia virus,grass carp reovirus and spring viremia of carp virus.The detection limit was 3.2×10-12 ng·μL-1.The sensitivity of this method was 10-fold higher than that of a previously published RT-LAMP assay for detecting the Spajarup(Sp)serotype of IPNV.This method,aimed at detecting IPNV isolates that were currently prevalent in China,possessed the characteristics of strong specificity,high sensitivity and direct interpretation by the naked eyes.The IPNV RT-LAMP was successfully applied to determine the clinical samples,which indicated the IPNV RT-LAMP assay was suitable for the rapid and large-scale detections of IPNV in China.
