A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAM...A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.展开更多
猫疱疹病毒I型(FHV-1)是威胁猫科动物健康的重要传染性疾病,本研究旨在研发一种灵敏、高效的FHV-1检测技术。依据GenBank上FHV-1 US6保守区域的基因序列,设计合成1对针对FHV-1 gD基因的引物,建立了一种SYBR Green I荧光定量PCR的检测方...猫疱疹病毒I型(FHV-1)是威胁猫科动物健康的重要传染性疾病,本研究旨在研发一种灵敏、高效的FHV-1检测技术。依据GenBank上FHV-1 US6保守区域的基因序列,设计合成1对针对FHV-1 gD基因的引物,建立了一种SYBR Green I荧光定量PCR的检测方法,构建标准曲线后分别验证该方法的特异性、敏感性、重复性,并将其进一步应用于人工感染猫产生的临床样本检测。结果:该特异性引物与猫杯状病毒(FCV)、猫细小病毒(FPV)和猫冠状病毒(FCoV)均未出现交叉反应,检测下限为14.78 copies/μL,组内和组间重复试验的变异系数均低于2%;该方法对临床样本的检出率比常规PCR高出25.46%;通过该方法检测人工感染FHV-1强毒后猫的每日排毒量,结果呈现上升趋势,与临床发病程度相符,猫的脏器病毒载量存在个体差异,但集中在心脏、肺脏、肠道和膀胱中检出。综上,该研究建立的SYBR Green I荧光定量PCR方法对FHV-1具有较好的特异性、灵敏度和重复性,为FHV-1感染的快速诊断以及疾病的防控提供方法支持。展开更多
[Objectives]To develop methods for the early and rapid detection of tomato gray mold.[Methods]Utilizing the ACTIN gene of Botrytis cinerea as the target,a set of specific primers for loop-mediated isothermal amplifica...[Objectives]To develop methods for the early and rapid detection of tomato gray mold.[Methods]Utilizing the ACTIN gene of Botrytis cinerea as the target,a set of specific primers for loop-mediated isothermal amplification(LAMP)was designed and screened.Subsequently,the reaction system and conditions were optimized to achieve rapid isothermal amplification of B.cinerea.[Results]Through agarose gel electrophoresis and SYBR GreenⅠvisualization analysis,the optimal dosages of BstⅡDNA polymerase and dNTPs,as well as the optimal ratio of internal to external primers,were determined to be 0.6 U/μL,1.25 mmol/L,and 2:1,respectively.The specific detection of B.cinerea was accomplished at 61℃ for 40 min,achieving a sensitivity of 100 ag/μL,which is 106 times greater than that of conventional PCR detection.When this method was applied to the detection of tomato diseases,the detection limit for B.cinerea spores reached 20 spores/mL.Furthermore,the pathogen was detectable in tomato leaves that had been infected for 4 d,despite the absence of visible phenotypic symptoms of gray mold.[Conclusions]This method is suitable for the early,rapid,sensitive,and visual detection of tomato gray mold,thereby offering technical support for its early diagnosis,prevention,and control.展开更多
基金funded by the National Key Science and Technology Projects of China(2012ZX10004219 and 2013ZX10004001)
文摘A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.
文摘猫疱疹病毒I型(FHV-1)是威胁猫科动物健康的重要传染性疾病,本研究旨在研发一种灵敏、高效的FHV-1检测技术。依据GenBank上FHV-1 US6保守区域的基因序列,设计合成1对针对FHV-1 gD基因的引物,建立了一种SYBR Green I荧光定量PCR的检测方法,构建标准曲线后分别验证该方法的特异性、敏感性、重复性,并将其进一步应用于人工感染猫产生的临床样本检测。结果:该特异性引物与猫杯状病毒(FCV)、猫细小病毒(FPV)和猫冠状病毒(FCoV)均未出现交叉反应,检测下限为14.78 copies/μL,组内和组间重复试验的变异系数均低于2%;该方法对临床样本的检出率比常规PCR高出25.46%;通过该方法检测人工感染FHV-1强毒后猫的每日排毒量,结果呈现上升趋势,与临床发病程度相符,猫的脏器病毒载量存在个体差异,但集中在心脏、肺脏、肠道和膀胱中检出。综上,该研究建立的SYBR Green I荧光定量PCR方法对FHV-1具有较好的特异性、灵敏度和重复性,为FHV-1感染的快速诊断以及疾病的防控提供方法支持。
基金Supported by Central Guiding Local Science and Technology Development Fund Project of Hebei Province(226Z6501G)Science and Technology Program of Hebei Academy of Sciences(23306,24306,25306).
文摘[Objectives]To develop methods for the early and rapid detection of tomato gray mold.[Methods]Utilizing the ACTIN gene of Botrytis cinerea as the target,a set of specific primers for loop-mediated isothermal amplification(LAMP)was designed and screened.Subsequently,the reaction system and conditions were optimized to achieve rapid isothermal amplification of B.cinerea.[Results]Through agarose gel electrophoresis and SYBR GreenⅠvisualization analysis,the optimal dosages of BstⅡDNA polymerase and dNTPs,as well as the optimal ratio of internal to external primers,were determined to be 0.6 U/μL,1.25 mmol/L,and 2:1,respectively.The specific detection of B.cinerea was accomplished at 61℃ for 40 min,achieving a sensitivity of 100 ag/μL,which is 106 times greater than that of conventional PCR detection.When this method was applied to the detection of tomato diseases,the detection limit for B.cinerea spores reached 20 spores/mL.Furthermore,the pathogen was detectable in tomato leaves that had been infected for 4 d,despite the absence of visible phenotypic symptoms of gray mold.[Conclusions]This method is suitable for the early,rapid,sensitive,and visual detection of tomato gray mold,thereby offering technical support for its early diagnosis,prevention,and control.
