Pectobacterium carotovorum is the causal agent of bacterial soft rot in a wide range of vegetable host species.Once P.carotovorum infects the plant,the spread of the disease is difficult to control.In this study,a rap...Pectobacterium carotovorum is the causal agent of bacterial soft rot in a wide range of vegetable host species.Once P.carotovorum infects the plant,the spread of the disease is difficult to control.In this study,a rapid and sensitive method based on loop-mediated isothermal amplification(LAMP)was developed for detecting P.carotovorum in celery with soft rot using a primer set designed from the pmrA conserved sequence of P.carotovorum.The specificity of the LAMP primer set for P.carotovorum was extensively validated on both P.carotovorum strains and nontarget strains.The sensitivity was 1 pg of P.carotovorum genomic DNA,which demonstrated 10 times more sensitive than the conventional PCR assay.LAMP was also used to detect P.carotovorum in bacterial suspension.The lowest detection concentration was 104 CFU·mL^−1.In addition,a LAMP assay,in conjunction with a crude DNA extraction method,was successfully performed on P.carotovorum-infected samples derived from both artificially and naturally infected plants.In summary,the LAMP assay established in this study constitutes a simple,sensitive,and rapid method for the detection of P.carotovorum,and has potential application in the control of celery soft rot disease through early detection.展开更多
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
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
In the last decade,some disease occurred on our experimental farms that had caused serious losses.They were not caused by fungi,bacteria or viruses.By loop-mediated isothermal amplification(LAMP)technique,the detectio...In the last decade,some disease occurred on our experimental farms that had caused serious losses.They were not caused by fungi,bacteria or viruses.By loop-mediated isothermal amplification(LAMP)technique,the detection results pointed to the possible pathogen as phytoplasma.The investigation results implied that phytoplasmas could cause more than 13 kinds of symptoms in almost all parts of plants in B.napus L.,including witches’broom,multi-stems,aggregate main inflorescences,and flat stems.The incidences of these phytoplasma-associated diseases in our experimental farms rose from 1.61%in 2010 to 6.00%in 2021.Some phytoplasma infected plants died without any growing points.These studies would be helpful for detecting phytoplasmas diseases,selecting disease resistant germplasm and improving varieties with disease resistances in B.napus L.展开更多
Objective:To develop a loop-mediated isothermal amplification(LAMP) assay for the detection of Entamoeba histolytica(E.histolytica),the causative agent of amebiasis.Methods:The LAMP primer set was designed from E.hist...Objective:To develop a loop-mediated isothermal amplification(LAMP) assay for the detection of Entamoeba histolytica(E.histolytica),the causative agent of amebiasis.Methods:The LAMP primer set was designed from E.histolytica hemolysin gene HLY6.Genomic DNA of E.histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions.Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye.Results:Positive LAMP reactions turned turbid while negative ones remained clear.Upon addition of a fluorescent dye,all positive reactions turned green while the negative control remained orange under ambient light After elecrophoresis in 1.5% agarose gels,a ladder of multiple bands of different sizes can be oliserved in positive samples while no bands were detected in the negative control.The sensitivity of the assay was found to be S parasites per reaction which corresponds to approximately 1S.8 ng/μL DNA.The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species,Entamoeba dispar. and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used.Conclusions:The I.AMP assay we have developed enables the detection of E.histolytica with rapidity and ease,therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.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
With the increasing global threat of various diseases and infections,it is essential to develop a fast,low-cost,and easy-to-use point-of-care testing(POCT)system for inspections at all levels of medical institutions a...With the increasing global threat of various diseases and infections,it is essential to develop a fast,low-cost,and easy-to-use point-of-care testing(POCT)system for inspections at all levels of medical institutions and self-examination at home.In this work,gold magnetic nanoparticles(GMNPs)are used as the key material,and a rapid visual detection method is designed through integrating loop-mediated isothermal amplification(LAMP)and lateral flow assay(LFA)biosensor for detecting a variety of analytes which includes whole blood,buccal swabs,and DNA.It is worth to note that the proposed method does not need DNA extraction.Furthermore,uracil DNA glycosylase(UDG)is employed to eliminate carrier contamination for preventing false positive results.The whole detection process can be finished within 25 min.The accuracy of detection is measured by assessing the polymorphisms of the methylenetetrahydrofolate reductase(MTHFR)C677T.The detection limit of the newly developed extraction-free detection system for MTHFR C677T is 0.16 ng/μL.A preliminary clinical study of the proposed method is carried out by analyzing 600 clinical samples(including 200 whole blood samples,100 buccal swabs,and 300 genomic DNA samples).The results indicate that the proposed method is 100%consistent with the sequencing results which provides a new choice for POCT and shows a broad application prospect in all levels of medical clinics and at home.展开更多
BackgroundSchistosomiasis remains a public health issue and the need for accurate and affordable diagnostics is crucial in the elimination of the disease. While molecular diagnostics are highly effective, they are exp...BackgroundSchistosomiasis remains a public health issue and the need for accurate and affordable diagnostics is crucial in the elimination of the disease. While molecular diagnostics are highly effective, they are expensive, with the main costs been associated with DNA extraction. The DNA dipstick is a rapid, affordable and simple purification method that allows DNA to be extracted from diagnostic samples within 30 s. We aimed to optimise the DNA dipstick method for samples from mice and egg-spiked human samples.MethodsUrine, blood and faeces were collected from mice exposed to Schistosoma japonicum infection at weekly intervals from Day 0 to Day 42. Urine and faecal samples were also collected from volunteer, uninfected humans and spiked with S. japonicum eggs. All samples were subject to several optimisation procedures and DNA extracted with the DNA dipstick. Amplification of the target DNA was carried out using LAMP and visualised using agarose gel electrophoresis and flocculation.ResultsThe DNA dipstick successfully identified S. japonicum from infected mice and human clinical samples spiked with cracked eggs or genomic DNA from S. japonicum. Amplification was observed from week 4 post infection in infected mice. For human samples, amplification was observed in sieved faecal samples, filtered urine samples heated at 95 ℃ for 30 min, and sera samples heated at 95℃ for 30 min.ConclusionsThe DNA dipstick combined with LAMP has huge potential in providing cost-effective, simple and accurate detection of schistosomiasis infection in endemic regions. This will allow for rapid treatment, tracking outbreaks—such as occur after typhoons, leading to better health outcomes and contributing to control and eventual elimination of schistosomiasis.展开更多
The infrastructure needed to detect Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)infection(COVID-19)that complies completely withWHO guidelines is lacking across many parts of the globe,especially in dev...The infrastructure needed to detect Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)infection(COVID-19)that complies completely withWHO guidelines is lacking across many parts of the globe,especially in developing countries,including Nepal.We outline the problems faced by such countries and suggest that the national and international community should collaborate in the development and adoption of novel protocols for the rapid detection of COVID-19 according to locally available infrastructure,in order to fight against the outbreak.展开更多
基金the earmarked fund for Beijing Innovation Consortium of Agriculture Research System(Grant No.BAIC-2019)Key Laboratory of Biology and Genetic Improvement of Horticultural Crops,Ministry of Agriculture,P.R.China,and Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(Grant No.CAAS-ASTIPIVFCAAS).
文摘Pectobacterium carotovorum is the causal agent of bacterial soft rot in a wide range of vegetable host species.Once P.carotovorum infects the plant,the spread of the disease is difficult to control.In this study,a rapid and sensitive method based on loop-mediated isothermal amplification(LAMP)was developed for detecting P.carotovorum in celery with soft rot using a primer set designed from the pmrA conserved sequence of P.carotovorum.The specificity of the LAMP primer set for P.carotovorum was extensively validated on both P.carotovorum strains and nontarget strains.The sensitivity was 1 pg of P.carotovorum genomic DNA,which demonstrated 10 times more sensitive than the conventional PCR assay.LAMP was also used to detect P.carotovorum in bacterial suspension.The lowest detection concentration was 104 CFU·mL^−1.In addition,a LAMP assay,in conjunction with a crude DNA extraction method,was successfully performed on P.carotovorum-infected samples derived from both artificially and naturally infected plants.In summary,the LAMP assay established in this study constitutes a simple,sensitive,and rapid method for the detection of P.carotovorum,and has potential application in the control of celery soft rot disease through early detection.
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 financial support provided by project of Science and Technology Tackling Key Problems in Henan Province(Grant No.182102110430)the National Key Research and Development Program of China(Grant No.2016YFD0101300)by China Agricultural Research system(CARS-12).
文摘In the last decade,some disease occurred on our experimental farms that had caused serious losses.They were not caused by fungi,bacteria or viruses.By loop-mediated isothermal amplification(LAMP)technique,the detection results pointed to the possible pathogen as phytoplasma.The investigation results implied that phytoplasmas could cause more than 13 kinds of symptoms in almost all parts of plants in B.napus L.,including witches’broom,multi-stems,aggregate main inflorescences,and flat stems.The incidences of these phytoplasma-associated diseases in our experimental farms rose from 1.61%in 2010 to 6.00%in 2021.Some phytoplasma infected plants died without any growing points.These studies would be helpful for detecting phytoplasmas diseases,selecting disease resistant germplasm and improving varieties with disease resistances in B.napus L.
基金supported financially by a research grant from the Natural Sciences Research Institute,University of the Philippines ) BIO 1 l-l-05) to W.L.R.
文摘Objective:To develop a loop-mediated isothermal amplification(LAMP) assay for the detection of Entamoeba histolytica(E.histolytica),the causative agent of amebiasis.Methods:The LAMP primer set was designed from E.histolytica hemolysin gene HLY6.Genomic DNA of E.histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions.Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye.Results:Positive LAMP reactions turned turbid while negative ones remained clear.Upon addition of a fluorescent dye,all positive reactions turned green while the negative control remained orange under ambient light After elecrophoresis in 1.5% agarose gels,a ladder of multiple bands of different sizes can be oliserved in positive samples while no bands were detected in the negative control.The sensitivity of the assay was found to be S parasites per reaction which corresponds to approximately 1S.8 ng/μL DNA.The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species,Entamoeba dispar. and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used.Conclusions:The I.AMP assay we have developed enables the detection of E.histolytica with rapidity and ease,therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.
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.
基金This work was supported by the National Natural Science Foundation of China(Nos.31771083 and 81772289).
文摘With the increasing global threat of various diseases and infections,it is essential to develop a fast,low-cost,and easy-to-use point-of-care testing(POCT)system for inspections at all levels of medical institutions and self-examination at home.In this work,gold magnetic nanoparticles(GMNPs)are used as the key material,and a rapid visual detection method is designed through integrating loop-mediated isothermal amplification(LAMP)and lateral flow assay(LFA)biosensor for detecting a variety of analytes which includes whole blood,buccal swabs,and DNA.It is worth to note that the proposed method does not need DNA extraction.Furthermore,uracil DNA glycosylase(UDG)is employed to eliminate carrier contamination for preventing false positive results.The whole detection process can be finished within 25 min.The accuracy of detection is measured by assessing the polymorphisms of the methylenetetrahydrofolate reductase(MTHFR)C677T.The detection limit of the newly developed extraction-free detection system for MTHFR C677T is 0.16 ng/μL.A preliminary clinical study of the proposed method is carried out by analyzing 600 clinical samples(including 200 whole blood samples,100 buccal swabs,and 300 genomic DNA samples).The results indicate that the proposed method is 100%consistent with the sequencing results which provides a new choice for POCT and shows a broad application prospect in all levels of medical clinics and at home.
文摘BackgroundSchistosomiasis remains a public health issue and the need for accurate and affordable diagnostics is crucial in the elimination of the disease. While molecular diagnostics are highly effective, they are expensive, with the main costs been associated with DNA extraction. The DNA dipstick is a rapid, affordable and simple purification method that allows DNA to be extracted from diagnostic samples within 30 s. We aimed to optimise the DNA dipstick method for samples from mice and egg-spiked human samples.MethodsUrine, blood and faeces were collected from mice exposed to Schistosoma japonicum infection at weekly intervals from Day 0 to Day 42. Urine and faecal samples were also collected from volunteer, uninfected humans and spiked with S. japonicum eggs. All samples were subject to several optimisation procedures and DNA extracted with the DNA dipstick. Amplification of the target DNA was carried out using LAMP and visualised using agarose gel electrophoresis and flocculation.ResultsThe DNA dipstick successfully identified S. japonicum from infected mice and human clinical samples spiked with cracked eggs or genomic DNA from S. japonicum. Amplification was observed from week 4 post infection in infected mice. For human samples, amplification was observed in sieved faecal samples, filtered urine samples heated at 95 ℃ for 30 min, and sera samples heated at 95℃ for 30 min.ConclusionsThe DNA dipstick combined with LAMP has huge potential in providing cost-effective, simple and accurate detection of schistosomiasis infection in endemic regions. This will allow for rapid treatment, tracking outbreaks—such as occur after typhoons, leading to better health outcomes and contributing to control and eventual elimination of schistosomiasis.
文摘The infrastructure needed to detect Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)infection(COVID-19)that complies completely withWHO guidelines is lacking across many parts of the globe,especially in developing countries,including Nepal.We outline the problems faced by such countries and suggest that the national and international community should collaborate in the development and adoption of novel protocols for the rapid detection of COVID-19 according to locally available infrastructure,in order to fight against the outbreak.