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
The rapid and accurate detection of peanuts and soybeans allergen is important to the food safety. In this study, Cu-TCPP nanosheet, a kind of ultra-thin metal-organic framework(MOF)was synthesized and applied in loop...The rapid and accurate detection of peanuts and soybeans allergen is important to the food safety. In this study, Cu-TCPP nanosheet, a kind of ultra-thin metal-organic framework(MOF)was synthesized and applied in loop-mediated isothermal amplification(named Cu-TCPP@LAMP), which can inhibit the non-specific amplification by absorbing and precise temperature releasing of single primer. As thus, Cu-TCPP@LAMP can achieve high sensitivity and specific amplification of the target gene. As a result, peanut and soybean allergens genes contained in food were successfully detected with a favorable detection sensitivity(5 ng/μL for peanuts and 10 ng/μL for soybeans)and reliable repeatability(The coefficient of variation was 3.38% for peanuts and 3.33% for soybeans). Moreover, the established method was utilized for detection of several commercial products, and had a high consistency with the standard method. Apart from food allergens, this novel assay can be widely used in other areas, such as pathogen detection, tumor nucleic acid detection and so on.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the Science and Technology Joint Project of the Yangtze River Delta (19395810100)Shanghai Agriculture Project (19391901500)+2 种基金the National Key Research and Development Program of China (2016YFD0501101)the Shanghai Science and Technology Innovation Foundation (19441903900 and 19XD1433000)Project of National Natural Science Foundation of China (82003705)。
文摘The rapid and accurate detection of peanuts and soybeans allergen is important to the food safety. In this study, Cu-TCPP nanosheet, a kind of ultra-thin metal-organic framework(MOF)was synthesized and applied in loop-mediated isothermal amplification(named Cu-TCPP@LAMP), which can inhibit the non-specific amplification by absorbing and precise temperature releasing of single primer. As thus, Cu-TCPP@LAMP can achieve high sensitivity and specific amplification of the target gene. As a result, peanut and soybean allergens genes contained in food were successfully detected with a favorable detection sensitivity(5 ng/μL for peanuts and 10 ng/μL for soybeans)and reliable repeatability(The coefficient of variation was 3.38% for peanuts and 3.33% for soybeans). Moreover, the established method was utilized for detection of several commercial products, and had a high consistency with the standard method. Apart from food allergens, this novel assay can be widely used in other areas, such as pathogen detection, tumor nucleic acid detection and so on.
