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Establishment of an Improved loop-mediated isothermal amplification Technique for Visual Detection of Brucella

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Medicinal Plant 2022年第2期13卷 18-20,36页

作者： Sixi YANG Kexin ZHANG Jinxia LIU Experimental Center of Pathogenic Biology Chengde Medical University Chengde 067000 China

[Objectives]loop-mediated isothermal amplification(LAMP)was developed and optimized for rapid and specific diagnosis of Brucella infection.[Methods]The conserved sequence of the brucellosis standard strain Omp25 gene fragment was selected,primers needed in the loop-mediated isothermal amplification system were designed,and nucleic acid of the standard strain was ***,reaction system,reaction conditions and stain types were repeatedly optimized to establish an improved LAMP detection kit for *** tests were performed on blood samples from clinically confirmed cases and their sensitivity and specificity were ***,the detection technology and kit were applied to the detection and screening of suspected *** in pastoral ***,SPSS21.0 was used to evaluate the consistency of LAMP and Q-PCR.[Results]LAMP system was optimized,and the optimal final concentrations of MgSO_(4) and betaine were 8 and 0.8 mmol/L,*** sensitivity and specificity of the modified LAMP technique were verified by using human blood samples with confirmed *** LAMP specific test,there was no nonspecific amplification,indicating good *** the LAMP sensitivity test,the minimum detection line for ***,***,and *** was 10 copies/μL,which was an order of magnitude higher than that for *** consistency between modified LAMP and Q-PCR was *** established Brucella LAMP kit was used to detect the blood samples of 200 sheep suspected to be infected with *** LAMP kit detected 42 positive samples,with a positive rate of 21%and a detection sensitivity of 100%.Negative specimens were detected in 158 cases,with a specificity of 100%.There were 41 negative samples detected by PCR,with a positive rate of 20.5%.The detection specificity was consistent with that of LAMP kit.[Conclusions]The modified LAMP for Brucella has been successfully *** technology is characterized by good sens

关键词： Brucella loop-mediated isothermal amplification Omp25 Diagnostic kit Development

SHORT REPORT:Rapid detection of sacbrood virus (SBV) by one-step reverse transcription loop-mediated isothermal amplification assay

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SHORT REPORT:Rapid detection of sacbrood virus (SBV) by one-...

Establishment of a loop-mediated isothermal amplification Assay for Rapid Detection of Xanthomonas oryzae ***

生态环境与畜牧业可持续发展学术研讨会暨中国畜牧兽医学会2012年学术年会和第七届全国畜牧兽医青年科技工作者学术研讨会会议

作者： Jin-Long Yang 1 ,Rui Yang 1 ,Ke-Fei Shen 1 ,Xiang-Wei Peng 1 ,Tao-Xiong 2 ,Zuo-Hua Liu 1 Address: 1 Chongqing Academy of Animal Science,Chongqing 402460,Chongqing China 2 Rongchang Bureau of Animal Husbandry,Chongqing,402460,China

Background:Sacbrood virus (SBV) primarily infects honeybee broods, and in order to deal with the problem cost effective detection methods are required. Findings:A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid identification of SBV. The data demonstrated that, in a simple water bath, SBV RNA could be detected as early as 20 min at 65°C, and a positive amplification reaction was visible to the naked eye due to a color change brought on by the addition of nucleic acid stain SYBR Green. Conclusions: The current study presents a method for the rapid and simple detection of SBV by RT-LAMP with high sensitivity and analytic specificity.

关键词： Sacbrood virus loop-mediated isothermal amplification SYBR Green honeybee

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Establishment of a Loop-Mediated Isothermal Amplification As...

Detection of Target Genes in Viable Bacteria and Extracellular DNA Using loop-mediated isothermal amplification Assay

中国植物病理学会2016年学术年会

作者： ZHANG Xiao-fang FU Lina LIU Ya-ting ZHANG Hai-yan LI Xue JI Guang-hai College of Plant Protection Yunnan Agricultural University College of Agronomy And Xbiotechnology Yunnan Agricultural University

A visual loop-mediated isothermal amplification detection of Xanthomonas oryzae ***,the causal agent of bacterial leaf streak disease was developed with SYBR Green I as a fluorescence indicator in this *** special loop-mediated isothermal amplification primers and two loop primers were designed based on putative glycosyltransferase gene loci which is unique for *** ***-mediated isothermal amplification assay efficiently amplified the target element in less than 60 min at 65 ℃.LAMP assay was evaluated for specificity and sensitivity.A positive colour(Fluorescent Green) was only observed in the presence of Xooc by addition of SYBR Green Ⅰ to amplification,whereas none of other closely related Xanthomonas showed a colour *** lower limit of detection of this LAMP assay was about 100 fg/μL DNA and 3×10CFU/mL Bacterium Suspension,which was 100 times more sensitive than PCR *** of LAMP provides a new alternative method for onsite quarantining and large-scale monitoring of this pathogen.

关键词： Xanthomonas oryzae pv.oryzicola loop-mediated isothermal amplification Detection

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渔业科学进展 2020年第2期41卷 41-50页

作者： YANG Qianqian ZHANG Xuzhi JIANG Xiaoyu LI Yang ZHAO Jun HAO Zhihui WANG Pingping QU Keming College of Marine Sciences Shanghai Ocean UniversityShanghai 201306 Yellow Sea Fisheries Research Institute Chinese Academy of Fishery SciencesQingdao 266071 School of Chemistry and Pharmaceutical Sciences Qingdao Agriculture UniversityQingdao 266109

When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many *** pretreatment(***)is enough to obtain rather sensitive *** test samples without any pretreatment can be used as *** feature suggests that LAMP is superior to PCR in developing point-of-care test *** this study,using Stx1 gene from *** as model,we verified that viable cells,dead cells and extracellular DNA could function as template in the LAMP *** the incubation at 63℃,viable bacteria in the LAMP reaction mixture lysed completely within 2 min,providing DNA template for nucleic acid *** 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 *** found that the complex substances in real sample(*** seawater)exhibited considerable inhibitory effect on the sensitivity of the LAMP *** outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring,bio-resource surveys,food safety,*** particular those based on environmental DNA.

关键词： loop-mediated isothermal amplification DNA extraction-free Direct gene detection Viable cell Extracellular DNA

Super-assembly of integrated gold magnetic assay with loopmediated isothermal amplification for point-of-care testing

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Nano Research 2023年第1期16卷 1242-1251页

作者： Jianping Liang Jie Zeng Xiaojuan Huang Tengteng Zhu Yonglong Gong Chen Dong Xiangrong Wang Lingzhi Zhao Lei Xie Kang Liang Qiongxiang Tan Yali Cui Biao Kong Wenli Hui The College of life science Northwest UniversityXi’an 710069China Shaanxi Provincial Engineering Research Center for Nano-Biomedical Detection Xi’an 710077China Department of Chemistry Shanghai Key Lab of Molecular Catalysis and Innovative MaterialsCollaborative Innovation Center of Chemistry for Energy MaterialsFudan UniversityShanghai 200438China Key Laboratory of Resource Biology and Biotechnology in Western China Ministry of EducationNorthwest UniversityXi’an 710069China Provincial Key Laboratory of Biotechnology of Shaanxi Province Northwest UniversityXi’an 710069China School of Chemical Engineering Graduate School of Biomedical Engineeringand Australian Centre for NanoMedicineUniversity of New South WalesNSW 2052Australia

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 *** 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 *** is worth to note that the proposed method does not need DNA ***,uracil DNA glycosylase(UDG)is employed to eliminate carrier contamination for preventing false positive *** whole detection process can be finished within 25 *** accuracy of detection is measured by assessing the polymorphisms of the methylenetetrahydrofolate reductase(MTHFR)*** 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.

关键词： gold magnetic nanoparticles loop-mediated isothermal amplification lateral flow assay system free extraction singlenucleotide polymorphisms genotyping

Smartphone-Imaged HIV-1 Reverse-Transcription loop-mediated isothermal Ampliflcation(RT-LAMP) on a Chip from Whole Blood

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Engineering 2015年第3期1卷 324-335页

作者： Gregory L.Damhorst Carlos Duarte-Guevara Weili Chen Tanmay Ghonge Brian T.Cunningham Rashid Bashir Department of Bioengineering The University of Illinois at Urbana-Champaign Micro and Nanotechnology Laboratory The University of Illinois at Urbana-Champaign Department of Electrical and Computer Engineering The University of Illinois at Urbana-Champaign

Viral load measurements are an essential tool for the long-term clinical care of human immunodeficiency virus （HIV）-positive individuals. The gold standards in viral load instrumentation, however, are still too limited by their size, cost, and sophisticated operation for these measurements to be ubiquitous in remote settings with poor healthcare infrastructure, including parts of the world that are disproportionately affected by HIV infection. The challenge of developing a point-of-care platform capable of making viral load more accessible has been frequently approached but no solution has yet emerged that meets the practical requirements of low cost, portability, and ease-of-use. In this paper, we perform reverse-transcription loop-mediated isothermal amplification （RT-LAMP） on minimally processed HIV-spiked whole blood samples with a microfluidic and silicon microchip platform, and perform fluorescence measurements with a consumer smartphone. Our integrated assay shows amplification from as few as three viruses in a - 60 nL RT- LAMP droplet, corresponding to a whole blood concentration of 670 viruses per μL of whole blood. The technology contains greater power in a digital RT-LAMP approach that could be scaled up for the determination of viral load from a finger prick of blood in the clinical care of HIV-positive individuals. We demonstrate that all aspects of this viral load approach, from a drop of blood to imaging the RT-LAMP reaction, are compatible with lab-on-a-chip components and mobile instrumentation.

关键词： human immunodeficiency virus (HIV) viral load loop-mediated isothermal amplification smartphone point-of-care

Ultrathin metal-organic framework nanosheets (Cu-TCPP)-based isothermal nucleic acid amplification for food allergen detection

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Food Science and Human Wellness 2023年第5期12卷 1788-1798页

作者： Jiale Gao Xiaodong Sun Yongxin Liu Bing Niu Qin Chen Xueen Fang Shanghai Key Laboratory of Bio-Energy Crops School of Life SciencesShanghai UniversityShanghai 200444China Shanghai Engineering Research Center of Organ Repair School of MedicineShanghai UniversityShanghai 200444China Department of Chemistry and Institutes of Biomedical Sciences Fudan UniversityShanghai 200433China

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.

关键词： Cu-TCPP nanosheet Plant allergen Nucleic acid detection loop-mediated isothermal amplification

DETECTION AND APPLICATION OF MICROFLUIDIC isothermal amplification ON CHIP

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Journal of Innovative Optical Health Sciences 2008年第2期1卷 257-265页

作者： GUOLIANG HUANG XIAOYONG YANG JIANG ZHU SHUKUAN XU CHENG DENG CHAO HAN Medical Systems Biology Research Center Tsinghua University School of Medicine Beijing 100084China National Engineering Research Center for Beijing Biochip Technology Beijing 102206China

loop-mediated isothermal amplification(LAMP)is a novel nucleic acid amplification *** with the widely utilized polymerase chain reaction(PCR),LAMP has higher speed and efficiency as well as lower requirement for system temperature control because the whole amplification process is isothermal and no efforts are needed to switch between different *** this paper,we designed and fabricated different kinds of polycarbonate(PC)microfluid chips,explored appropriate reaction condition for LAMP in microenvironment(1 nL→10μL),and developed a microfluidic isothermal amplification detection *** DNA optimal amplification temperature is obtained;the starting time of exponential amplification of DNA is put forward *** optimal condition of DNA amplification in microenvironment,with a little reaction materials and early starting exponential amplification time of DNA are very important for clinic DNA detection and the application of Lab-on-a-Chip.

关键词： loop-mediated isothermal amplification Lab-on-a-Chip microfluid chips polymerase chain reaction DNA amplification

Research progress and prospects of nucleic acid isothermal amplification technology

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BIOCELL 2023年第11期47卷 2385-2395页

作者： SHUHUI WU PING XU XIANGBIN XU SONG-BAI LIU Suzhou Key Laboratory of Medical Biotechnology Suzhou Vocational Health CollegeSuzhou215009China School of Food Science and Engineering Hainan UniversityHaikou570228China The Affiliated Infectious Diseases Hospital Suzhou Medical College of Soochow UniversitySuzhou215100China

Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular *** the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so *** paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.

关键词： isothermal amplification Rolling circle amplification Nucleic acid sequence-based amplification Strand displacement amplification loop-mediated isothermal amplification Helicase-dependent amplification Recombinase polymerase amplification Cross-primer amplification