One New Method of Nucleic Acid Amplification-Loop-mediated Isothermal Amplification of DNA

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.

The Development of a Loop-Mediated Isothermal Amplification (LAMP) Procedure for Plague Diagnostic

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.

Preliminary investigation and detection based on loop-mediated isothermal amplification(LAMP)of phytoplasmas associated with diseases in B.napus L.

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.

Rapid and Visualized Detection Method of Canine Parvovirus Using Loop-Mediated Isothermal Amplification

[Objective] To develop a rapid and visualized detection method using loop-mediated isothermal amplification （LAMP）, improve the detection rate of canine parvovirus （CPV） and reduce the testing cost. [ Method] According to the conserved regions of the VP2 gene of CPV, six primers were designed to amplify the special DNA sequences by LAMP. In addition, the reaction conditions of LAMP were optimized, and the sensitivity, specificity, repeatability and stability were verified. [ Result] The optimal reaction time of the LAMP method for CPVwas 60 min. The products obtained by LAMP had high specificity without cross-reaction with other generic viruses. The sensitivity of the LAMP was 100 times higher than that of PCR. [ Conclusion] The LAMP method for detecting CPV has high practical value. It has many advantages such as high specificity, high sensitivity, simple operation, low cost and rapid analysis, and it does not require special equipment. Therefore, this method is more suitable for the detection of CPV.

Establishment of a Loop-mediated Isothermal Amplification Method for Rice Bacterial Leaf Brown Spot Disease

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.

Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2

In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10-5 ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.

Development and Evaluation of a Loop-mediated Isothermal Amplification Assay for the Rapid Detection and Identification of Pectobacterium carotovorum on Celery in the Field

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.

Rapid,sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification （LAMP） assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase （empA） gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65℃ in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non-V, anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.

Sensitive and rapid detection of two toxic microalgae Alexandrium by loop-mediated isothermal amplification

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.

Development of an in situ loop-mediated isothermal amplification technique for chromosomal localization of DNA sequences

In situ loop-mediated isothermal amplification （in situ LAMP） combines in situ hybridization and loop-mediated isothermal amplification （LAMP） techniques for chromosomal localization of DNA sequences. In situ LAMP is a method that is generally more specific and sensitive than conventional techniques such as fluorescence in situ hybridization （FISH）, primed in situ labeling （PRINS）, and cycling primed in situ labeling （C-PRINS）. Here, we describe the development and application of in situ LAMP to identify the chromosomal localization of DNA sequences. To benchmark this technique, we successfully applied this technique to localize the major ribosomal RNA gene on the chromosomes of the Zhikong scallop （ Chlarnys farreri）.

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

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.

Establishment of Reverse-transcription Loopmediated Isothermal Amplification Method for Detection of Wheat Streak Mosaic Virus

A reverse-transcription loop-mediated isothermal amplification （RT-LAMP） method was established for the detection of wheat streak mosaic virus （WSMV）. Ac-cording to the conservative regions of the genes that encode the coat protein of WSMV, 2 pairs of primers were designed. Final y, the 1st pair of primers was select-ed through the specificity test. The sensitivity test showed the sensitivity of RT-LAMP method was 10 times higher than that of RT-PCR. In addition, the amplifica-tion of target gene could be judged visual y from the presence of fluorescence （cal-cein） in the final reaction system. The RT-LAMP method, established in this study, was rapid, easy, specific and sensitive. Moreover, it did not require sophisticated equip-ment. The RT-LAMP was suitable for the rapid detection of WSMV.

牛病毒性腹泻病毒LAMP检测方法的建立与应用

针对牛病毒性腹泻病毒(BVDV)5′UTR基因(GenBank登录号:AY278459.1)序列设计4条特异性环介导等温扩增(LAMP)引物,特异性识别靶基因序列上4个独立区域,采用LAMP技术,利用实时浊度仪实时检测LAMP反应过程中所产生的焦磷酸镁白色沉淀,实时监测反应液浊度来判断反应结果,实现对扩增反应全过程的监控,建立BVDV的LAMP快速检测方法。通过实时浊度仪在恒温63℃下50min完成检测,对方法的特异性、灵敏度、重复性进行了评价。结果显示,经优化该方法只检测BVDV阳性,特异性强;病毒10-6倍稀释时仍能被检测到,比PCR方法灵敏度至少高100倍;重复性良好。LAMP实时浊度法具有简单、快速、灵敏度高、特异性强的优势,为BVDV的临床检测提供了一种简单快速的试验手段。

改良LAMP检测鸡肉中单增李斯特氏菌的研究

本研究建立一种改良环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术检测鸡肉中单增李斯特氏菌(Listeria monocytogenes,L. monocytogenes)的方法。针对单增李斯特氏菌的溶血素基因(hlyA)设计LAMP引物,对单增李斯特氏菌进行特异性检测,通过荧光曲线和肉眼观察荧光颜色来判定检测结果。试验结果表明:改良LAMP方法检测单增李斯特氏菌具有良好的特异性,7株单增李斯特氏菌呈阳性结果,24株非单增李斯特氏菌呈阴性结果。与聚合酶链反应(polymerase chain reaction,PCR)方法相比,改良LAMP方法具有灵敏度高(5.4×100 fg/μL)、检出限低(3.9×100 CFU/g)的特点,均是PCR结果的100倍。对66份鸡肉样品进行检测,得到改良LAMP方法的敏感性为100%,特异性为98.41%,符合率为98.48%。综上所述,改良LAMP方法能够快速、准确的检测单增李斯特氏菌,具有很好的应用前景。

十足目虹彩病毒(DIV1)环介导等温扩增(LAMP)检测方法的建立及应用

本研究以十足目虹彩病毒(Decapod iridescent virus 1,DIV1)主要衣壳蛋白基因为靶序列设计引物,建立了DIV1的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)检测方法,并以pMD18-DIV1质粒标准品为模板对该方法的检测灵敏度、检测特异性等进行了评估。结果显示,此方法最适反应温度为64.4℃,优化后的25μl反应体系中包含2.5μl 10×Isothermal amplification buffer、4.0 mmol/L Mg^2+、1.2 mmol/L dNTPs、6.4 U Bst 2.0 WarmStart■DNA聚合酶、0.8μmol/L EvaGreen■和4.4μl ddH2O。该方法检测灵敏度下限为3.54×10^2拷贝/反应;与虾肝肠胞虫(EHP)、致急性肝胰腺坏死病副溶血弧菌(VpAHPND)、对虾偷死野田村病毒(CMNV)、传染性皮下及造血组织坏死病病毒(IHHNV)、白斑综合征病毒(WSSV)、桃拉综合征病毒(TSV)和黄头病毒(YHV)等主要虾类病原没有交叉反应;具有较好的重复性和稳定性。以GeneFinder■替换EvaGreen■并将其预置于反应管内,结合上述扩增方法可实现对DIV1的现场快速高灵敏检测。本研究建立的DIV1-LAMP实时荧光定量和现场检测方法具有灵敏、特异和快速等特点,为近几年新发虾类病原DIV1的定性、定量以及现场快速检测提供了新的技术选择,有利于对虾养殖业中开展DIV1的监测、预警和防控。

环介导等温可视扩增(LAMP)检测禽肺炎病毒方法的建立

建立一种快速简单检测禽肺炎病毒的方法。根据已发表的禽肺炎病毒的F基因序列,设计并合成6条特异扩增禽肺炎病毒F基因片段的引物,通过条件优化,成功建立了针对禽肺炎病毒的环介导等温扩增(LAMP)检测法,测定其特异性和敏感性,并对采集的禽临床样品的DNA分别进行了检测。结果表明该法只检出禽肺炎病毒;敏感性扩增结果表明,Lamp检测禽肺炎病毒为100fg的RNA模板。该LAMP方法有简便、快速和特异性高的优点,可用于临床上对禽肺炎病毒的快速检测。

LAMP法与PCR法在食品动物源性成分检测中的应用

目的:探讨和对比食品动物源性成分检测中环介导等温扩增法(LAMP)与实时荧光定量法(PCR)的应用价值。方法:于湖南省汨罗市各大超市抽取样品并将其作为试验材料,共抽检13份。抽检样品分别采用LAMP法与PCR法进行检测,观察并对比两种检测方法各样品提取DNA浓度的测定结果。结果:3种已知动物源性成分样品经LAMP检测显示阳性,且PCR检测也为阳性;LAMP法检测时13份样品中与标签明示肉源不符的样品共3份,而PCR法检测时有4份。两种检测方法检测结果显示1份样品检测结果不同。结论:LAMP法与PCR法在食品动物源性成分检测中均表现出显著的应用价值,准确性高,并且具备较好特异性,但本研究中LAMP总用时80min,较PCR总用时140 min少,更方便进行现场快速抽检。

樱桃小果病毒1 RT-LAMP检测方法的建立与应用

樱桃小果病毒1(LChV-1)已成为严重影响樱桃产量与质量的重要因素之一。本研究基于环介导的等温扩增技术建立LChV-1的快速检测体系。依据NCBI数据库中LChV-1外壳蛋白基因序列设计了3组特异性引物,经优化,筛选获得1组特异性引物。以感染LChV-1的甜樱桃叶片总RNA为模板,构建RT-LAMP检测体系为:6.0 mM Mg^(2+),0.2μM的外引物和1.2μM的内引物,在57℃条件下反应60 min。使用RT-LAMP方法对35个疑似樱桃小果病的甜樱桃样品进行检测,发现有13个样品感染了LChV-1,检测结果与RT-PCR法一致。RT-LAMP法具有特异性强、快速等特点,适合对LChV-1田间样品的快速检测与鉴定。

环介导等温扩增技术及扩增产物分析方法的比较

环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)自开发以来,以其操作简便、灵敏度高、特异性好、扩增效率高等优点得到广泛地研究,在细菌、病毒、寄生虫、转基因成分等检测和鉴定中发挥重要作用,检测样品涵盖食品、环境、医疗、动植物检疫鉴定领域。LAMP技术最大的优点之一是扩增产物分析方法简便快速、不需要特殊的设备,特别适合在基层检验机构和现场检验推广应用。随着分析方法的研究,LAMP技术与相关技术偶联,使得产物分析方法灵敏度大大提高,检测速度更快、更方便。该文对LAMP相关技术及LAMP扩增产物检测分析方法的研究进行比较与分析,以期为LAMP方法的应用和开发提供建议和方向。

马铃薯后代材料晚疫病的抗性鉴定

为筛选出农艺性状优良且抗病性较好的后代材料,本试验对60份马铃薯“青薯9号”与“大西洋”杂交后代材料的离体叶片进行4种不同菌株的接种,分析4种菌株接种后的病斑面积及抗性等级,并利用LAMP技术对接种不同天数的离体叶片进行扩增检测。结果表明在60份后代材料中,高抗有6份,抗性有11份,中抗有17份,感病有24份,高感有2份。表现抗病的后代材料一共有34份,均具有广谱性,其中18份后代材料对4种菌株均产生抗病,16份后代材料对3种或2种菌株产生抗病,对4种菌株表现抗性的后代材料的广谱性比对3种或2种菌株表现抗性的后代材料高。利用LAMP技术检测接种1~5 d内及5 d后离体叶片的晚疫病菌,其扩增检测结果一致,均呈蓝色,为阳性。综上,本研究筛选的马铃薯后代材料可为抗病育种提供品种参考,LAMP技术能够对马铃薯晚疫病达到早期鉴定及预警的目的,为马铃薯晚疫病早期防治提供技术支持。