Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea)

In recent years, the large yellow croaker (Pseudosciaena crocea), an important marine fish farmed in the coastal areas of Zhejiang province, east China, has become severely endangered as a result of the bacterial pathogen Pseudomonas putida. This paper reports the development of a visual loop-mediated isothermal amplification (LAMP) assay for rapid detection of the pathogen. Four primers, F3, B3, FIP and BIP, were designed on the basis of DNA sequence of the rpoN gene of P. putida. After optimization of the reaction conditions, the detection limit of LAMP assay was 4.8cfu per reaction, 10-fold higher than that of conventional PCR. The assay showed high specificity to discriminate all P. putida isolates from nine other Gram-negative bacteria. The assay also successfully detected the pathogen DNA in the tissues of infected fish. For visual LAMP without cross-contamination, SYBR Green I was embedded in a microcrystalline wax capsule and preset in the reaction tubes; after the reaction the wax was melted at 85°C to release the dye and allow intercalation with the amplicons. The simple, highly sensitive, highly specific and cost-effective characteristics of visual LAMP may encourage its application in the rapid diagnosis of this pathogen.     

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℃ with a LAMP real-time turbidimeter.Furthermore,PCV2 DNA templates,with a detection limit of 5.5×10-5ng 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 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 di...     

Molecular diagnostic methods for detection of Thelohania contejeani (Microsporidia), the causative agent of porcelain disease in crayfish

Diagnosis of Thelohania contejeani in the crayfish Astacus astacus is currently based on observation of gross clinical signs--opaque appearance of the abdomen and whitish colouration of the musculature--and confirmed by microscopic examination of histological sections of muscle. We have developed 2 molecular diagnostic methods for sensitive and rapid detection of porcelain disease in its early stages: PCR and loop-mediated isothermal amplification (LAMP). The PCR test utilises a primer based on the T. contejeani small subunit ssu ribosomal RNA (ssu rRNA) gene and amplified parasite DNA with high specificity and a detection limit of 10(-5) dilution. The LAMP assay involves incubation of the target DNA with a set of 6 primers and Bst DNA polymerase for 60 min at 65 degrees C in a water bath or heating block, followed by visualisation of the reaction products with the SYBR Green I stain; sensitivity of visual detection with SYBR Green I is equivalent to that with agarose gel electrophoresis. The LAMP assay can detect T. contejeani DNA to a dilution of 10(-7). The LAMP assay is 100 times more sensitive than the PCR test and is the method we recommend as an alternative to traditional means of diagnosing T. contejeani.     

A modified visual loop-mediated isothermal amplification method for diagnosis and differentiation of main pathogens from Mycobacterium tuberculosis complex

This study was aimed to rapidly identify and differentiate two main pathogens of the Mycobacterium tuberculosis complex: Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium bovis by a modified loop-mediated isothermal amplification (LAMP) assay. The reaction results could be evaluated by naked eye with two optimized closed tube detection methods as follows: adding the modified fluorescence dye in advance into the reaction mix so as to observe the color changes or putting a tinfoil in the tube and adding the SYBR Green I dye on it, then making the dye drop into the bottom of the tube by centrifuge after reaction. The results showed that the two groups of primers used jointly in this assay could successfully identify and differentiate Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium tuberculosis bovis. Sensitivity test displayed that the modified LAMP assay with the closed tube system could determine the minimal template concentration of 1 copy/μl, which was more sensitive than that of routine PCR. The advantages of this LAMP method for detection of the Mycobacterium tuberculosis complex included high specificity, high sensitivity, simplicity, and superiority in avoidance of aerosol contamination. The modified LAMP assay would provide a potential for clinical diagnosis and therapy of tuberculosis in the developing countries and the resource-limited areas.     

Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for periodontal pathogen, Porphyromonas gingivalis. A set of six primers was designed by targeting the 16S ribosomal RNA gene. By the detection system, target DNA was amplified and visualized on agarose gel within 30 min under isothermal condition at 64 degrees C with a detection limit of 20 cells of P. gingivalis. Without gel electrophoresis, the LAMP amplicon was directly visualized in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. Detection limits of these naked-eye inspections were 20 cells and 200 cells, respectively. Although false-positive DNA amplification was observed from more than 10(7) cells of Porphyromonas endodontalis, no amplification was observed in other five related oral pathogens. Further, quantitative detection of P. gingivalis was accomplished by a real-time monitoring of the LAMP reaction using SYBR Green I with linearity over a range of 10(2)-10(6) cells. The real-time LAMP was then applied to clinical samples of dental plaque and demonstrated almost identical results to the conventional real-time PCR with an advantage of rapidity. These findings indicate the potential usefulness of LAMP for detecting and quantifying P. gingivalis, especially in its rapidity and simplicity.     

Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis

Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons during DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the first detailed evaluation of the intercalating dye SYTO9. Our findings demonstrate that SYTO9 produces highly reproducible DNA melting curves over a broader range of dye concentrations than does SYBR Green I, is far less inhibitory to PCR than SYBR Green I, and does not appear to selectively detect particular amplicons. The low inhibition and high melting curve reproducibility of SYTO9 means that it can be readily incorporated into a conventional PCR at a broad range of concentrations, allowing closed tube analysis by DNA melting curve analysis. These features simplify the use of intercalating dyes in real-time PCR and the improved reproducibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.     

Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification

Vibrio vulnificus is a serious bacterial pathogen for humans and aquatic animals. We developed a rapid, sensitive and specific identification method for V. vulnificus using loop-mediated isothermal amplification (LAMP) technique. A set of primers, composed of two outer primers and two inner primers, was designed based on the cytolysin gene sequence of V. vulnificus. The LAMP reaction was processed in a heat block at 65 °C for 60 min. The amplification products were detected by visual inspection using SYBR Green I, as well as by electrophoresis on agarose gels. Our results showed that the LAMP reaction was highly specific to V. vulnificus. This method was 10-fold more sensitive than conventional PCR. In conclusion, the LAMP assay was extremely rapid, simple, cost-effective, sensitive and specific for the rapid identification of V. vulnificus.     

普通牛及其近缘种特异性序列筛选及环介导等温扩增检测方法

近年来,肉制品掺假事件频频发生,亟需建立快速、可靠的肉制品动物源性成分检测方法,保障肉类食品安全。与常规PCR等检测方法相比,环介导等温扩增（loop-mediated isothermal amplification,LAMP）方法具有高特异性、高灵敏度、反应快、易操作等优势。基于此,利用生物信息算法筛选普通牛及其近缘种（牦牛、水牛、美洲野牛）的全基因组序列,以获得种间相似性序列,进而建立并优化可用于检测普通牛及其近缘种成分的特异性LAMP方法。最终优化后的LAMP反应体系为：10 μmol·L-1外引物F3和B3各0.25 μL,10 μmol·L-1内引物FIP和BIP各2 μL,2 mmol·L-1 dNTP 2.5 μL,25 mmol·L-1 MgSO4 4 μL,5 mol·L-1 甜菜碱 3.5 μL,8.0 U·μL-1 Bst DNA 聚合酶 1 μL,10×ThermoPol Buffer 2.5 μL,DNA模板2 μL,加双蒸水至25 μL;LAMP反应条件为：65 ℃恒温扩增1 h,80 ℃ 10 min。在扩增产物中加入SYBR GreenⅠ荧光染料后,检测结果可用肉眼直接观察。优化后的LAMP方法在1 h内能特异性地检测出普通牛、牦牛、水牛及美洲野牛成分,检测灵敏度达到0.020 ng·μL-1,且可特异性地检测出市售肉制品中含有牛肉成分的样品。研究所建立的LAMP方法具有高特异性、高灵敏度、反应快速、操作简便和无需精密仪器等特点,可应用于肉制品中普通牛及其近缘种成分的实际检测,为我国肉类食品安全提供了有力保障。     

Tetraalkylammonium derivatives as real-time PCR enhancers and stabilizers of the qPCR mixtures containing SYBR Green I

Tetraalkylammonium (TAA) derivatives have been reported to serve as stabilizers of asymmetrical cyanine dyes in aqueous solutions and to increase the yield and efficiency of polymerase chain reaction (PCR) detected by end-point analysis. In this study, we compared the ability of various TAA derivatives (with alkyl chain ranging from 1 to 5 carbons) and some other compounds to serve as enhancers of real-time PCR based on fluorescence detection from intercalating dye SYBR Green I (SGI). Our data indicate that TAA chlorides and some other TAA derivatives serve as potent enhancers of SGI-monitored real-time PCR. Optimal results were obtained with 10-16 mM tetrapropylammonium chloride. The effect of TAA compounds was dependent on the nature of counter ions present and composition of the reaction mixtures used. Based on measurements of SGI-generated fluorescence signal in the presence of PCR-amplified DNA fragments, oligonucleotide primers and/or various additives, we propose that TAA-derivatives reduce the binding of SGI to oligonucleotide primers and thus enhance primer-template interactions during annealing phase. Furthermore, these compounds serve as stabilizers of SGI-containing PCR mixtures. The combined data indicate that TAA derivatives might be a new class of additives contributing to robustness of real-time PCR monitored by asymmetrical cyanine dye SGI.     

单管可视化环介导等温扩增技术快速检测恶性疟原虫

目的:建立一种基于环介导等温核酸扩增技术(Loop-mediated Isothermal Amplification,LAMP)的恶性疟原虫高灵敏可视化闭管检测方法。方法:针对恶性疟原虫核糖体DNA的序列保守区设计LAMP引物,通过优化LAMP体系中的Mg2+、甜菜碱浓度和反应温度等因素,建立环介导等温扩增法;并结合蜡封反应管对产物进行检测,检测结果可直接通过肉眼观察SYBR Green I荧光显色进行判定。结果:本方法可检测到70个拷贝/管的恶性疟原虫核酸片段,并具有高特异性,可区分检测常见的血液病毒。该法具有如下优点:1、整个反应恒温进行,无需热循环仪;2、闭管检测,极大降低了扩增产物交叉污染的风险;3、检测速度快,整个检测过程只需30 min。结论:该法的建立为恶性疟原虫的现场快速筛检提供了一种简便、高灵敏、高特异的工具。     

Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae . Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (χ² test, P <0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection.     

Rapid identification of Bactrocera latifrons (Dipt., Tephritidae) by real-time PCR using SYBR Green chemistry

Abstract:  The solanum fruit fly, Bactrocera latifrons (Hendel), is a major agricultural pest in Asia and Hawaii, and it is important to prevent its widespread invasion in plant quarantine. In this study we introduced a real-time polymerase chain reaction (PCR) essay, using SYBR Green I dye, to rapidly identify B. latifrons on an ABI PRISM 7700 sequence detection system. A latifron-specific PCR primer set was obtained based on mtDNA COI gene of B. latifrons . Nine Bactrocera fruit flies, B. latifrons , Bactrocera dorsalis , Bactrocera papayae , Bactrocera carambolae , Bactrocera philippinensis , Bactrocera occipitalis , Bactrocera correcta , Bactrocera cucurbitae and Bactrocera tau , were used to determine the specificity of primers lati1 and lati2. A series of genomic DNA dilutions of B. latifrons (0.01, 0.1, 1, 10, 20, 40 and 100 ng) were used to assess the sensitivity of the SYBR Green PCR. Template DNA concentration was one of the sources of variability in cycle threshold values (CT) and the optimum DNA concentration was between 1 and 20 ng. Genomic DNA isolated from larvae, pupae and adult specimens of B. latifrons were used to assess the specificity of the SYBR Green PCR. Melting curve analysis and agarose gel electrophoresis was employed to check the specificity of PCR products. Similar amplification plots were obtained using DNA from the three different stages of B. latifrons with primer set lati1/lati2. The melting temperature ( T _m) of PCR products was 77.5 ± 0.1°C, and the length of the amplified fragment 366 bp. Given the specificity and sensitivity of the assay, combined with high speed, low cost and the possibility of automating, SYBR Green PCR can be used as a rapid and specific technique for pest species identification in plant quarantine.     

African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA

Control of human African trypanosomiasis (HAT) is dependent on accurate diagnosis and treatment of infected patients. However, sensitivities of tests in routine use are unsatisfactory, due to the characteristically low parasitaemias in naturally infected individuals. We have identified a conserved sequence in the repetitive insertion mobile element (RIME) of the sub-genus Trypanozoon and used it to design primers for a highly specific loop-mediated isothermal amplification (LAMP) test. The test was used to analyse Trypanozoon isolates and clinical samples from HAT patients. The RIME LAMP assay was performed at 62 degrees C using real-time PCR and a water bath. DNA amplification was detectable within 25min. All positive samples detected by gel electrophoresis or in real-time using SYTO-9 fluorescence dye could also be detected visually by addition of SYBR Green I to the product. The amplicon was unequivocally confirmed through restriction enzyme NdeI digestion, analysis of melt curves and sequencing. The analytical sensitivity of the RIME LAMP assay was equivalent to 0.001 trypanosomes/ml while that of classical PCR tests ranged from 0.1 to 1000 trypanosomes/ml. LAMP detected all 75 Trypanozoon isolates while TBR1 and two primers (specific for sub-genus Trypanozoon) showed a sensitivity of 86.9%. The SRA gene PCR detected 21 out of 40 Trypanosoma brucei rhodesiense isolates while Trypanosoma gambiense-specific glycoprotein primers (TgsGP) detected 11 out of 13 T. b. gambiense isolates. Using clinical samples, the LAMP test detected parasite DNA in 18 out of 20 samples which included using supernatant prepared from boiled blood, CSF and direct native serum. The sensitivity and reproducibility of the LAMP assay coupled with the ability to detect the results visually without the need for sophisticated equipment indicate that the technique has strong potential for detection of HAT in clinical settings. Since the LAMP test shows a high tolerance to different biological substances, determination of the appropriate protocols for processing the template to make it a user-friendly technique, prior to large scale evaluation, is needed.     

Loop-mediated isothermal amplification method for rapid detection of the toxic dinoflagellate Alexandrium, which causes algal blooms and poisoning of shellfish

The marine dinoflagellate genus Alexandrium includes a number of species that produce potent neurotoxins responsible for paralytic shellfish poisoning, which in humans may cause muscular paralysis, neurological symptoms and, in extreme cases, death. Because of the genetic diversity of different genera and species, molecular tools may help to detect the presence of target microorganisms in marine field samples. Here we employed a loop-mediated isothermal amplification (LAMP) method for the rapid and simple detection of toxic Alexandrium species. A set of four primers were designed based upon the conserved region of the 5.8S rRNA gene of members of the genus Alexandrium . Using this detection system, toxic Alexandrium genes were amplified and visualized as a ladder-like pattern of bands on agarose gels under isothermal condition within 60 min. The LAMP amplicons were also directly visualized by eye in the reaction tube by the addition of SYBR Green I. This LAMP assay was 10-fold more sensitive than a conventional PCR method with a detection limit of 5 cells per tube when targeting DNA from Alexandrium minutum . The LAMP assay reported here indicates the potential usefulness of the technique as a valuable simple, rapid alternative procedure for the detection of target toxic Alexandrium species during coastal water monitoring.     

实时荧光定量PCR方法快速检测转基因大豆

针对转基因大豆中普遍含有的35S启动子进行引物设计,以双链DNA染料SYBR GreenⅠ为荧光标记物,利用实时荧光定量PCR方法对大豆样品进行检测。该法检测转基因大豆的检测低限为0.005 nmol/L的35S启动子,线性范围达3个数量级,可快速区分转基因大豆和非转基因大豆,具有快速、简便、灵敏、安全、高通量、低成本等优点,可推广用于转基因植物产品的快速定量检测。     

Development and evaluation of a loop‐mediated isothermal amplification assay for rapid detection of chicken anaemia virus

Aim: Chicken anaemia virus (CAV) causes an economically important viral disease in chickens worldwide. The main aim of this study was to establish a rapid, sensitive and specific loop‐mediated isothermal amplification (LAMP) assay for detecting CAV infection. Methods and Results: A set of four specific LAMP primers were designed based on the nucleotide sequence of the CAV VP2 gene, which encodes a nonstructural protein. These were used for the amplification of a specific target region of the VP2 gene. LAMP amplicons were successfully amplified and detected by DNA electrophoresis and by direct naked eye SYBR Green I visualization. A sensitivity test systematically demonstrated that the LAMP assay was superior to a conventional PCR assay with a minimum concentration limit of 100 fg compared to 10 ng for the conventional PCR. The specificity of the LAMP assay for CAV detection is consistent with conventional PCR. Using this established LAMP assay, infected and uninfected clinical samples obtained from an experimental farm were fully verified. Conclusions: A novel nucleic acid‐based approach of LAMP assay was successfully developed for detecting CAV infection. Significance and Impact of the Study: In this study, these results indicate that the developed LAMP assay herein for CAV detection is a time‐effective, simple, sensitive and specific test that can be used as an alternative approach in the future for large‐scaled diagnosis on the farm of CAV infection.