环介导恒温扩增法检测肺炎链球菌的研究

目的 建立环介导恒温扩增(LAMP)检测肺炎链球菌的方法.方法 用LAMP技术扩增肺炎链球菌菌株,并应用50例临床标本采用传统培养法、PCR法、LAMP法进行检测,比较3种方法的检出率,同时检测方法特异性和灵敏度.结果 所测肺炎链球菌均获扩增产物,对其他非肺炎链球菌无交叉反应.LAMP检测灵敏度可达102 CFU/mL.50例临床标本使用LAMP法检出9例肺炎链球菌阳性(18.0％),使用传统培养法检出阳性4例(8.0％).结论 LAMP法较传统培养检测方法特异性强、灵敏度高、操作方便、快速,适合临床标本的肺炎链球菌检测.     

Detection of Botrytis cinerea by loop-mediated isothermal amplification

Aims: To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop‐mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting. Methods and Results: A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross‐reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real‐time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in <15 min. Conclusions: The LAMP assay that was developed is suitable for rapid detection of B. cinerea in infected plant material. Significance and Impact of the Study: The LAMP method combines the sensitivity and specificity of nucleic acid‐based methods with simplified equipment and a reduced reaction time. These features make the method potentially suitable for on‐site use, where the results of testing could help to inform decisions regarding the storage and processing of commodities affected by B. cinerea, such as cut flowers, fruit and vegetables.     

Detection of fish nocardiosis by loop-mediated isothermal amplification

AIMS: Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a protocol for detecting Nocardia seriolae which is a causative agent of fish nocardiosis, was designed. METHODS AND RESULTS: A set of four primers, two inner and two outer, were designed based on the sequence of the 16S-23S ribosomal RNA internal transcribed spacer region of N. seriolae. Time and temperature conditions for detection of N. seriolae were optimized for 60 min at 65 degrees C. Other fish pathogen was not amplified by this LAMP system. The detection of N. seriola using LAMP was found to be more sensitive than that by polymerase chain reaction. CONCLUSIONS: LAMP is a highly sensitive and rapid diagnostic procedure for detection of N. seriolae. SIGNIFICANCE AND IMPACT OF THE STUDY: LAMP is a useful diagnostic method for fish nocardiosis.     

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.     

Detection of Vibrio parahaemolyticus in food samples using in situ loop-mediated isothermal amplification method

A novel in situ loop-mediated isothermal amplification (in situ LAMP) technique for rapid detection of the food-borne Vibrio parahaemolyticus strains had been developed and evaluated in this study. The sensitivity of the in situ LAMP assay was detected to be 10 CFU/reaction via test in serial 10-fold dilutions of V. parahaemolyticus cells, and high specificity had also been obtained through confirmation with 14 reference gram-positive and -negative strains. Application of the established in situ LAMP assay had been performed on 58 strains previously isolated from seafood samples, including 48 V. parahaemolyticus and 10 non-V. parahaemolyticus strains. Of 48 V. parahaemolyticus strains, 48, 45 and 34 strains were detected as positive by in situ LAMP, regular LAMP and PCR, respectively, with the detection rate and negative predictive value (NPV) found to be 100% vs 93.8% vs 70.8% and 100% vs 76.9% vs 41.7%. In addition, none of the tested non-V. parahaemolyticus strains showed positive result, indicating a 100% positive predictive value (PPV) for all of 3 assays. Compared with regular LAMP methods and PCR-based methods, the in situ LAMP assay is advantageous on rapidity, high specificity, less time consumption and ease in operation, and may provide a novel, useful and practical detection platform for pathogens in food safety laboratories.     

Detection of phytoplasma by loop-mediated isothermal amplification of DNA (LAMP)

Napier stunt phytoplasma (16SrXI and 16SrIII) in eastern Africa is a serious threat to the expansion of Napier grass (Pennisetum purpureum) farming in the region, where it is widely cultivated as fodder in zero grazing livestock systems. The grass has high potential for bio-fuel production, and has been adopted by farmers as a countermeasure to cereal stem borer Lepidoptera, since it attracts and traps the insect. Diagnosis of stunt phytoplasma have been largely by nested polymerase chain reaction (nPCR) targeting the 16S rRNA gene. However, the method is laborious, costly and technically demanding. This investigation has developed a simpler but effective phytoplasma diagnostic tool, called; loop-mediated isothermal amplification of DNA (LAMP). The assay was tested on 8 symptomatic and 8 asymptomatic plants, while its detection limit was compared to nested PCR using samples serially diluted from 3 ng/μl to 0.38 pg/μl. Molecular typing of LAMP products was determined by BsrI restriction digestion and Southern blot analysis. The assay sensitivity, positive and negative predictive values were estimated, while the specificity was tested on 11 phytoplasma groups. LAMP was specific to 5 phytoplasma groups: 16SrVI, X, XI and XVI. BsrI restriction digestion produced two predicted fragments, and there was specific binding of probe DNA to the LAMP amplicons in Southern blot analysis. The assay sensitivity was 100%, while the positive and negative predictive values were 63 and 100% respectively. LAMP was 20-fold more sensitive than nested PCR. This study validates LAMP for routine diagnosis of Napier stunt and other closely related phytoplasmas.     

Detection of porcine circovirus type 1 in commercial porcine vaccines by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) method with a real-time monitoring system was developed for the detection of porcine circovirus type 1 (PCV1) in commercial swine vaccines. This method was highly specific for PCV1. No cross-reaction to porcine circovirus type 2, porcine parvovirus, pseudorabies virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus was observed. The analytical sensitivity of the LAMP for PCV1 DNA was 10 copies/μl in the case of positive recombinant plasmid comparable to that obtained from the nested polymerase chain reaction (nested PCR). Furthermore, 25 commercial swine vaccines were tested by both the LAMP and the nested PCR, and three of them were tested positive for PCV1 DNA. These results indicate that PCV1 DNA can be real-time detected by the LAMP; the method was highly specific, sensitive, and rapid for the detection of PCV1 DNA, particularly in commercial swine vaccines.     

多重环介导等温扩增技术研究进展

环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)因其扩增速度快、灵敏度和特异性高、仪器要求低等优点而被广泛应用于核酸诊断领域。为充分利用LAMP技术优势、提高诊断检测的效率与可靠性、扩展其应用范围,同时节约试剂成本,近年来多重LAMP技术的研究成为一大热点。常规的LAMP扩增产物检测方法多数以聚合反应的双链DNA产物或其副产物为基础,只能判断有无扩增反应发生,而难以识别多重扩增产物的靶标来源及其特异性。为实现多重扩增产物的高特异检测,各国学者通过对该技术巧妙的改进或与其他技术相偶联,发展了一系列多重LAMP扩增检测技术。然而上述狭义的多重LAMP技术依然存在因引物间相互干扰、扩增效率存在差异而引发歧视性扩增的局限,限制了多重扩增的重数。近年研究活跃的微型扩增技术以其实现多个平行、互不干扰的小体积单重扩增的技术优势打破了这一局限,由此产生了新型的广义多重LAMP扩增技术。这些技术还具有试剂消耗少、自动化程度较高、交叉污染风险更小以及更适合对较多靶标进行现场快速检测等优势。本文分别从狭义多重LAMP的方法原理及其扩增反应体系优化、广义多重LAMP的方法原理以及多重LAMP技术在诊断检测中的应用等方面对近年来多重LAMP技术的研究进展进行了综述。     

Visualization and enumeration of bacteria carrying a specific gene sequence by in situ rolling circle amplification

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 10(6)-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.     

Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.

The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity.     

环介导等温扩增技术在临床上的应用

环介导等温扩增技术是一种在恒温条件下特异、高效、快速扩增DNA的新技术.不需要专业设备和人员,在一个反应体系中,通过四条特异性引物识别靶基因6个不同的位点,1h内完成整个扩增.扩增产物是一系列反向重复的靶序列构成的茎环结构和多环花椰菜样结构的DNA片段的混合物.通过肉眼观察浊度或加入荧光染料SYBR Green Ⅰ后的颜色变化即可判定结果.     

Detection of gp43 of Paracoccidioides brasiliensis by the loop-mediated isothermal amplification (LAMP) method

Paracoccidioidomycosis is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. We detected the species specific gp43 gene of P. brasiliensis by loop-mediated isothermal amplification (LAMP) in 22 clinical and seven armadillo-derived isolates. The amplified DNA appeared as a ladder with a specific banding pattern. The advantage of the LAMP method is speed; only 3 h were necessary for identification of the organism and diagnosis of the disease. We were also able to obtain positive results from DNA extracted from a paraffin-embedded tissue sample of paracoccidioidomycosis, suggesting that this method may achieve clinical application in the near future.     

Detection of Bacillus anthracis from spores and cells by loop-mediated isothermal amplification without sample preparation

Loop-mediated isothermal amplification (LAMP) is a technique capable of rapidly amplifying specific nucleic acid sequences without specialized thermal cycling equipment. In addition, several detection methods that include dye fluorescence, gel electrophoresis, turbidity and colorimetric change, can be used to measure or otherwise detect target amplification. To date, publications have described the requirement for some form of sample nucleic acid extraction (boiling, lysis, DNA purification, etc.) prior to initiating a LAMP reaction. We demonstrate here, the first LAMP positive results obtained from vegetative cells and spores of Bacillus anthracis without nucleic acid extraction. Our data show that the simple addition of cells or spores to the reaction mixture, followed by heating at 63°C is all that is required to reproducibly amplify and detect target plasmid and chromosomal DNA via colorimetric change. The use of three primer sets targeting both plasmids and the chromosome of B. anthracis allows for the rapid discrimination of non-pathogenic bacteria from pathogenic bacteria within 30 min of sampling. Our results indicate that direct testing of B. anthracis spores and cells via LAMP assay will greatly simplify and shorten the detection process by eliminating nucleic acid purification. These results may allow more rapid detection of DNA from pathogenic organisms present in field and environmental samples.     

Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification

In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.     

Monitoring the progression of loop-mediated isothermal amplification using conductivity

Loop-mediated isothermal amplification (LAMP) yields a large amount of DNA, as well as magnesium pyrophosphate precipitate, causing a decrease in ionic strength that can be measured with a conductivity meter. There is a clear relationship between the conductivity of the LAMP mixture solution and the duration of biochemical reaction. Moreover, there is also a clear relationship between the change in conductivity and the amount of initial template DNA over the range of 0.08 to 3.2 ng. These results demonstrate the feasibility not only for detecting the LAMP product qualitatively but also for real-time monitoring the biochemical reaction progression quantitatively using conductivity measurements.     

Rapid detection of Brucella spp. by the loop-mediated isothermal amplification method

Aims:  To develop a rapid and sensitive method for detecting Brucella spp.
Methods and Results:  Two sets of six Brucella -specific primers for loop-mediated isothermal amplification (LAMP) were designed from the sequence of the Brucella abortus BCSP31 gene. The specificity and sensitivity were examined for six Brucella species (22 strains) and 18 non- Brucella species (28 strains). The LAMP assay was specific to Brucella spp. in 35 min at 63°C and sensitive (detected 10 fg of genomic DNA). The assay was also applied for the detection of Brucella DNA in contaminated milk and infected mouse organs.
Conclusions:  We developed a sensitive and specific LAMP assay for Brucella spp., with the test appearing to be useful for the detection of the pathogen from clinical and food samples.
Significance and Impact of the Study:  This is the first report of the development of LAMP for the detection of Brucella spp. As the LAMP assay can be performed at a constant temperature and its reactivity is directly observed with the naked eye without electrophoresis, our assay should be useful for the diagnosis of brucellosis as well as the detection of the bacteria in environmental or food samples.