Colorimetric DNA-based assay for the specific detection and quantification of Ostreopsis cf. ovata and Ostreopsis cf. siamensis in the marine environment

Ostreopsis is a toxic benthic dinoflagellate largely distributed worldwide in tropical and temperate areas. In the Mediterranean Sea, periodic summer blooms have been reported and have become a serious concern due to their direct impact on human health and the environment. Current microalgae identification is performed via light microscopy, which is time-consuming and is not able to differentiate among Ostreopsis species. Therefore, there is mature need for rapid, specific and easy-to-use detection tools. In this work, a colorimetric assay exploiting a combination of recombinase polymerase amplification (RPA) and a sandwich hybridisation assay was developed for O. cf. ovata and O. cf. siamensis detection and quantification. The specificity of the system was demonstrated by cross-reactivity experiments and calibration curves were successfully constructed using genomic DNA, achieving limits of detection of 10 and 14 pg/μL for O. cf. ovata and O. cf. siamensis, respectively. The assay was applied to the analysis of planktonic and benthic environmental samples from different sites of the Catalan coast. Species-specific DNA quantifications were in agreement with qPCR analysis, demonstrating the reliability of the colorimetric approach. Significant correlations were also obtained between DNA quantifications and light microscopy counts. The approach may be a valuable tool to provide timely warnings, facilitate monitoring activities or study population dynamics, and paves the way towards the development of in situ tools for the monitoring of harmful algal blooms.     

环介导恒温扩增法快速检测海产品中的副溶血弧菌

副溶血弧菌广泛分布于海水或海产品中,人类摄入或接触污染的水源和食物易引起感染。近年来,有关致病性弧菌引起腹泻的报道逐渐增多,但GB标准的细菌学诊断方法检测周期长达1周左右,而且操作较为复杂,难以满足控制疾病暴发和传播的需要,就这一现状,建立了一套不仅快速、准确,而且操作简便、不依赖昂贵仪器的检测方法,应用于海产品中副溶血弧菌快速检测。采用环介导恒温扩增方法(LAMP),针对副溶血弧菌的gyrB基因设计特异引物,进行恒温扩增。使用该方法最低检出限达到101CFU/mL,灵敏度可以达到0.1pg副溶血弧菌基因组DNA,为海产品中副溶血弧菌的检测提供了一个新的辅助方法。     

Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances

To simplify the molecular detection of micro-organisms, we evaluated the tolerance of loop-mediated isothermal amplification (LAMP) to a culture medium and some biological substances. The sensitivity of LAMP was less affected by the various components of the clinical samples than was polymerase chain reaction (PCR); therefore, DNA purification from samples could be omitted.     

Specific,sensitive and rapid Curcuma longa turmeric powder authentication in commercial food using loop-mediated isothermal nucleic acid amplification

Turmeric (Curcuma longa) is a rhizomatous plant of the ginger family Zingiberaceae that is usually dried and ground into powder for use as a seasoning. Because turmeric has become increasingly popular in the functional food market, adulteration of C. longa by other turmeric species is becoming an increasingly significant problem. In this study, loop-mediated isothermal amplification (LAMP) was developed for the detection of C. longa DNA for turmeric authentication. ITS2-26S rDNA was used for the LAMP primer designation. The results demonstrated that the specific primers exhibited high specificity, authenticated C. longa DNA within 30 min at 65 °C isothermally and had no cross-reaction with other adulterants. LAMP was sensitive to 0.1 ng of turmeric C. longa DNA, and only 0.01% of C. longa turmeric powder in the sample was required for DNA amplification. The sensitivity of LAMP was 10-fold higher than that of PCR (0.1%) from a previous report. Moreover, all the collected commercial turmeric products were positively detected by LAMP and RtF-LAMP (real-time fluorescence LAMP). The developed LAMP assay not only had higher specificity and rapidity than that of other methods but could also be applied to authenticate turmeric to prevent adulteration in food products.     

Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3 × 10⁻⁵) was roughly similar to that of Pfu DNA polymerase (4.8 × 10⁻⁵), but much lower than those of wild-type Neq DNA polymerase (57.2 × 10⁻⁵), Neq A523R DNA polymerase (13.1 × 10⁻⁵), and Neq N540R DNA polymerase (37.7 × 10⁻⁵). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.     

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.     

环介导等温扩增核酸技术及其在食品安全检测领域的应用

环介导等温扩增(Loop-mediated isothermal amplification,简称LAMP)是利用能识别靶序列上6个位点的4个特殊设计的引物和一种具有链置换活性的DNA聚合酶,在恒温条件下,特异、高效、快速地扩增核酸的新技术。该技术在1h内扩增效率可达到109-1010个数量级,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片段混合物,电泳后在凝胶上显现出由不同大小的区带组成的阶梯式图谱。近年来LAMP技术以其特异性强、等温灵敏、操作简单、产物易检测等优点已经应用于食品安全检测领域的多个方面。     

水产品中创伤弧菌DNA环介导恒温扩增快速检测方法的建立及初步应用

创伤弧菌是一种重要的食源性致病菌,主要存在于河口和海洋环境中,严重危害水产养殖业的发展和人类健康。建立快速、准确、易操作的检测方法对防控创伤弧菌的传染,保障水产养殖业发展和增强食品安全意义重大。基于创伤弧菌vvHA基因,利用一种新型的核酸扩增技术-环介导恒温扩增(loop-mediated isothermal amplification,LAMP),建立了创伤弧菌LAMP快速检测方法。对11种共46株细菌进行扩增,仅创伤弧菌为LAMP阳性结果,说明LAMP方法具有高度特异性。灵敏度试验结果表明,对创伤弧菌纯培养菌的检测灵敏度为15CFU/ml,对污染食品中创伤弧菌的检测灵敏度为24CFU/g。此法40~60min内即可完成检测,检验检疫实践证明:LAMP方法操作简便、特异性强、灵敏度高且成本低廉,具有良好的应用前景。     

环介导等温扩增技术在致病微生物检测中的应用

环介导等温扩增(LAMP)是一种新兴的核酸扩增方法,由于其具有高灵敏、高特异、操作简单、检测快速、价格低廉等诸多优势,现已成为分子生物学诊断技术的重要组成部分,并广泛应用于基层实验室及野外致病微生物的在快速检测致病微生物中的应用及新进展,希望能为其发展提供参考。     

Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

Abstract

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies—examining the critical parameters and variations and their widespread applications—giving the strengths and limitations of these methodologies.     

Rapid and simple detection of eight major periodontal pathogens by the loop-mediated isothermal amplification method

Loop-mediated isothermal amplification (LAMP) was applied to develop a rapid and simple detection system for eight periodontal pathogens: Aggregatibacter (Actinobacillus) actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola and Tannerella forsythia. Primers were designed from the 16S ribosomal RNA gene for each pathogen, and the LAMP amplified the targets specifically and efficiently under isothermal condition at 64 degrees C. To simplify the manipulation of LAMP examination, boiled cells and intact cells suspended in phosphate-buffered saline (PBS) were tested as templates besides extracted DNA template. The detection limits were 1-10 cells per tube using extracted DNA template. However, LAMP methods using boiled cells and intact cells required 10-100 and 100-1000 cells per tube, respectively. LAMPs for A. actinomycetemcomitans, P. gingivalis and P. intermedia were then applied to clinical plaque samples, and the method demonstrated equal or higher sensitivity compared with the conventional real-time PCR method. These findings suggest the usefulness of the LAMP method for the rapid and simple microbiological diagnosis of periodontitis, and the possibility of LAMP examination without the DNA extraction step.     

The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China

The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies with high sensitivity, efficiency, and rapidity, deoxyribonucleic acid (DNA) under isothermal condition in simple incubators. Two primer sets for the LAMP method were designed using the nucleotide sequences of 18S rRNA gene of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 isolated in China. The primers were used to detect parasite DNA extracted from infected blood and purified parasites by LAMP. The specific ladder bands were amplified from the autologous genomic DNA of two Babesia species, respectively, and did not cross-react with the genomic DNA of Theileria sp. China 1, Theileria sp. China 2, B. bovis, Theileria sp. (Japan) and sheep. The LAMP was sensitive enough to detect 0.02 pg and 0.2 pg genomic DNA of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005, respectively, from 10-fold serially diluted samples corresponding to the amount of DNA present in 50 μl of 0.000002% and 0.00002% parasitemic erythrocytes. Furthermore, DNA extracted from blood of intact (non-splenectomized) sheep experimentally infected with Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 was amplified by the LAMP from week 1 to 9 and week 2 and 3 post-infection, respectively, demonstrating the high sensitivity of these primers. Of 365 samples collected from Gansu province, 14.3% (52/365) were positively detected by the LAMP. Of 145 samples collected on filter papers (Whatman) from the grazing sheep in Xinjiang province, 3.5% (5/145) were positive. These results show that the LAMP could be an alternative diagnostic tool for the detection of babesial infection in sheep and goats.     

Ectomycorrhizae of Lactarius lignyotus on Norway spruce,characterized by anatomical and molecular tools

The ectomycorrhizae of Lactarius lignyotus on Norway spruce are comprehensively described by morphological and anatomical characteristics. Identification of ectomycorrhizae was performed by tracing mycelia to the fruitbodies and also by molecular tools, using polymerase chain reaction (PCR) amplification of the fungal DNA. The newly described ectomycorrhiza is compared to ectomycorrhiza of the related Lactarius picinus. The amplified DNA products of the two fungi and their ectomycorrhizae could be distinguished by characteristic fragments after digestion with Hinf1.Considered as part LV of the series Studies on ectomycorrhizae of the Institute for Systematic Botany, Munich; part LIV: Agerer et al. (1994)     

Sperm Mediated Transfer of Genes into Goldfish and Detection by Polymerase Chain Reaction

Goldfish sperms were mixed with eggs for fertilization after incubation with antifreeze protein gene(AFP)from ocean pout for 30 min.A number of embryos and 145 adult goldfish were obtained.DNA from adult goldfish and embryos was extracted separately.Results of the amplification by PCRand Southern blot molecular hybridization indicate the integration of exogenous antifreeze gene intothe genome of a part of the recipient goldfish.Of the 45 samples detected by PCR,twelve showedpositive reaction with distinct hybridization band.The positive rate was 26%.     

Sensitive and rapid detection of Karenia mikimotoi (Dinophyceae) by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was developed to detect the genomic DNA of Karenia mikimotoi using a set of four specific primers based on a ribosomal DNA internal transcribed spacer (ITS). The sensitivity of this LAMP assay was 100-fold higher than regular PCR, and its specificity was validated using other algae as a comparison. Two visual detection approaches were feasible to interpret the positive or negative results. This technology may have the potential to aid in forecasting red-tides on the scene because of its high sensitivity, specificity and rapid detection.     

The influence of quality of primers on the formation of primer dimers in PCR

Abstract

Polymerase chain reaction (PCR) is the most commonly used method for nucleic acids amplification. PCR performance depends on several causes, among which the quality of primers is one of the main determinants affecting specificity, sensitivity and reliability of the reaction. Here, we report on the results of the detailed study devoted to the dimerization of the primers during PCR. The course and specificity of the reaction were studied on the model DNA templates as well as genomic DNA using primers that form amplifiable heterodimeric structures with different thermodynamic stability. It was confirmed that more than two 3′-overlapping nucleotides cause a considerable accumulation of primer dimers. It turned out that the presence of any DNA promotes the formation of dimers even for primers, which do not tend to nonspecific amplification in the absence of DNA. It was shown that dimerization could not be eliminated by commonly used techniques. Even the use of hot-start DNA polymerases does not prevent PD formation if primers with stable 3′-overlapping are employed. Despite several advantages of PCR with abutting primers, their close disposition has no benefits regarding the formation of PD if low-quality primers are utilized.     

Detection of Various Variant Verotoxin Genes in Escherichia coli by Polymerase Chain Reaction

We constructed common primers for the polymerase chain reaction to detect the genes for various Verotoxins reported, that is, VT1 (or SLT-I), VT2 (or SLT-II), VT2vha, VT2vhb, SLT-IIv (or VT2vp1, VTe) and SLT-IIva (or VT2vp2). A total of 80 Verocytotoxin-producing Escherichia coli strains isolated from humans, domestic animals and meats gave a positive result by PCR with the designed common primers. Digestion by restriction endonucleases BglII and EcoT14I of the amplicon of the VT2vp2 gene gave specific bands of the expected sizes, but not of the amplicons of other VT genes, suggesting a possible method for identification of the VT2vp2 gene. Application of the PCR with the designed primers in diagnostic and epidemiological studies on VTEC infection is also discussed.     

The challenge to quantify Listeria monocytogenes--a model leading to new aspects in molecular biological food pathogen detection

In this work, we discuss the latest insights concerning advantages and disadvantages and the nature of microbiological and molecular methods for quantitative food pathogen detection. The assessment of molecular methods must be brought on a basis that considers the nature of molecular methods and their underlying mechanism. A potential approach to setting up the development, validation and structure of an analytical chain is presented based on quantitative real-time PCR (qPCR). This is analysed exemplary on the basis of recent work using the model organism Listeria monocytogenes. Several prerequisites for successful quantitative detection of this pathogen will be discussed. In particular, sample preparation, controls for all methodical steps and the validation of the core assay qPCR are addressed, which constitute the basis for a reliable analytical detection chain for molecular biological pathogen detection from food. Microbiological methods are analysed based on growth of the single cell, which is the fundament of these traditional methods.