Isothermal DNA Amplification Technique Research Articles

Real-time MBDi-RPA using methyl-CpG binding protein 2: A real-time detection method for simple and rapid estimation of CpG methylation status

BackgroundAnalysis of CpG methylation is informative for cancer diagnosis. Previously, we developed a novel method to discriminate CpG methylation status in target DNA by blocking recombinase polymerase amplification (RPA), an isothermal DNA amplification technique, using methyl-CpG binding domain (MBD) protein 2 (MBD2). The method was named MBD protein interference-RPA (MBDi-RPA). In this study, MBDi-RPA was performed using methyl-CpG binding protein 2 (MeCP2), another MBD family protein, as the blocking agent. ResultsMBDi-RPA using MeCP2 detected low levels of CpG methylation, showing that it had higher sensitivity than MBDi-RPA using MBD2. We also developed real-time RPA, which enabled rapid analysis of DNA amplification without the need for laborious agarose gel electrophoresis and used it in combination with MBDi-RPA. We termed this method real-time MBDi-RPA. The method using MeCP2 could determine the abundance ratio of CpG-methylated target DNA simply and rapidly, although highly sensitive detection was challenging. Significance and noveltyReal-time MBDi-RPA using MeCP2 could be potentially useful for estimating CpG methylation status in target DNA prior to more detailed analyses.

453 Rapid SARS-CoV-2 testing with duplexed recombinase polymerase amplification and a bacteriophage internal control

OBJECTIVES/GOALS: Current COVID-19 rapid molecular tests require cartridge-reader detection, expensive circuitry, and complex microfluidics making the most accurate tests unavailable to the masses. Here we present a rapid molecular diagnostic leveraging isothermal amplification and paper-based microfluidics for a low-cost ultra-sensitive COVID-19 assay. METHODS/STUDY POPULATION: We designed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of SARS-CoV-2 and bacteriophage MS2 RNA. RT-RPA is a sequence specific, ultrasensitive, rapid isothermal DNA amplification technique that is well suited to home based testing due to its rapid assay time, robustness, ease of use, and readout options. RT-RPA reagents are added to a tube and incubated at 39°C in a fluorometer. Realtime fluorometer data gives results in under 15 minutes. This assay also provides visual detection via lateral flow readout with results in 23 minutes. RESULTS/ANTICIPATED RESULTS: We have developed a rapid multiplexed nucleic acid amplification assay with an internal process control for SARS-CoV-2 using single-pot RT-RPA. We screened 21 primer combinations to select primers that demonstrated excellent performance and target specificity against common respiratory viruses. We demonstrate the ability to multiplex SARS-CoV-2 and MS2 detection, utilizing MS2 as an internal process control for lysis, reverse transcription, amplification, and readout. We show duplexed detection using both fluorescence readout and visual readout using lateral flow strips. Duplexed fluorescence detection shows a limit of detection of 25 copies per reaction. Duplexed lateral flow readout shows a limit of detection of 50 copies per reaction DISCUSSION/SIGNIFICANCE: We developed a duplexed RT-RPA assay for SARS-CoV-2 with fluorescence or lateral flow readout. Our assay does not re-quire expensive reader, circuity, or fluid handling. The low material cost, temperature, and robustness make it ideal for a more accurate home-based COVID-19 diagnostic.

Rapid whole genome sequencing methods for RNA viruses.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

Rolling Circle Amplification on a Bead: Improving the Detection Time for a Magnetic Bioassay.

Detection of pathogens has become increasingly important, especially in the face of outbreaks and epidemics all over the world. Nucleic acid detection techniques provide a solid base to detect and identify pathogens. In recent years, magnetic sensors and magnetic labels have become of more interest due to their simplicity of use, low cost, and versatility. In this work, we have used the isothermal DNA amplification technique of rolling circle amplification (RCA) in combination with oligo-functionalized magnetic nanoparticles. Detection of RCA products takes place through specific binding between magnetic nanoparticles and RCA products. Upon binding, the relaxation frequency of the nanoparticle changes. This change was measured using an AC susceptometer. We showcase that the RCA time can be reduced for a quicker assay when performing the RCA on the surface of micrometer-sized beads, which consequently increases the hydrodynamic volume of the RCA products. This, in turn, increases the Brownian relaxation frequency shift of the nanoparticles upon binding. We performed optimization work to determine the ideal quantity of micrometer-sized particles, oligo-functionalized nanoparticles, and the amplification time of the RCA. We show that the detection of 0.75 fmol of preamplification synthetic target is possible with only 20 min of amplification time. Finally, we showcase the high specificity of the assay, as the functionalized nanoparticles are unable to bind to amplified DNA that does not match their labels. Overall, this paves the way for a simple bioassay that can be used without expensive laboratory equipment for detection of pathogens in outbreak settings and clinics around the world.

Recent advances in biological detection with rolling circle amplification: design strategy, biosensing mechanism, and practical applications.

Rolling circle amplification (RCA) is a simple and isothermal DNA amplification technique that is used to generate thousands of repeating DNA sequences using circular templates under the catalysis of DNA polymerase. Compared to alternating temperature nucleic acid amplification such as polymerase chain reaction (PCR) amplification, RCA is more suitable for on-spot detection without the need for an expensive thermal cycler. In this study, the principle and classification of RCA are introduced, and the applications of RCA in the detection of pathogenic bacteria, nucleic acid tumor markers, viruses, and proteins are reviewed. Finally, the perspectives of RCA in biological detection are anticipated. The RCA method has a great potential for biological detection. This review aims to provide references for the further development and application of the RCA technique in biosensors.

A stoichiometric and pseudo kinetic model of loop mediated isothermal amplification

Loop mediated isothermal amplification (LAMP) is one of the most popular isothermal DNA amplification techniques for research and commercial applications, enabling amplification of both DNA and RNA (with the assistance of reverse transcriptase). The LAMP mechanism is powered by strategic primer design and a strand displacement polymerase, generating products that fold over, creating loops. LAMP leads to generation of products of increasing length over time. These products containing multiple loops are conventionally called cauliflower structures. Existing literature on LAMP provides extremely limited understanding of progression of cascades of reactions involved in the reaction and it is believed that cauliflower structures of increasing length constitute a majority of the product formed in LAMP. This study presents a first of its kind stoichiometric and pseudo kinetic model to comprehend LAMP reactions in deeper depth by (i) classifying LAMP reaction products into uniquely identifiable categories, (ii) generating a condensed reaction network to depict millions of interconnected reactions occurring during LAMP, and (iii) elucidating the pathways for amplicon generation. Despite the inherent limitations of conventional stoichiometric modelling for polymerization type reactions (the network rapidly becomes too large and intractable), our model provides new theoretical understanding of the LAMP reaction pathway. The model shows that while longer length products are formed, it is the smaller length recycle amplicons that contribute more towards the exponential increase in the amount of double stranded DNA. Prediction of concentration of different types of LAMP amplicons will also contribute substantially towards informing design of probe-based assays.

Progress in rolling circle amplification in biological detection

Rolling circle amplification is a rapid, sensitive and isothermal single-stranded DNA amplification technique that can be used with staining or probes to amplify the detection signal. This technology has been widely used in biological detection and other aspects. The present paper introduces how to design rolling circle amplification, summarize its application in the detection of pathogens, nucleic acid tumor markers, proteins, biological small biomolecules, and viruses in recent years and prospects for future development.

A CRISPR\u2013Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection

Although polymerase chain reaction (PCR) is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications. Isothermal DNA amplification techniques are hence valuable for on-site diagnostic applications in place of traditional PCR. Here we describe a true isothermal approach for amplifying and detecting double-stranded DNA based on a CRISPR–Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification method (namely CRISDA). CRISDA takes advantage of the high sensitivity/specificity and unique conformational rearrangements of CRISPR effectors in recognizing the target DNA. In combination with a peptide nucleic acid (PNA) invasion-mediated endpoint measurement, the method exhibits attomolar sensitivity and single-nucleotide specificity in detection of various DNA targets under a complex sample background. Additionally, by integrating the technique with a Cas9-mediated target enrichment approach, CRISDA exhibits sub-attomolar sensitivity. In summary, CRISDA is a powerful isothermal tool for ultrasensitive and specific detection of nucleic acids in point-of-care diagnostics and field analyses.

A one-pot, isothermal DNA sample preparation and amplification platform utilizing aqueous two-phase systems.

Infectious diseases remain one of the major causes of death worldwide in developing countries. While screening via conventional polymerase chain reaction (PCR) is the gold standard in laboratory testing, its limited applications at the point-of-care have prompted the development of more portable nucleic acid detection systems. These include isothermal DNA amplification techniques, which are less equipment-intensive than PCR. Unfortunately, these techniques still require extensive sample preparation, limiting user accessibility. In this study, we introduce a novel system that combines thermophilic helicase-dependent amplification (tHDA) with a Triton X-100 micellar aqueous two-phase system (ATPS) to achieve cell lysis, lysate processing, and enhanced nucleic acid amplification in a simple, one-step process. The combined one-pot system was able to amplify and detect a target gene from whole-cell samples containing as low as 102cfu/mL, and is the first known application of ATPSs to isothermal DNA amplification. This system's ease-of-use and sensitivity underlie its potential as a point-of-care diagnostic platform to detect for infectious diseases. Graphical abstract ᅟ.

Discovery and Biosensing Applications of Diverse RNA-Cleaving DNAzymes.

DNA-based enzymes, or DNAzymes, are not known to exist in Nature but can be isolated from random-sequence DNA pools using test tube selection techniques. Since the report of the first DNAzyme in 1994, many catalytic DNA molecules for catalyzing wide-ranging chemical transformations have been isolated and studied. Our laboratory has a keen interest in searching for diverse DNAzymes capable of cleaving RNA-containing substrates, determining their sequence requirements and structural properties, and examining their potential as biosensors. This Account begins with the description of an accidental discovery on the sequence adaptability of a small DNAzyme known as "8-17", when we performed 16 parallel selections to search for DNAzymes that targeted each and every possible dinucleotide junction of RNA for cleavage. DNAzyme 8-17 dominated all the selection pools targeting purine-containing junctions. In-depth sequence analysis revealed that 8-17 could manifest itself in many sequence options defined by the requirement of four absolutely conserved nucleotides. This study also exposed the fact that 8-17 had poor activity toward pyrimidine-pyrimidine junctions. With this information in hand, we proceeded to the discovery of diverse non-8-17 DNAzymes that exhibited robust catalytic activity under physiological conditions. These DNAzymes were found to universally interact with their substrates through two Watson-Crick binding arms and have a catalytic core of varying length and secondary-structure complexity. RNA-cleaving DNAzymes were also isolated to function at acidic conditions (pH 3-5), and these molecules exhibited intriguing pH profiles, with the highest activity precisely matching the pH used for their selection. Interestingly, these DNAzymes appear to use non-Watson-Crick interactions in defining their structures. More recently, we have embarked on the development of ligand-responsive RNA-cleaving fluorogenic DNAzymes that can recognize specific bacterial pathogens, such as Escherichia coli and Clostridium difficile, using a method that does not require a priori identification of a specific biomarker. Instead, the crude extracellular mixture as a whole is used as the target to drive the DNAzyme isolation. High recognition specificity can be achieved with a double-selection approach in which a DNA library is negatively selected against the cellular mixture prepared from unintended bacteria, followed by positive selection against the same mixture derived from a specific species or strain of bacterial pathogen. Finally, we have shown that DNAzymes' compatibility with DNA replication can benefit the design of amplification mechanisms that uniquely link the action of RNA-cleaving DNAzymes to rolling circle amplification, an isothermal DNA amplification technique. These methods are well suited for translating the target-binding and cleavage activity of an analyte-activated RNA-cleaving DNAzyme into the production of massive amounts of DNA amplicons to achieve ultrahigh detection sensitivity. Given the high chemical stability of DNA, our ability to discover catalytic DNA sequences by simultaneously evaluating as many as 1016 different DNA sequences, the accessibility to diverse RNA-cleaving DNAzymes in a single DNA pool, and the availability of methods for designing simple biosensors that incorporate RNA-cleaving DNAzymes, we believe we are moving closer to employing RNA-cleaving DNAzymes for exciting applications, such as point of care diagnostics or field detection of environmental toxins.

On-nylon membrane detection of nucleic acid molecules by rolling circle amplification

Positively-charged nylon membrane (NM) is a general solid-phase support for nucleic acid detection due to its convenient immobilization of nucleic acid materials by direct electrostatic adherence and simple UV crosslinking. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification technique for nucleic acid detection. Near-infrared fluorescence (NIRF) is a new fluorescence technique with high sensitivity due to low background. This study developed a simple method for detecting nucleic acid molecules by combining the advantages of NM, RCA and NIRF, named NIRF-based solid phase RCA on nylon membrane (NM-NIRF-sRCA). The detection system of this method only need two kinds of nucleic acid molecules: target-specific probes with a RCA primer (P) at their 3′ end and a rolling circle (RC). The detection procedure consists of four steps: (1) immobilizing detected nucleic acids on NM by UV crosslinking; (2) hybridizing NM with specific probes and RC; (3) amplifying by a RCA reaction containing biotin-dUTP; (4) incubating NM with NIRF-labeled streptavidin and imaging with a NIRF imager. The method was fully testified by detecting oligonucleotides, L1 fragments of various HPV subtypes cloned in plasmid, and E.coli genomic DNA. This study thus provides a new facile method for detecting nucleic acid molecules.

G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection

Real-time PCR has revolutionized PCR from qualitative to quantitative. As an isothermal DNA amplification technique, rolling circular amplification (RCA) has been demonstrated to be a versatile tool in many fields. Development of a simple, highly sensitive, and specific strategy for real-time monitoring of RCA will increase its usefulness in many fields. The strategy reported here utilized the specific fluorescence response of thioflavin T (ThT) to G-quadruplexes formed by RCA products. Such a real-time monitoring strategy works well in both traditional RCA with linear amplification efficiency and modified RCA proceeded in an exponential manner, and can be readily performed in commercially available real-time PCR instruments, thereby achieving high-throughput detection and making the proposed technique more suitable for biosensing applications. As examples, real-time RCA-based sensing platforms were designed and successfully used for quantitation of microRNA over broad linear ranges (8 orders of magnitude) with a detection limit of 4 aM (or 0.12 zmol). The feasibility of microRNA analysis in human lung cancer cells was also demonstrated. This work provides a new method for real-time monitoring of RCA by using unique nucleic acid secondary structures and their specific fluorescent probes. It has the potential to be extended to other isothermal single-stranded DNA amplification techniques.

Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples.

Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings.

An integrated direct loop-mediated isothermal amplification microdevice incorporated with an immunochromatographic strip for bacteria detection in human whole blood and milk without a sample preparation step

We have developed an integrated direct loop-mediated isothermal amplification (Direct LAMP) microdevice incorporated with an immunochromatographic strip (ICS) to identify bacteria contaminated in real samples. The Direct LAMP is a novel isothermal DNA amplification technique which does not require thermal cycling steps as well as any sample preparation steps such as cell lysis and DNA extraction for amplifying specific target genes. In addition, the resultant amplicons were colorimetrically detected on the ICS, thereby enabling the entire genetic analysis process to be simplified. The two functional units (Direct LAMP and ICS) were integrated on a single device without use of the tedious and complicated microvalve and tubing systems. The utilization of a slidable plate allows us to manipulate the fluidic control in the microchannels manually and the sequential operation of the Direct LAMP and ICS detection could be performed by switching the slidable plate to each functional unit. Thus, the combination of the direct isothermal amplification without any sample preparation and thermal cycling steps, the ICS based amplicon detection by naked eyes, and the slidable plate to eliminate the microvalves in the integrated microdevice would be an ideal platform for point-of-care DNA diaganotics. On the integrated Direct LAMP-ICS microdevice, we could analyze Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) contaminated in human whole blood or milk at a single-cell level within 1h.

A rapid MZI-IDA sensor system for EGFR mutation testing in non-small cell lung cancer (NSCLC).

Epidermal growth factor receptor (EGFR) is a non-small-cell lung cancer biomarker, based on which several near-patient-testing methods have been developed and applied to predict treatment response on individual patients. Existing methods for detection of EGFR mutation are costly, labor-intensive and time-consuming. In this paper, we report a novel EGFR mutation testing system, which is based on Mach-Zehnder Interferometer (MZI) sensor and isothermal solid-phase DNA amplification (IDA) technique, called MZI-IDA sensor system. The system can deliver results within 30 min and shows high sensitivity to detect trace amounts of genomic DNA (<1 copy). In addition, the system is able to detect a L858R mutation in a 99:1 mixture of wild-type and mutant cells. In a pilot clinical study, the system is compared with conventional methods (PCR and direct sequencing) by using tissue biopsy samples from NSCLC patients. The MZI-IDA sensor system is proved to be capable of fast and accurate detection of the L858R mutation of EGFR gene in clinical samples. This may greatly help the clinicians develop an appropriate treatment plan.

Optimal DNA templates for rolling circle amplification revealed by in vitro selection.

Rolling circle amplification (RCA) has been widely used as an isothermal DNA amplification technique for diagnostic and bioanalytical applications. Because RCA involves repeated copying of the same circular DNA template by a DNA polymerase thousands of times, we hypothesized there exist DNA sequences that can function as optimal templates and produce more DNA amplicons within an allocated time. Herein we describe an in vitro selection effort conducted to search from a random sequence DNA pool for such templates for phi29 DNA polymerase, a frequently used polymerase for RCA. Diverse DNA molecules were isolated and they were characterized by richness in adenosine (A) and cytidine (C) nucleotides. The top ranked sequences exhibit superior RCA efficiency and the use of these templates for RCA results in significantly improved detection sensitivity. AC-rich sequences are expected to find useful applications for setting up effective RCA assays for biological sensing.

Rapid screening for human-pathogenic Mucorales using rolling circle amplification.

Mucormycosis has emerged as a relatively common severe mycosis in patients with haematological and allogeneic stem cell transplantation. Source of transmission is from unidentified sources in the environment. Early diagnosis of infection and its source of contamination are paramount for rapid and appropriate therapy. In this study, rolling circle amplification (RCA) is introduced as a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of six of the most virulent species (Rhizopus microsporus, R. arrhizus var. arrhizus, R. arrhizus var. delemar, Mucor irregularis, Mucor circinelloides, Lichtheimia ramosa, Lichtheimia corymbifera). DNAs of target species were successfully amplified, with no cross reactivity between species. RCA can be considered as a rapid detection method with high specificity and sensitivity, suitable for large screening.

Loop‐mediated isothermal amplification of single pollen grains

The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental requirements of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplification methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop-mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pollen analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field-based, high-throughput analysis.

Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis.

Hepatitis C virus (HCV) infection represents a significant health problem and represents a heavy load on some countries like Egypt in which about 20% of the total population are infected. Initial infection is usually asymptomatic and result in chronic hepatitis that give rise to complications including cirrhosis and hepatocellular carcinoma. The management of HCV infection should not only be focus on therapy, but also to screen carrier individuals in order to prevent transmission. In the present, molecular detection and quantification of HCV genome by real time polymerase chain reaction (PCR) represent the gold standard in HCV diagnosis and plays a crucial role in the management of therapeutic regimens. However, real time PCR is a complicated approach and of limited distribution. On the other hand, isothermal DNA amplification techniques have been developed and offer molecular diagnosis of infectious dieses at point-of-care. In this review we discuss recombinase polymerase amplification technique and illustrate its diagnostic value over both PCR and other isothermal amplification techniques.

The detection of Plasmodiophora brassicae using loop-mediated isothermal DNA amplification

&lt;em&gt;Plasmodiophora brassicae&lt;/em&gt;, the cause of clubroot, is a very serious problem preventing from successful and profitable cultivation of oilseed rape in Poland. The pathogen was found in all main growing areas of oilseed rape; it also causes considerable problems in growing of vegetable brassicas. The aim of this work was to elaborate fast, cheap and reliable screening method to detect &lt;em&gt;P. brassicae&lt;/em&gt;. To achieve this aim the Loop-mediated isothermal DNA amplification (LAMP) technique has been elaborated. The set of three primer pairs was designed using LAMP software. The detection was performed with the GspSSD polymerase, isolated from bacteria &lt;em&gt;Geobacillus&lt;/em&gt; sp., with strand displacement activity. DNA extraction from clubbed roots obtained from farmers’ fields of oilseed rape infected by &lt;em&gt;P. brassicae&lt;/em&gt; was done using a modified CTAB method. The reaction was performed for 60 min at 62&lt;sup&gt;o&lt;/sup&gt;C. The visual detection was done using CFX96 Real Time PCR Detection System (BioRad) or Gerie II Amplicatior (Optigen). The detection with LAMP proved its usefulness; it was easy, fast and accurate and independent of plant age. The detection limit was 5 spores per 1 µl of the spore suspension, so LAMP was less sensitive than quantitative PCR tests reported in the literature. However, the method is cheap and simple, so it is a good alternative, when it comes to practical use and the assessment of numerous samples.