Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

A simple and sensitive method for detection of Bacillus anthracis by loop‐mediated isothermal amplification

Aims: To develop a rapid and simple system for detection of Bacillus anthracis using a loop‐mediated isothermal amplification (LAMP) method and determine the suitability of LAMP for rapid identification of B. anthracis infection. Methods and Results: A specific LAMP assay targeting unique gene sequences in the bacterial chromosome and two virulence plasmids, pXO1 and pXO2, was designed. With this assay, it was possible to detect more than 10 fg of bacterial DNA per reaction and obtain results within 30–40 min under isothermal conditions at 63°C. No cross‐reactivity was observed among Bacillus cereus group and other Bacillus species. Furthermore, in tests using blood specimens from mice inoculated intranasally with B. anthracis spores, the sensitivity of the LAMP assay following DNA extraction methods using a Qiagen DNeasy kit or boiling protocol was examined. Samples prepared by both methods showed almost equivalent sensitivities in LAMP assay. The detection limit was 3·6 CFU per test. Conclusions: The LAMP assay is a simple, rapid and sensitive method for detecting B. anthracis. Significance and Impact of the Study: The LAMP assay combined with boiling extraction could be used as a simple diagnostic method for identification of B. anthracis infection.     

Specific detection of Pectobacterium carotovorum by loop‐mediated isothermal amplification

Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre‐ and post‐harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non‐pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop‐mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non‐target genera of plant‐associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular‐based detection assays.     

Loop‐mediated isothermal amplification for the rapid detection of Gardnerella vaginalis

The aim of this study was to develop a method for the rapid detection of Gardnerella vaginalis, which is proposed to play a key role in the pathogenesis of bacterial vaginosis. Specific loop‐mediated isothermal amplification (LAMP) primers were designed and used to detect target DNA within 45 min under isothermal conditions. Comparative screening indicated that the LAMP assay is superior to PCR in terms of rapidity, and is equivalent in sensitivity and specificity. This LAMP assay can be used for rapid screening and detection of G. vaginalis in vaginal samples; the limit of detection is 10 fg DNA.

     

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.     

Development of a loop‐mediated isothermal amplification assay for rapid and sensitive detection of Sporisorium scitamineum in sugarcane

Smut disease caused by Sporisorium scitamineum is one of the most destructive sugarcane diseases worldwide. The pathogen spreads primarily through infected sugarcane setts, and hence, the use of disease‐free planting materials is essential for preventing disease development in the field. In this study, a species‐specific loop‐mediated isothermal amplification (LAMP) assay was developed for rapid and accurate detection of S. scitamineum. Based on the differences in internal transcribed spacer (ITS) sequences of S. scitamineum, a set of four species‐specific primers, F3, B3, FIP and BIP, were designed by using a panel of fungal and bacterial species as controls. After optimization of the reaction conditions, the detection limit of LAMP assay was about 2 fg of the S. scitamineum genomic DNA in 25 µL reaction solution, 100‐fold lower than that of conventional polymerase chain reaction. The assay showed high specificity to discriminate all S. scitamineum isolates from nine other fungal and bacterial pathogens. The LAMP assay also detected smut infection from young sugarcane leaves with no visible smut‐disease symptoms. The findings from this study provide a simple, highly sensitive, rapid and reliable technique for early detection of S. scitamineum, which may be useful for sugarcane quarantine and production of smut‐free seedcanes. This is the first report of LAMP‐based assay for the detection of S. scitamineum in sugarcane.     

Rapid and simple identification of Beijing genotype strain of Mycobacterium tuberculosis using a loop‐mediated isothermal amplification assay

Beijing genotype strains of Mycobacterium tuberculosis are geographically widespread and pose a notorious public health problem, these strains causing outbreaks of multidrug‐resistant tuberculosis (TB); some studies have reported an association with drug resistance. Because the prevalence of Beijing strain has a substantial impact on TB control programs, the availability of a rapid and reliable method for detecting these strains is important for epidemiological monitoring of their circulation. The main methods currently used to identify Beijing genotype strains are IS6110 DNA fingerprinting, spoligotyping and PCR to detect specific deletions such as region of difference (RD)207. More recently, multiplex PCR assay using a Beijing‐specific single nucleotide polymorphism (SNP) has been developed for detecting Beijing lineage strains. However, these methods are time‐consuming and technically demanding. In the present study, a loop‐mediated isothermal amplification (LAMP) assay that allows specific identification of Beijing genotype strain was developed. This Beijing genotype strain‐identifying LAMP assay was performed 214 clinical isolates and the results compared with those of conventional PCR that targeted RD207 and Rv0679c‐targreting multiplex PCR for Beijing lineage identification. LAMP assay showed 100% sensitivity and specificity compared with RD207‐PCR. Furthermore, the sensitivity and specificity were 99.3% and 100%, respectively, compared with Rv0679c‐multiplex PCR. This LAMP assay could be used routinely in local laboratories to monitor the prevalence of the Beijing genotype strain and thereby used to help control the spread of these potentially highly virulent and drug resistant strains.     

Rapid detection of mecA and spa by the loop‐mediated isothermal amplification (LAMP) method

Aim: To develop a detection assay for staphylococcal mecA and spa by using loop‐mediated isothermal amplification (LAMP) method. Methods and Results: Staphylococcus aureus and other related species were subjected to the detection of mecA and spa by both PCR and LAMP methods. The LAMP successfully amplified the genes under isothermal conditions at 64°C within 60 min, and demonstrated identical results with the conventional PCR methods. The detection limits of the LAMP for mecA and spa, by gel electrophoresis, were 10² and 10 cells per tube, respectively. The naked‐eye inspections were possible with 10³ and 10 cells for detection of mecA and spa, respectively. The LAMP method was then applied to sputum and dental plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples, although frequency in detection of mecA and spa by the LAMP was relatively lower for the sputum samples when compared to the PCR methods. Conclusion: Application of the LAMP enabled a rapid detection assay for mecA and spa. The assay may be applicable to clinical plaque samples. Significance and Impact of the Study: The LAMP offers an alternative detection assay for mecA and spa with a great advantage of the rapidity.     

Use of loop‐mediated isothermal amplification to detect six groups of pathogens causing secondary lower respiratory bacterial infections in horses

Microbial substitution occasionally occurs following the administration of antimicrobials to horses that have pneumonia or pleuropneumonia. Four specific loop‐mediated isothermal amplification (LAMP) assays were developed to detect some equine respiratory pathogens, namely strains of the Bacteroides–Prevotella group, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and Staphylococcus aureus. These four LAMP assays and two previously published LAMP assays targeting Escherichia coli or Pseudomonas aeruginosa were used on clinical respiratory specimens and a high accordance found between the results of the LAMP assays and bacterial culture. Use of these LAMP assays could enable rapid detection of pathogenic bacteria and swift administration of the appropriate antimicrobials.