Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop‐mediated isothermal amplification

Aim: The objective of this study is to develop a serovar‐specific loop‐mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. Methods: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real‐time polymerase chain reaction (FQ‐PCR). Results: The results were as follows. (1) Serovar‐specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20 min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies μl^?1; thus, the sensitivity and specificity of this assay is similar to those of the FQ‐PCR. Conclusions: LAMP is a high‐throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. Significance and impact of the study: This is the first study involving the use of LAMP to detect Salmonella serovar‐specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.     

Loop‐mediated isothermal amplification assay targeting the blaCTX‐M9 gene for detection of extended spectrum β‐lactamase‐producing Escherichia coli and Klebsiella pneumoniae

Extended‐spectrum β‐lactamases (ESBLs) produced by Enterobacteriaceae are one of the resistance mechanisms to most β‐lactam antibiotics. ESBLs are currently a major problem in both hospitals and community settings worldwide. Rapid and reliable means of detecting ESBL‐producing bacteria is necessary for identification, prevention and treatment. Loop‐mediated isothermal amplification (LAMP) is a technique that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. This study was aimed to develop a convenient, accurate and inexpensive method for detecting ESBL‐producing bacteria by a LAMP technique. ESBLs‐producing Escherichia coli and Klebsiella pneumoniae were isolated from a tertiary hospital in Bangkok, Thailand and reconfirmed by double‐disk synergy test. A set of four specific oligonucleotide primers of LAMP for detection of bla_CTX‐M9 gene was designed based on bla_CTX‐M9 from E. coli (GenBank Accession No. AJ416345). The LAMP reaction was amplified under isothermal temperature at 63°C for 60 min. Ladder‐like patterns of band sizes from 226 bp of the bla_CTX‐M9 DNA target was observed. The LAMP product was further analyzed by restriction digestion with MboI and TaqI endonucleases. The fragments generated were approximately 168, 177 and 250 bp in size for MboI digestion and 165, 193, 229, 281 and 314 bp for TaqI digestion, which is in agreement with the predicted sizes. The sensitivity of the LAMP technique to bla_CTX‐M9 was greater than that of the PCR method by at least 10,000‐fold. These results showed that the LAMP primers specifically amplified only the bla_CTX‐M9 gene. Moreover, the presence of LAMP amplicon was simply determined by adding SYBR Green I in the reaction. In conclusion, this technique for detection of ESBLs is convenient, reliable and easy to perform routinely in hospitals or laboratory units in developing countries.     

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.

     

Rapid Detection of Banana Streak Virus by Loop‐mediated Isothermal Amplification Assay in South China

Banana streak virus (BSV) is a significant constraint to banana production and genetic improvement. It is necessary to develop and use BSV detection strategies that are both reliable and sensitive for the management of the virus. A loop‐mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of BSV. Four primers matching a total of six sequences of the conserved ORF III polyprotein genes were synthesized for developing a specific and sensitive LAMP for DNA extracts from field‐infected banana plants. LAMP assay could detect as low as 1 pg/μl template DNA. Test results of all field samples collected from different regions of South China showed that LAMP is more sensitive than PCR. This relatively simple and sensitive technique showed excellent potential with field‐collected samples and for routine screening of tissue culture materials in South China.     

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.     

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 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.     

Loop‐mediated isothermal amplification method for rapid detection of Vibrio alginolyticus,the causative agent of vibriosis in mariculture fish

Aims: The purpose of this study was to develop a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. Methods and Results: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64°C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3·7 × 10² CFU ml^?1 (3·7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross‐reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus‐infected fish tissues effectively. Conclusions: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.     

Establishment of a loop‐mediated isothermal amplification system for on‐site diagnosis of Oryctes rhinoceros nudivirus in Allomyrina dichotoma (Coleoptera: Scarabaeidae)

The Korean insect industry is rapidly developing, with the Korean horn beetle Allomyrina dichotoma as one of the most popular insects kept as pets. Korean horn beetles reared in local farms are suffering from Oryctes rhinoceros nudivirus (OrNV). In a nationwide investigation on the early death of young larvae due to this virus, 70 to 77% mortality was found. It was also confirmed that several larvae collected from some wild horn beetle habitats were infected with the virus. Thus, it was concluded that the virus could be transmitted vertically from an infected adult to the offspring and that the OrNV disease is a very serious threat to the wild horn beetle in Korea. With local farmers expecting that an early detection technique would be helpful for the quick removal of infected larvae, an on‐site diagnosis method for the viral disease using loop‐mediated isothermal amplification (LAMP) assay was thus tested. To avoid the DNA extraction process, the LAMP assay used a 50‐fold diluted hemolymph was set for diagnosis standardization and convenient on‐site application to infected larvae from local farms. A. dichotoma larvae were assayed for the OrNV infectivity with LAMP primers targeting the OrNV_gp102 gene. To evaluate the LAMP specificity, two bacterial pathogens, Bacillus thuringiensis and Serratia marcescens, causing disease in A. dichotoma were tested along with OrNV. The positive results were detected only from the OrNV‐infected larvae.     

DNA amplification in the field: move over PCR,here comes LAMP

It would not be an exaggeration to say that among molecular technologies, it is PCR (polymerase chain reaction) that underpins the discipline of molecular ecology as we know it today. With PCR, it has been possible to target the amplification of particular fragments of DNA, which can then be analysed in a multitude of ways. The capability of PCR to amplify DNA from a mere handful of copies further means that conservationists and ecologists are able to sample DNA unobtrusively and with minimal disturbance to the environment and the organisms of interest. However, a key disadvantage of PCR‐based methods has been the necessity for a generally non‐portable, laboratory setting to undertake the time‐consuming thermocycling protocols. LAMP (loop‐mediated isothermal amplification) offers a logistically simpler protocol: a relatively rapid DNA amplification reaction occurs at one temperature, and the products are visualized with a colour change within the reaction tubes. In the first field application of LAMP for an ecological study, Centeno‐Cuadros et al. ( 2016 ) demonstrates how LAMP can be used to determine the sex of three raptor species. By enabling DNA amplification in situ and in ‘real‐time’, LAMP promises to revolutionize how molecular ecology is practised in the field.     

环介导等温扩增核酸技术及其应用

环介导等温扩增(loop-mediated isothermal amplification,简称LAMP)是利用4个特殊设计的引物和具有链置换活性的DNA聚合酶,在恒温条件下特异、高效、快速地扩增DNA的新技术。该技术在1h内能扩增出109靶序列拷贝,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,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。LAMP技术以其特异性强、灵敏度高、快速、准确和操作简便等优点在核酸的科学研究、疾病的诊断和转基因食品检测等领域得到了日益广泛的应用。     

Detecting amplicons of loop‐mediated isothermal amplification

Loop‐mediated isothermal amplification (LAMP) assays are used to detect diverse pathogens. Initially, LAMP amplicons were detected using electrophoresis; later, real‐time monitoring based on turbidity was developed to overcome the problem of contamination with environmental DNA. Recently, real‐time monitoring of fluorescence signals using a quenching primer and probe has improved the reliability of amplification signals. Here, methods of detecting LAMP amplicons are reviewed.     

Sex determination in the wild: a field application of loop‐mediated isothermal amplification successfully determines sex across three raptor species

PCR‐based methods are the most common technique for sex determination of birds. Although these methods are fast, easy and accurate, they still require special facilities that preclude their application outdoors. Consequently, there is a time lag between sampling and obtaining results that impedes researchers to take decisions in situ and in real time considering individuals’ sex. We present an outdoor technique for sex determination of birds based on the amplification of the duplicated sex‐chromosome‐specific gene Chromo‐Helicase‐DNA binding protein using a loop‐mediated isothermal amplification (LAMP). We tested our method on Griffon Vulture (Gyps fulvus), Egyptian Vulture (Neophron percnopterus) and Black Kite (Milvus migrans) (family Accipitridae). We introduce the first fieldwork procedure for sex determination of animals in the wild, successfully applied to raptor species of three different subfamilies using the same specific LAMP primers. This molecular technique can be deployed directly in sampling areas because it only needs a voltage inverter to adapt a thermo‐block to a car lighter and results can be obtained by the unaided eye based on colour change within the reaction tubes. Primers and reagents are prepared in advance to facilitate their storage at room temperature. We provide detailed guidelines how to implement this procedure, which is simpler (no electrophoresis required), cheaper and faster (results in c. 90 min) than PCR‐based laboratory methods. Our successful cross‐species application across three different raptor subfamilies posits our set of markers as a promising tool for molecular sexing of other raptor families and our field protocol extensible to all bird species.     

Development and application of a simple,rapid and sensitive method for detecting moderately carbendazim‐resistant isolates in Botrytis cinerea

Sustainable disease management depends on the ability to monitor the development of fungicide resistance in pathogen populations. A point mutation resulting in an alteration (F200Y) at codon 200 of the target protein β‐tubulin leads to a moderate level of resistance to carbendazim in Botrytis cinerea. Although traditional methods remain a cornerstone in detection of fungicide resistance, molecular methods that do not require the isolation of pathogens, can detect the presence of resistance alleles at low frequencies, and require less time and labour than traditional methods. In this study, we present an efficient, rapid, and highly specific method for detecting the moderately carbendazim‐resistant isolates in B. cinerea based on loop‐mediated isothermal amplification (LAMP). By using specific LAMP primers, we detected the resistance‐conferring mutation underlying β‐tubulin F200Y. The concentrations of LAMP components and LAMP parameters were optimised, resulting in reaction temperatures and times of 61–65°C and 45 min, respectively. The feasibility of the LAMP assay was verified by assaying the diseased samples with artificial inoculation in the different hosts. The LAMP assay developed in the current study was specific, stable, repeatable and sensitive, and was successfully applied for detection of moderately carbendazim‐resistant isolates of B. cinerea in plant samples.     

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.     

Development of a Loop‐Mediated Isothermal Amplification Assay to Detect Fusarium oxysporum

We report the development of a loop‐mediated isothermal amplification (LAMP) assay targeting the CYP51C element for visual detection of F. oxysporum which caused Fusarium wilt in soybean. The CYP51C‐LAMP assay efficiently amplified the target gene in 60 min at 62°C. And specificity was evaluated against F. oxysporum, Fusarium spp. and other fungal species. The detection limit of the CYP51C‐specific LAMP assay for F. oxysporum was four conidia per gram soil. The assay also detected F. oxysporum from inoculated soybean tissues and residues. These results suggest that this CYP51C‐LAMP assay can be used to detect residues on plants in the field.