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Development of a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay for detection of African swine fever virus (ASFV)

PDF HTML阅读 XML下载 导出引用 引用提醒 一种快速检测微囊藻毒素mcyG基因的新技术——环介导恒温扩增 DOI: 10.5846/stxb201308112059 作者: 作者单位: 宁波大学,宁波大学,宁波大学,宁波大学,宁波大学,宁波大学 作者简介: 通讯作者: 中图分类号: 基金项目: 浙江省重大科技专项重点农业项目(2012C12009-4); 宁波市海洋藻类资源高效开发利用创新团队项目(2011B81007) A novel assay for rapid detection of microcystin mcyG gene: loop-mediated isothermal amplification Author: Affiliation: Ningbo University,Ningbo University,,,, Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:利用环介导恒温扩增(LAMP)技术,以微囊藻毒素(Microcystins,MCs)合成基因簇中的mcyG基因为靶序列,设计了1 套LAMP引物,建立了LAMP反应体系并进行灵敏度和特异性实验。结果表明mcyG基因的最低检测限为:24 cfu/mL,远低于常规PCR(Polymerase Chain Reaction)。整个检测过程仅需40 min,且可直接目测结果。特异性实验中, 13 株淡水常见水华蓝藻分属:色球藻属(Chroococcus)、念珠藻属(Nostoc)、鱼腥藻属(Anabaena)、束丝藻属(Aphanizomenon)、微囊藻属(Microcystis),其中10 株呈阳性反应, 3 株为阴性。在野外样品检测中,来自太湖与黄庆苗池塘的水样PCR检测显示阴性反应,而LAMP检测均呈阳性反应,提示此两处水样中可能含有产毒微囊藻,显示出了LAMP检测方法良好的野外检测和预警能力。综合上述,LAMP检测方法能够快速检测产微囊藻毒素的关键基因,且结果可视化。该方法简便、快捷、不依赖特殊检测设备,极具推广前景。 Abstract:Microcystin-producing cyanobacteria blooms, as well as their increasing global occurrence, are of worldwide concern because they have been implicated in the death of animals and humans. Currently, many techniques are used to evaluate whether toxins are present in water samples, including high-performance liquid chromatography (HPLC), mouse bioassays, protein phosphatase inhibition assays (PPIA), enzyme linked immunosorbent assay (ELISA), and mass spectrometry. While some of these techniques are quite sensitive, they are of use only once the toxin is present in the water source. However, microcystins remain within cyanobacterial cells until being released in high concentrations during cell lysis. Hence, identifying potential microcystin-producing strains in the environment prior to the release of toxins into the water body is more useful than detection of toxins. Many microcystin-producing strains cannot be differentiated from non-toxic strains by traditional microscopy or with ribosomal gene sequences. Synthetase genes (mcy) are responsible for the biosynthesis of microcystin and are only present in the genome of toxic strains. Polymerase chain reaction (PCR) assays based on mcy gene sequences have been reported. Though these assays for rapid, sensitive, and specific detection appear to be promising, PCR applied as a rapid diagnostic test in the field has been seriously constrained owing to the complexity of the amplification conditions and the need for specialized high-cost instruments. Recently, loop-mediated isothermal amplification (LAMP), a new technology for amplifying DNA, has been developed. Unlike PCR, which requires special instruments to control reaction temperature, LAMP requires only a temperature-controlled water bath. In this manuscript, we designed a set of LAMP primers to target the sequence of the mcyG gene, which is responsible for the first step in the biosynthesis of Adda (3-amino-9-methoxy-2, 6, 8-trim ethyl-10-phenyldeca-4, 6-dienoic acid), an important part of microcystin. The conditions of amplification were optimized to implement the sensitivity and specificity experiments. We found that 61 ℃ for 40 min was the most suitable reaction condition. We then selected thirteen cyanobacterial strains, including Microcystis, Aphanizomenon, Chroococcus, Nostoc, Oscillatoria, and Anabaena, to determine the specificity and sensitivity of the primers in the LAMP reactions. The results of amplification were positive with ten microcystin-producing strains and negative with three non-toxic strains, which demonstrated that the primers had suitable levels of specificity. The sensitivity of LAMP, with a detection limit of 24 cfu/ml, was 1000-fold higher than that of PCR. For verifying the application of this LAMP assay in the aquatic ecosystem, seven environmental samples from ponds and lakes in Ningbo and one from Tai Lake were analyzed using both the LAMP assay targeting the mcyG gene and a PCR assay. The LAMP assay detected amplification in two water samples that the PCR assay did not detect, demonstrating that the LAMP assay is more effective and more suitable for field testing. The LAMP method is fast, simple, and low in cost making it advantageous for the detection of potential toxic cyanobacterial blooms. By employing the LAMP assay, workers at the Environmental Monitoring Station will be able to detect toxic cyanobacterial cells at low levels, before they reach an unsafe concentration. This will allow the workers, through routine monitoring of potential toxin status in the freshwater, to have enough time to implement a series of control strategies for avoiding human exposure to cyanobacterial hepatotoxins at dangerous levels. 参考文献 相似文献 引证文献

Acanthamoeba, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. Acanthamoeba keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by Acanthamoeba. In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to Acanthamoeba 18S rRNA gene to detect more rapidly the presence of Acanthamoeba in clinical and environmental samples. Acanthamoeba strain grown in culture was diluted in 200 μL as 1x106 trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 °C in a total volume of 25 μL. To determine the sensitivity of the test, positivity of Acanthamoeba genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%. It has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of Acanthamoeba has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of Acanthamoeba in environmental and clinical samples.

A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, sensitive and specific detection of African swine fever virus (ASFV). A set of LAMP primers was designed based on the sequence of the ASFV gene K205R. Reaction temperature and time were optimized to 64 oC and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or visually with the addition of fluorescent dye. The detection limit of the LAMP assay was approximately 6 copies of the target gene per microliter, 100 times more sensitive than conventional PCR. LAMP is a simple and inexpensive molecular assay format for ASFV detection. To date, African swine fever has not been reported in China. LAMP can be used to monitor ASFV spread into China, thereby reducing the threat of ASF.

Accurately identifying and targeting the human reservoir of malaria parasitemia is critical for malaria control, and requires a reliable and sensitive diagnostic method. Loop-mediated isothermal amplification (LAMP) is increasingly used to diagnose submicroscopic parasitemia. Although most published studies report the sensitivity of LAMP compared with nested polymerase chain reaction (PCR) as ≥ 80%, they have failed to use a consistent, sensitive diagnostic as a comparator. We used cross-sectional samples from children and adults in Tororo, Uganda, a region with high but declining transmission due to indoor residual spraying, to characterize the sensitivity and specificity of pan-Plasmodium LAMP for detecting submicroscopic infections. We compared LAMP results targeting a mitochondrial DNA sequence conserved in all Plasmodium species, performed on DNA extracted from dried blood spots, to those of a gold standard quantitative PCR assay targeting the var gene acidic terminal sequence of Plasmodium falciparum (varATS qPCR), performed on DNA extracted from 200 µL of whole blood. Using LAMP and varATS qPCR increased the detection of parasitemia 2- to 5-fold, compared with microscopy. Among microscopy-negative samples, the sensitivity of LAMP was 81.5% for detecting infection ≥ 1 parasites/µL. However, low density infections were common, and LAMP failed to identify more than half of all infections diagnosed by varATS qPCR, performing with an overall sensitivity of 44.7% for detecting submicroscopic infections ≥ 0.01 parasites/µL. Thus, although the LAMP assay is more sensitive than microscopy, it missed a significant portion of the submicroscopic reservoir. These findings have important implications for malaria control, particularly in settings where low-density infections predominate.

Prompt and accurate diagnosis of gastrointestinal tuberculosis (GITB) is highly challenging. Current conventional techniques lack sensitivity and are time-consuming. Multi targeted loop-mediated isothermal amplification (LAMP) using two targets (IS6110 and MPB64) is a promising technique for rapid diagnosis of TB. LAMP test using IS6110 and MPB64 targets for M. tuberculosis complex was performed on ileocecal biopsy samples of 35 clinically suspected patients of GITB and 30 ileocecal biopsy samples of non-tuberculosis control subjects. Results of IS6110 LAMP and MPB64 LAMP were compared with IS6110 PCR and culture INFERENCE: Overall LAMP test (using any of the two targets) had a sensitivity and specificity of 100 and 100%, respectively, for confirmed GITB (five culture positive) cases and 85.71 and 100%, respectively, for clinically suspected 30 ileocecal biopsy samples of GITB. Sensitivity of IS6110 LAMP, MPB64 LAMP and IS6110 PCR for clinically suspected cases was 22 (73.33%), 24 (80%) and 21 (70%), respectively. In total 35 GITB patients, the overall sensitivity of microscopy, culture, IS6110 PCR, IS6110 LAMP, MPB64 LAMP and the multi-targeted LAMP assay (if any of the two targets were used) were 5.71, 14.28, 68.57, 74.28, 82.85 and 85.71%, respectively. Specificity of all the tests was 100%. There were three cases which were missed by IS6110 LAMP and two cases by MPB64 LAMP. Multi-targeted LAMP is a promising technique for rapid and accurate diagnosis of GITB.

Introduction: Community-acquired pneumonia (CAP) caused by M. pneumoniae affect 3%-10% of children. It is not possible to spot M. pneumoniae infection depending entirely on clinical signs and symptoms. Correct identification of M. pneumoniae infections is crucial to initiate proper antibiotic therapy. The role of culture is questionable as this organism is fastidious. Diagnostic accuracy of serology depends on the specimen collection time. IgM can usually be tracked within one week after the onset of clinical illness, followed by IgG two weeks later. Nucleic Acid Amplification Techniques (NAATs) are proved to be the “new gold standard”. Loop-Mediated Isothermal Amplification (LAMP) amplifies DNA of mycoplasma in less than an hour under isothermal conditions with the use of six primers in a single tube. The amplified products can be visualized by naked eye as turbidity or fluorescence. Aim: To assess the diagnostic value of LAMP assay compared to serum Mycoplasma IgM for rapid detection of M. pneumoniae. Materials and methods: A 6-months study was conducted on hospitalized children diagnosed with community acquired pneumonia (CAP) admitted to Cairo University specialized pediatric hospital (CUSPH). M. pneumoniae IgM was done on serum samples by RD-Ratio Diagnostics ELISA kit, Germany. LAMP assay was done on nasopharyngeal swabs using Loopampᵀᴹ RNA/DNA, Eiken Chemical, Japan. Kappa measure of agreement was calculated to assess the concordance between LAMP and Mycoplasma IgM. Results: 90 hospitalized children with CAP were included in the study. Serum IgM was positive in 27(30%) of cases. LAMP assay was positive in 33 cases (36.7%). The sensitivity of LAMP was 85.2%, the specificity was 84.1%, PPV was 69.7%, NPV 93.0% with AUC=0.847. The agreement between serology and LAMP was good K=0.652 with 95% CI (0.487-0.816). Conclusion: LAMP technique is a rapid, sensitive and specific method for diagnosis of M. pneumoniae. Combination of LAMP assay and serology may be optimal for the diagnosis of M. pneumoniae infection.

Simple SummaryContagious agalactia (CA) is an infectious disease of small ruminants endemic in the Mediterranean countries, causing significant socioeconomic impacts predominantly on small-scale farmers who still subsist on marginal lands. Mycoplasma agalactiae is historically considered the principal etiological agent of CA, especially in sheep. Clinical signs are characterised by mastitis, arthritis, keratoconjunctivitis and occasionally, abortion. Rapid, accurate and cost-effective field tests are urgently needed for effective control of M. agalactiae mastitis. Our study illustrated the validation of a Loop-Mediated Isothermal Amplification (LAMP) test for the detection of M. agalactiae in dairy sheep in order to confirm its application as a diagnostic tool in the field level.Contagious agalactia (CA), an infectious disease of small ruminants, caused by Mycoplasma agalactiae, is responsible for severe losses to dairy sheep production with substantial socioeconomic impacts on small-scale farmers. The diagnosis of CA is still problematic, time-consuming and requires well-equipped labs for confirmation of outbreaks. Therefore, rapid, accurate and cost-effective diagnostic tests are urgently needed. This work aims to validate a novel Loop-Mediated Isothermal Amplification (LAMP) test, based on the p40 target gene, for the detection of M. agalactiae in dairy sheep in order to confirm its potential practical use as a rapid and cheap field test. The LAMP system proposed in this study consists of a portable device composed of real-time fluorometer with the automatic interpretation of results displayed in a tablet. A total of 110 milk samples (90 positives and 20 negatives) were analysed to optimise the analysis procedure and to investigate the efficacy and robustness of the LAMP method. All samples were analysed using LAMP and conventional real-time PCR to compare the diagnostic sensitivity of the methods. The sensitivity of the LAMP was 10-fold higher than that of real-time PCR, with a detection limit up to 103 CFU/ml. The LAMP assay was able to detect M. agalactiae in 81 of 90 (90%, 95%CI 0.84–0.96) positive milk samples compared to 69 (77%, 95%CI 0.59–0.95) positive samples detected by real-time PCR; no positive signal occurred for any of the negative milk samples in either test. Therefore, the LAMP assay was found to be more sensitive than real-time PCR, low-cost, easy to perform, fast and not affected by contamination, indicating its potential as an effective diagnostic tool in the field level for the diagnosis of CA.

Haemonchus contortus is one of the most significant strongylid nematodes infecting small ruminants, and it causes great economic losses to the livestock industry worldwide. Accurate diagnosis of H. contortus is crucial to control strategies. Traditional microscopic examinations are the most common methods for the diagnosis of H. contortus , but they are time-consuming and inaccurate. Molecular methods based on PCR are more accurate, but need expensive machines usually only used in the laboratory. Loop-mediated isothermal amplification (LAMP) is a rapid, simple, specific, and sensitive method that has been widely used to detect viruses, bacteria, and parasites. In the present study, a LAMP method targeting ribosomal ITS-2 gene for detection of the H. contortus in goat fecal samples has been established. The established LAMP method was H. contortus specific, and the sensitivity of LAMP was the same as that of the H. contortus species-specific PCR, with the lowest DNA level detected as being 1 pg. Examination of the clinical samples indicated that the positive rate of LAMP was higher than that of PCR, but no statistical difference was observed between LAMP and PCR (χ2 = 17.991, P = 0.053). In conclusion, a LAMP assay with a high specificity and a good sensitivity has been developed to detect H. contortus infection in goats. The established LAMP assay is useful for clinical diagnosis of H. contortus .

Background: Trypanosoma evansi, the causative agent of surra, poses a major veterinary concern in tropical regions, particularly affecting cattle and buffalo. The disease leads to reproductive failures, including abortion, and significant economic losses. Early and accurate diagnosis is crucial for effective control, especially in endemic, resource-limited areas. Objectives: This study aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid detection of T. evansi and evaluate its diagnostic performance in comparison with conventional polymerase chain reaction (PCR) and the CATT/T. evansi card agglutination test. Materials and methods: Four LAMP primers were designed to target the RoTat 1.2 variant surface glycoprotein (VSG) gene of T. evansi. Optimal reaction parameters, including temperature and incubation time, were established. The LAMP assay, conventional PCR, and the CATT/T. evansi card agglutination test were performed on 79 blood samples collected from cattle with suspected trypanosomiasis in northern Thailand (Lamphun and Chiang Mai). Diagnostic sensitivity, specificity, and agreement between tests were statistically analyzed. Results: The LAMP assay detected T. evansi in 32 (40.5%, 95% CI: 29.8-51.9%) samples, slightly outperforming PCR, which detected 30 (37.9%, 95% CI: 27.6-49.0%). However, this difference was not statistically significant (McNemar’s test, p=0.724). The CATT/T. evansi test yielded 45 (56.9%) positives but lacked the ability to differentiate active infection from prior exposure. Conclusion: The LAMP assay demonstrated high sensitivity, specificity, and rapid detection capabilities under simplified conditions, making it highly suitable for field applications. When paired with colorimetric or lateral flow readouts, LAMP offers a promising point-of-care diagnostic tool for improving trypanosomiasis control in endemic regions.

Sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD) are caused by subspecies of the capripox virus (CaPVs). They are significant pathogens of sheep, goats, and cattle. The causative agent is the capripox virus (CaPV), which was first isolated in South Africa. The viruses responsible for sheep pox (SP), goat pox (GP), and lumpy skin disease (LSD) are morphologically indistinguishable and have been adapted to different host species (4). Serologically, distinguishing between these viruses is challenging, and cross-immunity exists among them (2). The present study reports the evaluation and optimization of a novel loop-mediated isothermal amplification (LAMP) technique for the rapid detection of capripox viruses (CaPVs) and compares it with the polymerase chain reaction (PCR) method. LAMP primers were selected from the P32-protected gene of CaPV. The Safe-Red fluorescent dye was used to monitor the color change from red to bright yellow at a wavelength of 320 nm in positive cases, and the final results were documented through electrophoresis. The proposed LAMP test for the capripox virus demonstrated high specificity and did not cross react with other viruses in the Poxviridae family that present similar clinical symptoms. The optimized LAMP test was then compared with the PCR. The diagnostic sensitivity of LAMP and PCR was found to be identical (100%). The specificity of the LAMP test was evaluated using 30 samples of cow skin that were suspected of lumpy skin disease, along with16 additional samples, including nine positive references, fivenegative references, and two negative controls. A negative reference sample was used to assess the diagnostic sensitivity of LSDV. The proposed LAMP test is simple to implement, cost-effective, and highly sensitive, making it particularly well-suited for the detection of the capripox virus in less developed regions, laboratories, and facilities with limited resources.

Ortleppascaris sinensis is the dominant nematode species infecting the gastrointestinal tract of the captive Chinese alligator, a critically endangered species. Gastrointestinal nematode infection may cause a loss of appetite, growth, a development disorder, and even mortality in alligators, especially young ones. This research first establishment a loop-mediated isothermal amplification (LAMP) assay in rapidly identifying O. sinensis, upon the basis of the complete internal transcribed spacers (ITS) gene. Eight sets of primers were designed for recognition of the unique conserved ITS gene sequences, and one set was selected to be the most suitable primer for rapid detection. The specific as well as the sensitive features of the most appropriate primer in LAMP reactions for O. sinensis, and feces specimens of Chinese alligators suffering from O. sinensis were determined. Turbidity monitoring and Te Visual Reagent methods were used for determining negative and positive consequences. According to this study, amplification and visualization of the target DNA could be realized through two detection approaches during 50 min at 65 °C isothermal temperature. The sensitivity of LAMP was a detecting limitation of 3.46 pg/µl DNA. No cross-reactions were found between O. sinensis and any other of the nine heterologous nematode parasites, which shows the outstanding specific features of the primers. The LAMP assay could also perform a detection of target DNA of O. sinensis in the feces samples of Chinese alligators. This LAMP assay is useful for directly detecting O. sinensis in the Chinese alligator breeding centers, particularly due to its rapidity, simplicity and low cost.

Enteric fever is one of the major causes of death and morbidity around the world, especially in resource limited health care facility. The primary reason for empirical therapy in enteric fever management is a lack of rapid diagnostic testing. To improve typhoid fever diagnosis and treatment, as well as to reduce antibiotic overuse, we attempted to develop a loop-mediated isothermal amplification (LAMP) for rapid detection of enteric fever. We designed and evaluated a LAMP assay that targets the STY2879 gene, which is found only in S. Typhi and encodes for reverse transcriptase protein. LAMP utilises three sets of primers to complete the reaction in 60 minutes at 65°C. The LAMP assay procedure in our study for detecting S. Typhi by targeting the STY2879 gene was rapid and more sensitive than the Polymerase Chain Reaction (PCR) method. Among the 107 blood samples that have been tested, the sensitivity and specificity of the assay we obtained were 100% and 87.65% respectively. Also, we demonstrated lower limit of detection (LOD) of target DNA concentration ranging from 10 pg to 5 ng that could be readily detected by a LAMP assay within 60 min. The findings of our study suggest that the LAMP assay is superior to PCR and can be used as a rapid alternative diagnostic tool for the diagnosis of enteric fever in the aspects of specificity and sensitivity. As a result, with some additional enhancements and modifications, this reliable and cost-effective assay can be promptly used to enhance disease management and surveillance.

Tea leaf spot caused by Didymella segeticola is an important disease that threatens the healthy growth of tea plants (Camellia sinensis) and results in reductions in the productivity and quality of tea leaves. Early diagnosis of the disease is particularly important for managing the infection. Loop-mediated isothermal amplification (LAMP) assay is an efficient diagnostic technique with the advantages of simplicity, specificity, and sensitivity. In this study, we developed a rapid, visual, and high-sensitivity LAMP assay for D. segeticola detection based on sequence-characterized amplified regions. Two pairs of amplification primers (external primers F3 and B3 and internal primers FIP and BIP) were designed based on a specific sequence in D. segeticola (NCBI accession number: OR987684). Compared to common pathogens of other genera in tea plants and other species in the Didymella genus (Didymella coffeae-arabicae, Didymella pomorum, and Didymella sinensis), the LAMP method is specific for detecting the species D. segeticola. The assay was able to detect D. segeticola at a minimal concentration of 1 fg/μL genomic DNA at an optimal reaction temperature of 65 °C for 60 min. When healthy leaves were inoculated with D. segeticola in the laboratory, the LAMP method successfully detected D. segeticola in diseased tea leaves at 72 h post inoculation. The LAMP assays were negative when the DNA samples were extracted from healthy leaves. Leaf tissues with necrotic lesions from 18 germplasms of tea plants tested positive for the pathogen by the LAMP assay. In summary, this study established a specific, sensitive, and simple LAMP method to detect D. segeticola, which provides reliable technical support for estimating disease prevalence and facilitates sustainable management of tea leaf spot.

Loop-mediated isothermal amplification (LAMP) assay for speedy diagnosis of tubercular lymphadenitis: The multi-targeted 60-minute approach