Optimization of isothermal amplification method for Mycobacterium tuberculosis detection and visualization method for fieldwork

The purpose of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish. A set of four primers, two outer and two inner primers, were designed from the toxR gene sequence of V. harveyi. The LAMP reaction was conducted at 65 degrees C for 60 min. There were no cross-reactions with other bacterial strains indicating a high specificity of the LAMP. The detection sensitivity of the LAMP assay for V. harveyi with both of pure cultures and added shellfish cultures is about 10(-5) dilution level (equivalent to 17.2 cells per reaction). The amplification products were detected by visual inspection using SYBR Green I. The detection sensitivity using the LAMP method was 10 times higher than that of conventional PCR. The LAMP assay established in this study is an extremely specific, sensitive and rapid for identification of V. harveyi in mariculture shellfish. This LAMP technique provides an important detecting tool for the detection of V. harveyi infection both in the laboratory and field. This technique is recommended as an applied protocol for health management programme and disease surveillance of in hatcheries as well as in grow-out pond, to prevent the disease outbreak.

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.

Harmful algal blooms frequently occur in various coastal regions worldwide, deteriorating marine ecology and causing huge economic losses. Therefore, developing a potential method for rapid detection of harmful algae species is highly necessitated. In this study, a loop-mediated isothermal amplification (LAMP) method coupled with a lateral flow dipstick (LFD) was developed for detecting the harmful algae Karenia mikimotoi. In this method, the internal transcribed spacer (ITS) sequence of K. mikimotoi was used as the template, and the corresponding specific primers were designed by the online software PrimerExplorer V5. Biotin was labeled on the 5' end of forward inner primer (FIP), and the LAMP reaction was performed under the determined optimal conditions of 63℃ and 60min. The lowest concentration of K. mikimotoi DNA tested using LAMP was 3.3 × 10-1pg/μL. Additionally, a 6-FAM-labeled probe was designed and displayed on the LFD after hybridization of the amplified product with the probe. The results demonstrated that LAMP-LFD could be a promising approach for detecting and monitoring harmful algae due to its high sensitivity and specificity.

Staphylococcus pseudintermedius is a global animal pathogen. Traditional identification methods are time-consuming necessitating a more efficient approach. This study validated and enhanced the loop-mediated isothermal amplification (LAMP) technique by integration it with a lateral flow dipstick (LFD) assay for the detection of S. pseudintermedius and methicillin-resistant S. pseudintermedius (MRSP) strains. Conventional identification methods were compared with LAMP and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The isolates were tested for MRSP detection using oxacillin and cefoxitin disk diffusion tests alongside the LAMP assay targeting the mecA gene, a marker for methicillin resistance. Results showed that LAMP combined with LFD effectively detected S. pseudintermedius and MRSP. This study identified 53 isolates as S. pseudintermedius by conventional and LAMP methods, with MALDI-TOF MS correctly identifying 39.62% (21/53). The mecA gene, crucial for methicillin resistance, was detected in all PCR-positive isolates (n = 33) by LAMP, while the disk diffusion method identified 69.70% (23/33) of mecA-positive strains. LAMP and conventional methods exhibited superior accuracy, sensitivity, and specificity (all 100%) compared to MALDI-TOF MS, which showed lower sensitivity (39.62%) for S. pseudintermedius identification. Similarly, the LAMP assay demonstrated higher accuracy, sensitivity, and specificity (all 100%) for MRSP detection compared to the disk diffusion method (83.33%, 69.70%, and 94.87%, respectively). The LAMP assay coupled with the LFD method proved suitable for routine bacterial identification in laboratories, offering adequate sensitivity and specificity with simple steps and short reaction time.

Human tuberculosis caused by Mycobacterium tuberculosis and Mycobacterium bovis shows similar clinical symptoms; however, the treatment differs because M. bovis is inherently resistant to pyrazinamide, a key first-line drug effective against M. tuberculosis. Most available molecular tools cannot distinguish the two biovars. This study addresses this gap by introducing a multiplex loop-mediated isothermal amplification (LAMP) method coupled with dipstick chromatography that can simultaneously and differentially detect M. bovis and M. tuberculosis within 60 min. The LAMP method does not require sophisticated high-cost equipment and can be easily implemented in resource-limited settings. Our LAMP facilitates rapid and accurate tuberculosis diagnosis, enabling appropriate therapeutic agents to be selected in areas where bovine and human tuberculosis coexist. It can also screen for M. bovis infection in humans and livestock, providing prevalence data in areas where such information is lacking.

Toxoplasma gondii: Sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method

Development and evaluation of a novel loop-mediated isothermal amplification (LAMP) method targeting Theileria parasites infecting Yezo sika deer.

Edwarsiela ictaluri is a potential bacteria as a pathogen causing Enteric Septicemia of Catfish (ESC) and also still a big problem in catfish because it can cause death to 100%. This study aims to develop diagnosis DNA based on molecular using Loop Mediated Isothermal Amplification. Method (LAMP). Detection DNA of E. ictaluri bacteria from 9 isolates was extracted at Fish Health Laboratory, in Depok. used four pieces of specific primers from Eip 18 gene of E. ictaluri. DNA amplification, for LAMP reactions without a thermocycler machine, using only an incubator/water bath/oven, at 65°C for 1 hour. The result of detection with LAMP, showed that 8 isolates were positive of E. ictaluri marked by SYBR green I color change from orange to green. While 1 isolate was negatively marked with SYBR Green remain orange. The LAMP results are also verified with PCR using outer primer F3 and B3 which showed positive E. ictaluri. LAMP can detect ESC disease simple, quickly, easily, economically in 1 hour compared by PCR and LAMP method can be used as an alternative diagnostic test for fish diseases, especially in areas with limited laboratory infrastructure.

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

Plant-based food ingredients such as garlic, Chinese leek, Chinese onion, green onion and onion are widely used in many cuisines around the world. However, these ingredients known as the “five forbidden vegetables” (FFVs) are not allowed in some vegetarian diets. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of FFVs using five respective LAMP primer sets. The specific primers targeted the ITS1-5.8S-ITS2 nuclear ribosomal DNA sequence regions among the five vegetables. The results demonstrated that the identification of FFVs using the newly developed LAMP assay is more sensitive than the traditional PCR method. Using pepper, basil, parsley, chili and ginger as references, established LAMP primer sets showed high specificity for the identification of the FFV species. Moreover, when FFVs were mixed with other plant ingredients at different ratios (100:0, 50:50, 20:80, 10:90, 5:95, 2:98, and 1:99), no cross-reactivity was evident using LAMP. Finally, genomic DNAs extracted from boiled and steamed FFVs in processed foods were used as templates; the performance of the LAMP reaction was not influenced using validated LAMP primers. Not only can FFV ingredients be identified but commercial foods containing FFVs can also be authenticated. This LAMP method will be useful for the authentication of FFVs in practical food markets in the future.

Qu, G., Fu, S., Shen, N., Wang, J., Xiao, Y., Guan, Y., Tang, N., Chen, L., Gao, S. and Shen, Z. 2010. Rapid and sensitive diagnosis of porcine parvovirus by loop-mediated isothermal amplification (LAMP) method. J. Appl. Anim. Res., 37: 113–116. To form a detection model for the porcine parvovirus (PPV), six special primers were designed based on the PPV gene NS1. After optimization only 45 min were needed to amplify PPV DNA by incubation at 63C. The specificity of the LAMP assay was validated by the absence of any cross-reaction with six other genetically related viruses, followed by restriction digestion and was observed to be similar to PCR assay. The LAMP assay demonstrated 100-fold higher sensitivity compared to PCR, with a detection limit of 12fg DNA. Thus the LAMP can be well-applied to laboratories, as a portable device and valuable tool for differential diagnosis of PPV in the countryside.

Preliminary establishment and validation of a loop-mediated isothermal amplification assay for convenient screening of 13 types of high-risk human papillomaviruses in cervical secretions

We developed a novel loop-mediated isothermal amplification (LAMP) method to detect Rickettsia spp., including Rickettsia prowazekii and R. typhi. Species-specific LAMP primers were developed for orthologous genes conserved among Rickettsia spp. The selected modified primers could detect all the Rickettsia spp. tested. The LAMP method was successfully used to detect 100 DNA copies of Rickettsia spp. within approximately 60 min at 63℃. Therefore, this method may be an excellent tool for the early diagnosis of rickettsiosis in a laboratory or in the field.

The current methods for detecting Mycobacterium tuberculosis (Mtb) are not clinically optimal. Standard culture methods (SCMs) are slow, costly, or unreliable, and loop-mediated isothermal amplification (LAMP) cannot differentiate live Mtb. This study compared reverse transcription (RT)-LAMP, LAMP, and an SCM for detecting Mtb. A first experiment tested the sensitivity and specificity of primers for 9 species of Mycobacterium (H37Rv, M. intracellulare, M. marinum, M. kansasii, M. avium, M. flavescens, M. smegmatis, M. fortuitum, and M. chelonae); and 3 non-Mycobacterium species (Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae). A second experiment tested sputum specimens for the presence of Mtb, from 100 patients with tuberculosis (clinical) and 22 from patients without tuberculosis (control), using Roche solid culture (SCM), LAMP, and RT-LAMP. In the clinical samples. The rates of positivity for Mtb of the SCM, LAMP, and RT-LAMP methods were 88%, 92%, and 100%, respectively. The difference in detection rate was significant between RT-LAMP and SCM, but RT-LAMP and LAMP were comparable. In the control group, the detection rates were nil for all three methods. The specificities of the methods were similar. The sensitivity of RT-LAMP was ~10-fold higher than that of LAMP for detecting Mtb. Unlike LAMP, RT-LAMP could identify viable bacteria, and was able to detect a single copy of Mtb. Among SCM, LAMP, and RT-LAMP, the latter is the most suitable for wide use in the lower-level hospitals and clinics of China for detecting Mtb in sputum samples.

During the last 10 years, with the development of loop-mediated isothermal amplification (LAMP) method, it has been widely applied in nucleic acid analysis because of its simplicity, rapidity, high efficiency, and outstanding specificity. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. Expensive equipment are not necessary to acquire a high level of precision, and there are fewer preparation steps compared to conventional PCR and real-time PCR assays. This paper briefly summarized the applications of LAMP method in pathogenic microorganisms, genetically modified ingredients, tumor detection, and embryo sex identification.