scholarly journals q-LAMP Assays for the Detection of Botryosphaeria dothidea Causing Chinese Hickory Canker in Trunk, Water, and Air Samples

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3142-3149
Author(s):  
Q. W. Wang ◽  
C. Q. Zhang
Keyword(s):  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Botryosphaeria Dothidea ◽  
Canker Disease ◽  
Air Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Optimum Reaction ◽  
Epidemiological Characteristics ◽  
Trunk Canker

Trunk canker disease caused by Botryosphaeria dothidea with a prolonged latent infection phase poses a serious threat to Chinese hickory production. To further understand the epidemiological characteristics and develop reasonable management techniques, a quantitative loop-mediated isothermal amplification (q-LAMP) assay was developed to quantitatively monitor B. dothidea in hickory plants, water, and air samples. Specific primers were designed based on the different sites of the β-tubulin sequence between B. dothidea and other fungi commonly found on Chinese hickory. At the optimum reaction temperature of 65.9°C, this loop-mediated isothermal amplification (LAMP) assay can specifically distinguish B. dothidea from other tested fungi. The limit of detection of LAMP assays for B. dothidea was 0.001 ng/µl of pure genomic DNA and 10 spores per 1 ml of water. The q-LAMP assay enables rapid detection of B. dothidea within 60 min in hickory trunk, water in hickory forests, and spores captured on tapes. These results provide a powerful and convenient tool for monitoring B. dothidea, which could be applied widely in epidemiology, forecast, and management of tree canker disease.

scholarly journals Rapid detection of cytochrome cd1-containing nitrite reductase encoding gene nirS of denitrifying bacteria with loop-mediated isothermal amplification assay

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xuzhi Zhang ◽  
Qianqian Yang ◽  
Qingli Zhang ◽  
Xiaoyu Jiang ◽  
Xiaochun Wang ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Denitrifying Bacteria ◽  
Isothermal Amplification ◽  
Target Sequence ◽  
Bacterial Cells ◽  
Loop Mediated Isothermal Amplification ◽  
Order Of Magnitude

Abstract The cytochrome cd1-containing nitrite reductase, nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance of nirS is a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a rapid method for detecting nirS gene with loop-mediated isothermal amplification (LAMP) was developed, using Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1) as model microorganism to optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, it was validated that P. aeruginosa PAO1 cells as well as genomic DNA could be directly used as template. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success. The nirS gene of P. aeruginosa PAO1 in spiked seawater samples could be detected with both DNA-template based LAMP assay and cell-template based LAMP assay, demonstrating the practicality of in-field use.

scholarly journals Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Matthew A Lalli ◽  
Joshua S Langmade ◽  
Xuhua Chen ◽  
Catrina C Fronick ◽  
Christopher S Sawyer ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Viral Detection ◽  
Loop Mediated Isothermal Amplification

Abstract Background Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44–104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

scholarly journals Rapid detection of cytochromecd1-containing nitrite reductase encoding genenirSwith loop-mediated isothermal amplification assay

2018 ◽  
Author(s):  
Qianqian Yang ◽  
Xuzhi Zhang ◽  
Xiaoyu Jiang ◽  
Xiaochun Wang ◽  
Yang Li ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Target Sequence ◽  
Bacterial Cells ◽  
Conventional Pcr ◽  
Loop Mediated Isothermal Amplification

AbstractThe cytochromecd1-containing nitrite reductase,nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance ofnirSis a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a new molecular biology technique termed loop-mediated isothermal amplification (LAMP) was developed to rapidly detectnirSgene using those ofPseudomonas aeruginosato optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The specificity of the assay was confirmed by the lack of amplification when using DNA from 15 other bacterial species lackingnirSgene. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, a cell-template based LAMP assay was also developed for detectingnirSgene that directly used bacterial cells as template rather than genomic DNA. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success, and bulky and sophisticated equipment were not needed. Further, thenirSgene ofP. aeruginosain spiked seawater samples could be detected with both the DNA-template based LAMP assay and the cell-template based LAMP assay, thereby demonstrating the practicality of in-field use of them. In summary, the LAMP assays described here represent a rapid, user-friendly, and cost-effective alternative to conventional PCR.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) Assay for Detecting Burkholderia cepacia Complex in Non-Sterile Pharmaceutical Products

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1071
Author(s):  
Soumana Daddy Gaoh ◽  
Ohgew Kweon ◽  
Yong-Jin Lee ◽  
John J. LiPuma ◽  
David Hussong ◽  
...  
Keyword(s):  
Burkholderia Cepacia ◽  
Limit Of Detection ◽  
Free Water ◽  
Lamp Assay ◽  
Pharmaceutical Products ◽  
Isothermal Amplification ◽  
Burkholderia Cenocepacia ◽  
Burkholderia Cepacia Complex ◽  
Loop Mediated Isothermal Amplification ◽  
Consumer Safety

Simple and rapid detection of Burkholderia cepacia complex (BCC) bacteria, a common cause of pharmaceutical product recalls, is essential for consumer safety. In this study, we developed and evaluated a ribB-based colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of BCC in (i) nuclease-free water after 361 days, (ii) 10 μg/mL chlorhexidine gluconate (CHX) solutions, and (iii) 50 μg/mL benzalkonium chloride (BZK) solutions after 184 days. The RibB 5 primer specifically detected 20 strains of BCC but not 36 non-BCC strains. The limit of detection of the LAMP assay was 1 pg/μL for Burkholderia cenocepacia strain J2315. Comparison of LAMP with a qPCR assay using 1440 test sets showed higher sensitivity: 60.6% in nuclease-free water and 42.4% in CHX solution with LAMP vs. 51.3% and 31.1%, respectively, with qPCR. These results demonstrate the potential of the ribB-based LAMP assay for the rapid and sensitive detection of BCC in pharmaceutical manufacturing.

scholarly journals Rapid Visualization and Detection of Staphylococcus Aureus Based on Loop-Mediated Isothermal Amplification

2021 ◽  
Author(s):  
Chuan Wu ◽  
Yuanyuan Zeng ◽  
Yang He
Keyword(s):  
Staphylococcus Aureus ◽  
Limit Of Detection ◽  
Reaction System ◽  
Bacterial Pathogen ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Diagnostic Efficacy ◽  
Diverse Range ◽  
Loop Mediated Isothermal Amplification

Abstract Staphylococcus aureus is a common clinical bacterial pathogen that can cause a diverse range of infections. The establishment of a rapid and reliable assay for the early diagnosis and detection of S. aureus is of great significance. In this study, we developed a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus using the colorimetric indicator hydroxy naphthol blue (HNB). The LAMP reaction was optimized by adjusting the amplification temperature, the concentrations of Mg2+, dNTP, and HNB, and the incubation time. In the optimized reaction system, the specificity of LAMP for S. aureus was 100%. The results established that this method accurately identified S. aureus, with no cross-reactivity with 16 non-S. aureus strains. The limit of detection (LOD) of LAMP was 8 copies/reaction of purified plasmid DNA or 400 colony-forming units/reaction of S. aureus. Compared with conventional PCR, LAMP lowered the LOD by 10-fold. Finally, 220 clinically isolated strains of S. aureus and 149 non-S. aureus strains were used to evaluate the diagnostic efficacy of LAMP. The findings indicated that LAMP is a reliable test for S. aureus and could be a promising tool for the rapid diagnosis of S. aureus infections.

scholarly journals A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

2020 ◽  
Vol 21 (8) ◽  
pp. 2826 ◽  
Author(s):  
Renfei Lu ◽  
Xiuming Wu ◽  
Zhenzhou Wan ◽  
Yingxue Li ◽  
Xia Jin ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
N Gene ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

scholarly journals Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus

2020 ◽  
Vol 21 (5) ◽  
pp. 1756 ◽  
Author(s):  
Sumyya Waliullah ◽  
Kai-Shu Ling ◽  
Elizabeth J. Cieniewicz ◽  
Jonathan E. Oliver ◽  
Pingsheng Ji ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Visual Observation ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Infected Leaf ◽  
Loop Mediated Isothermal Amplification ◽  
Efficient Detection ◽  
Field Samples ◽  
Optimum Reaction ◽  
Cucurbit Leaf Crumple Virus

A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie® III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.

scholarly journals Rapid and Visual Differentiation of Mycobacterium tuberculosis From the Mycobacterium tuberculosis Complex Using Multiplex Loop-Mediated Isothermal Amplification Coupled With a Nanoparticle-Based Lateral Flow Biosensor

2021 ◽  
Vol 12 ◽  
Author(s):  
Xinggui Yang ◽  
Junfei Huang ◽  
Xu Chen ◽  
Ziyu Xiao ◽  
Xiaojuan Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Limit Of Detection ◽  
Clinical Specimen ◽  
Lamp Assay ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Whole Process ◽  
Specimen Processing ◽  
Mycobacterium Africanum

Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (MTB), but other members of the Mycobacterium tuberculosis complex (MTBC), especially Mycobacterium bovis (pyrazinamide-resistant organisms), may also be involved. Thus, the ability to rapidly detect and identify MTB from other MTBC members (e.g., M. bovis, Mycobacterium microti, Mycobacterium africanum) is essential for the prevention and treatment of TB. A novel diagnostic method for the rapid detection and differentiation of MTB, which employs multiplex loop-mediated isothermal amplification (mLAMP) combined with a nanoparticle-based lateral flow biosensor (LFB), was established (mLAMP-LFB). Two sets of specific primers that target the IS6110 and mtp40 genes were designed according to the principle of LAMP. Various pathogens were used to optimize and evaluate the mLAMP-LFB assay. The optimal conditions for mLAMP-LFB were determined to be 66°C and 40 min, and the amplicons were directly verified by observing the test lines on the biosensor. The LAMP assay limit of detection (LoD) was 125 fg per vessel for the pure genomic DNA of MTB and 4.8 × 103 CFU/ml for the sputum samples, and the analytical specificity was 100%. In addition, the whole process, including the clinical specimen processing (35 min), isothermal amplification (40 min), and result confirmation (1–2 min), could be completed in approximately 80 min. Thus, mLAMP-LFB is a rapid, reliable, and sensitive method that is able to detect representative members of MTBC and simultaneously differentiate MTB from other MTBC members, and it can be used as a potential screening tool for TB in clinical, field, and basic laboratory settings.

scholarly journals Validation of a Commercial Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Anisakis spp. DNA in Processed Fish Products

Foods ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 92 ◽  
Author(s):  
Gaetano Cammilleri ◽  
Vincenzo Ferrantelli ◽  
Andrea Pulvirenti ◽  
Chiara Drago ◽  
Giuseppe Stampone ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Allergic Diseases ◽  
Isothermal Amplification ◽  
Fish Products ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Fish Samples ◽  
Anisakis Spp

Parasites belonging to the Anisakis genera are organisms of interest for human health because they are responsible for the Anisakiasis zoonosis, caused by the ingestion of raw or undercooked fish. Furthermore, several authors have reported this parasite to be a relevant inducer of acute or chronic allergic diseases. In this work, a rapid commercial system based on Loop-Mediated Isothermal Amplification (LAMP) was optimised and validated for the sensitive and rapid detection of Anisakis spp. DNA in processed fish products. The specificity and sensitivity of the LAMP assay for processed fish samples experimentally infected with Anisakis spp. larvae and DNA were determined. The LAMP system proposed in this study was able to give positive amplification for all the processed fish samples artificially contaminated with Anisakis spp., giving sensitivity values equal to 100%. Specificity tests provided no amplification for the Contracaecum, Pseudoterranova, or Hysterothylacium genera and uninfected samples. The limit of detection (LOD) of the LAMP assay proposed was 102 times lower than the real-time PCR method compared. To the best of our knowledge, this is the first report regarding the application of the LAMP assay for the detection of Anisakis spp. in processed fish products. The results obtained indicate that the LAMP assay validated in this work could be a reliable, easy-to-use, and convenient tool for the rapid detection of Anisakis DNA in fish product inspection.

scholarly journals Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Glaesserella (Haemophilus) parasuis

2020 ◽  
Vol 9 (1) ◽  
pp. 41
Author(s):  
Veronika Pilchová ◽  
Diana Seinige ◽  
Isabel Hennig-Pauka ◽  
Kathrin Büttner ◽  
Amir Abdulmawjood ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Economic Losses ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Glaesserella parasuis is a fastidious pathogen that colonizes the respiratory tract of pigs and can lead to considerable economic losses in pig production. Therefore, a rapid detection assay for the pathogen, preferably applicable in the field, is important. In the current study, we developed a new and improved detection method using loop-mediated isothermal amplification (LAMP). This assay, which targets the infB gene, was tested on a collection of 60 field isolates of G. parasuis comprising 14 different serovars. In addition, 63 isolates from seven different closely related species of the family Pasteurellaceae, including A. indolicus, A. porcinus, and A. minor, and a species frequently found in the respiratory tract of pigs were used for exclusivity experiments. This assay showed an analytical specificity of 100% (both inclusivity and exclusivity) and an analytical sensitivity of 10 fg/µL. In further steps, 36 clinical samples were tested with the LAMP assay. An agreement of 77.1 (95% CI: 59.9, 89.6) and 91.4% (95% CI: 75.9, 98.2) to the culture-based and PCR results was achieved. The mean limit of detection for the spiked bronchoalveolar lavage fluid was 2.58 × 102 CFU/mL. A colorimetric assay with visual detection by the naked eye was tested to provide an alternative method in the field and showed the same sensitivity as the fluorescence-based LAMP assay. Overall, the optimized LAMP assay represents a fast and reliable method and is suitable for detecting G. parasuis in the laboratory environment or in the field.

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