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 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 Methylation-Specific Loop-Mediated Isothermal Amplification for Detecting Hypermethylated DNA in Simplex and Multiplex Formats

2010 ◽  
Vol 56 (8) ◽  
pp. 1287-1296 ◽  
Author(s):  
Francesco Zerilli ◽  
Cinzia Bonanno ◽  
Erlet Shehi ◽  
Giulia Amicarelli ◽  
Daniel Adlerstein ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Kinase Inhibitor ◽  
Methylation Status ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Target Sequence ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Isothermal Method ◽  
Loop Mediated Isothermal Amplification

BACKGROUND Aberrant DNA methylation of gene promoters and the associated silencing of tumor suppressor genes are recognized as mechanisms contributing to tumor development. Therefore, detection of promoter hypermethylation is becoming important for diagnosis, prognosis, and aiding the design of cancer therapies. We describe a novel isothermal method for the detection of DNA hypermethylation. METHODS Methylation-specific loop-mediated isothermal amplification (MS-LAMP) is a novel adaptation of LAMP. MS-LAMP was used for the highly specific detection of hypermethylated CpGs in the promoters of the CDKN2A [cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)], GATA5 (GATA binding protein 5), and DAPK1 (death-associated protein kinase 1) genes. The reactions occurred under isothermal conditions with 3 primer sets specific for methylated promoters. Both turbidimetry and fluorescence were used for detection. The MS-LAMP assay was validated with bisulfite-treated plasmid and genomic DNA controls of known methylation status and was applied to detect hypermethylation in 18 clinical tumor samples. A multiplex MS-LAMP for CDKN2A, GATA5, and DAPK1 was also validated with the aid of synthetic positive and negative controls. RESULTS The MS-LAMP assay showed high specificity with plasmid and genomic DNA targets in reactions carried out in <1 h. The assay had a detection limit of approximately 30 copies of methylated target sequence and a selectivity of 0.5% methylated DNA in a mixture with unmethylated DNA. Compared with methylation-specific PCR, the MS-LAMP assay detected lower rates of methylation in lung adenocarcinoma samples. Simultaneous multiplex detection of hypermethylation in the 3 targets (CDKN2A, GATA5, and DAPK1) was readily achieved with the MS-LAMP assay in both the turbidimetric and fluorescence detection formats. CONCLUSIONS MS-LAMP provides a highly specific isothermal method for methylation detection and is well suited for multiplex approaches.

scholarly journals Highly Sensitive Loop-Mediated Isothermal Amplification for the Detection of Leptospira

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Hua-Wei Chen ◽  
Giulia Weissenberger ◽  
Erin Atkins ◽  
Chien-Chung Chao ◽  
Yupin Suputtamongkol ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
Rickettsia Conorii ◽  
Bartonella Bacilliformis ◽  
Loop Mediated Isothermal Amplification ◽  
Different Strains

Leptospirosis is a worldwide zoonosis caused by an infection with the pathogenic species of Leptospira. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the DNA of Leptospira spp. Six sets of primers targeting the gene of the subsurface protein, lipL32, were evaluated for their detection sensitivity. The best primer set detected less than 25 copies of lipL32 per reaction of both plasmid DNA template and purified leptospiral genomic DNA. By combining primers targeting lipL32 with the previously published primer set targeting lipL41, the sensitivity of the assay was improved to 12 copies of L. interrogans. The specificity of the LAMP assay was evaluated by using the genomic DNA from other clinically encountered bacterial species such as different strains of Orientia tsutsugamushi, Rickettsia typhi, Rickettsia conorii, Rickettsia rickettsii, Coxiella burnetii, and Bartonella bacilliformis. These genomic DNA samples were all negative in our LAMP assay. The sensitivity of the LAMP assay was very similar to that of quantitative real time PCR. Several detection methods for the amplified product of LAMP assay were performed to demonstrate the simplicity of the assay. In summary, our results have suggested that this assay is rapid, robust, and easy to perform and has the potential to be used in endemic locations.

scholarly journals Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Somayyeh Sedaghatjoo ◽  
Monika K. Forster ◽  
Ludwig Niessen ◽  
Petr Karlovsky ◽  
Berta Killermann ◽  
...  
Keyword(s):  
Test Performance ◽  
Genomic Dna ◽  
Lamp Assay ◽  
Fungal Species ◽  
Isothermal Amplification ◽  
Genome Comparison ◽  
Performance Study ◽  
Loop Mediated Isothermal Amplification ◽  
Tilletia Controversa ◽  
Genomic Regions

AbstractTilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

Loop-mediated Isothermal Amplification for Rapid Detection of Mating Types of Villosiclava virens

Plant Disease ◽  
2021 ◽  
Author(s):  
Xiayan Pan ◽  
Xiao Wang ◽  
Junjie Yu ◽  
Mina Yu ◽  
Huijuan Cao ◽  
...  
Keyword(s):  
Mating Type ◽  
Genomic Dna ◽  
High Efficiency ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Villosiclava Virens ◽  
False Smut ◽  
Mating Type Genes ◽  
Polymerase Chain

Rice false smut (RFS), caused by Villosiclava virens, is an important fungal disease in panicle of rice. V. virens is a heterothallic ascomycete that controlled by two opposite idiomorphs, MAT1-1 and MAT1-2. Previous study showed sexual reproduction of V. virens plays an important role in the epidemic of RFS. In this study, we have developed a loop-mediated isothermal amplification (LAMP) assay to detect mating type of V. virens easily and rapidly by using specific primers designed on the mating type genes MAT1-1-2 and MAT1-2-1, respectively. The LAMP assay required only a water/dry bath and could recognize the mating type of V. virens in just 45 min. The LAMP assay was so sensitive that could detect small amounts of V. virens genomic DNA (as low as 2.0 pg of MAT1-1, and 200.0 pg of MAT1-2), which was 10-fold more sensitive than polymerase chain reaction (PCR). In addition, the application of mating type using LAMP assay was demonstrated feasibly by assessing the genomic DNA of V. virens isolated from rice fields. The high efficiency and specificity of this LAMP assay suggested it can be used as a rapid testing tool in mating type recognition of V. virens isolates in the field.

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 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.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

2005 ◽  
Vol 54 (11) ◽  
pp. 1037-1041 ◽  
Author(s):  
Ryoichi Saito ◽  
Yoshiki Misawa ◽  
Kyoji Moriya ◽  
Kazuhiko Koike ◽  
Kimiko Ubukata ◽  
...  
Keyword(s):  
Real Time ◽  
Mycoplasma Pneumoniae ◽  
Rapid Detection ◽  
Clinical Laboratory ◽  
Direct Sequencing ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Nasopharyngeal Swab ◽  
Loop Mediated Isothermal Amplification

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Mycoplasma pneumoniae was developed and evaluated. The assay specifically amplified only M. pneumoniae sequences, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The detection limit for this assay was found to be 2 × 102 copies, corresponding to 2–20 colour changing units of M. pneumoniae in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100 % was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of M. pneumoniae infection in the clinical laboratory.

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.

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