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

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

Clinical Chemistry ◽

10.1093/clinchem/hvaa267 ◽

2020 ◽

Cited By ~ 5

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.

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A quite sensitive fluorescent loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus

The Journal of Infection in Developing Countries ◽

10.3855/jidc.11549 ◽

2019 ◽

Vol 13 (12) ◽

pp. 1135-1141 ◽

Cited By ~ 2

Author(s):

Yihong Hu ◽

Zhenzhou Wan ◽

Yonglin Mu ◽

Yi Zhou ◽

Jia Liu ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Rapid Detection ◽

Limit Of Detection ◽

Isothermal Amplification ◽

Clinical Samples ◽

High Morbidity ◽

Rt Pcr ◽

Loop Mediated Isothermal Amplification ◽

Syncytial Virus ◽

Syto 9

Introduction: Human respiratory syncytial virus (hRSV) is a common respiratory virus closely related to respiratory tract infection (RTI). Rapid and accurate detection of hRSV is urgently needed to reduce the high morbidity and mortality due to hRSV infection. Methodology: Here, we established a highly sensitive and specific reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of A and B group hRSV simultaneously. The specific primer sets for hRSV A and B groups were designed in the M and M2-2 gene, respectively. SYTO 9 was used as the fluorescent dye for real-time monitoring of the amplification of hRSV RNA without cross reaction between hRSV A and B. Results: The limit of detection (LOD) of our new method was 281.17 50% tissue culture infective doses (TCID50)/mL for hRSV A and 1.58 TCID50/mL for hRSV B. Using 90 clinical samples, a comparison to traditional RT-PCR was performed to validate this assay. The positivity rate of RT-LAMP and RT-PCR were 67.8% and 55.6%, respectively, and the positivity rate of RT-LAMP was significantly higher than RT-PCR (χ2 test, P < 0.01). Conclusions: Compared with traditional RT-PCR method, the newly developed fluorescent RT-LAMP combined with well-designed primers and SYTO 9 is quite sensitive, specific, rapid and well applicable to hRSV clinical diagnosis.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21082826 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2826 ◽

Cited By ~ 44

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.

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Rapid Detection of Clostridium botulinum in Food Using Loop-Mediated Isothermal Amplification (LAMP)

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18094401 ◽

2021 ◽

Vol 18 (9) ◽

pp. 4401

Author(s):

Yufei Chen ◽

Hao Li ◽

Liu Yang ◽

Lei Wang ◽

Ruyi Sun ◽

...

Keyword(s):

Rapid Detection ◽

High Throughput Screening ◽

Clostridium Botulinum ◽

Lamp Assay ◽

High Specificity ◽

Isothermal Amplification ◽

Clinical Samples ◽

Loop Mediated Isothermal Amplification ◽

Specificity And Sensitivity ◽

Target Dna

Botulinum neurotoxins are considered as one of the most potent toxins and are produced by Clostridium botulinum. It is crucial to have a rapid and sensitive method to detect the bacterium Clostridium botulinum in food. In this study, a rapid detection assay of C. botulinum in food using loop-mediated isothermal amplification (LAMP) technology was developed. The optimal primers were identified among three sets of primers designed specifically based on the partial ntnh gene encoding nontoxic-nonhaemagglutinin (NTNH) for rapid detection of the target DNA in plasmids. The optimal temperature and reaction time of the LAMP assay were determined to be 64 °C and 60 min, respectively. The chemical kit could be assembled based on these optimized reaction conditions for quick, initial high-throughput screening of C. botulinum in food samples. The established LAMP assay showed high specificity and sensitivity in detecting the target DNA with a limit of 0.0001 pg/ul (i.e., ten times more sensitive than that of the PCR method) and an accuracy rate of 100%. This study demonstrated a potentially rapid, cost-effective, and easy-operating method to detect C. botulinum in food and clinical samples based on LAMP technology.

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Development and evaluation of a multiplex loop-mediated isothermal amplification (LAMP) assay for differentiation of Mycobacterium tuberculosis and non-tuberculosis mycobacterium in clinical samples

PLoS ONE ◽

10.1371/journal.pone.0244753 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0244753

Author(s):

Jeeyong Kim ◽

Borae G. Park ◽

Da Hye Lim ◽

Woong Sik Jang ◽

Jeonghun Nam ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Positive Reaction ◽

Real Time Pcr ◽

Lamp Assay ◽

Isothermal Amplification ◽

Clinical Samples ◽

Published Data ◽

Analytical Sensitivity ◽

Loop Mediated Isothermal Amplification

Introduction The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time PCR–based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples. Material and methods For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method. Results Of 123 NTM samples, 121 were identified as NTM and 72/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72/72) for MTB, and 98.4% (121/123) for NTM. And the specificity of assay was 100% (138/138) for all. Conclusions Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.

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Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

Journal of Clinical Microbiology ◽

10.1128/jcm.01751-15 ◽

2016 ◽

Vol 54 (4) ◽

pp. 950-955 ◽

Cited By ~ 11

Author(s):

Qing Tang ◽

Shuguang Tian ◽

Nong Yu ◽

Xi Zhang ◽

Xiaodong Jia ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Rapid Detection ◽

Lamp Assay ◽

Isothermal Amplification ◽

Clinical Samples ◽

Content Type ◽

Loop Mediated Isothermal Amplification ◽

Real Time Quantitative Pcr ◽

Amplification Method ◽

Positive Results

Aspergillus fumigatusis a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection ofA. fumigatusinfection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection ofA. fumigatus. The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatusstrains, including 5 species of theAspergillusgenus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 102copies for the same target. Clinical samples from a total of 69 patients with probable IA (n =14) and possible IA (n= 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection ofA. fumigatusin clinical testing has been developed.

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

Foods ◽

10.3390/foods9010092 ◽

2020 ◽

Vol 9 (1) ◽

pp. 92 ◽

Cited By ~ 2

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.

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Loop Mediated Isothermal Amplification as a Rapid Molecular Method for Pseudomonas aeruginosa T3SS ExoY Gene Septicemia Detection in Beta- Lactamase Species Co-Infections

Current Journal of Applied Science and Technology ◽

10.9734/cjast/2021/v40i2631521 ◽

2021 ◽

pp. 19-29

Author(s):

J. Mageto Ombega ◽

Zhao-Hua Zhong

Keyword(s):

Pseudomonas Aeruginosa ◽

Real Time ◽

Limit Of Detection ◽

Uv Light ◽

Lamp Assay ◽

Chronically Ill ◽

Isothermal Amplification ◽

Clinical Samples ◽

Molecular Technique ◽

Loop Mediated Isothermal Amplification

Background: Pseudomonas aeruginosa is among the most important causative agent of infection in chronically ill patients admitted in hospitals globally. Coupled with its, mixed symptomatology, rapid drug resistance tendency and its causation of severe disease, a fast, reliable and affordable diagnostic technique is required to enable healthcare providers expeditiously mitigate its progression and eventual treatment. The Loop-Mediated Isothermal Amplification (LAMP) technique has the potential to serve as a simple, rapid, specific, sensitive and cost-effective point-of-care diagnostic tool. Broad Objective: To investigate Loop Mediated Isothermal Amplification as a molecular technique for microbial diagnostic and prognostic predictor. Study Design: This study was aimed at evaluating LAMP assay against Simple Polymerase chain reaction and Multiplex PCR on the diagnosis of P. aeruginosa in mixed clinical samples. Materials and Methods: This study developed P. aeruginosa Loop Mediated Isothermal Amplification (PaLAMP) assay to target the ExoY gene with appropriate primer testing and validation procedures. Culture of patient bacterial samples was done on MHA and MHB medium, grown overnight in an Incubator and a incubating shaker at 37oc respectively. Housekeeping gene were identified through online bioinformatics and blasted against known sequences. A set of 6 primers, comprising 2 outer primers (F3 and B3), 2 inner primers (FIP and BIP), and 2 loop primers (FLP and BLP), were designed. Microbial DNA extraction was done followed by PCR amplification as a classical identification using LAMP outer primers 9(F3 and B3). LAMP amplicons were detected by real time turbidimetry (LA-500) at 64°C for 40 minutes as well as under UV light with 1.0 μl of 1/10-diluted original SYBR Green I. Results: LAMP validation against traditional PCR shows a high limit of detection at 10-6ng/µl compared to 10-5ng/µl for PCR. The findings are consistent with outcomes for real time turbidimetric outcomes. Real time LAMP turbidimetric results was cross validated by direct observation through SYBR fluorescence under UV light for positive P. aeruginosa detection through positive amplification. Conclusion: Thus far, Loop mediated isothermal amplification show significantly high limit of detection comparable to standard PCR, its use in field based diagnosis offers an opportunity for a cheap, reliable and faster method to determine disease trends and therapy approaches. This method can be applied in primary care to enhance accuracy in diagnosis and thereby prompt initiation of mitigation treatment regimens.

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Development of a Novel Loop Mediated Isothermal Amplification Assay (LAMP) for the Rapid Detection of Epizootic Haemorrhagic Disease Virus

Viruses ◽

10.3390/v13112187 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2187

Author(s):

Paulina Rajko-Nenow ◽

Emma L. A. Howson ◽

Duncan Clark ◽

Natasha Hilton ◽

Aruna Ambagala ◽

...

Keyword(s):

Real Time ◽

Rapid Detection ◽

High Throughput Screening ◽

Limit Of Detection ◽

Lamp Assay ◽

Cost Effective ◽

Virus Genome ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Haemorrhagic Disease

Epizootic haemorragic disease (EHD) is an important disease of white-tailed deer and can cause a bluetongue-like illness in cattle. A definitive diagnosis of EHD relies on molecular assays such as real-time RT-qPCR or conventional PCR. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a cost-effective, specific, and sensitive technique that provides an alternative to RT-qPCR. We designed two sets of specific primers targeting segment-9 of the EHD virus genome to enable the detection of western and eastern topotypes, and evaluated their performance in singleplex and multiplex formats using cell culture isolates (n = 43), field specimens (n = 20), and a proficiency panel (n = 10). The limit of detection of the eastern and western RT-LAMP assays was estimated as ~24.36 CT and as ~29.37 CT in relation to real-time RT-qPCR, respectively, indicating a greater sensitivity of the western topotype singleplex RT-LAMP. The sensitivity of the western topotype RT-LAMP assay, relative to the RT-qPCR assay, was 72.2%, indicating that it could be theoretically used to detect viraemic cervines and bovines. For the first time, an RT-LAMP assay was developed for the rapid detection of the EHD virus that could be used as either a field test or high throughput screening tool in established laboratories to control the spread of EHD.

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Rapid detection of Betacoronavirus 1 from clinical fecal specimens by a novel reverse transcription loop-mediated isothermal amplification assay

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638711425937 ◽

2011 ◽

Vol 24 (1) ◽

pp. 174-177 ◽

Cited By ~ 3

Author(s):

Jun Qiao ◽

Qingling Meng ◽

Xuepeng Cai ◽

Chuangfu Chen ◽

Zaichao Zhang ◽

...

Keyword(s):

Reverse Transcription ◽

Rapid Detection ◽

Lamp Assay ◽

High Specificity ◽

Isothermal Amplification ◽

Clinical Samples ◽

Lamp Method ◽

Nucleocapsid Gene ◽

Loop Mediated Isothermal Amplification ◽

Amplification Assay

Betacoronavirus 1 (BCoV-1) is an important pathogen causing diarrhea in calves. In the current study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of BCoV-1 was successfully developed. The primers were designed to target the highly conserved fragment of BCoV-1 nucleocapsid gene. The assay displayed high specificity detecting only BCoV-1 with no cross reaction with other viruses. When 418 clinical samples from 6 different geographical areas of Xinjiang province were tested by the RT-LAMP method, the results indicated that this test is a simple, rapid, accurate, and sensitive method for the detection of BCoV-1.

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