Rapid and effective diagnosis of pulmonary tuberculosis with novel and sensitive loop-mediated isothermal amplification (LAMP) assay in clinical samples: A meta-analysis

ABSTRACTBackgroundCoronavirus Disease 2019 (COVID-19) has caused a severe outbreak and become a global public health priority. Rapid increment of infection number along with significant deaths have placed the virus as a serious threat to human health. Rapid, reliable, and simple diagnostic methods are critically essential for disease control. While Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) is the current diagnostic gold standard, Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) appears as a compelling alternative diagnostic test due to its more simplicity, shorter time to result, and lower cost. This study examined RT-LAMP application for rapid identification of SARS-CoV-2 infection compared to RT-PCR assay.MethodsA systematic review and meta-analysis (2020) was conducted in 6 scientific databases following the PRISMA Guideline. Original published studies on human clinical samples in English were included. Articles evaluated sensitivity and specificity of RT-LAMP relative to RT-PCR were considered eligible. Quality assessment of bias and applicability was examined based on QUADAS-2.ResultsA total of 351 studies were found based on the keywords and search queries. 14 eligible case control studies fitted the respective criteria. Quality assessment using QUADAS-2 indicated low risk bias in all included studies. All case studies, comprises 2,112 samples, had the cumulative sensitivity of 95.5% (CI 97.5%=90.8-97.9%) and cumulative specificity of 99.5% (CI 97.5%=97.7-99.9%).ConclusionRT-LAMP assay could be suggested as a reliable alternative COVID-19 diagnostic method with reduced cost and time compared to RT-PCR. RT-LAMP could potentially be utilized during the high-throughput and high-demand critical situations.

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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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Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay during an Outbreak in Timor-Leste

Viruses ◽

10.3390/v12121444 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1444

Author(s):

Peter T. Mee ◽

Shani Wong ◽

Kim J. O’Riley ◽

Felisiano da Conceição ◽

Joanita Bendita da Costa Jong ◽

...

Keyword(s):

African Swine Fever Virus ◽

African Swine Fever ◽

Lamp Assay ◽

Fever Virus ◽

Isothermal Amplification ◽

Clinical Samples ◽

Loop Mediated Isothermal Amplification ◽

Field Verification ◽

Extraction Step ◽

Timor Leste

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

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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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First report on optimization of loop-mediated isothermal amplification (LAMP) for the diagnosis of Babesia felis

Indian Journal of Animal Research ◽

10.18805/ijar.b-567 ◽

2017 ◽

Cited By ~ 1

Author(s):

Muhammad Awais Salim ◽

Raheela Akhtar ◽

Muhammad Lateef ◽

Imran Rashid ◽

Harron Akbar ◽

...

Keyword(s):

Lamp Assay ◽

Cost Effective ◽

Conventional Polymerase Chain Reaction ◽

18S Rrna Gene ◽

Isothermal Amplification ◽

Rrna Gene ◽

Chain Reaction ◽

Loop Mediated Isothermal Amplification ◽

Polymerase Chain ◽

Effective Diagnosis

The objective of present study was to optimize loop mediated isothermal amplification (LAMP) assay for the diagnosis of Babesia felis in cats. LAMP primers were designed recognizing four sections of 18SribosomalRNA (18S rRNA) gene of B. felis. The blood samples of cats microscopically positive for Babesia felis were further used to extract deoxyribo neuclic acid (DNA) and the reaction mixture of 25 µL was standardized at 63°C temperature for 1 hour. LAMP assay provided more positive samples than conventional polymerase chain reaction (PCR). The prevalence of B. felis was also determined in cats using this optimized LAMP assay and it was found that the prevalence was more in younger cats as compare to adults. The application of LAMP can be helpful in rapid, reliable and cost effective diagnosis of B. felis in field.

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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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Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

PeerJ ◽

10.7717/peerj.5993 ◽

2018 ◽

Vol 6 ◽

pp. e5993 ◽

Cited By ~ 5

Author(s):

Shao-Xin Cai ◽

Fan-De Kong ◽

Shu-Fei Xu ◽

Cui-Luan Yao

Keyword(s):

Real Time ◽

Clinical Signs ◽

Lamp Assay ◽

Isothermal Amplification ◽

Clinical Samples ◽

Lamp Method ◽

The Real ◽

Loop Mediated Isothermal Amplification ◽

Enterocytozoon Hepatopenaei ◽

Time Loop

Background Enterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP. Methods A rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples. Results Positive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP. Discussion In this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.

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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 of a multiplex Loop-Mediated Isothermal Amplification (LAMP) assay for on-site diagnosis of SARS CoV-2

PLoS ONE ◽

10.1371/journal.pone.0248042 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0248042

Author(s):

Woong Sik Jang ◽

Da Hye Lim ◽

Jung Yoon ◽

Ahran Kim ◽

Minsup Lim ◽

...

Keyword(s):

South Korea ◽

Real Time ◽

Real Time Pcr ◽

Detection Limits ◽

Lamp Assay ◽

Isothermal Amplification ◽

Clinical Samples ◽

Lamp Primer ◽

Loop Mediated Isothermal Amplification ◽

Primer Sets

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).

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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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