A Sensitive and Specific Fluorescent RT-LAMP Assay for SARS-CoV-2 Detection in Clinical Samples

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a system was established in one tube for the detection of the ASFV p72 gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporters and CRISPR-derived RNA (crRNAs) were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples, a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. In the current study, a LAMP coupled with the CRISPR detection method was developed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Rapid isothermal amplification and portable detection system for SARS-CoV-2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2014739117 ◽

2020 ◽

Vol 117 (37) ◽

pp. 22727-22735 ◽

Cited By ~ 11

Author(s):

Anurup Ganguli ◽

Ariana Mostafa ◽

Jacob Berger ◽

Mehmet Y. Aydin ◽

Fu Sun ◽

...

Keyword(s):

Limit Of Detection ◽

Detection System ◽

Alternative Pathway ◽

Rna Extraction ◽

Lamp Assay ◽

Isothermal Amplification ◽

Clinical Samples ◽

Complete Agreement ◽

Rt Pcr ◽

Viral Transport Medium

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

Download Full-text

Development and validation of direct RT-LAMP for SARS-CoV-2

10.1101/2020.04.29.20075747 ◽

2020 ◽

Cited By ~ 5

Author(s):

Abu Naser Mohon ◽

Jana Hundt ◽

Guido van Marle ◽

Kanti Pabbaraju ◽

Byron Berenger ◽

...

Keyword(s):

Sindbis Virus ◽

Limit Of Detection ◽

Point Of Care ◽

Extraction Procedure ◽

Lamp Assay ◽

Cold Chain ◽

Clinical Samples ◽

Lamp Method ◽

Rt Pcr ◽

Percent Agreement

AbstractWe have developed a reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2. The LAMP assay achieves comparable limit of detection as commonly used RT-PCR protocols based on artificial targets, recombinant Sindbis virus, and clinical samples. Clinical validation of single-target (S gene) LAMP (N=120) showed a positive percent agreement (PPA) of 41/42 (97.62%) and negative percent agreement (NPA) of 77/78 (98.72%) compared to reference RT-PCR. Dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 44/48 (91.97%) and NPA 72/72 (100%) when including discrepant samples. The assay can be performed without a formal extraction procedure, with lyophilized reagents which do need cold chain, and is amenable to point-of-care application with visual detection.

Download Full-text

A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

10.21203/rs.3.rs-56856/v3 ◽

2020 ◽

Author(s):

Boon-Teong Teoh ◽

Kim-Ling Chin ◽

Nur-Izyan Samsudin ◽

Shih-Keng Loong ◽

Sing-Sin Sam ◽

...

Keyword(s):

Detection Limit ◽

Sensitivity And Specificity ◽

Reverse Transcription ◽

Zika Virus ◽

Lamp Assay ◽

Isothermal Amplification ◽

Clinical Samples ◽

Pcr Assay ◽

Zikv Infection ◽

Qrt Pcr

Abstract Background: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.Methods: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.Results: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6 – 98.2) and 100% (95% CI = 78.5 – 100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (k = 0.913, P < 0.001).Conclusion: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

Download Full-text

A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

10.21203/rs.3.rs-56856/v2 ◽

2020 ◽

Author(s):

Boon-Teong Teoh ◽

Kim-Ling Chin ◽

Nur-Izyan Samsudin ◽

Shih-Keng Loong ◽

Sing-Sin Sam ◽

...

Keyword(s):

Detection Limit ◽

Sensitivity And Specificity ◽

Reverse Transcription ◽

Zika Virus ◽

Lamp Assay ◽

Isothermal Amplification ◽

Clinical Samples ◽

Pcr Assay ◽

Zikv Infection ◽

Qrt Pcr

Abstract Background: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.Methods: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.Results: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6 – 98.2) and 100% (95% CI = 78.5 – 100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (k = 0.913, P < 0.001).Conclusion: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text