Optimizing the NEB LAMP Test

Optimizing the NEB LAMP Test

#nebLAMPtest

NEB recently published a preprint of a new test protocol that requires very little equipment. Can it be optimized and work across many labs?

Created on: March 26, 2020

by Ellen Jorgensen

Participating to challenge(s): Covid19 Diagnostic and Detection

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Introduction 

Project title Optimizing the NEB LAMP Test

Short name NEB LAMP


Problem and Background (200 words max)


There is a shortage SARS-CoV-2 testing throughout the world. Test protocols that diagnose infections or detect environmental contamination by the virus are not yet simple, affordable or widely accessible. Currently most diagnostic testing is accomplished by Reverse Transcription–quantitative Polymerase Chain Reaction (RT-qPCR). RT-qPCR is expensive, laborious and not scalable or accessible to many people living in low-resource settings. Over-reliance on this complicated technique of molecular amplification limits people in many regions of the globe from knowing whether the virus is present and hinders public health efforts. Loop-mediated Isothermal Amplification (LAMP) is an affordable, robust, simple and open detection method which requires only a single incubation temperature and can be done in a cup of hot water. A recent preliminary report (see Documents section) from New England Biolabs (NEB) used LAMP to detect SARS-CoV-2 in conjunction with a pH indicator to allow visualization of results by a simple color change in 30 minutes or less. If this method can be optimized and compares favorably to the current RT-qPCR test, it will be single-tube nucleic acid amplification method offering rapid, accurate, and cost-effective detection of SARS-CoV-2 that could be deployed anywhere. 


Solution summary in simple terms (150 words max)


Preliminary data on a LAMP-based method suggests it is sensitive and can be done without complicated equipment, with a simple +/- color change readout, using the commercially-available reagents from NEB. We will optimize and validate a version of this across several labs. This is the first stage of development of a SARS-CoV-2 test that can be run anywhere, even with limited facilities, and by people without a specific background in medicine or biology.   


Solution summary in technical terms (200 words max)


New England Biolabs (NEB) recently published preliminary results on a simple SARS-CoV-2 test based on RT-LAMP, a molecular detection method that allows identification of specific nucleic acids in samples with colorimetric visualization of results . The reaction is run for 15-45 min at a single temperature (65 degrees Celsius), and can be done in a cup of hot water with results read by naked eye or cell phone. The price per test is less than 3€. [3] [4] LAMP reagents have been successfully freeze-dried by NEB for detection of other diseases, potentially eliminating the need for a cold chain to deploy it [5].

 

Initial reports on this test are promising, but the pH -dependent reporter mechanism could potentially be interfered with by sample conditions and the reaction itself is very sensitive to variables such as reaction time. Thus, more work is needed if the test is to become widely accessible and users are of different skill levels.

           

We would optimize the test protocol by varying primer ratios, magnesium concentration, sample buffers and purification of RNA. Furthermore we would test the ruggedness of the method through an interlab study across at least four labs internationally.


State of advancement of the project (100 words max)

Some initial experiments with the Colorimetric NEB LAMP Master Mix and set of N gene primers (from the nucleocapsid region of the viral genomic sequence) have been conducted. Simple experiment by NYC lab with plasmid positive control and water negative control yielded expected results. Chicago lab has achieved more efficiency by changing primer ratios, and a collaboration between several labs has formed where we validate each other's results (ruggedness in analytical method validation terminology) including Chris Monaco at CDC.


Project Timeline

●  Week 0 - Order reagents (primers, enzymes, buffers, positive control DNA/RNA)

●  Week 1 - Modifications of ratio of primers and magnesium concentrations to assess maximal efficiency of the reaction and its detection. Order synthetic RNA positive controls.

●  Week 2 - Test relating to buffers and treatments for the sample transfer/processing/use using synthetic RNA controls

●  Weeks 3 & 4 - Final Validations of Sensitivity and Specificity with Controls (by day 28)

●  Week 5 - Write up data and transfer methodology to partner lab with BSL2 facilities (CDC partner lab and possibly others)

●  By Week 8 - Conclusions (Is this method robust enough for dealing with samples in this pandemic?) and broad dissemination of the open information.




Project Implementation

Research: describe hypothesis and research objectives (1000 words max)


We will test the hypothesis that an inexpensive, easy to use kit for detecting SARS-CoV-2 based on isothermal amplification with colorimetric output can be optimized and simplified beyond what is currently commercially available. If the test could be simplified and made more reliable it could allow much wider use by people with minimal training and resources, potentially having a very broad impact. 


Environmental monitoring would be facilitated for anyone, although use for any patient sample should be run in an appropriate laboratory setting (BSL3). 


The form of isothermal amplification known as Loop-mediated Isothermal Amplification (LAMP) was developed in 2000 (6), and later made even more rapid and able to detect RNA by adding a reverse transcriptase RT-LAMP. LAMP is an established single tube nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases (8) at isothermal conditions. It employs a strand displacing DNA polymerase and 4–6 primers at 60-73 degrees Isothermally, and is done in less than an hour (9). The RT-LAMP assay has already been applied for detection of many RNA viruses in humans, such as West Nile, severe acute respiratory syndrome coronavirus, influenza A, mumps, measles, dengue, HIV, and respiratory syncytial virus (10). LAMP can amplify a few copies of the target to 10^9 in less than one hour, even when large amounts of non-target DNA are present or other biological substances (11). RT-LAMP has been shown to be sensitive and specific enough to detect RNA viruses from biological samples such as blood, serum, urine, cerebrospinal fluid, feces, tissue, and nasal swabs, without the need for RNA purification (12). Several recent significant improvements to RT-LAMP have been made, and New England Biolabs (NEB) has recently published a protocol using their WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA).


This is a short-term project with several clear objectives:


Objective 1. Validate and optimize NEB’s published protocol. 

We will perform parallel experiments across several international labs, varying conditions and reagents to achieve results that are faster, more reliable and less expensive than the current protocol.


Objective 2. Collect ruggedness data on the optimized protocol

We will ascertain the reliability of the test in the hands of people across several international labs to see if results are consistent.


Objective 3. Test the optimized protocol on inactivated Covid-19 patient samples.

At least one of our participating labs (US Centers for Disease Control) has the requisite biosafety level facilities for testing the improved protocol on patient samples. This is relevant for potential deployment of the test in areas of the world where the machines for the current tests used (WHO and US tests both require a qPCR machine) are scarce.


Methodology (500 words max)


Interlab cooperation and participation is a key part of this methodology. All partner labs will attempt to carry out all of the experiments as their facilities and personnel permit. The objective is to validate the improved protocol across many different labs with varying capabilities and geographic locations.


  1. Reproduce the original protocol across several labs. The original NEB publication using RT-LAMP for the detection of SARS-CoV-2 tested five sets of primers. We will start by using the one that worked the best in their hands and for which there is a commercially-available, safe positive control in the form of plasmid DNA. The target is a portion of nucleic acid sequence of the nucleocapsid protein N. The protocol will be as described in Basic NEB LAMP Protocol found in the Document section of this project. 


  1. Varying parameters to optimize sensitivity and specificity. We plan to optimize and validate our RT-LAMP primers against two different commercially-available synthetic positive controls (plasmid and RNA) and commercially available controls to check for potential cross-reactivity with MERS and previous SARS. We will measure their sensitivity, specificity, cross-reactivity and TTR (time to response ), and vary primer concentration, incubation times, and magnesium concentrations.


  1. Effect of sample buffers. Test samples are collected in various buffers. SInce purification of RNA is often a limiting step in areas with few resources, and since even in developed countries these kits have been in short supply, it is important to determine if testing samples without pre-purification of RNA can yield acceptable results. We will test the effect of different sample buffers on the sensitivity and specificity of the optimized test.


  1. Initial validation against current methods using patient samples. This portion of the project will only be carried out by laboratories meeting the biosafety level standards of their country for handling inactivated infected patient samples. Inactivation will be via heating (30 min at 54C was used in the NEB paper) or the use of a medium such as RNAlater. For this first phase of the project a limited number of patient samples will be used to compare the results of the optimized test with those obtained through currently used RT-qPCR-based tests.


Results/Expected results (500 words max)


At the end of this phase of the project we will have


  1. Developed an optimized version of the RT-LAMP protocol from NEB fro SARS-CoV-2 detection.
  2. Demonstrated that the protocol can be used across labs of varying resources and by persons with varied levels of previous molecular biology training
  3. Documented the process and resulting data that led to 1 & 2 above in a clear and accessible fashion on JOGL
  4. Executed preliminary testing of the optimized method on human samples in a partner lab with appropriate biosafety measures.


Safety, quality assurance and regulation


What steps have you taken to ensure your solution’s safety? 

Each participating lab is following biosafety level guidelines in their respective country. Most of the work will be using synthetic positive control samples in a BSL1 area. If the project proceeds to the point where a higher level of biosafety is required (e.g. inactivated human samples) it will proceed only in the participating labs that are equipped to handle samples at that biosafety level. All personnel performing the experiments have received appropriate safety training through their host laboratories to handle the minimal-risk chemical reagents involved in this protocol and use good laboratory hygiene practices.


In this first (research only) phase of the project we will not be engaging with the regulatory systems or medical staff. We will not be distributing test kits.


Impact, issues and risks


What impact do you feel your product could have? (100 words max)


There is a shortage SARS-CoV-2 testing throughout the world. New test methods are urgently needed that are simple, affordable and widely accessible. Over-reliance on this complicated technique of molecular amplification limits people in many regions of the globe from knowing whether the virus is present and hinders public health efforts. If we can optimize a simple RT-LAMP method that compares favorably to the current RT-qPCR test, it will offer rapid, accurate, and cost-effective detection of SARS-CoV-2 that could be deployed anywhere.


What do you think would make your project a success?(100 words max)

Success would be completion of the following objectives:


  1. Develop an optimized version of the RT-LAMP protocol from NEB fro SARS-CoV-2 detection that is sensitive and specific.
  2. Demonstrate that the protocol can be used across labs of varying resources and by persons with varied levels of previous molecular biology training.
  3. Document the process and resulting data that led to 1 & 2 above in a clear and accessible fashion on JOGL.
  4. Execute preliminary testing of the optimized method on human samples in a partner lab with appropriate biosafety measures, showing that it performs as well or better than currently used methods.



Please list the known issues, potential risks, grey-areas, etc in your project


-Optimization with synthetic controls may result in a test that will not work as well with patient samples

-Some of the reagents are available only from single sources, we need to plan for alternatives such as DIY master mixes. This would also be true if the regents end up unobtainable or costing too much per test to be useful in areas with few resources.

-It may prove impossible to achieve good sensitivity when using crude samples without RNA extraction. If so we may need to segue into alternate purification methods or new sample buffers.

-The test may not be easily shippable despite the reports that these types of reagents can be freeze dried.


List of participating labs and their team members


BioBlaze Community Bio Lab, West Chicago Il, USA.

Sarah Ware has a doctorate degree and has 20 years of research experience as a geneticist/molecular biologist. She is the founder of two independent labs in the Chicagoland area: BioBlaze Community Bio Lab and Lizzy Blossom Ag Services. Sarah also teaches biology and humanities at the university level.

Isabella Zorra has a Masters in Construction Engineering and Management and a Bachelor of Architecture with a focus on fabrication. She currently works as a Project Manager in the Energy Sector. Additionally she is passionate about the sciences and making a lasting improvement on the human condition. Isabella is also with BioBlaze Community Bio Lab.

Lizzy Blossom Ag Services, West Chicago, IL, USA.

Aubry Field has a Masters in Horticulture & Crop Science from The Ohio State University in the Plant Transformation Lab. Her work focused on transient expression and stable transformation using GFP as a reporter gene for soybean. She is a research scientist/lab manager with over 20 years of experience in a molecular laboratory setting with a strong background in plant viruses.  


Aanika Biosciences, Brooklyn NY, USA.

Ellen Jorgensen has a Ph.D. in molecular biology and has worked in the biotechnology industry for over 30 years, including in FDA-regulated assay development. She has founded two community labs, Genspace and Biotech Without Borders. Her thesis work was on Newcastle Disease virus, a negative strand RNA paramyxovirus. She was a founding staff member of the Sabin Vaccine Foundation, and currently is the Chief Science Officer at Aanika Biosciences, a company offering environmental testing for SARS-CoV-2. https://www.linkedin.com/in/ellenjorgensen/


Hackuarium, Lausanne, Switzerland.

Rachel Aronoff worked on retroviral RNA packaging for her PhD thesis project, cloned an mRNA ‘quality control’ gene from C. elegans, used lentivectors and AAV to study mouse brain circuits, and got implicated in the world of Open Science and Participatory Research when she founded a non-profit - AGiR! Action for Genomic integrity through Research!, but never expected to be involved in anything like this Open Covid19 Initiative. Currently president of the Association Hackuarium and CSO for AGiR!, she loves providing information and promoting research.


Centers for Disease Control, Atlanta GA, USA.

Chris Monaco earned his Masters degree in bioinformatics at the Georgia Institute of Technology. He is currently a microbiologist in the CDC's Division of Scientific Resources' Biotechnology Core Facility Branch (BCFB). https://www.linkedin.com/in/cmonaco/


Center for Research and Interdisciplinarity, Paris, France

Guy Aidelberg is completing his PhD on democratizing DNA detection using related techniques and has experience developing similar systems for Zika, Dengue and Chikungunya viruses. He holds a Masters degree in system biology from the Weizmann Institute of Science.


openFIESTA community lab, Madrid, Spain

Francisco Javier Quero: Biologist by training. Tsinghua University Master's degree student and the manager of openFIESTA community lab. Two years working with synthetic biology as the coordinator of Madrid iGEM teams 2018 and 2019. Experience developing isothermal amplifications for infectious diseases. 


Funding 


All partner labs (except the CRI in France; Guy is contributing through protocol design etc. until his institute reopens) are contributing their lab spaces and donating the use of core equipment and person-hours to this project. The funding we are asking for will go towards the purchase of reagents and consumables, and a few pieces of key lab equipment to fill gaps.


Some labs require no funding from JOGL, but those who do have each submitted a separate budget. It is understood that there will be sharing of many reagents as shipping regulations permit.


Please see the individual lab budgets in the Documents section (tab above).