Novel strategies for single step molecular diagnostics assays with full dynamic range quantitation
Lead Research Organisation:
CARDIFF UNIVERSITY
Department Name: School of Biosciences
Abstract
Molecular diagnostics is the most sensitive technique to detect specific organisms, pathogens or other biological material that contains DNA, and is widely used in diagnostic laboratories worldwide. It relies on specific primers to recognize target DNA sequences unique to the organism being detected, making it highly specific. Most molecular diagnostics uses the polymerase chain reaction (PCR), which involves repeated rounds of heating and cooling to make many copies of the target DNA, amplifying it so it can be detected. However the need for temperature cycling, and for sophisticated fluorescence-based systems to detect the amplified DNA, means that it is largely restricted to laboratory use. Moreover PCR is rather sensitive to inhibition by a number of common chemicals found in the environment and body fluids, requiring samples to be extensively purified before analysis.
As a result the instruments required for PCR are vulnerable and relatively expensive to engineer. Molecular methods are therefore largely confined to large and expensive equipment within diagnostic laboratories requiring skilled operators. The limited equipment available for out-of-laboratory use is expensive, relatively large and unsuited to widespread application.
The best solution to these problems is a recently developed approach based on amplification at a constant temperature, so-called isothermal amplification, coupled with a read-out of light as the specific target DNA sequence is amplified. This "bioluminescent assay in real-time" or BART results in a emission of a continuous light signal that reaches a peak at a time proportional to the amount of DNA present. BART was invented by the applicant and the CEO of the company started to commercialise such assays, Lumora Ltd. BART has been licensed by Lumora to the global partner 3M, who have commercialised assays for detecting food pathogens. This demonstrates the effectiveness and robustness of the approach.
BART produces a light output that is simple and cheap to monitor using a camera chip or photodiodes in a solid-state device. This substantially reduces instrument costs and open up new applications for diagnostics and disease monitoring in resource-poor settings such as in the developing world, where there are extensive requirements for cheap molecular assays for disease diagnosis.
A major challenge in molecular diagnostics remains the accurate measurement of the numbers of disease organisms (and hence target DNA molecules) in the sample. This can be done both by PCR and using BART, but the ability to accurately determine the number of molecules of the target becomes much more difficult below about 50 copies for both techniques. For certain diseases and situations, it is critical to be able to accurately measure the numbers of targets at this level. The proposed project is based on new methods discovered by the applicant and the company partner which are able to accurately determine the number of molecules down to a single molecule of target DNA. Moreover, remarkably they can distinguish between numbers of copies accurately in the range 1-10. The proposal is to test and develop these new approaches, and to use them with fluidic chips that would allow accurate measurement of numbers of molecules from 1 up to 100's of billions in a single set of assays carried out on a single plastic chip simultaneously- a so-called full dynamic range assay. Purpose designed algorithms will be developed and explored to analyse the data allowing the numbers to be quantified throughout the dynamic range using the most appropriate combination of methods for each part of the range.
If successful, this would represent a breakthrough in molecular diagnostics with significant implications as a full dynamic range quantification method, which would see widespread application in research, medical diagnostics, disease monitoring, and environmental protection, with potential economic, health and societal benefits.
As a result the instruments required for PCR are vulnerable and relatively expensive to engineer. Molecular methods are therefore largely confined to large and expensive equipment within diagnostic laboratories requiring skilled operators. The limited equipment available for out-of-laboratory use is expensive, relatively large and unsuited to widespread application.
The best solution to these problems is a recently developed approach based on amplification at a constant temperature, so-called isothermal amplification, coupled with a read-out of light as the specific target DNA sequence is amplified. This "bioluminescent assay in real-time" or BART results in a emission of a continuous light signal that reaches a peak at a time proportional to the amount of DNA present. BART was invented by the applicant and the CEO of the company started to commercialise such assays, Lumora Ltd. BART has been licensed by Lumora to the global partner 3M, who have commercialised assays for detecting food pathogens. This demonstrates the effectiveness and robustness of the approach.
BART produces a light output that is simple and cheap to monitor using a camera chip or photodiodes in a solid-state device. This substantially reduces instrument costs and open up new applications for diagnostics and disease monitoring in resource-poor settings such as in the developing world, where there are extensive requirements for cheap molecular assays for disease diagnosis.
A major challenge in molecular diagnostics remains the accurate measurement of the numbers of disease organisms (and hence target DNA molecules) in the sample. This can be done both by PCR and using BART, but the ability to accurately determine the number of molecules of the target becomes much more difficult below about 50 copies for both techniques. For certain diseases and situations, it is critical to be able to accurately measure the numbers of targets at this level. The proposed project is based on new methods discovered by the applicant and the company partner which are able to accurately determine the number of molecules down to a single molecule of target DNA. Moreover, remarkably they can distinguish between numbers of copies accurately in the range 1-10. The proposal is to test and develop these new approaches, and to use them with fluidic chips that would allow accurate measurement of numbers of molecules from 1 up to 100's of billions in a single set of assays carried out on a single plastic chip simultaneously- a so-called full dynamic range assay. Purpose designed algorithms will be developed and explored to analyse the data allowing the numbers to be quantified throughout the dynamic range using the most appropriate combination of methods for each part of the range.
If successful, this would represent a breakthrough in molecular diagnostics with significant implications as a full dynamic range quantification method, which would see widespread application in research, medical diagnostics, disease monitoring, and environmental protection, with potential economic, health and societal benefits.
Technical Summary
Molecular diagnostics (MD) involves detecting specific DNA/RNA sequences of a target through amplification, and offers the highest sensitivity of any diagnostic test, being theoretically capable of detecting a single molecule. Most MD uses quantitative PCR (qPCR) involving temperature cycling to drive the amplification and fluorescent detection of the amplified product. The alternative isothermal loop-mediated amplification (LAMP) is driven by strand-displacing polymerases. This can be coupled to a bioluminescent output in the "BART" assay, invented by the applicant and Lumora. BART involves conversion of pyrophosphate released during amplification to ATP using ATP sulphurylase and its simultaneous utilization by a thermostable firefly luciferase. This provides a continuous light output proportional to the DNA amplification. During amplification, the light signal rises to a sharp peak. Peak time is proportional to target DNA originally present.
However quantification in molecular diagnostics remains challenging at low copy numbers for both qPCR and LAMP-BART, and the LOQ is normally 20-100 copies. Recent digital approaches based on limiting dilution can accurately determine very low copy numbers, but require multiple reactions, extensive sample handling and complex apparatus.
Recent results have led to potential novel methods for accurate determination of copy number to near single digit precision using small numbers of replicates. These novel methods will be researched and analysed, and appropriate algorithms developed. A combination of measurement algorithms will be used (including time to light peak at higher copy numbers) to measure accurately sample number across the full dynamic range. Combined with integrated fluidic circuits allowing a single sample to be automatically partioned into multiple assays, this opens the possibility of a single sample load onto a single disposible providing quantification from single molecules across a full dynamic range.
However quantification in molecular diagnostics remains challenging at low copy numbers for both qPCR and LAMP-BART, and the LOQ is normally 20-100 copies. Recent digital approaches based on limiting dilution can accurately determine very low copy numbers, but require multiple reactions, extensive sample handling and complex apparatus.
Recent results have led to potential novel methods for accurate determination of copy number to near single digit precision using small numbers of replicates. These novel methods will be researched and analysed, and appropriate algorithms developed. A combination of measurement algorithms will be used (including time to light peak at higher copy numbers) to measure accurately sample number across the full dynamic range. Combined with integrated fluidic circuits allowing a single sample to be automatically partioned into multiple assays, this opens the possibility of a single sample load onto a single disposible providing quantification from single molecules across a full dynamic range.
Planned Impact
This project aims to develop improved strategies for quantitation of molecular diagnostic assays. Molecular diagnostics are applied in a wide range of sectors from disease diagnosis to public health, agriculture and environmental monitoring.
The development of improved molecular diagnostics with full dynamic range quantification has the potential to foster improved research by providing new, improved techniques. This project aims to offer significant improvements to current molecular diagnostics approaches and consequently would impact on the research, diagnostics and the related industry sectors. As a result, there are potential wider impacts in health, economic competitiveness and global development.
There would be an impact in academic and commercial laboratories engaged in experiments or assays to determine specific DNA or RNA sequence abundance for a range of purposes. The new developments would have the potential for widespread uptake, and could accelerate research and diagnosis and reduce its cost.
There is significant economic potential in these developments. Currently 60% of the commercial value in molecular diagnostics is in infectious disease testing, particularly human immunodeficiency virus (HIV), human papilloma virus (HPV), hepatitis B and C (HBV/HCV), and sexually transmitted disease agents, and these agents require quantitative tests. Improvements in such testing would generate new commercial activity within the UK by increasing the potential market share. Worldwide sales of molecular diagnostics totaled $4.1 billion in 2010, and Frost and Sullivan predict that by 2014 revenues will exceed $6.2 billion. Compound annual growth rate (CAGR) is projected at more than 11%.
The particular utility of the proposed developments are that they offer quantitation in a format that could be used in the field or at point of care. This would have particular relevance in settings such as epidemic or sporadic disease outbreaks in human or animal health, such as foot and mouth, blue tongue virus and classic swine fever. Improved and more rapid on-farm diagnosis would have impacts on agriculture through improved disease control. Control of infection of plant or animal material at ports of entry could help prevent or limit the spread of novel diseases, such as ash dieback, benefiting the environment and general society. Improved methods for monitoring GMO levels in non-GMO foodstuffs would assist in regulatory compliance at reduced cost, and hence consumers could ultimately benefit.
Organisations engaged in delivering healthcare in the developing world would also benefit. There is a substantial need for molecular diagnostics suitable for low resource settings, and in management of HIV/AIDS in particular there is a need for quantitation. There are reported to be only 8 sites in sub-Saharan Africa equipped for qPCR (WHO), and a quantitative diagnostic suitable for mobile clinics would be of major significance. Lumora, the commercial partner is already collaborating with the not-for-profit organisation PATH (http://www.path.org/), with the aim of delivering potential HIV diagnostics, and with the Foundation for Innovative New Diagnostics (FIND http://www.finddiagnostics.org) to develop a rapid, high-throughput malaria diagnostic assay for screening patients in the developing world (see http://www.lumora.co.uk/index.php/news2/37-news/2013/102-lumora-to-partner-with-find-to-develop-new-high-throughput-malaria-molecular-diagnostic-test-for-use-in-the-developing-world).
The development of improved molecular diagnostics with full dynamic range quantification has the potential to foster improved research by providing new, improved techniques. This project aims to offer significant improvements to current molecular diagnostics approaches and consequently would impact on the research, diagnostics and the related industry sectors. As a result, there are potential wider impacts in health, economic competitiveness and global development.
There would be an impact in academic and commercial laboratories engaged in experiments or assays to determine specific DNA or RNA sequence abundance for a range of purposes. The new developments would have the potential for widespread uptake, and could accelerate research and diagnosis and reduce its cost.
There is significant economic potential in these developments. Currently 60% of the commercial value in molecular diagnostics is in infectious disease testing, particularly human immunodeficiency virus (HIV), human papilloma virus (HPV), hepatitis B and C (HBV/HCV), and sexually transmitted disease agents, and these agents require quantitative tests. Improvements in such testing would generate new commercial activity within the UK by increasing the potential market share. Worldwide sales of molecular diagnostics totaled $4.1 billion in 2010, and Frost and Sullivan predict that by 2014 revenues will exceed $6.2 billion. Compound annual growth rate (CAGR) is projected at more than 11%.
The particular utility of the proposed developments are that they offer quantitation in a format that could be used in the field or at point of care. This would have particular relevance in settings such as epidemic or sporadic disease outbreaks in human or animal health, such as foot and mouth, blue tongue virus and classic swine fever. Improved and more rapid on-farm diagnosis would have impacts on agriculture through improved disease control. Control of infection of plant or animal material at ports of entry could help prevent or limit the spread of novel diseases, such as ash dieback, benefiting the environment and general society. Improved methods for monitoring GMO levels in non-GMO foodstuffs would assist in regulatory compliance at reduced cost, and hence consumers could ultimately benefit.
Organisations engaged in delivering healthcare in the developing world would also benefit. There is a substantial need for molecular diagnostics suitable for low resource settings, and in management of HIV/AIDS in particular there is a need for quantitation. There are reported to be only 8 sites in sub-Saharan Africa equipped for qPCR (WHO), and a quantitative diagnostic suitable for mobile clinics would be of major significance. Lumora, the commercial partner is already collaborating with the not-for-profit organisation PATH (http://www.path.org/), with the aim of delivering potential HIV diagnostics, and with the Foundation for Innovative New Diagnostics (FIND http://www.finddiagnostics.org) to develop a rapid, high-throughput malaria diagnostic assay for screening patients in the developing world (see http://www.lumora.co.uk/index.php/news2/37-news/2013/102-lumora-to-partner-with-find-to-develop-new-high-throughput-malaria-molecular-diagnostic-test-for-use-in-the-developing-world).
People |
ORCID iD |
James Murray (Principal Investigator) |
Publications
Jathoul A
(2022)
A higher spectral range of beetle bioluminescence with infraluciferin
in Frontiers in Bioengineering and Biotechnology
Hardinge P
(2019)
Reduced False Positives and Improved Reporting of Loop-Mediated Isothermal Amplification using Quenched Fluorescent Primers.
in Scientific reports
Hardinge P
(2019)
Lack of specificity associated with using molecular beacons in loop mediated amplification assays.
in BMC biotechnology
Hardinge P
(2018)
Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART).
in Scientific reports
Hardinge P
(2023)
Plant Genotyping - Methods and Protocols
Hardinge P
(2022)
Optimized Loop-Mediated Isothermal Amplification (LAMP) Allows Single Copy Detection Using Bioluminescent Assay in Real Time (BART).
in Methods in molecular biology (Clifton, N.J.)
Hardinge P
(2020)
Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells.
in Scientific reports
Halliwell LM
(2018)
?Flucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant.
in Biotechnology and bioengineering
Anderson JC
(2017)
Convergent synthesis and optical properties of near-infrared emitting bioluminescent infra-luciferins.
in RSC advances
Description | Discovery 1: Loop-mediated amplification (LAMP) has been widely used to amplify and hence detect nucleic acid target sequences from various pathogens, viruses and genetic modifications. Two distinct types of primer are required for LAMP; hairpin-forming LAMP and displacement. High specificity arises from this use of multiple primers, but without optimal conditions for LAMP, sensitivity can be poor. We confirm here the importance of LAMP primer design, concentrations and ratios for efficient LAMP amplification. We further show that displacement primers are non-essential to the LAMP reaction at certain concentrations providing accelerating loop primers are present. We investigate various methods to quantify DNA extracts from GM maize certified reference materials to calculate the target copy numbers of template presented to the LAMP reaction, and show that LAMP can amplify transgenic promoter/terminator sequences in DNA extracted from various maize GM events using primers designed to target the 35S promoter (35Sp) or NOS terminator (NOSt) sequences, detection with both bioluminescence in real-time (BART) and fluorescent methods. With prior denaturation and HPLC grade LAMP primers single copy detection was achieved, showing that optimised LAMP conditions can be combined with BART for single copy targets, with simple and cost efficient light detection electronics over fluorescent alternatives. Discovery 2: Loop mediated isothermal amplification of nucleic acid templates is a rapid, sensitive and specific method suitable for molecular diagnostics. However the complexity of primer design and the number of primers involved can lead to false positives from non-specific primer interactions. Standard methods of LAMP detection utilise the increasing concentrations of DNA or inorganic pyrophosphate and therefore lack specificity for identifying the desired LAMP amplification. Molecular beacons used in PCR reactions are target specific and may enhance specificity with LAMP. Results: Molecular beacons used in LAMP, despite providing a change in fluorescent intensity with amplification, appear not to anneal to the target sequences and therefore target specificity is not a benefit of this method. However, the molecular beacons appear to be specific to the LAMP amplification of the template. We present a molecular beacon approach to LAMP detection targeting the single stranded region between loops. We describe LAMP molecular beacons targeting the 35S promoter and NOS terminator sequences commonly used in GM crops, and optimisation of the molecular beacons for assay temperature, concentration and sensitivity. Conclusions: Molecular beacons can be used to detect LAMP amplification without an increase in specificity for the loop or stem regions of amplicons. The method is however unsuitable for multiplex LAMP reactions due to non-specific detection of LAMP amplification. |
Exploitation Route | These findings are of interest in the use of loop mediated isothermal amplification for diagnostic assays and tests, and hence in the molecular diagnostics industries. |
Sectors | Agriculture Food and Drink Environment Healthcare |
Description | The information has been used by our industrial collaborators to assist in design of molecular diagnostic assays and is leading to a further grant application. Subsequent to this application, emergency UKRI Covid funding was obtained to exploit the results of the award, as well as additional Welsh Govt Covid funding. A Follow on Fund application has now been funded. |
First Year Of Impact | 2017 |
Sector | Healthcare |
Impact Types | Economic |
Description | ALMOND: Agriculture Living Machine of Operational Nano Droplets |
Amount | £1,600,000 (GBP) |
Funding ID | BB/Y008537/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2024 |
End | 05/2026 |
Description | Development, evaluation and validation of a microfluidic device for pathogen detection and quantification using LAMP and BART |
Amount | £205,441 (GBP) |
Funding ID | 80762-CU-085 |
Organisation | Cardiff University |
Sector | Academic/University |
Country | United Kingdom |
Start | 08/2017 |
End | 08/2020 |
Description | Digital quantification of infectious diseases using micro droplets, single temperature DNA amplification and bioluminescent detection |
Amount | £19,988 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2021 |
End | 08/2021 |
Description | Novel technologies for point-of-care genetic testing for SARS-CoV-2 |
Amount | £311,979 (GBP) |
Organisation | Government of Wales |
Sector | Public |
Country | United Kingdom |
Start | 11/2020 |
End | 04/2021 |
Description | School of Pharmacy and Pharmaceutical Sciences, Cardiff University |
Organisation | Cardiff University |
Department | School of Pharmacy and Pharmaceutical Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Knowledge and practical experience with bioluminescent detection of isothermal nucleic acid amplification, detection methods and results analysis. Quantification of DNA at low copy number and full dynamic range. |
Collaborator Contribution | Knowledge and practical experience with microfluidics, the generation of nanolitre droplets and the formation of artificial cells/encapsulated droplet interface bilayers (eDIBs). |
Impact | Publication of research by Springer Nature in Scientific Reports 2020 'Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells'. Further funding to develop bioluminescent LAMP-BART droplet microfluidics from Wellcome Trust and the Welsh Government. |
Start Year | 2017 |
Description | Presentation at BBSRC showcase event |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Other audiences |
Results and Impact | Showcase event to show the range of BBSRC funded research in the School of Biosciences, Cardiff University with valuable networking opportunities. |
Year(s) Of Engagement Activity | 2015 |
Description | Presentation at SYNBIO conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Opportunity to present and discuss quantification utilising LAMP DNA amplification coupled to the bioluminescent assay in real-time (BART) to researchers, academics and industry. |
Year(s) Of Engagement Activity | 2016 |
Description | Presentation at qPCR conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | International conference on qPCR in London with the opportunity to present and discuss next generation quantification utilising LAMP DNA amplification coupled to the bioluminescent assay in real-time (BART). |
Year(s) Of Engagement Activity | 2014 |