Patent Application
20230126580
The present invention relates to a device and method for detecting a pathogen in water and in particular, although not exclusively, to a system for rapid monitoring of a pathogen in a water system such as a wastewater flow.
The novel coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 infection spread rapidly around the globe. Although public health authorities raced to contain the spread of the virus, cumulative deaths escalated. Some clinical studies found that some carriers of the virus are asymptomatic, with no fever, and no, or only slight symptoms of infection. Without the ability to screen asymptomatic patients quickly and effectively, these unsuspecting carriers have the potential to increase the risk of disease transmission if no early effective quarantine measures are implemented.
Accordingly, fast and accurate screening of potential virus carriers and diagnosis of asymptomatic patients would be an important step for intervention and prevention at an early stage. It remains a highly challenging logistical exercise for medical professionals to practically and effectively screen suspected infectious cases from individual households. Such an undertaking is time-consuming, labour intensive and is constrained by the availability of testing technologies.
Paper analytical devices have emerged as powerful tools for the rapid diagnosis of pathogens. These origami-type devices are small analytical tools having different functional paper areas or regions that may be created by printing a hydrophobic wax on to the paper using a wax printer. The devices integrate the various processes of extraction, enrichment, purification, elution, amplification, and visual detection that are required, for example, for nucleic acid testing. The testing process may be undertaken through simple folding of a paper-based device in different ways and the eluting of fluids through the device without a pump or power supply. The paper analytical devices enable multiplexed sensitive assays that rival polymerase chain reactions (PCR) laboratory-based assays to provide high-quality, fast precision diagnostics for pathogens. For example, a study has demonstrated the multiplexed determination of malaria from whole blood using a paper-based device in rural Uganda [Reboud, J.; Xu, G.; Garrett, A.; Adriko, M.; Yang, Z.; Tukahebwa, E. M.; Rowell, C.; Cooper, J. M. ‘Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities’, Proc. Natl. Acad. Sci. U.S.A 2019, 116 (11), 4834-4842]. The test could sensitively analyse multiplexed nucleic acid sequences of pathogens within 50 min, which gave a higher-quality and faster precision diagnosis for malaria than PCR. In addition, paper analytical devices are easy to stack, store, and transport as they are thin and lightweight. Visual analysis is made simple due to the strong contrast with a coloured substrate. These paper-based devices can also be incinerated after use, reducing the risk of further contamination. However, such earlier systems have utilised direct biological material samples which would require the abovementioned time-consuming and labour-intensive screening to collect an individual's blood or saliva. Accordingly, what is required is apparatus, a method and system that addresses these problems.
It is an objective of the present invention to provide a system for rapid analysis of pathogens such as microorganisms in water to enable early detection. It is a further specific objective to provide a device and method for detection of pathogens, microorganisms, infectious diseases, bacteria, a virus and the like within a water network such as a wastewater flow from a commercial or domestic building.
It is a yet further specific objective to provide an earlier identification and detection system for high infectious diseases including in particular coronavirus species in wastewater from buildings including community wastewater, so as to identify affected households, communities, local populations and to minimise pathogen spread and the risk to public health.
Accordingly, the subject invention provides an early warning system that includes a rapid analytical device and method for on-site detection of viruses in wastewater. The present system utilizes wastewater-based epidemiology (WBE), to provide an effective approach to predict the potential spread of the infection by testing for infectious agents in wastewater. The present system also finds application as an effective means to trace illicit drugs, obtain information on health, disease, and pathogens at a local population/community level.
Faeces and urine from disease carriers in a community will contain many biomarkers that can enter the sewer system. The coronavirus infection disease (SARS-CoV-2) is capable of being isolated from faeces and urine of infected people and it has been shown to survive for up to several days in an appropriate environment after exiting the human body. The present device and method is directed to the monitoring, detection and analysis of infectious disease such as coronavirus species e.g., COVID-19, in household and community wastewater so as to trace the pathogen sources through sewage pipe networks and determine whether there are potential pathogen carriers in certain local areas. The present system provides infectious diseases monitoring at a community/household level and at a very early stage through WBE thereby enabling effective intervention such as movement restrictions on local populations to minimize spread of the pathogen.
The inventors provide an efficient, transportable and robust analytical tools to accurately and quickly identify trace or low-level pathogen sources through WBE so as to screen asymptomatic infected cases without centralized laboratories. The present WBE early warning and intervention system utilises a rapid analytical method and device for on-site detection of viruses in water. In particular, the inventors provide a microfluidic cellulose/paper-based analytical device (μPAD) to detect pathogens in a water sample such as a sample of wastewater from a household or community wastewater outlet. The presently developed monitoring and detection tool provides a fast ‘sample-to-answer’ analysis system for quantitative monitoring of nucleic acids and genetic information through the analysis of sewage. The present μPADs are small and portable and can detect pathogens in wastewater on site.
The present μPADs provide a substrate with a plurality of microfluidic channels and reaction chambers that is inexpensive, lightweight, disposable, and can be manufactured conveniently and readily. Cellulose/paper is a suitable construction material for the present μPADs due to its physical characteristics and in particular its hydrophilicity and capability to allow various solutions to flow through its porous structure via capillary action.
The present μPADs and methods are biocompatible with biological samples and may be used with a variety of different sensing mechanisms such as colorimetric, electrochemical, chemiluminescence (CL), electro-chemiluminescence (ECL) and other signal detection with the results being used quantitatively and/or quantitatively for diagnostic testing. Such sensing mechanisms, techniques and methods may be integrated with the present μPADs specifically for the detection of pathogens. Additionally, such systems enable bioassays to be undertaken and the results obtained simultaneously. Moreover, the present μPADs and methods are compatible for use with a digital camera or camera-enabled phone (smart phone) to collect data and images conveniently. Such data may then be used directly (or locally) or transmitted wirelessly over communications networks to centralized laboratories for analysis and results processing in real-time.
According to a first aspect of the present invention there is provided a method of detecting a pathogen present in a water sample comprising: extracting nucleic acid of the pathogen from a nucleic acid solution derived from the water sample at a solid phase extraction structure mounted at a first layer of a multilayer device; eluting nucleic acid from the solid phase extraction structure to at least a second layer of the multilayer device having paper-based fluid flow channels; allowing the nucleic acid to flow thought the paper-based fluid flow channels to a further layer of the multilayer device having discrete reaction chambers, each of the chambers fed respectively by at least one of the fluid flow channels; performing LAMP reactions within each reaction chamber to obtain LAMP products; and detecting the LAMP products via an amplicon detection test.
Reference within the specification to the extraction, elution, flow etc of nucleic acid or a nucleic acid-based analogue includes the extraction, elution, flow etc of compounds having nucleotide repeating units such as DNA, RNA and/or nucleic acid analogues of a pathogen.
Optionally, the method comprises filtering the water sample through a filter membrane and adding a lysis buffer to the filtered sample to form the nucleic acid solution. Optionally, prior to said step of eluting the nucleic acid, the method comprises washing the nucleic acid at the solid phase extraction structure with a washing buffer. Optionally, the solid phase extraction structure comprises glass fibre or magnetic beads onto cellulous paper.
Optionally, the step of allowing the nucleic acid to flow comprises allowing the nucleic acid to flow along the paper-based fluid flow channels of the second layer into paper-based fluid flow channels of a third layer positioned adjacent the second layer.
Optionally, the method comprises allowing the nucleic acid to flow from the paper-based fluid flow channels of the third layer into paper-based fluid flow channels of a fourth layer positioned adjacent the third layer.
Optionally, the flow of the nucleic acid in the paper-based fluid flow channels is divided as it transfers between the respective layers.
Optionally, the discrete reaction chambers at the further layer comprises paper inserts positioned within respective holes in the third layer, the further layer comprising a plastic material.
Optionally, prior to said step of performing the LAMP reactions, the method comprises sealing the nucleic acid within the discrete reaction chambers by coating a film onto the further layer to cover the paper inserts within the holes. Optionally, the step of performing the LAMP reactions comprises adding at least one set of LAMP primers to the discrete reaction chambers to create respective LAMP assays. Optionally, the method comprises adding a plurality of different sets of LAMP primers to the discrete reaction chambers. Optionally, the step of performing the LAMP reactions further comprises heating the further layer and the LAMP assays at a predetermined temperature and for a predetermined time. Optionally, the predetermined temperature is in a range 40 to 80° C.; 50 to 75° C.; 55 to 75° C.; or 60 to 70° C. and the predetermined time is in a range 10 to 90 minutes; 20 to 60 minutes; 30 to 50 minutes; or 35 to 55 minutes.
Optionally, the step of detecting the LAMP products comprises monitoring and capturing a UV or colorimetric signal from the LAMP products emitted from the reaction chambers. Optionally the signal may be fluorescence, colorimetric or UV based. Optionally, the captured images are UV-torch luminated signals. Optionally, the signal emitted by the sample is a fluorescent signal.
Optionally, the step of capturing the UV signal comprises recording the fluorescent UV signal as a photographic image. Optionally, the method comprises analysing the at least one photographic image using software to obtain an average UV intensity of the LAMP products emitted from the respective reaction chambers. Optionally, the step of detecting the LAMP products comprises using one of the discrete reaction chambers as an internal positive control containing a predetermined genomic nucleic acid as a template and using one of the discrete reaction chambers as an internal negative control containing a predetermined genomic nucleic acid as a template. Optionally, the method comprises normalising the average signal intensity of the LAMP products using an average intensity of the positive control and the negative control respectively.
According to a second aspect of the present invention there is provided a multilayer device for detecting a pathogen present in a water sample comprising: a sample preparation part having at least one layer including a solid phase extraction structure mounted therein to receive a nucleic acid solution derived from the water sample; a fluid flow part comprising a plurality of layers each having paper-based fluid flow channels therein to enable fluid capillary flow from the solid phase extraction structure through the plurality of layers; and a reaction layer comprising a plurality of discrete reaction chambers each provided in fluid communication with the fluid flow channels to receive by capillary flow a fluid from the fluid flow channels.
Optionally, the device comprises a filter membrane positioned in a fluid flow direction upstream of the solid phase extraction structure to enable a pre-filtering of the water sample and a nucleic acid lysising of the pathogen to form the nucleic acid solution.
Optionally, the plurality of layers of the fluid flow part comprises a plurality of primary layers each of the primary layers divided into a plurality of secondary layers. Optionally, the primary layers and the secondary layers are integrally formed and coupled to one another by folded or hinge regions positioned at respective edges of the primary and secondary layers.
Optionally, the fluid flow channels within each primary layer are divided respectively at the folded or hinge regions that divide respectively the primary layers into the secondary layers.
Optionally, the sample preparation part further comprises a layer having a sample introduction port and a layer having a waste collection component. Optionally, the reaction layer comprises a plastic material having a plurality of holes and paper inserts positioned within the holes to define the discrete reaction chambers.
Optionally, the device comprises at least one set of LAMP primers for introduction to the discrete reaction chambers.
Optionally, the device comprises a lateral flow device having a plurality of lateral flow detection strips in fluid communication with the discrete reaction chambers respectively.
Optionally, the device comprises a camera to capture an image of the discrete reaction chambers and software to analyse the image captured by the camera. Optionally, the software is configured to analyse the images captured by the camera to determine an average intensity (fluorescence or UV) generated by the LAMP products derived from the LAMP primers.
The present pPADs may be fabricated by various methods, such as photolithography, inkjet printing, polydimethylsiloxane (PDMS) plotting, wax printing, wax dipping, wax screen printing and plasma treatment.
According to a further aspect of the present invention there is provided a use of the method and/or device as claimed herein to detect a pathogen in a water sample. Optionally, the pathogen is a microbe, an infectious disease, a bacteria or a virus. Optionally, the infectious disease is a coronavirus and optionally COVID-19.
A specific implementation of the present invention will now be described, by way of example only, and with reference to the accompanying drawings in which:
Referring to
In particular, sample preparation part 10 comprises a multilayer construction having a first layer 13, second layer 14 and third layer 15. The fluid flow part 11 comprises four primary layers 17, 18, 19 and 20 each divided into two secondary layers 17a, 17b; 18a, 18b; 19a, 19b; 20a, 20b, respectively. An intermediate layer 16 provides a bridging layer between the sample preparation part 10 and the fluid flow part 11. Each of the layers 13 to 20 are formed as a folded origami paper-based device with the layers 13 to 20 forming a single paper unit having multiple folds 26 that separate the paper unit into the individual layers 13 to 20. Primary layers 17 to 20 are divided by respective primary folds 24 and are each, in turn, subdivided into the secondary layers 17a to 20b by secondary folds 25. Each of the paper layers 13 to 20 (alternatively termed panels, plates or sheets), that form the present μPAD are constructed from a paper strip having dark regions that are created by printing the paper with hydrophobic wax leaving non-waxed paper regions that represent fluid flow channels. In particular, the fluid flow part 11 comprises fluid flow channels 24 present within at least some, most or all of the layers 17 to 20 that a conduit to direct sample flow along the layers and from layer to layer from the initial sample preparation part 10 to a final reaction layer 12.
First layer 13 comprises a glass fibre disc 21 mounted within the wax printed paper construction. The second layer 14 comprises a sample introduction port 22. Third layer 15 comprises a waste zone 23 defined as a region of blotting paper surrounded by the dark region (hydrophobic wax).
Reaction layer 12 is formed as a plastic plate and comprises a plurality of holes 27 to 31 each accommodating a paper insert (or spot). These holes/paper inserts define respective reaction chambers provided in fluid communication with the fluid flow channels 24 of part 11. Accordingly, a fluid sample introduced into port 22 is configured to flow onto the solid phase extraction structure (defined by glass fibre disc 21) and into the fluid flow channels 24. Within the fluid flow part 11, the fluid flows under capillary action and the flow path is divided or split at each region of the secondary folds 25. The flow is eventually feds into the respective reaction chambers 27 to 31.
As illustrated in
Referring to
To assess the analytical sensitivity and suitability of the present paper-based device, three target bacteria were investigated: Salmonella; E. coli and C. perfringens.
Referring to
Specifically, a lysis buffer was added of at stage 42. At stage 43, the DNA solution was then absorbed and introduced onto the present μPAD 47 at stage 44. This was followed by DNA washing using 25 μL of a washing buffer (70% ethanol) at stage 45 and then DNA elusion (using a DNA elusion buffer) at stage 46. The DNA solution containing the extracted DNA was then allowed to progress through part 11 of the μPAD 47 under the capillary flow (via fluid flow channels 24) where the flow was divided at stage 47 according to the folded configuration 35. The extracted DNA was then delivered to the reaction part 12 at stage 48 and into the respective reaction chambers 27 to 31. A set of loop-mediated outer-thermal amplification (LAMP) primers were then pipetted into the respective reaction chambers 27 to 31 to create the respective LAMP assays and to generate the LAMP products (amplicons). The reaction chambers 27 to 39 where heated at 65° C. on a hot plate to perform the multiplex LAMP reactions.
In one embodiment, fluorescence was used as the detection method. Referring to
Experimental
Materials
Whatman chromatography paper No. 1 (pure cellulose paper) purchased from GE Healthcare Worldwide (UK) was wax printed by a Xerox ColorQube 8580 digital wax printer from Xerox (UK). The Black Cast Acrylic obtained from Stockline Plastics (UK) was processed by a Laser cutter from Laserscript (UK). A Bio-Rad C1000 Thermal Cycler, a horizontal electrophoresis apparatus and a Gel Doc XR+ Imager for PCR assay and LAMP assay were from Bio-Rad Laboratories (UK). The hot plate, the Digital Drybath and the UV LAMP (366 nm) were purchased from Fisher Scientific (UK). The MicroAmp Optical Adhesive Film was from Thermo Scientific (UK), and the punchers were from kai Europe GmbH (Germany). PCR Master Mix and LAMP Master Mix were purchased from Agilent Technologies (UK) and OptiGene (UK), respectively. Evergreen was from Cambridge BioScience (UK), while Calcein, Manganese (II) chloride solution and double distilled H2O (ddH2O) were from SIGMA (UK). A MagaZorb DNA Mini-Prep Kit was from Promega (UK) and a Nucleopore DNA isolation Mini Kit was from Genetix (India). A Qubit 2.0 Fluorometer was from Thermo Fisher Scientific (UK). LAMP primers used in this work were synthesized by Eurofins (Germany). Bacterial strains of Salmonella, E. coli and C. perfringens were isolated and kindly supplied by the Scottish Water staff.
Loop Mediated Isothermal Amplification (LAMP) Assay
The optimization of the LAMP assay was performed on a Bio-Rad C1000 Thermal Cycler. The LAMP amplicons were analyzed on 3% agarose gel in 1×TAE buffer and the related image was recorded by a Gel Doc XR+ Imager (Figure S2). The LAMP primers sets for Salmonella, E. coli, C. perfringens and Brucella are detailed in Table S1. Besides LAMP primers, the 20 μL reaction mixture of LAMP assay also contains 0.4 mM dNTPs, 4.0 mM MgSO4, 1 M betaine, Ibuffer (20×), 25 μM calcein, 500 μM MnCl2, 0.4 U Bst Polymerase, 1 μL ddH2O and 2 μL DNA sample. Brucella genomic DNA was chosen as the target of the internal positive control while ddH2O was used as the template of the internal negative control with the same composition.
Salmonella
Escherichia coli
Clostridium
perfringens
Brucella
To confirm amplification of target sequence in Salmonella, Escherichia coli and Clostridium perfringens, the outer primers (F3 and B3) of each designed LAMP primer set were used for conventional PCR assay. DNA extracted from three organisms were subjected to amplification at a final volume of 20 μl containing 10 μl qPCR Master Mix (Agilent Technologies, UK), 0.2 μM of each primer and 2 μL of template DNA. Amplification cycles consisted of an initial denaturation step at 95° C. for 3 min, followed by 40 cycles of denaturation at 95° C. for 10 s, annealing at 55° C. for 30 s, an extension at 72° C. for 30 s. The PCR amplicons were analysed on 3% agarose gel electrophoresis and visualized in Gel Doc XR+ System (Bio-Rad, USA).
Results
The results of LAMP reactions were read out with a hand-held UV LAMP after incubation in a digital drybath at 65° C. In a LAMP assay, Calcein is used as a colorimetric indicator. Calcein molecules combine with Mn2+ before LAMP reaction, quenching calcein fluorescence. As LAMP reaction proceeds in the presence of target DNA, Mn2+ complexes with newly generated P2O74−, therefore calcein molecules recover green fluorescence. Moreover, calcein molecules will combine with residual Mn2+, enhancing green fluorescence signal. Eventually, the positive result can be determined from the color change of the LAMP reaction solution from yellow to green by the naked eye. Results can also be read out by a hand-held UV LAMP or digitally collected by a mobile-phone camera.
From previous investigations by the inventors, in Real-time PCR assay, a positive reaction is detected by the accumulation of a fluorescent signal. The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold. Therefore, the smaller Ct is, the more efficient the reaction is. In addition, Ct levels are inversely proportional to the amount of target nucleic acid, and a smaller Ct results in a higher DNA yield. It was noted, Ct decreases when the pore size increases (from 3.0 mm to 4.0 mm), given that a larger pore size results in a stronger adhesion ability and thus more DNA is attached to the sample pore and a higher DNA yield leads to a smaller Ct. However, when the pore size becomes too large, the DNA on the sample pore may not elute entirely and remain on the paper, which in turn causes Ct to become large (from 4.0 mm to 5.0 mm). With regard to channel width, a wider channel width caused a lower yield and a larger Ct. When fixing the pore size and the channel width, more DNA is attached to the channel due to a longer channel. As the channel length increases (from 1.0 mm to 2.0 mm), the yield decreases and Ct becomes larger. Because the difference in Cts obtained is quite small, a channel length with a smaller Error Bar and a smaller Ct was selected. On the basis of the results it was concluded that the optimal pore size, channel width and channel length of the paper-based device were 4.0 mm, 1.5 mm and 2.5 mm, respectively.
Within the present system and method, LAMP assay Calcein was used as a colorimetric indicator in the place of Evagreen. The concentration ratio of Calcein to Mn2+ was optimized by LAMP assay on Brucella DNA. Threshold Time is defined as the time corresponding to 10% of the maximum fluorescence intensity, which is a function of target concentration. The Threshold Time of Real-Time LAMP assay is analogous to the cycle threshold (CO of Real-time PCR assay. Time decreases when the concentration ratio increases, indicating a higher DNA yield. Nevertheless, Ratio (defined as the fluorescence intensity ratio between negative control and positive control) increases with an increasing concentration ratio. When the Ratio is 1:15 and 1:10, the two negative controls show light fluorescence, which will affect the results observed by the naked eye. From the results obtained, the inventors concluded that the optimal concentration ratio of Calcein to Mn2+ is 1:20. These optimal parameters were applied to the fabrication of the present μPAD and LAMP experiments.
The present μPAD enables a sample-to-answer assay within less than 1 hour. This was assessed using Salmonella, E. coli and C. perfringens, where different concentrations of organism samples were spiked into tap water (Salmonella at 3.3 fg μL−1-330 ng E. coli at 0.5 CFU-14 CFU; C. perfringens at 0.25 CFU-2.25 CFU). The feasibility of Paper-based LAMP assay was assess as above and established the paper-based device could detect Salmonella genomic DNA, E. coli and C. perfringens as low as 33 fg 1 CFU and 0.5 CFU, respectively. The analytical specificity of the LAMP primers sets for detection of bacteria spiked into tap water was confirmed. LAMP products were detected for the associated targets, while no LAMP products were detected for the other targets.
The results indicate that the limits of detection of the present paper-based device are similar to those obtained in a real-time configuration on a Bio-Rad C1000 Thermal Cycler. LOD is defined as the target concentration that can be reliably detected as a positive signal by Paper-based LAMP assay. The device enabled an LOD of 33 fg μL−1 for Salmonella determination, which is a 10-fold improvement in sensitivity compared with the LOD of 0.5 pg μL−1 in an assay for Salmonella detection using a facile cascade signal-on colorimetric DNAzyme LAMP (dLAMP) sensor that integrates the LAMP technique and the inherent catalytic activity of the DNAzyme for simple target analysis. E. coli has been measured with the device at levels as low as 1 CFU, which is comparable to the LOD of 1 CFU based on a platform for E. coli detection by combining carbon nanotube (CNT) multilayer biosensors and the microfluidic chip-based LAMP technique. The limit of detection for C. perfringens was identified as 0.5 CFU. The present μPAD demonstrated a detection limit 20 times lower than that in a previous study for detection of C. perfringens in food with an LOD of 10 CFU mL−1. Therefore, the present μPAD system and method has great potential for rapid detection of microbial/pathogen contamination in water or sewerage systems and networks especially in the resource-limited regions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2013248.6 | Aug 2020 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2021/052136 | 8/18/2021 | WO |
