Patent Application
20240131508
The present disclosure relates to enhancements in diagnostic assays for detection of analytes. The improved assays are suitable for management of infections and pandemics in humans and animal reservoirs, including but not limited to SARS-CoV-2 (CoV2), and for measurement of biomarkers of disease (both human and veterinary).
Numerous CoV2 diagnostic tests have been described, with the great majority comprising three classes: host immune response assays, also called serological or antibody tests; direct detection of viral proteins, also called antigen tests; and viral nucleic acid assays, also called molecular tests. While each class has advantages, molecular tests have been shown to provide the most reliable data on a subject's current viral load, which is important for prevalence assessment, for contact tracing, and for evaluation of efficacy of prophylactic or therapeutic interventions.
Molecular tests are typically based on PCR (polymerase chain reaction) and/or LAMP (loop-mediated isothermal amplification). The most widely used assay is based on PCR. LAMP was first described two decades ago, as recently reviewed {Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63; and U.S. Pat. Nos. 7,374,913; 7,851,186; 7,846,695 assigned to Eiken Chemical Co., Ltd}; {Wong Y P, Othman S, Lau Y L, Radu S, Chee HL Y. Loop-mediated isothermal amplification (LAMP): avers: the technique for detection of micro-organisms. J Appl Microbiol, 2018; 124(3):626-6431.
LAMP assays to detect CoV2, the causative agent of COVID-19, have been published by multiple investigators, including: {Ganguli A, Mostafa A, Berger J, Aydin M Y, Sun F, Ramirez S A S, Valera E, Cunningham B T, King W P, Bashir R. Rapid isothermal amplification and portable detection system for SARS-CoV-2. Proc Natl Acad Sci USA. 2020 Sep. 15; 117(37):22727-22735}, {Lalli M A, Langmade S J, Chen X, Fronick C C, Sawyer C S, Burcea L C, Wilkinson M N, Fulton R S, Heinz M, Buchser W J, Head R D, Mitra R D, Milbrandt J. Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric reverse-transcription loop-mediated isothermal amplification. Clin Chem. 2020 Oct. 24:hvaa267}, {Lee J Y H, Best N, McAuley J, Porter J L, Seemann T, Schultz M B, Sait M, Orlando N, Mercoulia K, Ballard S A, Druce J, Tran T, Catton M G, Pryor M J, Cui H L, Luttick A, McDonald S, Greenhalgh A, Kwong J C, Sherry N L, Graham M, Hoang T, Herisse M, Pidot S J, Williamson D A, Howden B P, Monk I R, Stinear T P. Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA. J Med Microbiol. 2020 September; 69(9):1169-1178}; {Huang X, Tang G, Ismail N, Wang X. Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva. EBioMedicine. 2022 January; 75:103736}; Heithoff D M, Barnes L 5th, Mahan S P, Fox G N, Arn K E, Ettinger S J, Bishop A M, Fitzgibbons L N, Fried J C, Low D A, Samuel C E, Mahan M J. Assessment of a Smartphone-Based Loop-Mediated Isothermal Amplification Assay for Detection of SARS-CoV-2 and Influenza Viruses. JAMA Netw Open. 2022 Jan. 4; 5(1):e2145669}.
The LAMP process begins with strand invasion by the forward outer and inner primers (F3, FIP) hybridizing to the target. A strand displacing DNA polymerase extends the primer and separates the target DNA duplex. The backward outer and inner primers (B3, BIP) then hybridize to the newly formed strand, enabling a further round of strand displacement replication. The reverse complementary sequences in the FIP and BIP lead to formation of self-hybridizing loops (dumbbell structure) which then becomes a seed for exponential amplification, with multiple sites for initiation of synthesis: from the 3′ ends of the open loops, from annealing sites for the inner primers, and from additional forward or backward loop primers (LF, FB). As amplification proceeds from these multiple sites, the products form long concatemers, each with more sites for initiation. The result is an exponential accumulation of double-stranded DNA (
While other assays may be useful, point of care (POC) assays for CoV2 have particular utility as has been recently reviewed {Choi J R (2020) Development of Point-of-Care Biosensors for COVID-19. Front. Chem. 8:517}. A recent study concluded that time to results for CoV2, which is indicative of COVID-19 status, was significantly shorter in the POC group than in the control group (p<0.0001). In a related publication, the same investigators noted that POC diagnosis of influenza led to substantially shorter median time to antiviral administration: 1.0 hour vs 6.0 hours in the control group (p<0.004) {Brendish N J, Poole S, Naidu V V, Mansbridge C T, Norton N J, Wheeler H, Presland L, Kidd S, Cortes N J, Borca F, Phan H, Babbage G, Visseaux B, Ewings S, Clark T W. Clinical impact of molecular POC testing for suspected COVID-19 in hospital (COV-19POC): a prospective, interventional, non-randomised, controlled study. Lancet Respir Med. 2020 December; 8(12):1192-1200}; {Clark T W, Beard K R, Brendish N J, Malachira A K, Mills S, Chan C, Poole S, Ewings S, Cortes N, Nyimbili E, Presland L. Clinical impact of a routine, molecular, point-of-care, test-and-treat strategy for influenza in adults admitted to hospital (FluPOC): a multicentre, open-label, randomised controlled trial. Lancet Respir Med. 2020 Dec. 4:S2213-2600(20)30469-0}.
In one embodiment, a microfluidic device includes a housing, a substrate, and a controller. The substrate is disposed within the housing and includes a sample well and a reaction well. The sample well has a sample port extending through the housing and is adapted to receive a sample and extract at least one analyte from the sample into a liquid assay sample. The reaction well is coupled to the sample well via a first microfluidic channel and a second microfluidic channel. The first microfluidic channel is coupled to the sample well at a proximal end, and the second microfluidic channel is coupled to the reaction well at a distal end. A distal end of the first microfluidic channel and a proximal end of the second microfluidic channel are isolated from each other via a fluid channel seal. The controller is adapted to connect the first and second microfluidic channels by breaking the fluid channel seal and to meter the liquid assay sample from the sample well into the reaction well for an assay.
In another embodiment, a method of performing an assay is provided. The method includes extracting an analyte from a sample received in a sample well via an extraction mixture, which may be in liquid form or dried form that is rehydrated by the sample. The first microfluidic channel is connected to a second microfluidic channel via a controller. The first microfluidic channel is coupled to the sample well at a proximal end, and the second microfluidic channel is coupled to a reaction well at a distal end. The controller breaks a fluid channel seal disposed between a distal end of the first microfluidic channel and a proximal end of the second microfluidic channel to connect the distal end of the first microfluidic channel and the proximal end of the second microfluidic channel. The method also meters the liquid assay sample from the sample well into the reaction well via the controller and assays the liquid assay sample in the reaction well.
The foregoing and other aspects, features, details, utilities, and advantages of the present invention will be apparent from reading the following description and claims, and from reviewing the accompanying drawings.
Diagnostic tests for detection of an analyte(s) are provided herein. In one embodiment, the analyte(s) are nucleic acids. Enhancements in nucleic acid amplification are also described herein, for example applicable to LAMP. The assays are suitable for management of infections and pandemics in humans and animal reservoirs, including but not limited to SARS-CoV-2 (CoV2), and for measurement of markers of disease (both human and veterinary). In one embodiment, the diagnostic tests of the present invention can improve speed and accuracy and reduce reliance on skilled operators, which enables decentralized testing.
Biological samples or specimens to be assayed may be obtained by nasal swabs, tongue or cheek swabs, saliva, respiratory condensate, or other biological sources, such as urine or blood. Saliva is a common source for CoV2 detection: {Wyllie A L, Fournier J, Casanovas-Massana A, Campbell M, Tokuyama M, Vijayakumar P, Warren J L, Geng B, Muenker M C, Moore A J, Vogels C B F, Petrone M E, Ott I M, Lu P, Venkataraman A, Lu-Culligan A, Klein J, Earnest R, Simonov M, Datta R, Handoko R, Naushad N, Sewanan L R, Valdez J, White E B, Lapidus S, Kalinich C C, Jiang X, Kim D J, Kudo E, Linehan M, Mao T, Moriyama M, Oh J E, Park A, Silva J, Song E, Takahashi T, Taura M, Weizman O E, Wong P, Yang Y, Bermejo S, Odio C D, Omer S B, Dela Cruz C S, Farhadian S, Martinello R A, Iwasaki A, Grubaugh N D, Ko A I. Saliva or Nasopharyngeal Swab Specimens for Detection of SARS-CoV-2. N Engl J Med. 2020 Sep. 24; 383(13):1283-1286}; {Sakanashi D, Asai N, Nakamura A, Miyazaki N, Kawamoto Y, Ohno T, Yamada A, Koita I, Suematsu H, Hagihara M, Shiota A, Kurumiya A, Sakata M, Kato S, Muramatsu Y, Koizumi Y, Kishino T, Ohashi W, Yamagishi Y, Mikamo H. Comparative evaluation of nasopharyngeal swab and saliva specimens for the molecular detection of SARS-CoV-2 RNA in Japanese patients with COVID-19. J Infect Chemother. 2021 January; 27(1):126-129}; {Rao M, Rashid F A, Sabri FSAH, Jamil N N, Zain R, Hashim R, Amran F, Kok H T, Samad M A A, Ahmad N. Comparing nasopharyngeal swab and early morning saliva for the identification of SARS-CoV-2. Clin Infect Dis. 2020 Aug. 6:ciaa1156}.
In some embodiments, the diagnostic assays comprise an extraction mixture for extracting the analyte (e.g., nucleic acid or protein) from the sample or specimen. In one embodiment, for example, an extraction mixture can include denaturants or lytic agents, such as non-ionic detergents, that act to liberate the viral nucleic acid as well as divalent cation chelators, such as EDTA, that suppress degradation of the liberated nucleic acid by nucleases. Addition of sample to the extraction mixture not only provides the genetic substrate for amplification (in the case of PCR or LAMP based assays) but also may be used to inactivate the virus. In some embodiments, the extraction mixture can be contained in a sample well that receives the sample for analysis.
In some embodiments, the extraction mixture can include broad specificity protease such as Proteinase K, glycosidase and lipase enzymes to reduce sample matrix interference and to assist in lytic extraction of the nucleic acid from the virus. For certain assays, such as those using colorimetric detection based on acidification of the medium as amplification proceeds (e.g., colorimetric LAMP), it may be beneficial to control buffering capacity. In one embodiment, control of buffering capacity can be accomplished according to the methods described in U.S. Pat. No. 9,580,748 {Detection of an Amplification Reaction Product using pH-sensitive Dyes}, which is incorporated by reference.
In another embodiment, concentrated assay reagents can be lyophilized to improve stability, which is useful for allowing storage and shipping without refrigeration. The lyophilized reagents can readily dissolve when in contact with an aqueous solution. A desiccant can be provided in an assay packaging to prevent reagent deterioration from moisture absorption. In one embodiment, lyophilized beads of 0.5-10 mm diameter may be used to provide the enzymes and other reagents needed for the amplification assay (the reaction mixture) as well as the analyte-specific DNA primers. In some embodiments, the assay reagents are contained within a reaction well. Similarly, the agents used to lyse the virus in a liquid sample can also be provided in dried form.
Lyophilized beads can further include a fluorescent or colorimetric indicator for quantifying an analyte, for example amplified DNA. An embodiment of the beads includes sufficient divalent cations, such as Mg′, to offset chelators arising from the sample dissolved in the extraction mixture. In embodiments using a LAMP based assay, the components of the LAMP reaction mixture are known in the art and available commercially, such as the WarmStart Master Mix from New England Biolabs (Ipswich, MA), which includes Bst DNA polymerase. For tests based on detecting RNA, reverse transcriptase is also included. Primers specific for the analytes to be detected are provided [FIP/BIP 1.0 μM, F3/B3 0.2 μM, and LF/LB 0.4 μM]. Optionally, RNase H, an enzyme that cleaves RNA in an RNA/DNA substrate, may be included for assays based on reverse transcription of RNA into DNA for further LAMP amplification, as removal of the RNA facilitates the DNA polymerase activity.
In another embodiment, concurrently run assays enable differential diagnosis of infections, such as a differential diagnosis of infection by CoV2 versus seasonal influenza which has utility since the two infections have similar initial clinical symptoms but are treated differently. Utility has been established by the CDC (US Centers for Disease Control) which has issued an EUA (emergency use authorization) for a PCR assay that provides such a differential diagnosis {CDC catalog #Flu SC2-EUA Influenza SARS-CoV-2 Multiplex Assay, LB-122 Rev 01, 21 Sep. 2020}. Specific primers are disclosed for a LAMP based embodiment.
Lyophilized beads for LAMP assays are useful in several embodiments, including 96-well microplate assays. In an embodiment, the diagnostic assay is a point of care (POC) test performed on a microfluidic device. In one embodiment, the microfluidic device is portable. In another embodiment, the microfluidic device is a compact bench top device or handheld device that is easily carried.
In various embodiments, the microfluidic device 10 includes a housing that differs depending on an application of the device. In a single use, disposable embodiment, for example, the housing may provide a structural enclosure surrounding a mounting substrate, a controller adapted to initiate and/or control an assay by performing one or more functions on components of the mounting substrate, and a printed circuit board (PCB) as described herein. In a reusable, portable embodiment, the housing may be adapted to accept cartridge(s) and provide components such as the controller and PCB for initiating and/or controlling an assay. Similarly, a benchtop instrument or other instrument typically fixed in a single location may be adapted to accept cartridges(s) and provide components such as a controller and PCB that assume some of the functionalities described for corresponding components described herein.
In one embodiment, the cover 12 includes at least one controller element 22 (e.g., a manual and/or electronic controller such as the two dials or knobs in the embodiment shown in
In one embodiment, a sample carrier 40 can be inserted directly into the microfluidic device 10 through a sample port as shown in
In one embodiment, the sample well 28 contains a volume (e.g., 1.5 mL) of an extraction mixture. The volume of the extraction mixture, for example, may represent a portion of the fluid volume of the sample well 28 (e.g., −50% of the well's capacity). After inserting the sample carrier 40, fluid displacement may occur raising the fluid level to a greater portion of the sample well volume (e.g., −75%). In some embodiments, the sample well 28 may include one or more fluid containment elements. For example, in one embodiment, the top of the sample well's opening can be smaller (e.g., −10% smaller) than the diameter of the sample well, which inhibits ejection of fluid from the well during mechanical mixing [see, e.g.,
In one embodiment, the substrate may contain at least one reaction well(s) 32 and a microfluidic channel(s) 30 allowing the reaction well(s) 32 and sample well 28 to be connected. In another embodiment, the microfluidic channel(s) 30 connects the sample well 28 to a series of reaction wells 32 arranged in sequential order as determined by the particular assay. In the embodiment shown in
In some embodiments, the pistons 42, 58 in steps (a) and (b) above travel in opposite directions. In one embodiment, the controller(s) 22 (e.g., dial or knob) contains a ramp that contacts the pistons as the controller is turned. The controller 22 may include ratcheting teeth that mate with the inside surface of the cover to restrict turning to one direction (e.g., clockwise). In another embodiment, a hard stop can be provided to prevent over-filling the reaction well. As shown in
In one embodiment, each piston forms a hermetic seal with a corresponding barrel. A flared bell bottom of the piston and a slightly raised collar at the top of the barrel can be provided [see, e.g.,
In one embodiment, the pair of pistons is duplicated (totaling 4 pistons) to provide a parallel path for a control or an independent assay. The same or a different controller may be used to control both pairs of pistons together or independently. As a further option, the quartet of pistons can be replicated to enable two additional assays, controlled by a second knob [see, e.g.,
In one embodiment, the reaction well 28 contains one or more lyophilized beads. The beads, in some embodiments, may have a diameter in the range of between about 0.5 to 10 mm. In another embodiment, a single bead has a diameter of about 0.5 mm. Each bead can contain reagents required for the assay in general (the reaction mixture) as well as for the specific assay being performed. The volume and concentrations used in the reaction well may be established using small test tubes, such as in a 96-well microplate. In one embodiment, for example, the reaction well volume is 25 microliters. Lyophilized beads may contain analyte specific reaction agents, including for example, analyte specific DNA primers. In other words, in this embodiment, all other reagents required for a reaction are fixed and not analyte specific. This feature facilitates reprogramming of the assay allowing a prompt response to emerging or mutating pathogens. In some embodiments, reaction agents may be validated prior to use. For example, in nucleic acid-based assays, probes can be validated, such as by using a 96-well microplate format assay, before manufacturing the new point of care assay.
In one embodiment, an assay readout can be accomplished using either colorimetric or fluorescent dyes. Illumination can be by an LED (light emitting diode) of an appropriate wavelength. Optionally, filters may also be used to enhance discrimination between the illumination and the assay signal. For visual readout, the reaction well can optionally be covered at a predetermined angle, for example, from 5-60 degrees by a magnification lens. In other embodiments, the predetermined angle is 45 degrees, the lens can be plano-convex, and the reaction well magnification can be 2-3× [see, e.g.,
In one embodiment, activation of the electrical switch turns on a microprocessor that provides several useful functions. For example, reaction well LEDs can be turned on in this manner. Also, power to reaction well heating elements (e.g., heating pad(s) under each reaction well) can be provided intermittently under the control of the microprocessor, based on a temperature-sensing thermistor or other temperature-sensing device, in order to maintain reaction well temperature in a predetermined range (e.g., a range of 60° to 70° Centigrade, such as an example range of 63-67° C. or 64-66° C.). Another microprocessor-controlled function turns on an additional LED at a preset time to indicate that the results are ready to be read [see, e.g.,
In one embodiment, the heating pad is a square piece of copper (e.g., 3×3 mm and 0.5 mm high). The copper pad can be heated, such as by a set of small resistors. The height of the copper pads can provide an air gap from the bulk of the device reducing heating other than at the reaction well. An air insulator around the reaction well further reduces the power consumption [see, e.g.,
In yet another embodiment, diagnostic assays can be formatted to enable both serological and molecular assays in a single device. A competitive immunoassay format is suitable for this purpose: a DNA-labelled detector antibody (or aptamer, or other antibody-like binding moiety) of moderate affinity for a viral antigen is precluded from binding to immobilized analyte (or analyte mimic) by presence of higher affinity or more abundant antibodies in the sample; for example, the immobilized analyte may be on beads present in the sample well that are too large to enter the microfluidic channel. The DNA labelled probe is detected via a LAMP amplification assay. In this manner, the same assay format can be used for both serological and molecular assays. Similarly, direct detection of a viral antigen can be accomplished by preparing a DNA-labelled version of the antigen and capturing it on agarose beads using an antibody. If the viral antigen is present in the patient sample, it will displace the labelled tracer from the bead-bound antibody allowing it to enter the microfluidics pathway. The resulting positive signal in the reaction well indicates presence of the antigen in the patient sample.
LAMP (loop-mediated isothermal amplification) assay: The LAMP assay shown schematically in
In one example, reactions are carried out in 25 μL volumes containing: WarmStart RT-LAMP master primer mix (New England Biolabs; Ipswich, MA); molecular grade water (Sigma); and template. Reactions are mixed and incubated at 65° C. for 30 min, e.g. in a thermocycler held at a constant temperature, such as the AriaMx Real-time PCR System from Agilent (Santa Clara, CA). These conditions mimic the conditions in a point-of-care assay reaction well.
Endpoint readout may be fluorescent for which an assay readout can use SYBR green which becomes fluorescent when intercalated between stacked DNA bases, making the signal proportional to the amount of DNA present {Hu Y, Wan Z, Mu Y, Zhou Y, Liu J, Lan K, Zhang C. A quite sensitive fluorescent loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus. J Infect Developing Countries. 2019 Dec. 31; 13(12):1135-1141; Le Thi N, Ikuyo T, Nguyen Gia B, Truong Thai P, Vu Thi T V, Bui Minh V, Dao Xuan C, Le Trung D, Phan Thu P, Do Duy C, Pham The T, Do V T, Pham Thi P T, Ngo Quy C, Dang Quoc T, Jin T, Shohei S, Takato O, Noriko N, Tsutomu K. A clinic-based direct real-time fluorescent reverse transcription loop-mediated isothermal amplification assay for influenza virus. J Virol Methods. 2020 March; 277:113801}. Optionally, for assay validation the LAMP products can be confirmed to be of correct size by running the amplified DNA on a 1-1.5% agarose gel in 5 mM lithium metaborate buffer at 3.5V/cm and visualized by ethidium bromide staining compared to a ladder of sizing standards.
Malachite Green is an intercalating dye which does not require UV excitation. This dye is compatible with LAMP assays {Nzelu C O, Gomez E A, Caceres A G, Sakurai T, Martini-Robles L, Uezato H, Mimori T, Katakura K, Hashiguchi Y, Kato H. Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection. Acta Trop. 2014 April; 132:1-6}.
An alternative to intercalating dyes and fluorescents is provided by use of fluorescein amidites (FAM). In this approach, a labelled loop probe is quenched in its unbound state but fluoresces when bound to its target. This approach reduces non-specific signals arising from unintentional duplex DNA formation when using a large mixture of primers, or degenerate primers, to encompass a family of targets {Gadkar V J, Goldfarb D M, Gantt S, Tilley P A G. Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes. Sci Rep. 2018 Apr. 3; 8(1):5548}. Conversely, the fluor can be quenched upon binding {Le Thi N, Ikuyo T, Nguyen Gia B, Truong Thai P, Vu Thi T V, Bui Minh V, Dao Xuan C, Le Trung D, Phan Thu P, Do Duy C, Pham The T, Do V T, Pham Thi P T, Ngo Quy C, Dang Quoc T, Jin T, Shohei S, Takato O, Noriko N, Tsutomu K. A clinic-based direct real-time fluorescent reverse transcription loop-mediated isothermal amplification assay for influenza virus. J Virol Methods. 2020 March; 277:113801}.
Colorimetric probes can also be used in LAMP assays, specifically a dye that changes color as the reaction mixture pH drops due to liberation of pyrophosphate and hydrogen ions during DNA polymerization {Tanner N A, Zhang Y, Evans T C Jr. Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. Biotechniques. 2015 Feb. 1; 58(2):59-68}. In one embodiment, cresol red can be used.
A FET (field effect transistor) chip can also be used for pH change detection in LAMP {See, e.g., Salm E, Zhong Y, Reddy B Jr, Duarte-Guevara C, Swaminathan V, Liu Y S, Bashir R. Electrical detection of nucleic acid amplification using an on-chip quasi-reference electrode and a PVC REFET. Anal Chem. 2014 Jul. 15; 86(14):6968-75}.
An alternative colorimetric readout relies on detection of free Mg++ which decreases after forming a complex with pyrophosphate as nucleotides become incorporated into DNA. Hydroxynaphthol blue and calcein have both been used for this purpose {Suebsing R, Kampeera J, Tookdee B, Withyachumnarnkul B, Turner W, Kiatpathomchai W. Evaluation of colorimetric loop-mediated isothermal amplification assay for visual detection of Streptococcus agalactiae and Streptococcus iniae in tilapia. Lett Appl Microbiol. 2013 October; 57(4):317-24}. Alternatively, the turbidity arising from precipitation of magnesium-pyrophosphate can be measured.
Extraction Mixture. Swabs are exposed to extraction mixture which can be formulated to include buffers and denaturants or lytic agents, such as non-ionic detergents, that act to liberate the viral nucleic acid (in some embodiments) from the viral capsid as well as divalent cation chelators, such as EDTA, to suppress nucleases in the sample. Broad specificity protease, glycosidase and lipase enzymes may also be included to reduce sample matrix interference; if such enzymes are temperature sensitive, inactivation is achieved when temperature is raised to 65° C. either in the reaction well or in transit from the sample well to the reaction well. Chaotropic agents, such as guanidine hydrocholoride may also be included. The insensitivity of the LAMP enzymes to interfering substances facilitates use of saliva as the sample, rather than the more invasive nasal swab. The extraction mixture may also serve to inactivate live virus. Regarding sample collection for CoV2 assays, the US Centers for Disease Control (CDC) has issued recommendations {See, e.g., https://www.cdc.gov/urdo/downloads/SpecCollectionGuidelines.pdf}.
Dried assay reagents: In one embodiment, the assay reagent mixture, dissolved in an aqueous solution of excipients to provide reagent stability as well as mechanical strength to the beads, includes the primers and 4× concentrated WarmStart assay reagents for a fluorescence assay and 5× concentrated WarmStart assay reagents for a colorimetric assay, and in some embodiments may optionally include positive controls. The liquid is delivered through a nozzle under positive pressure to create droplets of approximately 2.7 to 3.1 mm diameter (approximately 10 to 15 microliters, respectively) that fall into a liquid nitrogen receiving vessel. The frozen beads are distributed on a flat surface at low enough density that >90% of the beads are at least one 1 mm apart from any other bead. The liquid nitrogen is allowed to evaporate, after which the frozen droplets are lyophilized (freeze-dried) in place. Using these methods, −200,000 beads can be readily produced daily. The resulting beads are mechanically sturdy enough to be collected, including filtering through a meshwork to eliminate aggregates of two or more beads. Dispensing of the dried spherical beads into reaction wells can be accomplished using a vacuum tweezer or similar instrument.
Multiple analyte assay. All SARS-CoV-2 genomes in the virus pathogen database (viprbrc.org) were aligned using Clustal W in BioEdit to identify highly conserved regions >15 nucleotides in the alignment. Nucleic acid targets that are widely used to detect SARS-CoV-2 include SARS-CoV-2 nucleocapsid (N) gene (N gene) and the open reading frame of lab (ORFlab) either of which could be used. The conserved regions in the viral N gene are useful for selecting RT-LAMP primers using primerexplorer.jp/e ver 5.0. These RT-LAMP primers permit direct comparison with PCR data. See Table 1 for suitable CoV2 sequences. Primers for influenza have been previously described: {Le Thi N, Ikuyo T, Nguyen Gia B, Truong Thai P, Vu Thi T V, Bui Minh V, Dao Xuan C, Le Trung D, Phan Thu P, Do Duy C, Pham The T, Do V T, Pham Thi P T, Ngo Quy C, Dang Quoc T, Jin T, Shohei S, Takato O, Noriko N, Tsutomu K. A clinic-based direct real-time fluorescent reverse transcription loop-mediated isothermal amplification assay for influenza virus. J Virol Methods. 2020 March; 277:113801}.
The following examples are offered to illustrate but not to limit the invention.
Limit of Detection (LoD) testing was performed with genomic RNA from the N gene of SARS-CoV-2. The RNA was serially diluted in extraction mixture (0.2% Triton X-100, 1.0 mM EDTA, 50 mM guanidine HCl in sterile water) and tested in triplicate. The LoD was determined as the lowest concentration with ≥75% detection. For a fluorescent assay using SYBR green as the readout, the LoD was 25 viral genome copies per sample (25 μL). For a colorimetric assay using cresol red as the readout, the LoD was 100 viral genome copies per sample (25 μL).
Sensitivity was equivalent for SARS-CoV-2 B.1.1.7 variant (20I/501Y.V1) and wild-type SARS-CoV-2 (USA-WA1/2020). RNAs of coronaviruses from three other human coronaviruses, which are not associated with mortality, were used as negative controls at 500 copies per sample to test the specificity of the assay: OC43 (GenBank: AY585228.1), 229E (GenBank: AF304460.1), and NL63 (GenBank: AY567487.2). None of these samples scored as positive in the assay.
Samples containing live SARS-CoV-2 virus, from the Georgia COVID-19 Task Force, were assayed by RT-LAMP and results compared to PCR. The cutoff for PCR true positives, as recommended by the CDC, was set at Ct<30. As shown in Table 3, concordance was 97%; for Ct>30, concordance was 65%. For PCR true negatives (no signal), concordance was 98%; For 9 samples with Ct>40 (false positives), there was no concordance. In short, RT-LAMP is a more reliable assay than PCR. That is, PCR requires data analysis to distinguish true positives from false positives.
In a variation of the RT-LAMP Assay Validation example, improved signal to noise and sensitivity is provided by using fluorescein amidites (FAM) with a fluorescence quencher. In this approach, a labelled loop probe is quenched in its unbound state but fluoresces when bound to its target. Suitable primers and probes for CoV2 are provided in Table 4 [numbering according to CoV2 Wuhan-Hu-1 (GenBank: MN908947.3)]. See
To test this assay further, 50 μL of saliva (from a pre-pandemic archive) was pipetted into the well of a 96-well PCR plate with Proteinase K (10 U/mL) and adjusted to pH 7.5 with 30 mM Tris-HCl, then spiked with WA1-CoV2 cell culture supernatant. After incubation for 15 minutes at 50° C., the Proteinase K was inactivated by heating to 95° C. for 5 minutes; 10 μL of the material was transferred to the FQ-LAMP (15 μL) reaction (25 μL total volume), and LAMP analysis performed. The assay was conducted in 6 replicates, with two replicates of negative control (no added virus). The results are shown in
990 ± 75 (±7.7%)
A point of care device has been constructed that embodies the innovative elements noted above. The microfluidic device of the present invention may comprise a mounting substrate in which various microfluidic elements are disposed. The mounting substrate includes an exterior portion or surface, as well as an interior portion which defines the various microscale channels and/or chambers of the overall microfluidic device. For example, the mounting substrate of exemplary microfluidic devices typically employs a solid or semi-solid substrate that may be planar in structure, i.e., substantially flat or having at least one flat surface. Suitable substrates may be fabricated from any one of a variety of materials, or combinations of materials. Often, the planar substrates are manufactured using solid substrates common in the fields of microfabrication, e.g., silica-based substrates, such as glass, quartz, silicon, or polysilicon, as well as other known substrates, i.e., gallium arsenide. In the case of these substrates, common microfabrication techniques, such as photolithographic techniques, wet chemical etching, micromachining, i.e., drilling, milling and the like, may be readily applied in the fabrication of microfluidic devices and substrates. Alternatively, polymeric substrate materials may be used to fabricate the devices of the present invention, including, e.g., polydimethylsiloxanes (PDMS), polymethylmethacrylate (PMMA), polyurethane, polyvinylchloride (PVC), polystyrene, polysulfone, polycarbonate and the like. In the case of such polymeric materials, injection molding or embossing methods may be used to form the substrates having the channel and reservoir geometries as described herein. In such cases, original molds may be fabricated using any of the above-described materials and methods. The channels and chambers of an exemplary device are typically fabricated into one surface of a planar substrate, as grooves, wells or depressions in that surface. A second planar substrate, typically prepared from the same or similar material, is overlaid and bound to the first, thereby defining and sealing the channels and/or chambers of the device. Together, the upper surface of the first substrate, and the lower mated surface of the upper substrate, define the interior portion of the device, i.e., defining the channels and chambers of the device. In some embodiments, the upper layer may be reversibly bound to the lower layer. In an embodiment, a substrate material is clarified polypropylene. Polypropylene is heat sealable, provides a natural vapor barrier, and being clarified has good optical properties. Other Olefins (Polypropylene or Polyethylene) could also be used. The laminated films (foils) can be heat sealable or adhesive adhered. In one embodiment, the foils provide vapor barrier properties to reduce or minimize evaporation of the liquid reagents, and to protect the dry reagents from stability damaging humidity and oxygen. In one embodiment, the foil comprises 0.001″ or thick pore free aluminum foil. This can be laminated with a heat or adhesive seal layer on one side, and a non-electrically conductive layer on the other on a side facing the PCB.
To detect serum antibodies to an antigen (e.g. CoV2 Spike protein S1) from a saliva sample, the extraction mixture is augmented with reagents for a competitive binding immunoassay. A peptide derived from S1 is attached to agarose beads that are too large to enter the microfluidics pathway. A monoclonal antibody with moderate affinity for the peptide (Kd in the 0.1-10 μM range) is conjugated to a label comprising a DNA or RNA sequence suitable for LAMP amplification. If the patient sample contains antibodies with higher affinity for the peptide (tighter binding), then the labelled detector antibody will be displaced and then be able to enter the microfluidics pathway. The resulting positive signal in the reaction well (with suitable primers in the dried reagent bead) indicates presence of a host immune response to S1.
Using LAMP or RT-LAMP to amplify the DNA or RNA label provides a sensitive assay. Since the same LAMP reagents are used to amplify the label for a serological assay and the gene for a molecular assay, parallel detection of both protein and nucleic acid biomarkers of a pathogen can be accomplished. One useful application of such a dual detection assay is measuring escape rate of a virus under selection due to widespread vaccination or natural incidence. That is, escape from detection of the viral genome is qualitatively more difficult than escape from antibody binding to the viral antigen.
This LAMP-LISA assay format is quite different from a previously described “PCR-ELISA” in which the amplified PCR product incorporates a modified base (e.g. digoxigenin conjugated) which is then detected using an enzyme conjugated antibody to the modified base thereby increasing the sensitivity of the PCR assay to allow fewer amplification cycles and thus faster results {Sue M J, Yeap S K, Omar A R, Tan S W. Application of PCR-ELISA in molecular diagnosis. Biomed Res Int. 2014; 2014:653014}.
To detect a biomarker of the host immune response, an antibody against the biomarker is immobilized on the agarose beads in the extraction mixture. A DNA-labelled version of the biomarker protein is added to the extraction mixture, which is captured on the bead by the antibody. If the biomarker is present in the patient sample, it will displace the labelled tracer from the bead-bound antibody allowing it to enter the microfluidics pathway. The resulting positive signal in the reaction well indicates presence of the biomarker in the patient sample.
CoV2, influenza and RSV infections are each accompanied by host responses that contribute to the pathology. Variability in the host response may account for a substantial portion of the variability in disease severity. Measuring biomarkers of host response in conjunction with measuring viral presence allows improved assessment of prognosis. Precedent for measuring a key host response effector, interferon-gamma, in saliva has been reported in the context of biomarkers for host vs graft disease following organ transplant {Resende R G, Correia-Silva J D, Silva T A, Xavier S G, Bittencourt H, Gomez R S, Abreu M H. Saliva and blood interferon gamma levels and IFNG genotypes in acute graft-versus-host disease. Oral Dis. 2012 November; 18(8):816-22}. Similarly, a panel of inflammatory markers (including IL-6, IL-17a, and TNF-alpha) has been assayed in saliva to characterize patients with Sjögren's syndrome, an auto-immune condition {Hung Y H, Lee Y H, Chen P P, Lin Y Z, Lin C H, Yen J H. Role of Salivary Immune Parameters in Patients With Primary Sjögren's Syndrome. Ann Lab Med. 2019 January; 39(1):76-80}.
A biomarker of particular interest for concurrent assay of pathogen and host response is Class I interferons whose dysregulation has been implicated in the pathology of both RSV and CoV2 {Stephens L M, Varga S M. Function and Modulation of Type I Interferons during Respiratory Syncytial Virus Infection. Vaccines (Basel). 2020 Apr. 10; 8(2):177-193}; {Xia H, Shi P-Y. Antagonism of Type I Interferon by Severe Acute Respiratory Syndrome Coronavirus 2. Journal of Interferon & Cytokine Research 2020 December; 40(12):543-548}.
Although implementations have been described above with a certain degree of particularity, those skilled in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention. All directional references (e.g., upper, lower, upward, downward, left, right, leftward, rightward, top, bottom, above, below, vertical, horizontal, clockwise, and counterclockwise) are only used for identification purposes to aid the reader's understanding of the present invention, and do not create limitations, particularly as to the position, orientation, or use of the invention. Joinder references (e.g., attached, coupled, connected, and the like) are to be construed broadly and may include intermediate members between a connection of elements and relative movement between elements. As such, joinder references do not necessarily infer that two elements are directly connected and in fixed relation to each other. It is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative only and not limiting. Changes in detail or structure may be made without departing from the spirit of the invention as defined in the appended claims.
This application is a continuation of, and claims priority benefit to, International Patent Application PCT/US2022/026522, filed Apr. 27, 2022, which claims priority benefit to U.S. provisional application 63/180,391, filed Apr. 27, 2021. The contents of each of these applications are expressly incorporated by reference herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63180391 | Apr 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2022/026522 | Apr 2022 | US |
| Child | 18383777 | US |
