WO2024191684A1 - Detection of hepatitis c virus ribonucleic acid from whole blood using reverse transcription loop-mediated isothermal amplification - Google Patents

Abstract

Rapid, sensitive, and low-cost methods for the detection of hepatitis C virus (HCV) RNA in biological samples using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions and rationally designed primers are described. The RT-LAMP primers can amplify nucleic acid from all HCV genotypes. The methods, primers and kits of the disclosure are suitable for point-of-care diagnostics. Efficient methods for isolation of HCV RNA from whole blood are also described.

Description

DETECTION OF HEPATITIS C VIRUS RIBONUCLEIC ACID FROM WHOLE BLOOD USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/489,519, filed March 10, 2023, which is herein incorporated by reference in its entirety.

FIELD

This disclosure concerns oligonucleotide primers, kits, and methods for detecting hepatitis C virus (HCV) RNA in biological samples using reverse transcription loop-mediated isothermal amplification (RT- LAMP). This disclosure further concerns optimized methods for the isolation of HCV RNA from whole blood samples for use in RT-LAMP assays.

INCORPORATION OF ELECTRONIC SEQUENCE LISTING

The electronic sequence listing, submitted herewith as an XML file named 4239- 109635-02. xml (6,113 bytes), created on February 23, 2024, is herein incorporated by reference in its entirety.

BACKGROUND

Hepatitis C virus (HCV) is a major global health problem with an estimated 58 million infections in 2019 (World Health Organization, Global progress report on HIV, viral hepatitis and sexually transmitted infections, 2021, Accountability for the global health sector strategies 2016-2021: actions for impact, Web Annex 2, data methods). Many infections become chronic, potentially lasting for the life of the patient (Westbrook and Dusheiko, J Hepatol 61:S58-S68, 2014). Chronic infection with HCV is a major cause of hepatocellular carcinoma, cirrhosis, and other liver complications leading to an estimated 290,000 deaths globally each year (World Health Organization, 2021). Fortunately, highly effective direct acting antivirals (DAAs) with high cure rates are available for people with HCV infections (Pawlotsky et al., J Hepatol 73:1170-1218, 2020; Yin et al., J Manag Care Spec Pharm 25:195-210, 2019). However, most HCV infections go undiagnosed due to the lack of clinical symptoms or for lack of access to simple and affordable diagnostic testing. It is estimated that only 21% and 61% of people with a chronic HCV infection have been diagnosed, worldwide and in the United States, respectively (World Health Organization, 2021; Ryerson et al., MMWR Morb Mortal Wkly Rep 69:399-404, 2020). Additionally, when testing is available, there are a high proportion of infected persons who are lost to follow up and never successfully linked to care and treatment (Cooke et al., Lancet Gastroenterol Hepatol 4: 135-184, 2019; Konerman and Lok, Clin Transl Gastroenterol 7:el93, 2016). This may be due in part to the slow turnaround time of diagnostic testing for HCV infection which often needs to be performed in a laboratory capable of performing high-complexity testing. The World Health Organization (WHO) has established goals for reducing the global burden of viral hepatitis by the year 2030 (WHO, Global health sector strategy on viral hepatitis 2016-2021, WHO, 2016). If these goals are to be met, diagnostic testing for HCV infection will need to become simpler so it can be performed outside of high-complexity laboratories; faster so an accurate diagnosis can be made in a single health care visit; and less expensive so more people have the resources to access testing.

The standard testing algorithm for the diagnosis of current HCV infections starts with testing for anti-HCV antibodies followed by reflex testing of anti-HCV positive samples for HCV RNA (Centers for Disease Control and Prevention (CDC), MMWR Morb Mortal Wkly Rep 62:362-365, 2013). This two-tiered approach is used to economize testing and administer expensive HCV RNA testing only to patients who are likely to be infected. However, there are problems with this testing approach. People with acute or recent infections who have yet to seroconvert for anti-HCV antibodies will be missed by this testing algorithm. Additionally, this approach is incompatible with receiving a diagnosis during a single health care visit and, if reflex testing is not automatically requested, it could require a second visit for an additional blood draw for HCV RNA testing after a positive anti-HCV antibody result. These problems lead to incomplete testing or losing patients to follow up for HCV RNA testing and subsequent linkage of infected individuals to care and treatment. Failure to expeditiously diagnose infected individuals and inform them of their infection status will make achievement of HCV elimination goals more difficult. Direct testing for HCV RNA or HCV core antigen, which can also be used to diagnose current HCV infection, with an inexpensive and rapid assay could overcome these challenges.

Direct testing for HCV RNA or HCV core antigen should occur at or near the point of care while the patient waits. Existing tests with attributes that make them attractive for diagnosing current HCV infection at or near the point of care include the Cepheid GeneXpert HCV VL fingerstick assay and the Genedrive HCV ID kit (Tang et al., Diagnostics 12: 1255, 2022). Both of these tests are Conformite Europeenne (CE) marked and WHO prequalified for the detection of HCV RNA (Lamoury et al., J Infect Dis 217:1889-1896, 2018; Llibre et al., Gut 67:2017-2024, 2018). However, neither of these tests are currently approved or available in the United States. Moreover, there are no commercially available tests for the detection of HCV core antigen that are compatible with point-of-care use. Thus, a need exists for simple, low-cost, rapid, and sensitive methods for detection of HCV RNA.

SUMMARY

The present disclosure describes rapid and sensitive methods for the detection of HCV RNA in biological samples using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions and rationally designed primers for amplification of all major HCV genotypes. The disclosed methods, primers and kits are low-cost and suitable for point-of-care diagnostics.

Provided herein are methods of detecting HCV RNA in a biological sample. In some aspects, the method includes subjecting the biological sample to a RT-LAMP reaction using a set of primers specific for HCV nucleic acid to produce an HCV nucleic acid amplification product, and detecting the HCV nucleic acid amplification product, thereby detecting HCV RNA in the biological sample. In some examples, the set of primers includes six primers each respectively having a sequence at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. The HCV nucleic acid amplification product can be detected by, for example, fluorescence or a lateral flow assay.

Also provided herein are kits for detecting HCV RNA in a biological sample. In some aspects, the kit includes a set of oligonucleotide primers, wherein the set of primers includes six primers each respectively having a sequence at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. One or more of oligonucleotide primers can optionally include a detectable label, such as a fluorophore or biotin. In some examples, the kit further includes buffer, nuclease-free water, magnesium, betaine, ethanol, paramagnetic beads, a magnet (such as a magnetic wand or magnetic tube rack), reverse transcriptase, dNTPs, DNA polymerase, nucleic acid stain, lateral flow test strips, Eppendorf tubes, pipets, sample collection materials (such as a syringe, EDTA tube, needle, and/or lancet), or any combination thereof. In some examples, the magnet is a neodymium magnet. In some aspects, the kit further includes a mini-centrifuge, heat block, or both.

Further provided are isolated oligonucleotides having a sequence at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some aspects, the isolated oligonucleotide includes a detectable label, such as but not limited to, a fluorophore or biotin.

Methods of isolating HCV RNA from a whole blood sample are also provided. In some aspects, the method includes subjecting the whole blood sample to centrifugation to remove red blood cells from the sample; mixing the red cell-free sample with a lysis buffer and paramagnetic beads; applying a magnet (such as a neodymium magnet) to the mixture to isolate the beads; washing the isolated beads with an ethanol solution; resuspending the washed beads in water to elute nucleic acid bound to the beads; and removing the beads from the eluate, thereby isolating HCV RNA from the whole blood sample. In some examples, the ethanol solution is 70% ethanol.

The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C: Exemplary workflows for extraction of HCV RNA from whole blood samples. Three workflows were optimized and evaluated for preparing HCV RNA from whole blood samples for amplification by RT-LAMP. (FIG. 1A) In the water workflow, in some examples, whole blood is diluted four-fold in water. After five minutes at room temperature, the diluted sample is added to the RT-LAMP mix at 10% of the final volume. (FIG. IB) In the heat workflow, in some examples, whole blood is spun in a mini-centrifuge for 20 seconds. The red-cell free portion is diluted four-fold in 66% QuickExtract DNA extraction solution and then heated at 98°C for five minutes. The sample is then added to the RT-LAMP mix at 10% of the final volume. (FIG. 1C) In the bead workflow, in some examples, whole blood is spun in a mini-centrifuge for 20 seconds. Twenty microliters of the red-cell free portion are mixed with 170 pL of buffer AVL/SPRI bead solution. After five minutes, the sample tube is placed on a magnetic rack and the liquid portion is removed and discarded. Without removing the tube from the magnetic rack, the beads are washed once with 195 pL of 70% ethanol. The beads are resuspended in 20 pL of water and placed back on the magnetic rack. The bead-free elution is added to the RT-LAMP mix at 40% of the final reaction volume.

FIGS. 2A-2C: Time of RT-LAMP reactions with HCV RNA-positive whole blood samples. Eighty plasma samples positive for HCV RNA were diluted ten-fold in EDTA whole blood and tested using the water (FIG. 2A), heat (FIG. 2B), or bead (FIG. 2C) workflow with RT-LAMP. The time from the start of the RT-LAMP reaction at which an increase in SYTO9 fluorescence was detected is indicated on the y-axis. Samples that did not have an increase in fluorescence by 50 minutes (dashed line) were classified as target not detected and assigned a time of 52 minutes for inclusion on these graphs. Samples are color-coded by HCV genotype/subtype.

FIGS. 3A-3C: Lateral flow visual detection of low-level HCV RNA from whole blood. Three replicate whole blood samples containing the lowest HCV RNA level detected 10/10 times in the limit of detection analysis were evaluated for use with lateral flow dipsticks using each of the three workflows with RT-LAMP. These HCV RNA levels were 4.7 logio(IUZmL) for the water workflow (FIG. 3A), 4.2 logio(IU/mL) for the heat workflow (FIG. 3B), and 2.8 logio(IU/mL) for the bead workflow (FIG. 3C). The bi-thermal RT-LAMP reactions were run for 40 minutes for the water and heat workflows and 30 minutes for the bead workflow. These times were selected because they were the shortest that allowed for reliable detection of low-titer samples. Lateral flow was stopped 5 minutes after the insertion of the dipstick into the sample tube. Due to the nature of the lateral flow detection, the intensity of the control line is inversely proportional to the intensity of the test line. neg.= HCV RNA-negative plasma diluted in whole blood.

FIGS. 4A-4C: Bead workflow with magnetic wand processing. (FIG. 4A) A magnetic wand for purifying HCV RNA using paramagnetic SPRI beads was created using a 0.2 mL PCR tube, a 2 mL pipette tip, and a spherical magnet. (FIG. 4B) The magnetic wand was used to remove the beads from the lysed sample solution, dip the beads into the wash solution, transfer the beads to the elution solution, and remove the beads from the nucleic acid eluate. (FIG. 4C) Twenty HCV RNA-positive plasma samples were diluted ten-fold in EDTA whole blood and tested using the bead workflow with a magnetic wand replacing the magnetic rack and the pipetting steps for the wash and elution. The time from the start of the RT-LAMP reaction at which an increase in SYTO9 fluorescence was detected is indicated on the y-axis. Samples that did not have an increase in fluorescence by 50 minutes (dashed line) were classified as target not detected and assigned a time of 52 minutes for inclusion on these graphs. Samples are color-coded by HCV genotype.

FIG. 5: Probit analysis to determine RT-LAMP limit of detection. The proportion of replicates detected at each whole blood HCV RNA level was transformed to a probit value and plotted against the base- 10 logarithm of the HCV RNA. The best-fit linear regression equations (indicated on the graph) were used to calculate the limit of detection (95% detection rate, probit - 6.64) for the detection of HCV RNA from whole blood using each workflow. The calculated limits of detection were 4.4 logio(IUZmL) for the water workflow, 4.3 logio(IU/mL) for the heat workflow, and 2.9 logio(IU/mL) for the bead workflow.

FIG. 6: RT-LAMP products from different HCV genotypes. Plasma containing HCV genotype/subtypes la, lb, 2a, 2b, 3a, 4a, 5a, or 6 were diluted to 3.5 logio(IU/mL) in EDTA whole blood. Whole blood samples were processed using the bead workflow and tested by RT-LAMP. After 1 hour of bithermal amplification (55°C for 10 minutes followed by 65°C for 50 minutes) the reaction products were examined by gel electrophoresis, neg. = HCV RNA-negative plasma diluted in whole blood, MW = 1 Kb Plus DNA ladder (Invitrogen).

FIGS. 7A-7H: Bead workflow with a magnetic wand. (FIG. 7 A) Magnetic wand. (FIG. 7B) A 2 mL tube containing 20 pL of red-cell-free whole blood mixed with 170 pL of lysis/bead solution, a 1.5 mL tube containing 200 pL of 70% ethanol (wash solution) and a 1.5 mL tube containing 20 pL of nuclease-free water are prepared. (FIG. 7C) The magnetic wand is placed into the lysed sample. (FIG. 7D) The magnetic wand is left for one minute to attract the beads. (FIG. 7E) The wand is briefly dipped into the wash solution five times. (FIG. 7F) The wand is placed into the tube containing 20 pL of nuclease-free water. Another magnet or a metallic object is used to elevate the spherical magnet within the wand. The wand is gently twirled to resuspend the beads in the water. (FIG. 7G) The magnet is lowered to the bottom of the wand and kept in place for one minute to attract the beads. (FIG. 7H) The wand is removed, and a pipette is used to transfer the bead-free nucleic acid eluate to an RT-LAMP reaction.

SEQUENCES

The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NOs: 1-6 are oligonucleotide primer sequences.

DETAILED DESCRIPTION

I. Introduction

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a rapid method for nucleic acid amplification from an RNA template (Notomi et al., Nucleic Acids Res 28:63e-663, 2000). Several protocols for HCV RNA detection by RT-LAMP have been published during the past decade (Hongjaisee et al., Int J Infect Dis 102:440-445, 2021; Kargar et al., Indian J Virol 23: 18-23, 2012; Wang et al., FEMS Immunol Med Microbiol 63: 144-147, 2011; Witkowska et al., Nat Commun 12:6994, 2021; Yang et al., Arch Virol 156: 1387-1396, 2011; Zhao et al., J Med Virol 89:1048-1054, 2017). While these protocols suggest that RT-LAMP can sensitively detect HCV RNA, their performance using simple methods for the extraction of HCV RNA or use with whole blood samples are often not reported. Studies that have reported RT-LAMP use with simple methods for HCV RNA extraction from plasma show less-than-optimal sensitivity (Hongjaisee et al., Diagnostics 12: 1599, 2022; Nyan et al., hit J Infect Dis 43:30-36, 2016). A recent analysis of HCV infected individuals suggests that 97% have HCV RNA levels above 1,300 lU/mL (3.1 logio(IU/mL)) in their blood (Freiman et al., J Hepatol 71:62-70, 2019). To be an effective diagnostic tool, an HCV RNA test should be sensitive enough to detect HCV RNA at this level and also able to detect all 8 HCV genotypes and the many subtypes that are currently circulating globally (Hedskog et al., Open Forum Infect Dis 6:ofz076, 2019). Described herein is the development of three simple workflows for the extraction of HCV RNA from whole blood and a unique primer set for rapid and specific nucleic acid amplification from HCV RNA by RT-LAMP.

II. Abbreviations

EDTA ethylenediaminetetraacetic acid

HCV hepatitis C virus

LAMP loop-mediated isothermal amplification

RT reverse transcriptase

RT-LAMP reverse transcription loop-mediated isothermal amplification

SPRI solid phase reversible immobilization

WHO World Health Organization

III. Summary of Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

Amplification: Increasing the number of copies of a nucleic acid molecule, such as a gene or fragment of a gene, for example at least a portion of an HCV nucleic acid molecule. The products of an amplification reaction are called amplification products. An example of in vitro amplification is the polymerase chain reaction (PCR), in which a sample (such as a biological sample from a subject) is contacted with a pair of oligonucleotide primers, under conditions that allow for hybridization of the primers to a nucleic acid molecule in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid molecule. Other examples of in vitro amplification techniques include real-time PCR, quantitative real-time PCR (qPCR), reverse transcription PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), loop-mediated isothermal amplification (LAMP; see Notomi et al., Nucl. Acids Res. 28:e63, 2000); reversetranscription LAMP (RT-LAMP); strand displacement amplification (see U.S. Patent No. 5,744,311); transcription-mediated amplification (U.S. Patent No. 5,399,491) transcription-free isothermal amplification (see U.S. Patent No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see U.S. Patent No. 5,686,272); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889); and NASBA™ RNA transcription-free amplification (see U.S. Patent No. 6,025,134).

Biological sample: A sample obtained from a subject (such as a human or veterinary subject). Biological samples, include, for example, fluid, cell and/or tissue samples. In some aspects herein, the biological sample is a fluid sample. Fluid samples include, but are not limited to, whole blood, serum, plasma, urine, feces, saliva, cerebral spinal fluid (CSF) and bronchoalveolar lavage (BAL) fluid. In one example, the biological sample is whole blood. Biological samples can also refer to cells or tissue samples, such as biopsy samples, tissue sections, or isolated leukocytes.

Biotin: A molecule (also known as vitamin H or vitamin B?) that binds with high affinity to avidin and streptavidin. Biotin is often used to label nucleic acids and proteins for subsequent detection by avidin or streptavidin linked to a detectable label, or for subsequent isolation using avidin/ streptavidin or an antibiotin antibody linked to a solid support (such as a lateral flow assay paper strip). The term “biotin” includes derivatives or analogs that participate in a binding reaction with avidin. Biotin analogs and derivatives include, but are not limited to, N-hydroxysuccinimide-iminobiotin (NHS-iminobiotin), amino or sulfhydryl derivatives of 2-iminobiotin, amidobiotin, desthiobiotin, biotin sulfone, caproylamidobiotin and biocytin, biotinyl-e-aminocaproic acid-N-hydroxysuccinimide ester, sulfo-succinimide-iminobiotin, biotinbromoacetylhydrazide, p-diazobenzoyl biocytin, 3-(N-maleimidopropionyl) biocytin, 6-(6- biotinamidohexanamido)hexanoate and 2-biotinamidoethanethiol. Biotin derivatives are also commercially available, such as DSB-X™ Biotin (Invitrogen). Additional biotin analogs and derivatives are known (see, for example, U.S. Patent No. 5,168,049; U.S. Patent Application Publication Nos. 2004/0024197, 2001/0016343, and 2005/0048012; and PCT Publication No. WO 1995/007466).

Contacting: Placement in direct physical association; includes both in solid and liquid form. “Contacting” is often used interchangeably with “exposed.” For example, contacting can occur in vitro with one or more primers and a biological sample (such as a sample containing nucleic acids) in solution.

Control: A reference standard, for example a positive control or negative control. A positive control is known to provide a positive test result (e.g., known to include HCV nucleic acids). A negative control is known to provide a negative test result (e.g., known to not include HCV nucleic acids). However, the reference standard can be a theoretical or computed result, for example a result obtained in a population.

Detectable label: A compound or composition that is conjugated (e.g., covalently linked) directly or indirectly to another molecule (such as a nucleic acid molecule) to facilitate detection of that molecule. Specific non-limiting examples of labels include fluorescent and fluorogenic moieties (e.g., fluorophores), chromogenic moieties, haptens (such as biotin, digoxigenin, and fluorescein), affinity tags, and radioactive isotopes (such as ³²P, ³³P, ³⁵S, and ¹²³1) . The label can be directly detectable (e.g., optically detectable) or indirectly detectable (for example, via interaction with one or more additional molecules that are in turn detectable). Methods for labeling nucleic acids, and guidance on the choice of labels useful for various purposes, are discussed, e.g., in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Fourth Edition, 2012, and Ausubel et al., Short Protocols in Molecular Biology, Current Protocols, Fifth Edition, 2002.

DNA polymerase: An enzyme that catalyzes the synthesis of DNA molecules from nucleoside triphosphates. Isothermal DNA amplification methods typically employ a DNA polymerase with a high strand displacement activity and/or high processivity. In some aspects herein, the DNA polymerase is Bst 2.0 DNA polymerase (New England Biolabs), Bst 3.0 DNA polymerase (New England Biolabs), Bst 2.0 WARMSTART DNA polymerase (New England Biolabs), Bsm DNA polymerase (Thermo Fisher) or Phi29 DNA polymerase (available from several commercial sources).

Fluorophore: A chemical compound, which when excited by exposure to a particular wavelength of light, emits light (fluoresces), for example at a different wavelength than that to which it was exposed. Also encompassed by the term “fluorophore” are luminescent molecules, which are chemical compounds that do not require exposure to a particular wavelength of light to fluoresce; luminescent compounds naturally fluoresce. Therefore, the use of luminescent signals eliminates the need for an external source of electromagnetic radiation, such as a laser. An example of a luminescent molecule includes, but is not limited to, aequorin (Tsien, Ann Rev Biochem 67:509, 1998).

In some aspects herein, an oligonucleotide (such as a primer) is labeled with (e.g., has attached thereto) a fluorophore, such as at the 5' end and/or the 3' end of the oligonucleotide. Fluorophores suitable for use with nucleic acid amplification methods, such as PCR, LAMP, or RT-LAMP, include, but are not limited to, fluorescein, 6-carboxyfluorescein (FAM), tetrachloro fluorescein (TET), tetr methylrhodamine (TMR), hexachlorofluorescein (HEX), JOE, 6-carboxy-X-rhodamine (ROX), CAL Fluor™, Pulsar™, Quasar™, Texas Red™, Cy™3 and Cy™5.

Other examples of fluorophores that can be used in the methods provided herein are described in U.S. Patent No. 5,866,366. These include: 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)amino-naphthalene- 1-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]-naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l-naphthyl)-maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumaran 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"- dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4- methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethyl-amino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethyl-aminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron® Brilliant Red 3B-A); rhodamine and derivatives such as 6- carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N’- tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives.

Other fluorophores that can be used include thiol-reactive europium chelates that emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999).

Other fluorophores that can be used include cyanine, merocyanine, stryl, and oxonyl compounds, such as those disclosed in U.S. Patent Nos. 5,627,027; 5,486,616; 5,569,587; and 5,569,766. Specific examples of fluorophores disclosed in one or more of these patent documents include Cy™3 and Cy™5, for instance, and substituted versions of these fluorophores.

Other fluorophores that can be used include GFP, Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Patent No. 5,800,996) and derivatives thereof. Numerous fluorophores are commercially available from known sources.

Hepatitis C virus (HCV): A small, enveloped, positive-sense single-stranded RNA virus that can cause hepatitis and some forms of cancer, such as hepatocellular carcinoma and certain lymphomas. The HCV RNA genome has a single open reading frame (ORF) that is translated to produce a single polyprotein product. The polyprotein is processed to produce the structural proteins (core protein, El and E2 glycoproteins) and the non- structural proteins (NS1, NS2, NS3, NS4A, NS4B, NS5A, NS5B). There are eight known genotypes of HCV and numerous subtypes, including la, lb, 2a, 2b, 3a, 4a, 5a, and 6.

Isolated: An “isolated” or “purified” biological component (such as a nucleic acid, peptide, protein, protein complex, or virus) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component occurs, that is, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” or “purified” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. The term “isolated” or “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated biological component is one in which the biological component is more enriched than the biological component is in its natural environment within a cell, or other production vessel. Preferably, a preparation is purified such that the biological component represents at least 50%, such as at least 70%, at least 90%, at least 95%, or greater, of the total biological component content of the preparation.

Isothermal amplification: Nucleic acid amplification that is not dependent on significant changes in temperature (in contrast to PCR, for example). Isothermal amplification is performed substantially at about the same single temperature. In some examples, isothermal amplification is substantially isothermal, for example, amplification may include small variations in temperature, such as changes in temperature of no more than 1°C, no more than 2°C, or no more than 3°C during the amplification reaction. In some examples, isothermal amplification is performed at about 55°C, about 60°C, or about 65°C.

Lateral flow assay (LFA): A paper-based assay for the detection and quantification of analytes in a sample. LFA is a simple, rapid, portable and low-cost detection method. The LFA paper strip typically includes an absorbent pad at one end where the sample is added, a conjugate release pad that contains labelled antibodies that bind the target analyte, a test line containing analyte-specific antibodies and a control line that has antibodies specific for a control analyte (such as anti-biotin antibodies or anti-IgG antibodies). To perform an LFA, the sample is applied to the absorbent pad and the sample moves along the paper strip through capillary action. If the analyte of interest is present in the sample, it will bind to the labelled analyte-specific antibodies and the anti-analyte antibodies located at the test strip (see, e.g., Koczula and Estrela, Essays Biochem 60(1): 111-120, 2016; and Ma et al., BMC Infect Dis 19: 108, 2019).

Loop- mediated isothermal amplification (LAMP): A method for amplifying nucleic acid. The method is a single-step amplification reaction utilizing a DNA polymerase with strand displacement activity (e.g., Notomi et al., Nucl. Acids. Res. 28:E63, 2000; Nagamine et al., Mol. Cell. Probes 16:223-229, 2002; Mori et al., J. Biochem. Biophys. Methods 59:145-157, 2004). At least four primers, which are specific for six regions within a target nucleic acid sequence, are typically used for LAMP; however, in some examples, two additional primers may be used for LAMP. The primers include a forward outer primer (F3), a backward outer primer (B3), a forward inner primer (FIP), and a backward inner primer (BIP). A forward loop primer (Loop F or LF), and/or a backward loop primer (Loop B or LB) can also be included in some aspects. The amplification reaction produces a stem-loop DNA with inverted repeats of the target nucleic acid sequence. To amplify RNA sequences using LAMP, reverse transcriptase (RT) is added to the reaction. This variation is referred to as RT-LAMP. In contrast to PCR, LAMP and RT-LAMP can be carried out at a constant temperature and do not require a thermal cycler. In some examples herein, the RT-LAMP assay is bi-thermal, meaning the reaction is carried out at two different temperatures, such as at about 55°C followed by about 65°C.

Neodymium magnet: A type of rare-earth magnet made from an alloy of neodymium, iron and boron. Neodymium magnets are the strongest type of permanent magnet that is commercially available.

Primer: Primers are short nucleic acids, generally DNA oligonucleotides 10 nucleotides or more in length (such as 10-60, 15-50, 20-40, 20-50, 25-50, or 30-60 nucleotides in length). Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs or sets of primers (such as 2, 3, 4, 5, 6, or more primers) can be used for amplification of a target nucleic acid, e.g., by PCR, LAMP, RT-LAMP, or other nucleic acid amplification methods.

Sequence identity/similarity: The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

Subject: Living multi-cellular vertebrate organisms, a category that includes both human and nonhuman animals, such as non-human mammals, such as chimpanzees. In some examples, the subject is a human subject who is or is suspected of having an HCV infection, such as one who previously received a blood transfusion, blood product, or organ donation, or one who is or was an i.v. drug user. IV. Methods and Compositions for Isolation and Detection of HCV RNA

Accurate diagnosis of current HCV infection typically relies on testing plasma or serum collected by phlebotomy for HCV RNA using complex, expensive, and lengthy nucleic acid extraction and amplification procedures. The present disclosure describes and characterizes the performance of three workflows (water, heat and bead) for extracting HCV RNA from small volumes of whole blood that are compatible with a sensitive, specific, and rapid RT-LAMP procedure. The calculated limits of detection for the water, heat, and bead workflows were 4.4, 4.3, and 2.9 logio(IU/mL) of HCV RNA in whole blood, respectively. These limits of detection indicate that the water, heat and bead workflows would respectively be able to detect 88%, 89%, and 98% of global HCV RNA positive cases with at least a 95% probability (Freiman et al., J Hepatol 71:62-70, 2019). Additionally, results from the sensitivity panel showed that the water and heat workflows can detect the majority of the whole blood samples tested with HCV RNA levels between 3.0 and 4.0 logio(IUZmL). The tradeoff between workflow simplicity and limit of detection/sensitivity may make certain HCV RNA extraction workflows more attractive for different settings or patient populations.

Each of the disclosed HCV RNA extraction workflows can be used with blood volumes that are easily obtained using lancet finger pricks (Serafin et al., Postgrad Med 132:288-295, 2020). The studies disclosed herein used 5, 20, and 60 pL volumes of whole blood for the water, heat, and bead workflows, respectively. After sample processing by these workflows, the sample volume equivalents that were added as the template to 50 pL RT-LAMP reactions were 1.25, 1.25, and 20 pL. The 16-fold difference in amplification reaction template volume between the bead workflow and the water and heat workflows likely accounts for most of the difference in the limits of detection.

The water, heat, and bead HCV RNA extraction workflows take approximately 6, 7, and 15 minutes, respectively. The workflow used determined how long the RT-LAMP reaction should be run, as the more complex the extraction procedure is, the faster the amplification proceeds. For maximum sensitivity, the RT- LAMP reaction proceeds for 45-50 minutes when used with the water or heat workflows, but only 30 minutes are needed when used with the bead workflow. When lateral flow dipsticks are used for visual detection of amplification, an additional 5 minutes is generally needed to obtain a result. Each of these workflows with RT-LAMP can be performed in less than 60 minutes from sample to result.

The workflows disclosed herein can be performed using inexpensive reagents and minimal equipment. With real-time fluorescence detection of amplification, each sample can be tested for less than $5.00 (Table 6). Lateral flow visual detection includes an additional cost, but each sample could still be tested for less than $7.00. Costs per sample are similar for the three workflows because the largest portion of the cost comes from the RT-LAMP reagents which are the same for each. The equipment for HCV RNA extraction differs for each workflow. No equipment beyond pipettes is needed for the water workflow. The heat workflow utilizes a mini-centrifuge and a heat block. The bead workflow utilizes a mini-centrifuge and a magnet (such as a magnetic tube rack or magnetic wand). The cost of the bead workflow could be decreased by using alternative and less expensive carboxy lated paramagnetic beads (Joung et al., N Engl J Med 383: 1492-1494, 2020). Heat blocks can be used to incubate the RT-LAMP reactions if an endpoint method such as lateral flow dipstick is used. Fluorescence can also be used as an endpoint method, which would require an instrument that heats the RT-LAMP reaction and measures fluorescence. A real-time PCR thermal cycler for real-time fluorescence measurement was used in the studies disclosed herein, but portable and less expensive equivalents can be used (Aglietti et al., AMB Express 9:50, 2019; Buultjens et al., ACS Biomater Sei Eng 7:4982^4990, 2021).

Compared to the GeneXpert HCV VL fingerstick assay and Genedrive HCV ID kit, which have been CE marked for the rapid detection of HCV RNA, the bead workflow disclosed herein is faster (45 minutes), less expensive (<$5.00 per sample), and uses a smaller sample volume (60 pL of whole blood, spun to collect 20 pL of cell-free sample). The GeneXpert HCV VL fingerstick assay has a lower limit of detection (1.6 logio(IU/mL)) and less hands-on time than the bead workflow, but requires 100 pL of capillary blood from a fmgerstick and takes 60 minutes (Lamoury et al., J Infect Dis 217: 1889-1896, 2018). Insufficient sample volumes can result in invalid results with this test and the cost of the instrument and test cartridges may be prohibitive in certain markets (Agarwal et al., J Med Microbiol 70, 2021; Petroff et al., Viruses 13:2327, 2021). The Genedrive HCV ID kit has an analytical sensitivity of 3.37 logio(IU/mL) and uses 30 pL of plasma (Llibre et al., Gut 67:2017-2024, 2018; Lamoury et al., Diagnostics 11 :746, 2021). Additionally, this test takes 90 minutes to produce results and requires several manual pipetting steps for sample preparation, potentially limiting its utility in certain patient settings.

Simple, rapid, inexpensive, and point-of-care methods for the sensitive detection of HCV RNA from small volumes of whole blood are urgently needed. Tests that meet these criteria could facilitate faster diagnosis and linkage of HCV infected individuals to care and treatment, potentially in a single visit to a health care provider. The three workflows for the preparation of HCV RNA and subsequent detection of HCV RNA by RT-LAMP, as described herein, meet this need.

A. Methods for Detecting HCV RNA by RT-LAMP

Provided herein are methods of detecting HCV RNA in a biological sample. In some aspects, the methods include subjecting the biological sample to a reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction using a set of primers specific for HCV nucleic acid to produce an HCV nucleic acid amplification product, and subsequently detecting the HCV nucleic acid amplification product. In some aspects, the methods are capable of detecting the la, lb, 2a, 2b, 3a, 4a, 5a, and 6 genotypes of HCV. In some aspects, the methods detect at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% of HCV RNA positive samples with at least a 95% probability. In some aspects, the methods have a limit of detection of about 1-5 logio(IU/mL) of HCV RNA in whole blood, such as 3-5 logio(IU/mL), 2.5-4.5 logio(IU/mL), such as about 4.4, 4.3, or 2.9 logio(IU/mL). In some aspects, the method uses whole blood, such as a whole blood sample of less than 200 pL, less than 100 pL, or less than 75 pL, such as 1 to 100 pL, 1 to 75 pL, or 5 to 60 pL, such as 5, 20, or 60 pL of whole blood. In some examples, combinations of these effects are achieved. In some aspects, the set of primers includes six primers each respectively having a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 (HCV-LF), SEQ ID NO: 2 (HCV-F3), SEQ ID NO: 3 (HCV-FIP), SEQ ID NO: 4 (HCV-BIP), SEQ ID NO: 5 (HCV-B3), and SEQ ID NO: 6 (HCV-LB). In some examples, the set of primers includes six primers each respectively including or consisting of the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

In some aspects, the RT-LAMP reaction is performed at a temperature of about 55°C to about 65°C, such as about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C or about 65°C. In the context of the present disclosure “about” with respect to a recited temperature refers to temperatures ranging from 2°C less than to 2°C greater than a specified temperature. Thus, a temperature of “about 55°C” includes 53°C, 54°C, 55°C, 56°C and 57°C. Similarly, a temperature of “about 65°C” includes 63°C, 64°C, 65°C, 66°C and 67°C.

The RT-LAMP reaction can be isothermal or bi-thermal. In some examples in which the reaction is bi-thermal, the RT-LAMP reaction is initiated at a temperature of about 55°C following by a temperature of about 65°C. In specific examples, the RT-LAMP reaction is performed at a temperature of about 55°C for 5 to 15 minutes (such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minutes), followed by a temperature of about 65°C for 15 to 45 minutes (such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 minutes). In particular non-limiting examples, the RT-LAMP reaction is performed at a temperature of about 55°C for 10 minutes, followed by a temperature of about 65°C for 20 to 40 minutes (such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 minutes).

In some aspects, the RT-LAMP reaction is performed with a final concentration of Mg²⁺ of about 0.5 to 10 mM, such as about 2 mM to 6 mM, such as about 2 mM, about 3 mM, about 4 mM, about 5 mM, or about 6 mM.

In some aspects, betaine is added to the RT-LAMP reaction to increase specificity of primer and template interactions. In some examples, the RT-LAMP reaction includes about 0.1 to 1.5 mM betaine, such as 0.6 to IM betaine, such as about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M or about 1 M betaine. In particular examples, the RT-LAMP reaction includes about 0.8 M betaine.

In some aspects, the DNA polymerase included in the RT-LAMP reaction is one with high strand displacement activity. In some examples, the DNA polymerase is a DNA polymerase long fragment (LF) of a thermophilic bacterium such as Bacillus stearothermophilus (Bst), Bacillus Smithii (Bsm), Geobacillus sp. M (GspM) or Thermodesulfatator indicus (Tin), an engineered variant therefrom or a Taq DNA polymerase variant. In some examples, the DNA polymerase is Bst LF DNA polymerase, GspM LF DNA polymerase, GspSSD LF DNA polymerase, Tin exo-LF DNA polymerase or SD DNA polymerase (see, e.g., WO 2016/189490). In specific examples, the DNA polymerase is Bst 2.0 DNA polymerase (New England Biolabs), Bst 3.0 DNA polymerase (New England Biolabs), Bst 2.0 WARMSTART DNA polymerase (New England Biolabs), Bsm DNA polymerase (Thermo Fisher) or Phi29 DNA polymerase (available from several commercial sources). A skilled person is capable of selecting an appropriate DNA polymerase for use with the methods disclosed herein.

The reverse transcriptase (RT) used in the RT-LAMP reaction can be any RT enzyme appropriate for the assay, which can be selected by a skilled person. In some aspects, the RT is Warmstart RTx reverse transcriptase (New England Biolabs, Inc.). In other aspects, the RT is from avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (MMLV).

In some aspects, the HCV nucleic acid amplification product is detected by fluorescence, such as by including a fluorescent nucleic acid stain (e.g., SYTO 9, Thermo Fisher Scientific) in the RT-LAMP reaction and detecting fluorescence in real-time. In other aspects, the HCV nucleic acid amplification product is detected by a lateral flow assay (LFA). In some examples in which LFA is used to detect the amplification product, the loop-recognizing primers used in the RT-LAMP assay are modified by adding biotin to the 5’ end of one primer (such as the LF primer) and fluorescein to the 5’ end of a second primer (such as the LB primer). Alternative labels are known and can be used to render the primer(s) compatible with detection by LFA. Other means for detecting nucleic acid amplified by RT-LAMP are known and can be used in the methods disclosed herein (see, e.g., WO 2018/204175).

In some aspects, the methods further include isolating HCV RNA from the biological sample prior to performing the RT-LAMP assay. In some examples, the biological sample is whole blood. In some aspects, the whole blood sample used is less than 200 U.L, less than 100 U.L, or less than 75 (lL, such as 1 to 100 uL, 1 to 75 LlL, 5 to 60 llL, or 5 to 20 LlL, such as about 5, 20, or 60 pL of whole blood. A variety of methods can be used to isolate RNA from whole blood for the detection of HCV RNA. In some examples herein, RNA is isolated from whole blood using the “water workflow” which includes diluting the whole blood sample in water and incubating the diluted sample, such as at room temperature. In specific examples, the blood to water ratio used in this workflow is about 1:4, 1:6, 1:8, 1 : 12, 1: 16, 1 :20, 1:24, 1:28 or 1:32. In some aspects, the extraction portion of the methods takes no more than 30 minutes, no more than 20 minutes, no more than 10 minutes, such as about 5 to 20 minutes or about 6 to 15 minutes, such as 6, 7, or 15 minutes.

In other examples, RNA is isolated from whole blood using the “heat workflow” which includes heating the whole blood sample, such as at a temperature of about 95°C to about 100°C, for example about 95°C, 96°C, 97°C, 98°C, 99°C or 100°C. The heat workflow can also include treating the heated sample with QuickExtract DNA extraction solution (Lucigen Corporation) prior to heating. When QuickExtract buffer is used, the proportion of the buffer in the whole blood/buffer solution can vary. In some instances, the proportion of QuickExtract buffer is about 25% to about 75%, such as 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some examples in which the heat workflow is used, the whole blood sample is subjected to centrifugation to remove red blood cells prior to heating the sample.

In yet other examples, RNA is isolated from whole blood using the “bead workflow.” In particular examples, this workflow includes mixing the whole blood sample with a lysis buffer and paramagnetic beads; applying a magnet (such as a neodymium magnet) to the mixture to isolate the beads; washing the isolated beads with an ethanol solution; resuspending the washed beads in water to elute nucleic acid bound to the beads; and removing the beads from the eluate. In specific non-limiting examples, the lysis buffer includes guanidinium thiocyanate (such as AVL buffer from Qiagen). The lysis buffer may also include Tris HC1 pH 8.0, polyethylene glycol (PEG) 8000, NaCl, ethylenediaminetetraacetic acid (EDTA) and/or Tween- 20. In some examples, the paramagnetic beads are RNA clean XP beads (Beckman Coulter) or Ampure XP beads (Beckman Coulter). However, other paramagnetic beads are available and can be used with the disclosed methods. In some examples, the ethanol solution is about 40-95% ethanol, such as about 50-90% ethanol, such as about 50%, about 60%, about 70%, about 80%, or about 90% ethanol. In specific examples, the ethanol solution is about 70%. The isolated beads can be washed in the ethanol solution a single time, or multiple times (such as two times or three times). In some examples in which the bead workflow is used, the whole blood sample is subjected to centrifugation to remove red blood cells prior to mixing with the lysis buffer and paramagnetic beads. In alternative aspects, the bead workflow uses non-magnetic carboxylated beads in the isolation method. In these aspects, centrifugation is used for processing.

In some aspects of the disclosed methods, the method further includes detecting the presence or absence of HCV core antigen in the biological sample. For example, HCV core antigen can be detected using an immunoassay (e.g., Western blot, ELISA, radioimmunoassay, flow cytometry, or immunohistochemi stry).

B. Kits for Detecting HCV RNA

Also provided herein are kits for detecting HCV RNA in a biological sample. The kits include a set of oligonucleotide primers capable of amplifying nucleic acid from all major genotypes of HCV by RT- LAMP. In some aspects, the set of primers includes six primers each respectively having a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 (HCV-LF), SEQ ID NO: 2 (HCV-F3), SEQ ID NO: 3 (HCV-FIP), SEQ ID NO: 4 (HCV-BIP), SEQ ID NO: 5 (HCV-B3), and SEQ ID NO: 6 (HCV-LB). In some examples, the set of primers includes six primers each respectively having a sequence including or consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

In some aspects, one or more of the primers includes a detectable label, such as at the 5' and/or 3' end of the primer. In some examples, the detectable label is a fluorophore, radiolabel, hapten (such as biotin), or chromogen. In certain examples, a detectable label is attached e.g., covalently or non-covalently attached) to an oligonucleotide. The attachment may be to any portion of the oligonucleotide, including to a base, sugar, phosphate backbone, or 5' or 3' end of the oligonucleotide. The label may be directly attached to the oligonucleotide or indirectly attached, for example through a linker molecule. In particular examples, an RT-LAMP primer (e.g., one of SEQ ID NOs: 1-6) includes a fluorophore at the 5' or 3' end. In some examples, the fluorophore is HEX, FAM, TET, fluorescein, fluorescein isothiocyanate (FITC), or QFITC (XRITC). A skilled person can select additional suitable fluorophores (see, e.g., The Molecular Probes Handbook, Life Technologies, 11^th Edition, 2010). In specific non-limiting examples, the detectable label includes a fluorophore or biotin.

In some aspects, the kits further include buffer, nuclease-free water, magnesium, betaine, ethanol, paramagnetic beads, a magnet (such as a magnetic wand or magnetic tube rack), reverse transcriptase, dNTPs, DNA polymerase, nucleic acid stain, lateral flow test strips, or any combination thereof. In some aspects, the kits further include plastic microfuge tube(s), pipet(s), sample collection material(s) (such as a syringe, needle, and/or lancet), or any combination thereof. In some examples, the magnet is a neodymium magnet. In some aspects, the kits further include a mini-centrifuge, heat block, or both.

In the disclosed kits, one or more of the oligonucleotide primers (such as one or more of SEQ ID NOs: 1-6) are provided in one or more containers or in one or more individual wells of a multi-well plate or card. Nucleic acid primers may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. The container(s) in which the nucleic acid(s) are supplied can be any conventional container that can hold the supplied form, for instance, microfuge tubes, multi-well plates, ampoules, or bottles. The kits can include either labeled or unlabeled nucleic acid primers.

One or more positive and/or negative control primers and/or nucleic acids also may be supplied in the kit. Exemplary negative controls include non-HCV nucleic acids (such nucleic acids from other viruses). Exemplary positive controls include purified HCV nucleic acid or a vector or plasmid including the HCV target sequence. A skilled person can select suitable positive and negative controls for the assays disclosed herein.

In some examples, one or more primers are provided in pre-measured single use amounts in individual, typically disposable, tubes, wells, plates, cards, or equivalent containers. In some examples, a set of primers (such as each of SEQ ID NOs: 1-6) is included in a single container. In this example, the sample to be tested for the presence of the target nucleic acids can be added to the individual container(s) and amplification and/or detection can be carried out directly. The kit may also include additional reagents for the detection and/or amplification of HCV nucleic acids, such as buffer(s), nucleotides (such as dNTPs), enzymes (such as DNA polymerase and/or reverse transcriptase), or other suitable reagents. The additional reagents may be in separate container(s) from the one or more primers or may be included in the same container as the primer(s).

C. Oligonucleotide Primers

Primers (such as isolated nucleic acid primers) suitable for use in the disclosed methods and kits are also provided herein. In some aspects, the primers are suitable for detection of HCV nucleic acids by RT- LAMP, such as the assay described herein. RT-LAMP primer sets typically include a forward outer primer (F3), a backward outer primer (B3), a forward inner primer (FIP), a backward inner primer (BIP), a forward loop primer (Loop F or LF), and/or a backward loop primer (Loop B or LB). At least some of the RT- LAMP primers are non-naturally occurring nucleic acid molecules, for example, the primers have sequences that do not occur in nature. In particular, the FIP and BIP primers are composed of non-contiguous nucleic acid sequences and include a portion complementary to a first strand of a double-strand nucleic acid and another portion complementary to a second strand of a double-stranded nucleic acid (e.g., reverse complement of a first strand sequence). In addition, one or more of the disclosed primers include degenerate base positions to increase their complementarity to a broader range of HCV genome sequences.

In some aspects, provided herein are isolated oligonucleotides having a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 (HCV-LF), SEQ ID NO: 2 (HCV-F3), SEQ ID NO: 3 (HCV-FIP), SEQ ID NO: 4 (HCV-BIP), SEQ ID NO: 5 (HCV-B3), or SEQ ID NO: 6 (HCV-LB). In some examples, the nucleotide sequence of the oligonucleotides is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In other examples, the nucleotide sequence of the oligonucleotides is at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In yet other examples, the nucleotide sequence of the oligonucleotides is at least 98% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In specific examples, the nucleotide sequence of the oligonucleotide consists of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

In some aspects, one or more of the primers includes a detectable label, such as at the 5' and/or 3' end of the primer. In some examples, the detectable label is a fluorophore, radiolabel, hapten (such as biotin), or chromogen. In certain examples, a detectable label is attached (e.g., covalently or non-covalently attached) to an oligonucleotide. The attachment may be to any portion of the oligonucleotide, including to a base, sugar, phosphate backbone, or 5' or 3' end of the oligonucleotide. The label may be directly attached to the oligonucleotide or indirectly attached, for example through a linker molecule. In particular examples, an RT-LAMP primer (e.g., one of SEQ ID NOs: 1-6) includes a fluorophore at the 5' or 3' end. In some examples, the fluorophore is HEX, FAM, TET, fluorescein, fluorescein isothiocyanate (FITC), or QFITC (XRITC). A skilled person can select additional suitable fluorophores (see, e.g., The Molecular Probes Handbook, Life Technologies, 11^th Edition, 2010). In specific non-limiting examples, the detectable label includes a fluorophore or biotin.

The present application also provides primers that are slightly longer or shorter than the nucleotide sequences shown in any of SEQ ID NOs: 1-6, as long as such deletions or additions permit amplification and/or detection of the desired target nucleic acid molecule. For example, a primer can include a few nucleotide deletions or additions at the 5'- or 3 '-end of the primers shown in any of SEQ ID NOs: 1-6, such as addition or deletion of 1, 2, 3, 4, 5, or 6 nucleotides from the 5'- or 3'-end, or combinations thereof (such as a deletion from one end and an addition to the other end).

Also provided are primers that are degenerate at one or more positions (such as 1, 2, 3, 4, 5, or more positions), for example, a primer that includes a mixture of nucleotides (such as 2, 3, or 4 nucleotides) at a specified position in the primer. In other examples, the primers disclosed herein include one or more synthetic (e.g., non-naturally occurring) bases or alternative bases (such as inosine). In other examples, the primers disclosed herein include one or more modified nucleotides or nucleic acid analogues, such as one or more locked nucleic acids (see, e.g., U.S. Patent No. 6,794,499), an altered sugar moiety, an inter-sugar linkage, a non-naturally occurring nucleotide linkage, a phosphorothioate oligodeoxynucleotide, a peptide nucleic acid (PNA), or one or more superbases (Nanogen, Inc., Bothell, WA).

D. Methods for Isolating HCV RNA from Whole Blood

The present disclosure provides three different workflows for isolating HCV RNA from whole blood, referred to as the “water workflow,” “heat workflow,” and “bead workflow.” All three methods result in isolation of HCV RNA that can be used in RT-LAMP assays for the detection of HCV RNA. The water workflow uses the osmotic stress of diluting whole blood in water to release HCV RNA from viral particles (FIG. 1A). The heat workflow uses a brief heat treatment (such as at about 95°C to about 100°C, e.g., about 98°C) to release HCV RNA from viral particles (FIG. IB). Heating blood or plasma can result in the coagulation of blood proteins making subsequent liquid transfer steps difficult (Curtis et al., J Virol Methods 151 :264-270, 2008). However, the studies disclosed herein demonstrate that removing red blood cells from samples and treatment with QuickExtract DNA extraction solution prior to heating prevents sample solidification and improves downstream RNA detection sensitivity and reproducibility. The bead workflow allows for larger processed sample volumes to be added to the nucleic acid amplification reactions and can process whole blood samples for testing by RT-LAMP in less than 15 minutes (FIG. 1C).

In some aspects, the method for isolating HCV RNA from a whole blood sample includes diluting the whole blood sample in water and incubating the diluted sample, such as at room temperature, for a period of time sufficient to allow for release of HCV RNA from virions. In some examples, the diluted sample is incubated for about 3 minutes to about 7 minutes, such as about 3, about 4, about 5, about 6 or about 7 minutes at room temperature. In specific examples, the blood to water ratio used in this workflow is about 1 :4, 1:6, 1 :8, 1: 12, 1 :16, 1 :20, 1:24, 1:28 or 1:32. In some aspects, the water workflow method for isolating HCV RNA from a whole blood sample uses no more than 10 pL of whole blood, such as 1-10 pL or 3-8 pL, such as about 5 pL of whole blood.

In other aspects, the method for isolating HCV RNA from a whole blood sample includes heating the sample, such as at a temperature of about 95°C to about 100°C, for example about 95°C, 96°C, 97°C, 98°C, 99°C or 100°C, for a period of time sufficient to release HCV RNA from viral particles. In some examples, the sample is heated for about 3 minutes to about 7 minutes, such as about 3, about 4, about 5, about 6 or about 7 minutes. In specific examples, the sample is treated with QuickExtract DNA extraction solution (Lucigen Corporation), or the equivalent, prior to heating. When QuickExtract buffer is used, the proportion of the buffer in the whole blood/buffer solution can vary. In some instances, the proportion of QuickExtract buffer is about 25% to about 75%, such as 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some examples in which the heat workflow is used, the whole blood sample is subjected to centrifugation (such as, e.g., for 10 seconds, 20 seconds, or 30 seconds) to remove red blood cells prior to heating the sample and prior to treatment with QuickExtract DNA extraction solution. In some aspects, the heat workflow method for isolating HCV RNA from a whole blood sample uses no more than 30 pL of whole blood, such as 10-30 pL or 15-25 pL, such as about 20 pL of whole blood. In yet other aspects, the method for isolating HCV RNA from a whole blood sample includes mixing the whole blood sample with a lysis buffer and paramagnetic beads; applying a magnet (such as a neodymium magnet) to the mixture to isolate the beads; washing the isolated beads with an ethanol solution; resuspending the washed beads in water to elute nucleic acid bound to the beads; and removing the beads from the eluate. In some aspects, the bead workflow method for isolating HCV RNA from a whole blood sample uses no more than 100 LlL of whole blood, such as 40-100 LlL or 50-75 LlL, such as about 60 LlL of whole blood. In specific non-limiting examples, the lysis buffer includes guanidinium thiocyanate (such as AVL buffer from Qiagen). The lysis buffer may also include Tris HC1 pH 8.0, polyethylene glycol (PEG) 8000, NaCl, ethylenediaminetetraacetic acid (EDTA) and/or Tween-20. In some examples, the paramagnetic beads are RNA clean XP beads (Beckman Coulter) or Ampure XP beads (Beckman Coulter). However, other paramagnetic beads are available and can be used with the disclosed methods. In some examples, the ethanol solution is about 50%, about 60%, about 70%, about 80%, or about 90% ethanol. In specific examples, the ethanol solution is about 70%. The isolated beads can be washed in the ethanol solution a single time, or multiple times (such as two times or three times). In some examples in which the bead workflow is used, the whole blood sample is subjected to centrifugation to remove red blood cells prior to mixing with the lysis buffer and paramagnetic beads. In alternative aspects, the bead workflow uses nonmagnetic carboxylated beads in the isolation method. In these aspects, centrifugation is used for processing.

EXAMPLES

The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

Example 1: Materials and Methods

This example describes the materials and experimental procedures for the studies described in Examples 2-7.

Samples

The samples used for workflow evaluation were venous whole blood spiked with HCV RNA- positive or HCV RNA-negative plasma at a ratio of less than or equal to 10% of the final volume. Venous whole blood was collected from HCV RNA-negative volunteer donors in EDTA tubes, stored in a refrigerator, and used within two weeks of collection. For the limit of detection, and lateral-flow detection, whole blood was spiked with plasma from the Accuspan HCV RNA Linearity Panel (Seracare, Milford, MA). For the HCV genotype inclusivity experiment, venous whole blood was spiked with plasma from the Accuset HCV Worldwide Performance Panel (Seracare). Eighty HCV RNA-positive and 20 HCV RNA- negative plasma samples from unique anonymous donors were used to spike venous whole blood for the sensitivity and specificity experiments. The HCV RNA levels of the plasma samples used to spike the whole blood were measured using the AmpliPrep/COBAS TaqMan HCV Test (Roche, Indianapolis, IN). The genotypes/subtypes of the HCV RNA-positive samples were determined using either the GenMark Dx eSensor HCVg Direct Test (GenMark Diagnostics, Inc., Carlsbad, CA) or by sequencing the 5 ’-untranslated region of the HCV genome (Tejada-Strop et al., J Virol Methods 212:66-70, 2015).

Workflows for extraction of HCV RNA from whole blood

Water workflow. Whole blood samples were diluted 4-fold in nuclease-free water (ex. 5 pL of whole blood plus 15 pL of nuclease-free water) and incubated for five minutes at room temperature (FIG. 1 A).

Heat workflow. At least 20 pL of whole blood was spun for 20 seconds in a benchtop minicentrifuge (Fisher Scientific, Pittsburgh, PA). Five microliters of the supernatant was added to 15 pL of 66% QuickExtract DNA extraction solution (Lucigen Corp., Middleton, WI) and incubated for five minutes at 98°C in a programmable dry heat block (Fisher Scientific) (FIG. IB).

Bead workflow: At least 60 pL of whole blood was spun for 20 seconds in a benchtop minicentrifuge (FIG. 1C). Twenty microliters of the supernatant was mixed with 170 pL of lysis/bead solution (90 pL buffer AVL (Qiagen, Germantown, MD), 32 pL RNAclean XP or AMPure XP beads (Beckman Coulter, Brea, CA), and 48 pL solid phase reversible immobilization (SPRI) buffer (10 mM tris-HCl pH 8.0, 20% PEG-8000, 2.5 M NaCl, 1 mM EDTA, and 0.05% tween-20)) by pipette. After five minutes at room temperature, the sample was placed on a magnetic tube rack (Life Technologies Corp., Rockville, MD). The liquid portion was removed and discarded according to local waste regulations. The beads were washed once with 195 pL of 70% ethanol without removing the sample tube from the magnetic rack or resuspending the beads. The wash solution was removed and discarded. The sample tube was then removed from the magnetic rack and the beads were resuspended in 20 pL of nuclease-free water. The sample tube was then returned to the magnetic rack to separate the beads from the eluate.

To simplify the bead workflow, a magnetic wand was used to perform the bead capture, wash, and elution steps. The wand was made by cutting the cap off a 0.2 mL PCR tube (Thermo Scientific, Waltham, MA) and inserting it into a 2 mL pipette tip (Mettler Toledo, Columbus, OH) that has had both ends cut off. The large end of the 2 mL pipette tips was cut at the diameter that allowed for the PCR tube to fit snuggly. A nickel plated, one-eighth inch diameter spherical neodymium magnet (K and J Magnetics, Inc., Pipersville, PA) was placed inside of the wand. After sample lysis using the lysis/bead solution in a 2 mL microcentrifuge tube, the wand was inserted for one minute to attract the beads. The wand was then dipped five times into a tube containing 200 pL of 70% ethanol. After the wash, the wand was placed into a 1.5 mL microcentrifuge tube containing 20 pL of nuclease-free water. The spherical magnet was elevated within the wand using a cylindrical magnet (or other metallic object) and the wand was twirled gently to resuspend the beads in the water. The spherical magnet was then lowered to the bottom of the wand. The beads were allowed to attract to the magnet for one minute prior to removing the wand from the elution tube leaving the bead-free eluate. The spherical magnet was retrieved from the wand for reuse and the wand was discarded. Primers

An alignment of the whole genome sequences from 238 HCV isolates representing all genotypes and subtypes was downloaded from the International Committee on Taxonomy of Viruses (ICTV) website (cms.ictv.global/sg wiki/flaviviridae/hepacivirus/hcv files). RT-LAMP primers HCV-LF, HCV-F3, and HCV-FIP were manually designed to target conserved sequences within the 5’ untranslated region of the HCV genome. Primers HCV-BIP, HCV-B3, and HCV-LB were modified from previously published primer sequences (Nyan et al., Int J Infect Dis 43:30-36, 2016). The loop-recognizing primers were modified for lateral flow detection of amplification by adding biotin to the 5’ end of primer HCV-LF and fluorescein to the 5’ end of primer HCV-LB. All primer sequences are listed in Table 1.

RT-LAMP

The RT-LAMP reactions contained lx isothermal amplification buffer (20 mM tris-HCl, 10 mM (NH 2SO4, 50 mM KC1, 2 mM MgSCL, 0.1% tween-20, pH 8.8) (New England Biolabs, Inc., Ipswich, MA), 4 mM MgSOi, 1.4 mM dNTPs (Thermo Scientific), 0.8 M betaine (Thermo Scientific), 0.32 units/pL Bst 2.0 DNA polymerase (New England Biolabs, Inc.), 0.3 units/pL Warmstart RTx reverse transcriptase (New England Biolabs, Inc.), 0.4 pM primers HCV-LF and HCV-LB, 0.2 pM primers HCV-F3 and HCV- B3, and 1 .6 pM primers HCV-FIP and HCV-BIP. Unless specified otherwise, 50 pL RT-LAMP reactions were used for all experiments. The sample volume added to the reaction depended upon the sample processing workflow that was used. For samples processed using the water workflow, the water-lysed sample was added to the RT-LAMP reaction as 10% of the final reaction volume. For samples processed by the heat workflow, the heat-lysed sample was added to the RT-LAMP reaction as 10% of the final reaction volume. For samples processed using the bead workflow, bead-free eluate was added to the RT-LAMP reaction as 40% of the final reaction volume. Reactions included 0.5 pM of SYTO9 (Life Technologies Corp.) in experiments where real-time fluorescence was measured to identify positive amplification. These reactions were run and monitored on a LightCycler 480 II (Roche) using a bi-thermal temperature profile starting with 55°C for 10 minutes followed by 65°C for 40 minutes (50-minute total reaction time), measuring fluorescence once each minute. The time to a positive result was determined by the point at which fluorescence crossed a threshold.

For RT-LAMP reactions used with lateral-flow detection, primers HCV-LF and HCV-LB were replaced with lateral-flow HCV-LF and lateral-flow HCV-LB. These RT-LAMP reactions were run in a programmable dry heat block. Reactions using samples from the water and heat workflows were run at 55°C for 10 minutes followed by 65°C for 30 minutes (40-minute total reaction time), and reactions using samples from the bead workflow at 55°C for 10 minutes followed by 65°C for 20 minutes (30-minute total reaction time). At the end of the reaction time, the RT-LAMP reactions were removed from the dry heat block, 50 pL of HybriDetect assay buffer (Milenia Biotech, Giessen, Germany) was added, and a HybriDetect dipstick (Milenia Biotech) was inserted. After five minutes of lateral flow, the dipsticks were removed from the reaction tubes and imaged. Calculations and statistics

The limit of detection for RT-LAMP with each workflow was determined by probit analysis. The probit-transformed proportion of positive replicates was plotted against the base-10 logarithm of the HCV RNA level and best-fit linear regression equation was determined using R (version 4.2.1) (Clinical and Laboratory Standards Institute, EP17-A2 - Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures, 2nd ed., Clinical and Laboratory Standards Institute, Wayne, PA, 2012). Using this equation, the HCV RNA level at which 95% of replicates would be detected (probit= 6.64) was calculated. All statistical analyses were performed using R.

Example 2: Optimization of HCV RNA extraction workflows and RT-LAMP

Three simple and rapid workflows (water, heat, and bead) were developed and optimized for extracting HCV RNA from whole blood samples in a manner that is compatible with its use as an RT- LAMP amplification template. The simplest workflow, the water workflow, uses the osmotic stress of diluting whole blood in water to release HCV RNA from viral particles (FIG. 1 A). The heat workflow uses a brief treatment at 98°C to release HCV RNA from viral particles (FIG. IB). Typically, heating blood or plasma results in the coagulation of blood proteins making subsequent liquid transfer steps difficult (Curtis et al., J Virol Methods 151 :264-270, 2008). However, the present study demonstrates that removing red blood cells from samples and treatment with QuickExtract DNA extraction solution prior to heating prevents sample solidification and improves downstream RNA detection sensitivity and reproducibility. Spinning whole blood for 20 seconds in a mini-centrifuge is sufficient to collect enough volume that has been cleared of red cells to perform this workflow. Blood and plasma are known to contain molecules that inhibit reverse transcriptases and DNA polymerases. To minimize the presence of these inhibitors in the nucleic acid amplification reactions, the volume of samples processed using the water and heat workflows was limited to 10% of the final RT-LAMP reaction volume. The bead workflow removes the inhibitors and allows for larger processed sample volumes to be added to the nucleic acid amplification reactions (FIG. 1C). The bead workflow uses paramagnetic SPRI beads to bind nucleic acids from a chemically lysed sample followed by a single wash step and an elution step. A single wash was necessary and additional washes did not improve workflow performance. Due to proteins from lysed red blood cells aggregating to the SPRI beads, the bead workflow performs better on samples that have been cleared of red blood cells by a brief spin in a minicentrifuge. The bead workflow can process whole blood samples for testing by RT-LAMP in less than 15 minutes.

A set of RT-LAMP primers that are complementary to a broad range of HCV genotypes and sequences was designed (Table 1). These primers target conserved regions within the 5’ -untranslated region of HCV. Due to variability within these targeted regions, degenerate base positions were included in the primers to increase their complementarity to a broader range of viral genome sequences. The only notable mismatch between these primers and a representative set of HCV genomes are three consecutive nucleotides in the HCV-BIP primer that are not complementary to genotype 3 HCV sequences. These primers allow for faster and more sensitive amplification from an HCV RNA template than other primer sets tested (Table 5). A bi-thermal, rather than an isothermal, temperature procedure was used for the RT-LAMP reaction because starting the reaction at the optimal temperature for reverse transcriptase activity allowed for more sensitive detection of samples with low HCV RNA levels (Table 5) (Suarez et al., Anal Methods 14:378-382, 2022).

Example 3: Limit of detection

Serial 3-fold dilutions of venous EDTA whole blood spiked with HCV RNA-positive plasma were tested using each of the three HCV RNA extraction workflows and the optimized RT-LAMP assay. Using a probit analysis, the 95% limits of detection for HCV RNA from whole blood samples were determined to be 4.4, 4.3, and 2.9 logio(IU/mL) for the water, heat, and bead workflows, respectively (FIG. 5). In addition to slightly more sensitive detection of HCV RNA at low levels, the heat workflow yielded positive amplification detectable by real-time fluorescence measurements faster than the water workflow (Table 2). The bead workflow allowed for the fastest detection of HCV RNA, yielding positive results within 30 minutes from the start of the RT-LAMP reaction for all HCV RNA levels tested.

Example 4: Genotype inclusivity

To test the performance of the RT-LAMP assay for detecting diverse HCV viruses, a panel of eight viruses with genotypes/subtypes la, lb, 2a, 2b, 3a, 4a, 5a, and 6 diluted to 3.5 logio(IU/mL) in venous whole blood were tested using the bead workflow. All three replicates for each genotype and subtype were detected in less than 30 minutes (Table 3). The time from the start of the RT-LAMP reaction until positive amplification detected by real-time fluorescence was generally similar, but with significantly longer times for subtype 3a (21.7 minutes) than subtype la (18.0 minutes) (one-way ANOVA, Tukey correction, p < 0.01) and for subtype 2b (26.4 minutes) than each of the other genotypes/subtypes tested (one-way ANOVA, Tukey correction, p < 0.001 for each comparison). Each of the genotypes/subtypes tested yielded amplification products with identical banding patterns (FIG. 6). These results indicate that the RT-LAMP reaction can amplify HCV RNA from a broad range of HCV genotypes.

Example 5: Sensitivity and specificity

To evaluate the sensitivity and specificity of the three workflows with RT-LAMP, a panel of 80 HCV RNA-positive and 20 HCV RNA-negative plasma samples diluted in HCV RNA-negative venous whole blood were tested. A single test was performed for each sample. The diluted HCV RNA-positive samples included subtypes la, lb, 2b, and 3a and had RNA levels ranging from 2.0 to 7.4 logio(IU/mL) but biased towards samples with less than 5.0 logio(IU/mL) (Table 7). Each workflow was specific with no increase in fluorescence from any of the negative samples at 50 minutes after the start of the RT-LAMP reaction (Table 4). For the positive samples, the proportion of samples detected were 69%, 75%, and 94% for the water, heat, and bead workflows, respectively. The water workflow missed 3 samples (14%) with HCV RNA levels between 4.0 and 5.0 logio(IUZmL), while the heat workflow detected 100% of samples with HCV RNA levels above 4.0 logio(IU/mL) and the bead workflow detected 100% of samples with HCV RNA levels above 3.0 logio(IU/mL). For the water and heat workflows, positive amplification signal was detected between 23 and 46 minutes after the start of the RT-LAMP reaction (FIGS. 2A and B). Faster amplification was observed for the bead workflow with positive amplification detected between 15 and 26 minutes after the start of the RT-LAMP reaction (FIG. 2C). With all three workflows, the time to a positive result was generally longer for HCV subtype 2b and 3a samples than for subtype la and lb samples with similar HCV RNA levels.

Example 6: Visual detection of amplification

An additional study tested whether the three workflows and the RT-LAMP reaction would allow for visual detection by lateral flow dipstick. To allow for this detection method, the loop primers were modified to include biotin and fluorescein (Witkowska et al., Nat Commun 12:6994, 2021). HCV RNA-positive plasma was diluted in venous whole blood to the lowest HCV RNA levels that were detected in 10/10 replicates for each workflow in the limit of detection analysis. Three replicate HCV RNA-positive whole blood samples and one HCV RNA-negative whole blood sample were tested for each workflow. With the water workflow, a positive visual result was observed for all 4.7 logio(IUZmL) HCV RNA replicates after 40-minute RT-LAMP reactions (FIG. 3A). Positive lateral flow results were obtained using the heat workflow with 4.2 logio(IU7mL) whole blood samples after 40-minute RT-LAMP reactions (FIG. 3B). The bead method allowed for a faint but distinct positive visual result from all replicates of 2.8 logio(IUZmL) whole blood after 30-minute RT-LAMP reactions (FIG. 3C). Allowing the RT-LAMP reaction to proceed for longer than 30 minutes increased the intensity of the bands when used with these low titer samples. Importantly, no bands were observed at the test line of the lateral flow dipstick for HCV RNA-negative whole blood samples used with any of the three workflows. These results demonstrate that lateral flow dipsticks are a sensitive alternative to fluorescent detection of RT-LAMP amplification.

Example 7 : Bead workflow with magnetic wand

Magnetic wands have been proposed for simplifying the paramagnetic bead processing steps of SPRI protocols for purifying nucleic acids (Bektas et al., Viruses 13:742, 2021). A simple wand was developed using a nuclease-free PCR tube, a 2 mL pipette tip, and a small spherical magnet (FIG. 4A). The bead workflow was modified by replacing the magnetic rack and the pipetting steps for removal of the lysis solution, washing the beads, and eluting the nucleic acids from the beads with the magnetic wand (FIGS. 7A-7H). Twenty-five venous whole blood samples were tested using this modified bead workflow (Table 7). Out of 20 blood samples with HCV RNA levels below 4.0 logio(IUZmL), 19 were detected within 27 minutes of the start of the RT-LAMP reaction (FIG. 4C). No amplification was observed for any of the five HCV RNA-negative blood samples that were tested. These results indicate that a simple magnetic wand can be used to simplify the bead workflow while maintaining the sensitive detection of low-level HCV RNA from blood samples. TABLES

Tabic 1. Primers used in this study

Table 2. Limit of detection of HCV RNA from whole blood samples

TND= target not detected, NT= not tested. ¹ Average time from the start of the RT-LAMP reaction until the detection of a positive result. Table 3. HCV genotype inclusivity using the bead workflow with whole blood samples

¹ Average time from the start of the RT-LAMP reaction until the detection of a positive result.

Table 4. Sensitivity and specificity of HCV RNA detection from whole blood

Table 5. Performance of optimized primer set with bi-thermal RT-LAMP reaction

Whole blood samples containing 3.0 logio(IU/mL) of HCV RNA were tested using the bead workflow with 25 pL RT-LAMP reactions. ¹ Average time from the start of the RT-LAMP reaction until the detection of a positive result. Table 6. Cost analysis of workflows

Costs are from vendor websites as of 09/30/2022. Per sample costs are for 50 L RT-LAMP reactions with the workflow volumes described in figure 1. Items not including due to a negligible cost per reaction are tips, tubes, nuclease-free water, SPRI buffer (tris HO, EDTA, PEG-8000, NaCl, tween-20), primers, and SYTO9. Additional items not included are pipettes, blood collection devices (e.g., a lancet), a device for heating the RT-LAMP reaction (e.g., heat block), and a device for endpoint or real-time fluorescence measurements. Additional RT-LAMP reaction components such as the isothermal amplification buffer and MgSCL are included with the enzymes. Table 7. Data for sensitivity and specificity of HCV RNA detection from whole blood

¹ HCV genotypes and subtypes were determined using either the GenMark Dx eSensor HCVg Direct Test or sequencing of the 5 ’-untranslated region of the genome. ² HCV RNA levels of the parent plasma samples were determined using the AmpliPrcp/COBAS TaqMan HCV Test. TND = target not detected, NT = not tested It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

1. A method of detecting hepatitis C virus (HCV) RNA in a biological sample, comprising: subjecting the biological sample to a reverse transcription loop-mediated isothermal amplification

(RT-LAMP) reaction using a set of primers specific for HCV nucleic acid to produce an HCV nucleic acid amplification product, wherein the set of primers comprises six primers each respectively having a sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; and detecting the HCV nucleic acid amplification product, thereby detecting HCV RNA in the biological sample.

2. The method of claim 1, wherein the set of primers comprises six primers each respectively comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

3. The method of claim 1 or claim 2, wherein the set of primers consists of six primers each respectively having a sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

4. The method of any one of claims 1-3, wherein the RT-LAMP reaction is performed at a temperature of about 55°C to about 65 °C.

5. The method of any one of claims 1-4, wherein the RT-LAMP reaction is initiated at a temperature of about 55°C following by a temperature of about 65°C.

6. The method of any one of claims 1-5, wherein the RT-LAMP reaction is performed at a temperature of about 55°C for 5 to 15 minutes, followed by a temperature of about 65°C for 15 to 45 minutes.

7. The method of any one of claims 1-6, wherein the RT-LAMP reaction is performed at a temperature of about 55°C for 10 minutes, followed by a temperature of about 65 °C for 20 to 40 minutes.

8. The method of any one of claims 1-7, wherein the HCV nucleic acid amplification product is detected by fluorescence.

9. The method of any one of claims 1-7, wherein the HCV nucleic acid amplification product is detected by a lateral flow assay.

10. The method of any one of claims 1-9, further comprising isolating HCV RNA from the biological sample prior to performing the RT-LAMP assay.

11. The method of claim 10, wherein the biological sample is a whole blood sample.

12. The method of claim 11, wherein isolating HCV RNA from the whole blood sample comprises diluting the sample in water and incubating the diluted sample at room temperature.

13. The method of claim 11, wherein isolating HCV RNA from the whole blood sample comprises heating the sample.

14. The method of claim 13, wherein the sample is heated to about 95°C to about 100°C.

15. The method of claim 11, wherein isolating HCV RNA from the whole blood sample comprises: mixing the sample with a lysis buffer and paramagnetic beads; applying a magnet to the mixture to isolate the beads; washing the isolated beads with an ethanol solution; resuspending the washed beads in water to elute nucleic acid bound to the beads; and removing the beads from the eluate.

16. The method of claim 15, wherein the lysis buffer comprises Tris HC1 pH 8.0, polyethylene glycol (PEG) 8000, NaCl, ethylenediaminetetraacetic acid (EDTA) and/or Tween-20.

17. The method of any one of claims 13-16, further comprising subjecting the whole blood sample to centrifugation to remove red blood cells prior to isolation of the HCV RNA.

18. A kit for detecting hepatitis C virus (HCV) RNA in a biological sample, comprising a set of oligonucleotide primers, wherein the set of primers comprises six primers each respectively having a sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

19. The kit of claim 18, wherein the set of primers comprises six primers each respectively having a sequence comprising or consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

20. The kit of claim 18 or claim 19, wherein one or more of the primers comprises a detectable label.

21. The kit of claim 20, wherein the detectable label comprises a fluorophore or biotin.

22. The kit of any one of claims 18-21, further comprising buffer, nuclease-free water, magnesium, betaine, ethanol, paramagnetic beads, a magnet, reverse transcriptase, dNTPs, DNA polymerase, nucleic acid stain, lateral flow test strips, or any combination thereof.

23. An isolated oligonucleotide, wherein the nucleotide sequence of the oligonucleotide is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

24. The isolated oligonucleotide of claim 23, wherein the nucleotide sequence of the oligonucleotide consists of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

25. The isolated oligonucleotide of claim 23 or claim 24 comprising a detectable label.

26. The isolated oligonucleotide of claim 25, wherein the detectable label comprises a fluorophore or biotin.

27. A method of isolating hepatitis C virus (HCV) RNA from a whole blood sample, comprising: subjecting the whole blood sample to centrifugation to remove red blood cells from the sample; mixing the red cell-free sample with a lysis buffer and paramagnetic beads; applying a magnet to the mixture to isolate the beads; washing the isolated beads with an ethanol solution; resuspending the washed beads in water to elute nucleic acid bound to the beads; and removing the beads from the eluate, thereby isolating HCV RNA from the whole blood sample.

28. The method of claim 27, wherein the ethanol solution is 70% ethanol.

29. The method of any one of claims 1-17, further comprising detecting the presence or absence of HCV core antigen in the biological sample.