MPOX evolved branch II biomarker panel

Abstract

The present disclosure provides a set of primers and optional probes for confirming the presence of MPOX virus evolved branch II, the primers and optional probes may be included with (e.g., in a kit) or in a cartridge for automatic detection of these pathogens by nucleic acid amplification. The disclosure also provides related detection methods, as well as kits, systems, and kits useful in such methods.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 433,373, filed December 16, 2022, which is hereby incorporated by reference in its entirety.

[0003] field

[0004] The present invention generally relates to the field of detecting MPOX clade II viruses.

[0005] background

[0006] Monkeypox (MPXV or MPOX) is a viral zoonosis (a virus transmitted from animals to humans) with symptoms similar to those seen in patients with smallpox in the past, although clinically less severe. With the eradication of smallpox in 1980 and the subsequent cessation of smallpox vaccination, MPOX virus has become the most important orthopoxvirus (OPXV) for public health. MPXV infections occur primarily in Central and West Africa, often near tropical rainforests, and have increasingly emerged in urban areas.

[0007] MPXV, an enveloped, double-stranded DNA virus, belongs to the genus Orthopoxvirus in the family Poxviridae. MPXV has two distinct genetic clades: the Congo Basin clade (clade I) and the West African clade (clades IIa and IIb). Clade I, which historically caused more severe disease and is thought to be more transmissible, has not been detected in the recent global outbreak.

[0008] In 1970, human MPOX was first identified in humans in a 9-month-old boy in the Democratic Republic of the Congo, a region that had been free of smallpox in 1968. Since then, most case reports have come from rural, rainforest areas of the Congo Basin, particularly in the Democratic Republic of the Congo, with increasing reports of human cases throughout Central and West Africa. Since 1970, human cases of MPOX have been reported from 11 African countries: Benin, Cameroon, the Central African Republic, the Democratic Republic of the Congo, Gabon, Côte d'Ivoire, Liberia, Nigeria, the Republic of the Congo, Sierra Leone, and South Sudan. The true burden of MPOX is unknown. Cases continue to be reported.

[0009] MPOX is a disease of global public health importance, affecting not only West and Central African countries but also other regions of the world. The first MPOX outbreak outside of Africa occurred in the United States in 2003. This outbreak resulted in over 70 cases of MPOX in the United States. In May 2022, multiple cases of MPOX were confirmed in several non-endemic countries. As of October 4, 2022, 68,998 cases of MPOX had been reported from 100 countries, including 25,672 in the United States.

[0010] Overview

[0011] Various embodiments contemplated herein may include, but are not necessarily limited to, one or more of the following:

[0012] Embodiment 1: A set of primers and optional (multiple) probes for detecting and / or confirming the presence of MPOX evolutionary branch II virus in a sample, the set comprising: at least one primer pair and optional probe that selectively hybridizes to the OPG153 gene of MPOX evolutionary branch II, the OPG183 gene spacer of MPOX evolutionary branch II, or a combination thereof of the MPOX evolutionary branch II virus, wherein the primers and optional (multiple) probes of the set selectively hybridize to one or more conserved regions of nucleic acid from the MPOX evolutionary branch II virus.

[0013] Embodiment 2: The panel of embodiment 1, wherein each primer and optional probe has less than 90%, preferably less than 85%, sequence identity to a similar region, when present in MPOX clade I or a non-smallpox orthopoxvirus.

[0014] Embodiment 3: The panel of embodiment 1 or embodiment 2, wherein the primers and optional probe(s) do not selectively hybridize to MPOX clade I viruses or non-smallpox orthopoxviruses.

[0015] Embodiment 4: The panel of any one of Embodiments 1-3, further comprising additional primer pairs and optional probes for detecting and / or confirming the presence of one or more non-variola orthopoxviruses, wherein the additional primer pairs and optional probes comprise at least one primer pair and optional probe that selectively hybridize to the DNA polymerase (E9L) gene of a non-variola orthopoxvirus.

[0016] Embodiment 5: The panel of embodiment 4, wherein the additional primer pairs and optional probes for identifying one or more non-variola orthopoxviruses selectively hybridize to one or more conserved regions of nucleic acid from non-variola orthopoxviruses.

[0017] Embodiment 6: The group of any one of Embodiments 1-5, further comprising additional primer pairs and optional probes for detecting and / or confirming the presence of one or more MPOX evolutionary clade I viruses, wherein the additional primer pairs and optional probes comprise at least one primer pair and optional probe that selectively hybridize to one or more MPOX evolutionary clade I viruses.

[0018] Embodiment 7: The panel of any one of Embodiments 1-6, further comprising one or more additional primer pairs and optional probe(s) for detecting and / or confirming the presence of one or more of herpes simplex virus (HSV), varicella zoster virus (VZV), and syphilis.

[0019] Embodiment 8: The group of any one of Embodiments 4-6, wherein the group comprises: at least one primer pair and probe that selectively hybridizes to the OPG153 gene of MPOX clade II, at least one primer pair and probe that selectively hybridizes to the intergenic region of MPOX clade II, and at least one primer pair and probe that selectively hybridizes to the DNA polymerase (E9L) gene of a non-smallpox orthopoxvirus.

[0020] Embodiment 9: The group of any one of embodiments 1-8, wherein when the primers and optional probes that selectively hybridize to the OPG153 gene of MPOX evolutionary branch II are present, they comprise a sequence identical or complementary to at least 15 consecutive nucleotides of one or more of SEQ ID NOs: 1, 25, 26, 27 and 28; when the primers and optional probes that selectively hybridize to the OPG183 gene spacer of MPOX evolutionary branch II are present, they comprise a sequence identical or complementary to at least 15 consecutive nucleotides of one or more of SEQ ID NOs: 2, 29, 30, 31 and 32; when the primers and optional probes that selectively hybridize to the DNA polymerase (E9L) gene of a non-smallpox orthopoxvirus are present, they comprise a sequence identical or complementary to at least 15 consecutive nucleotides of one or more of SEQ ID NOs: 3, 33, 34, 35 and 36.

[0021] Embodiment 10: The panel of any one of Embodiments 1-9, wherein at least one of the primer and optional probe(s) comprises a detectable label.

[0022] Embodiment 11: The set of any one of Embodiments 1-10, wherein at least one probe, optionally each probe, of the set comprises a fluorescent dye and a quencher molecule.

[0023] Embodiment 12: The panel of any one of Embodiments 1-11, wherein the panel further comprises primer pairs that selectively hybridize to an exogenous control and / or an endogenous control, wherein the exogenous control is a sample processing control, and wherein the endogenous control is a sample adequacy control.

[0024] Embodiment 13: The set of any of Embodiments 1-12, wherein the set is contained within one or more boxes.

[0025] Embodiment 14: The set of Embodiment 13, wherein the set is contained in a box.

[0026] Embodiment 15: A kit for detecting and / or identifying MPOX clade II in a sample, the kit comprising:

[0027] A cartridge body comprising a plurality of chambers, wherein the plurality of chambers comprises:

[0028] i) a sample chamber having at least one fluid outlet in fluid communication with another chamber of the plurality of chambers; and

[0029] ii) optionally a lysis chamber in fluid communication with said sample chamber, optionally wherein said sample chamber and said lysis chamber are one and the same;

[0030] a reaction vessel fluidly connected to the plurality of chambers of the cartridge body and configured to:

[0031] i) nucleic acid amplification, and

[0032] ii) detecting and identifying one or more amplification products by real-time PCR, melting curve analysis, or a combination thereof;

[0033] A filter disposed in the fluid path between the lysis chamber and the reaction vessel, and a set of primers and / or probes according to any one of embodiments 1-11, for confirming the presence of MPOX clade II.

[0034] Embodiment 16: The cartridge of Embodiment 15, wherein the lysis chamber comprises one or more lysis reagents for releasing nucleic acids.

[0035] Embodiment 17: The cartridge of any of Embodiments 15-16, wherein the sample chamber and the lysis chamber are one and the same.

[0036] Embodiment 18: The box of any of Embodiments 15-17, wherein at least one of the multiple chambers contains the primers and / or probes or a subset thereof of the group, and at least one different chamber of the multiple chambers contains one or more lysis reagents for releasing nucleic acids from the sample.

[0037] Embodiment 19: The box of any of Embodiments 15-18, wherein the reaction vessel comprises one or more reaction chambers for detecting multiple amplification products.

[0038] Embodiment 20: The cartridge of any of Embodiments 15-19, wherein the reaction vessel comprises a reaction chamber.

[0039] Embodiment 21: The cartridge of Embodiment 19 or 20, wherein each reaction chamber is configured to detect a single amplification product.

[0040] Embodiment 22: The cartridge of Embodiment 19 or 20, wherein each reaction chamber is configured to detect a plurality of amplification products.

[0041] Embodiment 23: The cartridge of any of Embodiments 15-22, wherein the cartridge is configured to perform isothermal amplification.

[0042] Embodiment 24: The cartridge of any of Embodiments 15-23, wherein the cartridge is configured to perform non-isothermal amplification, optionally by thermal cycling or temperature oscillation.

[0043] Embodiment 25: The cartridge of any of Embodiments 15-24, wherein the cartridge is a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge.

[0044] Embodiment 26: A method for detecting and identifying MPOX clade II in a sample, the method comprising:

[0045] a) contacting nucleic acid from the sample with a set of primers and optional probe(s) according to any one of embodiments 1-12;

[0046] b) subjecting the nucleic acid, primers and optional probe(s) to amplification conditions, optionally followed by melting curve determination;

[0047] c) detecting the presence of any amplification product(s) by real-time PCR, melting curve analysis, or a combination thereof, and

[0048] d) confirming the presence of an MPOX clade II virus in the sample or determining the absence of an MPOX clade II virus detectable using the set of primers and / or probes based on the detection or absence of the amplification product(s), respectively.

[0049] Embodiment 27: The method of embodiment 26, wherein d) comprises differentially confirming the presence of MPOX clade II and / or non-variola orthopoxviruses in the sample based on the detection or absence of the amplification product(s), respectively, or determining that no MPOX clade II and non-variola orthopoxviruses are detectable using the set of primers and / or probes provided.

[0050] Embodiment 28: The method of any one of Embodiments 26-27, wherein:

[0051] a) contacting the nucleic acid from the sample with the set of primers and / or probes comprises:

[0052] placing the sample in a cartridge comprising a cartridge body having a plurality of chambers in fluid communication, a reaction vessel having one or more reaction chambers and configured for nucleic acid amplification and detection of one or more amplification products, a fluid path between the plurality of chambers and the reaction vessel, and a filter in the fluid path; and

[0053] if the sample comprises cells, lysing the cells in the sample with one or more lysing reagents present in at least one of the plurality of chambers;

[0054] b) subjecting the nucleic acid, primers and / or probes to amplification conditions comprises amplifying the nucleic acid using the primers and / or probes present in solution in the reaction vessel.

[0055] Embodiment 29: The method of Embodiment 28, wherein the amplification is isothermal.

[0056] Embodiment 30: The method of Embodiment 28, wherein the amplification is non-isothermal, optionally performed by thermal cycling or temperature oscillation.

[0057] Embodiment 31: The method of any one of Embodiments 26-30, wherein the detecting is performed in more than one reaction chamber.

[0058] Embodiment 32: The method of any one of Embodiments 26-31, wherein the detecting is performed in a single reaction chamber.

[0059] Embodiment 33: The method of any of Embodiments 26-32, wherein the sample is a skin sample, a lesion swab sample, a vesicular lesion fluid sample, a pustular lesion fluid sample, a rectal sample, a nasal aspirate sample, a nasal wash sample, a nasal swab sample, a nasopharyngeal swab sample, a saliva sample, an oropharyngeal swab sample, a throat swab sample, a bronchoalveolar lavage fluid sample, a bronchial aspirate sample, a bronchial wash sample, an endotracheal aspirate sample, an endotracheal wash sample, a tracheal aspirate sample, a nasal secretion sample, a mucus sample, a sputum sample, a plasma sample, a whole blood sample, or a combination thereof.

[0060] Embodiment 34: The method of any one of Embodiments 26-33, wherein the detecting is performed in the same facility where the sample is collected from the subject.

[0061] Embodiment 35: The method of any one of Embodiments 26-34, wherein the method is a point-of-care method.

[0062] Embodiment 36: The method of any of Embodiments 26-35, wherein the method is performed in a hospital, urgent care center, emergency room, doctor's office, health clinic, or at home.

[0063] Embodiment 37: The method of any one of Embodiments 26-36, wherein the method is a Clinical Laboratory Improvement Amendments (CLIA)-waived test.

[0064] Embodiment 38: The method of any of Embodiments 28-37, wherein the cartridge is a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge, is operated in accordance with CLIA, is operated by a CLIA-compliant laboratory, or is operated at a CLIA-compliant location.

[0065] Embodiment 39: The method of any one of Embodiments 26-38, wherein the method is performed to distinguish between virulent pathogens and less virulent pathogens.

[0066] Embodiment 40: The method of any one of Embodiments 26-39, wherein the method is implemented to facilitate a response to a pandemic, epidemic, and / or endemic pathogen.

[0067] Embodiment 41: The group of any one of embodiments 1-14, the box of any one of embodiments 15-25, or the method of any one of embodiments 26-40, wherein the box facilitates detection of MPOX evolutionary branch II virus in the sample within 60 minutes, within 45 minutes or within 30 minutes of collecting the sample from the subject and / or the method includes detecting MPOX evolutionary branch II virus in the sample within 60 minutes, within 45 minutes or within 30 minutes of collecting the sample from the subject.

[0068] Embodiment 42: The group of any one of embodiments 1-14, the box of any one of embodiments 15-25, or the method of any one of embodiments 26-40, wherein the box facilitates detection of MPOX evolutionary branch II virus in the sample within 60 minutes, within 45 minutes, within 30 minutes or within 20 minutes from the time the sample is placed in the box and / or the method includes detecting MPOX evolutionary branch II virus in the sample within 60 minutes, within 45 minutes, within 30 minutes or within 20 minutes from the time the sample is placed in the box.

[0069] Embodiment 43: The set, kit or method of any of Embodiments 41-42, wherein the one or more lysis reagents comprise a chaotropic agent, a chelating agent, a buffer and a detergent.

[0070] Embodiment 44: The set, kit or method of Embodiment 43, wherein the dissolution agent is selected from guanidine thiocyanate, guanidine hydrochloride, alkali metal perchlorate, alkali metal iodide, urea, formamide or a combination thereof.

[0071] Embodiment 45: The set, kit or method of Embodiment 43 or 44, wherein the one or more lysis reagents comprise a guanidinium compound, sodium hydroxide, EDTA, a buffer, and a detergent.

[0072] Embodiment 46: The set, cartridge or method of any one of Embodiments 41-45, wherein the filter is configured to bind the nucleic acid to be analyzed.

[0073] Embodiment 47: The group, box or method of any of Embodiments 41-46, wherein the filter comprises glass fibers and optionally a polymer binder, or the glass fibers are optionally modified with a DNA binding ligand, optionally with an alkylamine, a cycloalkylamine, an alkoxyamine, a polyamine moiety, an arylamine, an intercalator, a DNA groove binder, a peptide, an amino acid, a protein or a combination thereof.

[0074] Embodiment 48: The set, cartridge, or method of any of Embodiments 41-47, wherein the filter comprises a 500 to 2000 micron thick glass fiber disk having a pore size of 0.2 to 1 micron.

[0075] Embodiment 49: The set, cartridge or method of any of Embodiments 41-48, wherein the filter is configured to bind unwanted material and allow nucleic acid to pass therethrough.

[0076] Embodiment 50: The set, kit, or method of any one of Embodiments 41-49, wherein the kit further comprises a binding reagent, a washing reagent, an elution reagent, or a combination thereof.

[0077] Embodiment 51: The set, kit, or method of any of Embodiments 41-50, wherein the elution reagent comprises ammonia or an alkali metal hydroxide.

[0078] Embodiment 52: The set, kit or method of any of Embodiments 41-51, wherein the elution reagent has a pH greater than about 9, greater than about 10, or greater than about 11.

[0079] Embodiment 53: The set, kit or method of any of Embodiments 41-52, wherein the elution reagent comprises a polyanion, optionally carrageenan, a carrier nucleic acid or i-carrageenan, and KOH.

[0080] Embodiment 54: The set, cartridge, or method of any one of Embodiments 41-53, wherein the reaction vessel comprises up to 4 reaction chambers.

[0081] Embodiment 55: The set, cartridge, or method of any of Embodiments 41-54, wherein the reaction vessel comprises a reaction chamber.

[0082] Embodiment 56: The set, kit, or method of any of Embodiments 41-55, wherein at least one of the plurality of chambers comprises one or more lyophilized reagents.

[0083] Embodiment 57: The set, kit or method of Embodiment 56, wherein the one or more lyophilized reagents are in the form of one or more beads.

[0084] Embodiment 58: The set, kit or method of Embodiment 56 or 57, wherein the one or more lyophilized reagents are selected from primers, probes, salts, dNTPs, thermostable polymerases, reverse transcriptases, or combinations thereof.

[0085] Embodiment 59: The set, kit or method of Embodiment 58, wherein the one or more lyophilized reagents comprise lyophilized primers and probes.

[0086] Embodiment 60: The set, kit or method of any of Embodiments 41-55, wherein the reagents and components in the reaction vessel are in solution.

[0087] Embodiment 61: A system for detecting MPOX clade II in a biological sample, the system comprising:

[0088] A module having a receiving area for receiving the cartridge of any one of embodiments 15-25 and an instrument for docking with a reaction vessel interface, wherein the module includes one or more mechanisms within the receiving area for manipulating a fluid sample within the cartridge; and

[0089] A memory having programmable instructions recorded thereon, the programmable instructions being specifically configured to operate the module according to a monkeypox clade II assay protocol to determine the nucleic acid sequence characteristics of MPOX clade II.

[0090] Embodiment 62: The system of embodiment 61, wherein the module and / or system further comprises: a scanner or reader configured to read an identifier on the box; wherein the instructions are configured to determine an applicable protocol based on reading or scanning the identifier; and wherein the system operates the module according to the applicable protocol based on input from the scanner or reader.

[0091] Embodiment 63: The system of Embodiment 61 or 62, wherein the module and / or system further comprises: a housing; a plurality of modules comprising the module, wherein the modules are substantially identical and are configured to simultaneously perform assays on cartridges received therein.

[0092] Embodiment 64: The system of Embodiment 63 further comprising a network platform for transmitting results resulting from module operations.

[0093] BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 A-1C shows an overview of a sample cartridge having a valve assembly configured for performing various sample processing steps, including chemical lysis of a target, configured for PCR and optional integrated nucleic acid analysis of the MPOX clade II biomarker panel, according to some embodiments of the present invention. Figure 1 A shows a sample box body with a reaction container, Figure 1 B shows an exploded view of the sample box, Figure 1 C shows components of the valve assembly.

[0095] Figure 2 Various valve assemblies A, B, C, and D are shown, each suitable for one or more types of target lysis, any of which may be used in a respective sample cartridge.

[0096] Figures 3A-3C Illustrative, non-limiting embodiments of modules and systems (eg, processing units) for PCR detection and / or quantification of a biomarker panel and optional integrated nucleic acid analysis are shown. Figure 3A The figure shows a module configured to receive and interact with the valve assembly of the cartridge to operate the cartridge to facilitate sample preparation and analysis. Figure 3B The diagram illustrates a processing unit (eg, analytical testing unit) of the module that interacts with a fluid sample in a reaction vessel to facilitate sample processing and analytical testing (eg, PCR and optional nucleic acid analysis) for a biomarker panel. Figure 3C An analytical system is shown having a plurality of such modules within a housing for receiving a plurality of sample cartridges therein for testing for biomarker panels and / or various other targets or panels.

[0097] Figure 4 A non-limiting workflow for PCR and optional nucleic acid analysis (e.g., nucleic acid amplification) for a targeted assay is shown. In some embodiments, when PCR and nucleic acid analysis are performed, both are performed on the same sample. Thus, a single sample can be introduced into a single sample chamber. In other embodiments, the sample can be processed differently for PCR and nucleic acid analysis in a targeted assay panel.

[0098] Figure 5 An exploded view of a sample cartridge according to some embodiments is shown, illustrating its major components, including a lid, a multi-chamber body, a reaction vessel, a valve assembly, and a base. The chambers can be used to perform various processing on the sample (e.g., extraction, purification, deamidation, desulfonation).

[0099] Figures 6A-6B A diagram illustrates specialized components of a cartridge configured for a target assay panel, according to some embodiments. As shown, the specialized component is a valve assembly having a syringe barrel through which a sample is injected, and a plurality of ports on a valve body that facilitate transfer of the fluid sample between various processing chambers as the valve assembly rotates.

[0100] Figure 7 MPOX clade II is divided into clade IIa and clade IIb. The genomes from the recent 2022 outbreak are all from clade IIb. Figure 8 A tree from NextStrain (nextstrain.org / mpox / mpxv) illustrates this. Clade I corresponds to the lowest cluster of lines and points. Clade II branches off from Clade I on the left side of the tree. Clade II then divides into Clade IIa (the lower Clade II branch) and Clade IIB (the upper Clade II branch).

[0101] Figure 8 . Ct value for each analyte.

[0102] Figure 9 . End point fluorescence (EPF) for each analyte.

[0103] Figure 10 . Test results from GeneXpert software.

[0104] Figure 11 . Plot of fluorescence of 50 copies / ml NATtrol MPOX virus input versus cycle output.

[0105] Figure 12 . Plot of fluorescence of negative samples versus cycle output.

[0106] Details

[0107] The present disclosure describes methods, compositions, devices, and systems that facilitate the rapid detection of MPOX clade II viruses in a selective and specific manner that is amenable to automation and can be used in point-of-care testing devices, and in some embodiments, enables the differential identification of MPOX clade II viruses from clade I viruses in a subject. The methods rely on nucleic acid amplification to detect clade II biomarkers, which requires contacting sample nucleic acid with primers and / or optional probes that target clade II-specific nucleic acids (e.g., SEQ ID NOs: 1, 2, 3 below), subjecting the nucleic acids, primers, or optional probes to amplification conditions, and detecting the presence of any amplification product(s) to differentially confirm the presence of clade II and clade I viruses in the sample, or to determine the absence of MPOX viruses detectable using the set of primers in the sample.

[0108] definition

[0109] Unless otherwise stated, the terms used in the claims and the specification are defined as follows.

[0110] Orthopoxvirus is a genus of viruses in the Poxviridae family and the Chordopoxvirinae subfamily. Vertebrates, including mammals and humans, and arthropods serve as natural hosts. There are 12 species in this genus. Diseases associated with this genus include smallpox, cowpox, horsepox, camelpox, and MPOX. The most well-known member of this genus is variola virus, which causes smallpox.

[0111] As used herein, the term non-variola orthopoxvirus refers to viruses in the genus Orthopoxvirus other than variola virus.

[0112] Monkeypox virus (MPXV or MPOX virus) is an enveloped, double-stranded DNA virus belonging to the genus Orthopoxvirus in the family Poxviridae. First identified in 1958 in a population of Asian monkeys (cynomolgus macaques) and rhesus macaques (macaca fascicularis), MPOX is a viral zoonosis capable of human-to-human transmission. Two clades of MPOX have been described, and they recently underwent a nomenclature change. MPOX clade I (formerly the Congo Basin strain) is associated with a higher mortality rate than clade II (formerly the West African strain). MPOX has an incubation period of 5–21 days, with initial symptom onset typically lasting 1–5 days and consisting of fever, headache, myalgia, lymphadenopathy, and fatigue, followed by a vesicular rash that persists for approximately 2–3 weeks. Human-to-human transmission occurs through direct contact with lesions or body fluids, through contaminated infectious agents, or through respiratory secretions. In the spring of 2022, several cases of MPOX, specifically MPOX clade IIb, were identified in non-endemic areas.

[0113] "OPG153 gene of MPOX clade II" refers to the region of the clade II MPOX virus corresponding to gene accession number (GenBank) NC_063383.1, positions 133825-133927 (SEQ ID NO: 1). In this context, the term "corresponding to" refers to the region of the clade II MPOX virus genome that matches SEQ ID NO: 1 when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In various embodiments, the corresponding region has at least 70%, 80%, 90%, 92%, 94%, 96%, 98%, or 99% or more sequence identity to SEQ ID NO: 1.

[0114] "OPG183 intergenic region of MPOX clade II" refers to the region of the clade II MPOX virus corresponding to gene accession number (GenBank) NC_063383.1, positions 156408-156516. In this context, the term "corresponding to" refers to the region of the clade II MPOX virus genome that matches SEQ ID NO: 2 when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In various embodiments, the corresponding region has at least 70%, 80%, 90%, 92%, 94%, 96%, 98%, or 99% or more sequence identity to SEQ ID NO: 2.

[0115] The term "nucleic acid" refers to a polymer of nucleotides and, unless otherwise limited, includes analogs of natural nucleotides that can function in a manner similar to (eg, hybridize) naturally occurring nucleotides.

[0116] The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is the DNA representation of mRNA, typically obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; mRNA; and non-coding RNA.

[0117] The term nucleic acid includes double-stranded or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double-stranded or triple-stranded nucleic acid complexes, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).

[0118] The term nucleic acid also includes any modification thereof, such as any modification thereof by methylation and / or by capping. Nucleic acid modifications can include the addition of chemical groups that confer extra charge, polarizability, hydrogen bonding, electrostatic interactions, and functionality to individual nucleic acid bases or to the entire nucleic acid. Such modifications can include base modifications such as 2'-sugar modifications, 5-pyrimidine modifications, 8-purine modifications, modifications to the outer ring amine of cytosine, substitutions of 5-bromouracil, sugar-phosphate backbone modifications, unusual base pairing combinations such as the homotype bases isocytidine and isoguanidine, or the like.

[0119] More particularly, in some embodiments, nucleic acids can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing non-nucleotide backbones, for example, polyamides (e.g., peptide nucleic acids (PNA)) and polymorpholino polymers (see, e.g., Summerton and Weller (1997) “Morpholino Antisense Oligomers: Design, Preparation, and Properties,” Antisense & Nucleic Acid Drug Dev. 7:1817-195; Okamoto et al. (20020) “Development of electrochemically gene-analyzing method using DNA-modified electrodes,” Nucleic Acids Res. Supplement No. 2:171-172), and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that enables base pairing and base stacking, such as those found in DNA and RNA. The term nucleic acid also includes locked nucleic acids (LNA), which are described in US Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, which are incorporated herein by reference in their entireties for their disclosures regarding LNA.

[0120] The nucleic acid(s) can be derived from an entirely chemical synthesis process, such as solid phase-mediated chemical synthesis, from a biological source, such as by isolation from any nucleic acid-producing species, or from processes involving the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of these processes.

[0121] The term "sequence identity" in the context of two or more amino acid or nucleotide sequences refers to two or more sequences that are identical or have a specified percentage of amino acid residues or nucleotides that are identical, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.

[0122] For determining the sequence comparison of nucleotide or amino acid sequence identity percentage, usually a sequence serves as a "reference sequence" and a "test" sequence is compared thereto. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters. Sequence alignments for comparison can be performed using BLAST set to default parameters.

[0123] As used herein, about nucleotide sequence, the term "conserved" refers to a sequence that is identical or similar to a sequence that has been repeated for many generations of sequence in nature. In different embodiments, a given "conserved" sequence can have at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide sequence identity, or can be 100% identical for the entire data set. For these purposes, when all sequences in a data set meet this requirement when compared with a reference sequence from a data set, the sequence identity percentage requirement is met. A data set can include a sequence from a given virus strain or clade, for example, in the case described, it can be claimed that the sequence is conserved within the strain or clade. A data set can include a sequence from a variety of virus strains or clades, for example, in the case described, it can be claimed that the sequence is conserved for entire those virus strains or clades. For example, a sequence can be conserved in a strain or clade, but not in another strain and clade. In some embodiments, the sequence is not conserved in a strain if the sequence does not meet the conditions of the definition set forth in this paragraph. In some embodiments, the dataset may have at least 3, 5, 10, 15, 20, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more sequences. For example, a sequence can be at least 90% conserved across a data set of at least 500, at least 95% conserved across a data set of at least 400, at least 95% conserved across a data set of at least 300, at least 95% conserved across a data set of at least 200, at least 96% conserved across a data set of at least 150, at least 97% conserved across a data set of at least 100, at least 98% conserved across a data set of at least 50, at least 98% conserved across a data set of at least 10, at least 98% conserved across a data set of at least 5, at least 99% conserved across a data set of at least 10, at least 99% conserved across a data set of at least 5, and so forth.

[0124] As used herein, " conserved region " refers to the subsequence of the conservative longer nucleic acid sequence as defined above.The length of conserved subsequence can be at least 6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45 or more nucleotide lengths.In some embodiments, the length of conserved region can be less than 500,400,300,200 or one hundred nucleotides.Therefore, for example, the length of conserved region can be between 10 to 500,15 to 400,20 to 300,25 to 200 or 30 to 100 nucleotides, and has at least 90,91,92,93,94,95,96,97,98 or 99% nucleotide sequence identity relative to this subsequence.

[0125] As used herein, a "similar region" refers to a subsequence of a longer nucleic acid sequence that matches a region in a reference sequence. Similar regions can, and often do, have similar structure and / or function (e.g., a similar region can encode a protein domain that performs the same function in two protein isoforms).

[0126] As used herein, the term "gene" includes the coding sequence, introns, and any associated control sequences involved in the expression of the coding sequence.

[0127] As used herein, the term "complementarity" refers to the ability of two nucleotides to pair precisely; that is, if a nucleotide at a given position in a nucleic acid is capable of hydrogen bonding with a nucleotide in another nucleic acid to form a typical base pair, then the two nucleic acids are considered to be complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial," in which only some of the nucleotides bind, or it can be complete when there is complete complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0128] "Selective hybridization" or "selective annealing" refers to the binding of a nucleic acid to a target nucleic acid under defined stringent conditions without substantial binding to other nucleic acids present in the hybridization mixture. Those skilled in the art will recognize that relaxing the stringency of hybridization conditions allows for tolerance of sequence mismatches.

[0129] In some embodiments, hybridization is performed under stringent hybridization conditions. The phrase "stringent hybridization conditions" generally refers to conditions of defined ionic strength and pH, ranging from about 5°C to about 20°C or above the melting temperature (T) for the specific sequence. m ) is a temperature lower than 25°C. As used herein, T m It is the temperature at which a group of double-stranded nucleic acid molecules becomes semi-dissociated to form single strands. It is used to calculate the T of nucleic acids. mMethods for stringent hybridization are well known in the art (see, for example, Berger and Kimmel (1987) METHODS IN ENZYMOLOGY, VOL. 152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory, both of which are incorporated herein by reference for their description of stringent hybridization conditions). As indicated in standard references, when nucleic acids are in an aqueous solution of 1 M NaCl, T m A simple estimate of the value can be calculated using the following equation: m = 81.5 + 0.41 (% G + C) (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The melting temperature of the hybrid (and therefore the conditions for stringent hybridization) is affected by various factors, such as the length and nature of the primers or probes (DNA, RNA, base composition) and the nature of the target nucleic acid (DNA, RNA, base composition, present in solution or immobilized, etc.), as well as the concentrations of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and discussed in standard reference texts in the art. Exemplary stringent conditions suitable for achieving specific hybridization of most sequences are: a temperature of at least about 60°C and a salt concentration of about 0.2 molar at pH 7. T values ​​for oligonucleotide sequences based on nearest neighbor thermodynamics m Calculations can be performed as described in: "A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics" John SantaLucia, Jr., PNAS February 17, 1998 vol. 95 no. 4 1460-1465 (incorporated herein by reference for this description).

[0130] The term "oligonucleotide" is used to refer to a relatively short nucleic acid, typically shorter than 200 nucleotides, more particularly shorter than 100 nucleotides, and most particularly shorter than 50 nucleotides. Typically, an oligonucleotide is a single-stranded DNA molecule.

[0131] The term "primer" refers to an oligonucleotide that can hybridize to nucleic acids (also referred to as "annealing") and, under suitable conditions (i.e., at four different nucleoside triphosphates and reagents for polymerization, such as DNA or RNA polymerase or reverse transcriptase), be used as the starting site for nucleotide (RNA or DNA) polymerization. The appropriate length of a primer depends on the intended use of the primer, but a primer is typically at least 7 nucleotides long, and in some embodiments, a length range of 10 to 30 nucleotides, or in some embodiments, a length range of 10 to 60 nucleotides. In some embodiments, a primer can be, for example, 15 to 50 nucleotides long. Short primer molecules typically require cooler temperatures to form sufficiently stable hybrid complexes with templates. Primers do not need to reflect the precise sequence of the template, but must be sufficiently complementary to hybridize with the template.

[0132] A primer is said to "anneal to" or "hybridize to" another nucleic acid if it or a portion thereof hybridizes to a nucleotide sequence within the other nucleic acid. The statement that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer hybridizes completely or exclusively to that nucleotide sequence. For example, in some embodiments, an amplification primer as used herein is said to "anneal to" or be "specific for" a nucleotide sequence. This description encompasses primers that completely anneal to a nucleotide sequence, as well as primers that partially anneal to a nucleotide sequence.

[0133] The term "primer pair" refers to a set of primers comprising a 5' "upstream primer" or "forward primer" that hybridizes to the complementary sequence at the 5' end of the DNA sequence to be amplified and a 3' "downstream primer" or "reverse primer" that hybridizes to the 3' end of the sequence to be amplified. As will be appreciated by those skilled in the art, the terms "upstream" and "downstream" or "forward" and "reverse" are not intended to be limiting, but rather provide illustrative directions in some embodiments.

[0134] A "probe" is a nucleic acid that is capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, typically through complementary base pairing, typically through the formation of hydrogen bonds, thereby forming a double-stranded structure. The probe can be labeled with a detectable moiety to allow easy detection of the probe, particularly once the probe has hybridized to its complementary target. Alternatively, however, the probe can be unlabeled but detectable by specific binding to a labeled ligand, either directly or indirectly. Probes can vary widely in size.

[0135] As used herein, the term "specific for a nucleic acid" with respect to a portion of a primer or a nucleotide sequence within a primer refers to a primer or a nucleotide sequence that can specifically anneal to a target nucleic acid under appropriate annealing conditions.

[0136] The term "target" is used herein to refer to both "target nucleic acid" and "target organism." The former refers to the nucleic acid to be detected, while the latter refers to the organism to be detected. The term "target nucleic acid" is generally used herein to refer to the nucleic acid fragment that is defined by a primer pair and that results in the production of an amplicon in an amplification reaction; the term "amplification target" is also used herein to refer to this type of target nucleic acid. Primers and probes are also referred to as "targeting" nucleic acid sequences, and therefore these sequences may also be understood as "target nucleic acids." In addition, primers and probes are said to "target" a gene or be "specific" for a gene. In this usage, primers and probes can be used to detect the presence of a specific gene by specifically hybridizing to a portion of the gene that indicates the presence of the specific gene. The meaning of "target" and "target nucleic acid" will be clear to those skilled in the art based on the context in which the terms are used. In some embodiments, multiple target nucleic acids can be detected to detect a single target organism. In some embodiments, a single target nucleic acid can be detected to detect a single target organism. In some embodiments, an assay can use multiple target nucleic acids for one or more target organisms and a single target nucleic acid for one or more different target organisms.

[0137] According to the present teachings, amplification encompasses any method that generally replicates at least a portion of at least one target nucleic acid in a template-dependent manner, including but not limited to many techniques for linearly or exponentially amplifying nucleic acid sequences. Exemplary methods for performing the amplification step include PCR, nucleic acid strand-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), and the like, including multiplex versions and combinations thereof, such as, but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction—CCR), helicase-dependent amplification (HDA), and the like. Descriptions of such techniques can be found in, among other sources: Ausubel et al.; PCR Primer: A Laboratory Manual, Diffenbach ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34: 501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley ed., Humana Press, Totowa, NJ (2002); Abramson et al., Curr Opin Biotechnol. 1993 Feb.4(1): 41-7, U.S. Patent No. 6,027,998; U.S. Patent No. 6,605,451, Barany et al., PCT Publication No. WO97 / 31256; Wenz et al., PCT Publication No. WO01 / 92579; Day et al., Genomics, 29(1): 152-162 (1995), Ehrlich et al., Science 252: 1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18: 561-64 (2000); and Rabenau et al., Infection 28: 97-102 (2000); Belgrader, Barany and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the World Wide Web at: promega.com / geneticidproc / ussymp6proc / blegrad.html-); LCR kit instructions, catalog #200520, revision #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88: 188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25: 2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99: 5261-66 (2002); Barany and Gelfand, Gene 109: 1-11 (1991); Walker et al., Nucl. Acid Res. 20: 1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18- (2002); Lage et al., Genome Res. 2003 Feb.; 13(2):294-307, and Landegren et al., Science 241: 1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 Nov.; 2(6): 542-8., Cook et al., J Microbiol Methods. 2003 May; 53(2): 165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 Feb.; 12(1): 21-7, U.S. Patent No. 5,830,711, U.S. Patent No. 6,027,889, U.S. Patent No. 5,686,243, PCT Publication No. WO0056927A3 and PCT Publication No. WO9803673A1. .

[0138] In some embodiments, amplification comprises at least one cycle of the following sequential steps: annealing at least one primer to a complementary or substantially complementary sequence in at least one target nucleic acid; synthesizing at least one nucleotide strand using a polymerase in a template-dependent manner; and denaturing the newly formed nucleic acid duplex to separate the strands. The cycles may or may not be repeated. Amplification may include thermal cycling or may be performed isothermally.

[0139] As used herein, the term "amplification conditions" refers to conditions that promote the amplification of a target nucleic acid in the presence of appropriate primers.

[0140] As used herein, "in solution" means not immobilized on any type of substrate, eg, beads, or a surface in a cassette, such as a chamber wall.

[0141] A "multiplex amplification reaction" is a reaction in which two or more nucleic acids that are distinguishable by sequence are amplified simultaneously.

[0142] The term "qPCR" is used herein to designate quantitative real-time polymerase chain reaction (PCR), also known as "real-time PCR" or "kinetic polymerase chain reaction"; all terms refer to PCR with real-time signal detection.

[0143] The term "melting curve analysis" refers to the use of the dissociation characteristics of double-stranded nucleic acid fragments during heating. Initially, chain dissociation was observed using UV absorbance measurements, but techniques based on fluorescence measurements are now the most commonly used method. The temperature-dependent dissociation between two DNA strands can be measured in a "melting assay", for example, using DNA-intercalating fluorophores such as SYBR Green or EvaGreen, or fluorophore-labeled DNA probes. In the case of SYBR Green, which increases fluorescence intensity 1000-fold when intercalated into the minor grooves of both DNA strands, the dissociation of DNA during heating can be measured by the resulting large decrease in fluorescence intensity. Alternatively, juxtaposed probes (one featuring a fluorophore and the other featuring a suitable quencher) can be used to determine the complementarity of the probes to the target nucleic acid sequence.

[0144] "Reagent" broadly refers to any agent used in a reaction other than the analyte (e.g., the nucleic acid being analyzed). Exemplary reagents used in nucleic acid amplification reactions include, but are not limited to, buffers, metal ions, polymerases, reverse transcriptases, primers, template nucleic acids, nucleotides, labels, dyes, nucleases, dNTPs, and the like. Reagents used in enzymatic reactions include, for example, substrates, cofactors, buffers, metal ions, inhibitors, and activators.

[0145] As used herein, the term "label" refers to any atom or molecule that can be used to provide a detectable and / or quantifiable signal. Specifically, a label can be directly or indirectly attached to a nucleic acid or protein. Suitable labels that can be attached to a probe include, but are not limited to, radioisotopes, fluorophores, chromophores, mass markers, electron-dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0146] As used herein, the term "dye" generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation and produces a detectable signal (eg, a fluorescent signal).

[0147] As used herein, the term "quencher" generally refers to any organic or inorganic molecule that reduces the level of a detectable signal.

[0148] As used herein, the term "detection" refers to "determining the presence of something," such as determining the presence of a nucleic acid sequence, for example, a nucleic acid sequence that indicates the presence of an MPOX virus. Detection can include determining the presence of an MPOX virus without positively identifying the MPOX virus; determining the presence of one or more MPOX viruses belonging to an MPOX virus clade; determining the presence of a specific known MPOX virus strain; or determining the presence of a novel (previously undescribed) MPOX virus strain or other orthopoxvirus strain.

[0149] As used herein, the term "treatment regimen" refers to any medical intervention intended to alleviate the symptoms and / or pathology of a disorder. A treatment regimen may include one or more actions (e.g., bed rest, increased fluid intake), over-the-counter or prescription medications, supplements, foods, beverages, or the use of a medical device (e.g., a ventilator).

[0150] As used herein, "Clinical Laboratory Improvement Amendments (CLIA)" means the Clinical Laboratory Improvement Amendments (CLIA) regulations of 1988, which were in effect as of the original filing date of this application. CLIA regulations include federal standards that apply to all U.S. institutions or locations that test human samples for the purpose of health assessment or diagnosis, prevention, or treatment of disease. A "CLIA-compliant" test is one that complies with these regulations. A "CLIA-exempt" test includes tests that do not comply with all of these regulations. For example, CLIA-exempt tests include test systems approved by the U.S. Food and Drug Administration for home use and those tests that have been approved for exemption according to CLIA standards.

[0151] As used herein, the term "virulent" can refer to the degree of infectivity and / or severity of a disease caused by a pathogen, such as a virus. A "more virulent" virus is more contagious and / or causes more severe disease than a reference virus, and vice versa. In many embodiments, a "less virulent" virus generally does not cause disease requiring hospitalization.

[0152] As used herein, an "endogenous control" refers to a portion naturally present in a sample being assayed. In some embodiments, the endogenous control is a "sample adequacy control" (SAC), which can be used to determine whether the sample used in the assay is sufficient, or whether the sample contains sufficient biological material, such as cells. In some embodiments, the endogenous control is RNA (such as mRNA, tRNA, ribosomal RNA, etc.), such as human RNA for human samples. Non-limiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPK1a mRNA. In some embodiments, an endogenous control, such as a SAC, is selected that can be detected in the same manner as the target nucleic acid (e.g., RNA), and in some embodiments, can be detected simultaneously with the target nucleic acid (e.g., RNA).

[0153] As used herein, an "exogenous control" refers to a moiety added to a sample or assay, such as a "sample processing control" (SPC). In some embodiments, an exogenous control is included with the assay reagents. Exogenous controls are typically selected to be not expected to be present in the sample being tested, or to be present at very low levels in the sample, such that the amount of the moiety naturally present in the sample is either undetectable or detectable at a much lower level than the amount added to the sample as an exogenous control. In some embodiments, an exogenous control comprises a nucleotide sequence not expected to be present in the sample type used to detect the target nucleic acid (e.g., RNA). In some embodiments, an exogenous control comprises a nucleotide sequence not known to be present in the species from which the sample was collected. In some embodiments, an exogenous control comprises a nucleotide sequence from a species different from that of the subject from whom the sample was collected. In some embodiments, an exogenous control comprises a nucleotide sequence not known to be present in any species. In some embodiments, an exogenous control is selected to be detectable in the same manner as the target nucleic acid (e.g., RNA), and in some embodiments, is detected simultaneously with the target nucleic acid (e.g., RNA). In some embodiments, the exogenous control is RNA. In some such embodiments, the exogenous control is Armored RNA®, which comprises RNA encapsulated in a protective coat of a bacteriophage. See, e.g., Walker Peach et al., Clin. Chem. 45: 12: 2079-2085 (1999).

[0154] MPOX Clade II and Other Orthopoxvirus Biomarker Targets

[0155] In various embodiments, MPOX clade II viruses can be detected by nucleic acid amplification in a monkeypox (MPX or MPOX) panel assay, which can include detection of MPX clade II viruses and / or non-smallpox orthopoxviruses. In certain embodiments, the monkeypox panel assay can detect MPOX clade I viruses and / or non-smallpox orthopoxviruses by nucleic acid amplification.

[0156] In some embodiments, the viruses discussed above can be detected by nucleic acid amplification in an MPX clade II group assay, for example, in a multiplex amplification reaction, wherein the MPX clade II group assay can be designed to detect 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more target nucleic acids per amplification reaction mixture. This degree of multiplexing can be achieved by using primers and probes that do not substantially cross-react or bind off-target and can reliably identify the pathogens they target in the face of antigenic drift. Bioinformatics analysis of multiple databases can be performed to identify primers and probes for highly conserved regions in the genomes of these pathogens.

[0157] This article recognizes that currently available multiplex PCR methods present various challenges. For example, while multiplexing a large number of target amplification reactions (e.g., multiplex PCR) is possible, simultaneously detecting multiple amplicons is not straightforward. Multiplex q-PCR methods, defined as the simultaneous amplification and detection of multiple nucleic acid sequences in a single reaction chamber, have, to date, been implemented with a small number of amplicons. Efficiently performing multiple assays in the same reaction volume and enabling simultaneous amplification and detection of multiple targets in the same reaction chamber is of great interest. Such an approach would not only better utilize the original DNA sample but also significantly reduce any complexities associated with the fluidics and liquid handling procedures used to run multiple singleplex reactions.

[0158] Attempts to create multiplexed q-PCR methods have been plagued by practical challenges in simultaneously detecting different nucleic acid sequences in a single sample. One approach involves associating different reporter molecules (e.g., fluorescent dyes) with individual amplicons during the PCR reaction, enabling parallel detection of individual reporters using different "colors." While this approach theoretically offers parallelism, it is limited by: (i) the number of different reporter molecules available; (ii) the presence of crosstalk between channels due to optical signals in adjacent channels; and (iii) the availability of imagers and detectors capable of distinguishing between the different signals. Another potential approach to providing multiplexing capabilities is to separate the biological samples of interest and physically place them into separate, single, and isolated amplification chambers using a fluidics system. While this approach effectively creates multiplexed q-PCR by performing multiple singleplex (i.e., one amplicon per reaction chamber) q-PCR reactions, it can be suboptimal because it can reduce the number of target nucleic acid sequences in each chamber, which can generate random anomalies (Poisson noise) in the acquired data when the original sample has a low concentration. Furthermore, it requires complex fluidics processing.

[0159] Highly multiplexed detection of DNA sequences in a sample can be accomplished using analytical platforms such as DNA microarrays or next-generation DNA sequencers, but not q-PCR or equivalent technologies. Specifically, microarrays are massively parallel, affinity-based biosensors in which target nucleic acids are selectively captured from the same sample at distinct addressable coordinates (e.g., pixels) on a solid surface. Each addressable coordinate can have a unique capture DNA or RNA probe that is complementary to the target nucleic acid sequence to be detected in the sample. While microarrays can offer high multiplexing capabilities, they are semi-quantitative and, due to their endpoint nature (i.e., non-real-time detection) and the fact that they lack any target amplification, suffer from poor limits of detection (LOD) and detection dynamic range (DDR).

[0160] Because of the range of targets and target amplicons for primers and probes, a multiplex strategy is employed to screen and select primers and oligonucleotides. The MPX clade II group multiplex design strategy can involve the following steps: singleplex design of all primers / probes, multiplexing using background oligonucleotides and primer / probe matrix, multiplexing using primers and probes divided into different pools, sequencing of samples, redesign of primers / probes (if necessary), multiplexing using all sets of primers / probes together, and repeating one or more steps as needed.

[0161] In addition, to increase the number of target nucleic acids detected per channel, the following methods can be used: (i) using T m Melting probes below their annealing temperature (no amplification curve), it is possible to combine Taqman and melting probes in the same channel; and (ii) several melting targets in one channel; the melting window for each target will depend on the sequence variation of the target.

[0162] Since the high mutation rate of viral target organisms makes it difficult to find conserved regions, amplification detection is preferred to avoid mutation-related melting probes and T m Significant changes to the window.

[0163] Because combining large numbers of oligonucleotides in one reaction mixture can lead to undesirable interactions between them, in some embodiments, modified nucleotides can be used to reduce primer-primer interactions.

[0164] Several primers and probes for non-smallpox orthopoxviruses, including MPX, are known and include, for example, those shown in Table 1 below.

[0165]

[0166]

[0167] The novel primers and probes described herein target regions of the MPX clade II genome that are conserved within MPX clade II but sufficiently distinct from analogous regions within MPX clade I (and other orthopoxviruses) to enable unambiguous detection and identification of MPX clade II viruses in a sample using nucleic acid amplification assays. In some embodiments, MPX clade II can be identified based solely on the production of an amplification product; that is, primers specific for MPX clade II. In other embodiments, MPX clade II can be identified based on the use of amplification primers in combination with probes, e.g., probes with greater specificity for MPX clade II than the primers alone. Exemplary sequences of genomic regions determined to be sufficiently conserved within MPX clade II, but sufficiently distinct from MPX clade I (see Example 1), for use in identifying MPX clade II viruses in a sample are shown below. In an exemplary embodiment, sequences of genomic regions determined to be sufficiently conserved within MPX clade II, but sufficiently divergent from MPX clade I, for use in identifying MPX clade II viruses in a sample include the OPG153 gene (see, e.g., GenBank Gene ID: 72551553; Reference Sequence: NC_063383.1), which corresponds to the orthopoxvirus A26L / A30L protein. The A26 protein is contained in mature ORPV virions and facilitates MV binding to the plasma membrane and inhibition of virus-cell fusion. In some orthopoxviruses, it also participates in enclosing mature virions in dense inclusion bodies. A30L is a small virion phosphoprotein. In certain exemplary embodiments, the sequence of the genomic region determined to be sufficiently conserved within MPX clade II, but sufficiently divergent from MPX clade I, for use in identifying an MPX clade II virus in a sample includes the OPG183 intergenic region (see, e.g., GenBank accession number NC_063383.1; GeneID: 72551553), corresponding to an intergenic region of an orthopoxvirus. In certain exemplary embodiments, the sequence of the genomic region for identifying a non-variola orthopoxvirus in a sample includes the F8L gene (similar to the E9L gene of the Copenhagen strain of vaccinia virus; see, e.g., GenBank reference sequence: AF380138.1), corresponding to the catalytic subunit of the DNA polymerase of the MPX virus strain.

[0168] Table 2 below shows exemplary primers, probes, and amplicons that can be used in nucleic acid amplification assays to detect MPX Clade II and non-smallpox orthopoxviruses. "MPX CII" refers to MPX Clade II (also referred to herein as MPOX Clade II). The forward and reverse primers are designated "Fwd" and "Rev," respectively. The probes are shown immediately below the reverse primers in Table 2 and are labeled with a fluorescent marker and a quencher. The amplicon sequences are shown immediately below the probes.

[0169]

[0170]

[0171] Some embodiments of the MPX clade II biomarker panel assay can detect one or more additional viral targets, such as MPX clade I, MPX viruses of any clade (i.e., non-clade-specific MPX detection), or non-variola orthopoxviruses. Primers and probes for this purpose are known, and examples are shown in Table 2 above.

[0172] Other embodiments of the MPX Clade II biomarker panel assay can detect one or more targets in one or more additional viral pathogens, such as herpes simplex virus (HSV), varicella zoster virus (VZV), syphilis, chlamydia, gonorrhea, Haemophilus ducreyi, or a combination thereof. Infections caused by HSV, VZV, syphilis, chlamydia, Haemophilus ducreyi, and gonorrhea can lead to skin, oral, and genital lesions. In some embodiments, a genital lesion biomarker panel assay is described herein and can detect MPOX (Clade I and / or Clade II) and one or more additional viral pathogens selected from herpes simplex virus (HSV), varicella zoster virus (VZV), syphilis, chlamydia, gonorrhea, Haemophilus ducreyi, or a combination thereof. Primers and probes for this purpose are known, and examples are provided in Goldstein, EJ et al., Diagnostic Microbiology and Infectious Disease, 2021, 99, 2: 115221; Cotton, S. et al., Diagnostic Microbiology and Infectious Disease, 2021, 99, 4: 115262; and Bennett, S. et al., Journal of Virological Methods, 2013, 189, 1: 143-147, which are incorporated herein by reference for the purposes of this description.

[0173] The section below entitled "Exemplary Polynucleotides" describes in more detail considerations for primers and optional probes for detecting MPOX clade II biomarkers.

[0174] Host response biomarker targets

[0175] In some embodiments, detection of MPX clade II viruses is accompanied by detection of one or more host response biomarkers. As used herein, a host response biomarker refers to a biological compound, such as a polynucleotide or polypeptide, that is differentially expressed in samples taken from subjects with bacterial infection, viral infection, or non-infectious causes of fever, or in samples taken from subjects with both bacterial and viral infections, compared to comparable samples taken from control subjects (e.g., humans diagnosed negative, normal or healthy subjects, or uninfected subjects). A biomarker can be a detectable and / or quantifiable nucleic acid, nucleic acid fragment, polynucleotide, or oligonucleotide. Biomarkers for bacterial infection, viral infection, and non-infectious causes of fever include polynucleotides comprising nucleotide sequences from genes or RNA transcripts of genes, including but not limited to those described in U.S. Patent Application Publication No. 2022 / 0298572, which is incorporated herein by reference for this description.

[0176] Typically, primers for host biomarker targets are designed to avoid amplification of human genomic DNA (hgDNA) by having one of the primers span an exon-exon junction, or by having the amplicon span an exon-exon junction and rely on long introns, making PCR amplification of hgDNA unfavorable. In one exemplary design, the genomic sequence of the target nucleic acid is obtained from NCBI (National Center for Biotechnology Information), with the positions of exons and introns annotated and the presence of multiple transcript variants noted. Exon-exon junctions are selected, and ideally, introns are at least 2 kb in length, so that amplification of genomic sequence is less likely when using short (30 second) PCR extension times. Whenever possible, oligonucleotides are designed to minimize primer artifacts. General guidelines for primer design typically include the following: an amplicon length of less than 150 bp, a primer length of approximately 18-25 bp, a primer spanning the exon-exon junction and having fewer than 5 bases 3' to the second exon, and a primer with a T m : 60-65°C, hairpin T m s should preferably be no higher than 40-45°C for the forward primer and about 35°C for the reverse primer (below 30°C is ideal for both), self- and heterodimers should generally not be more negative than a ∆G (kcal / mol) of -9, and primers should not have more negative ∆G (kcal / mol) than -7 near their 3' end.

[0177] One, two, three, four, five, six or more host response biomarkers can be detected by nucleic acid amplification-based methods, such as those described herein. The test results can be used to determine whether the subject has a bacterial infection, a viral infection, both a viral infection and a bacterial infection, or a non-infectious cause of fever. U.S. patent application publication number 2022 / 0298572 describes these biomarkers and methods for performing such determinations in detail and is incorporated herein by reference for purposes of this description. Specifically, U.S. patent application publication number 2022 / 0298572 describes the determination of a bacterial infection score and / or a viral infection score, which is compared to a predetermined cutoff value to determine (respectively) whether the subject has a bacterial infection and / or whether the subject has a viral infection.

[0178] Primer and optional probe considerations for detecting host response biomarkers are no different than those for detecting MPX clade II group biomarkers, and such considerations are described in more detail below.

[0179] comparison

[0180] In some embodiments, the assays described herein include detecting the aforementioned MPX Clade II biomarkers and at least one endogenous control. In some embodiments, the endogenous control is a sample adequacy control (SAC). The SAC ensures that the sample contains human cells or human DNA. The assay includes primers and probes for detecting single copies of human genes. Only SAC signals are considered when the sample is negative for both MPX and OPX analytes. A negative SAC indicates the absence of human cells in the sample due to insufficient sample mixing or inadequate sample collection. In some such embodiments, if both the MPX Clade II biomarker and the SAC are not detected in the sample, the assay result is considered "invalid" because the sample may have been insufficient. While not wishing to be bound by any particular theory, insufficient samples may be too dilute, contain too little cellular material, or contain assay inhibitors. In some embodiments, the failure to detect the SAC may indicate assay reaction failure. In some embodiments, the endogenous control is RNA (such as mRNA, tRNA, ribosomal RNA, etc.). Non-limiting exemplary endogenous controls include ABL mRNA, GUSB mRNA, GAPDH mRNA, TUBB mRNA, and UPK1a mRNA.

[0181] In some embodiments, the assays described herein include detecting the aforementioned MPX clade II biomarkers and at least one exogenous control. In some embodiments, the exogenous control is a sample processing control (SPC). The SPC ensures that the sample was properly processed. The SPC verifies that sample processing was adequate. Furthermore, this control detects sample-related inhibition of real-time PCR assays, ensuring that PCR reaction conditions (temperature and time) are appropriate for the amplification reaction and that the PCR reagents are functional. The SPC should be positive in a negative sample and can be either negative or positive in a positive sample. If the SPC meets the validated acceptance criteria, the SPC is considered passed. In some such embodiments, if the aforementioned MPX clade II biomarkers are not detected in the sample and the SPC is also not detected in the sample, the assay result is considered "invalid" due to the possibility of an error in sample processing, including, but not limited to, assay failure. Non-limiting examples of errors in sample processing include inadequate sample processing, the presence of assay inhibitors, the presence of nucleases (such as RNases), or damaged reagents. In some embodiments, an exogenous control (such as an SPC) is added to the sample. In some embodiments, an exogenous control (such as an SPC) is added during an assay, for example, using one or more buffers or reagents. In some embodiments, when a GeneXpert® system is to be used, an SPC is included in a GeneXpert® cartridge. In some embodiments, an exogenous control (such as an SPC) is Armored RNA®, which is protected by a bacteriophage coat.

[0182] In some embodiments, an endogenous control and / or an exogenous control are detected simultaneously with detection of an MPX clade II biomarker, such as in the same assay. In some embodiments, an assay comprises reagents for simultaneously detecting the aforementioned MPX clade II biomarker and the SAC and / or the exogenous control in the same assay reaction mixture. In some such embodiments, for example, the assay reaction mixture comprises a primer set for amplifying the aforementioned MPX clade II biomarker, a primer set for amplifying the SAC, and / or a primer set for amplifying the exogenous control, and optionally a labeled probe (such as, for example, a TaqMan® probe) for detecting the amplification products.

[0183] In some embodiments, the assay includes a probe check control (PCC). In some such embodiments, before the PCR reaction begins, the system (e.g., GeneXpert system) measures the fluorescent signal from the probe to monitor bead rehydration, reaction tube filling, probe integrity, and dye stability. If it meets validated acceptance criteria, the PCC passes.

[0184] polynucleotides

[0185] In some embodiments, polynucleotides for detecting the above-mentioned biomarkers are provided. In some embodiments, synthetic polynucleotides are provided. As used herein, a synthetic polynucleotide refers to a polynucleotide that has been chemically or enzymatically synthesized in vitro. Chemical synthesis of polynucleotides includes, but is not limited to, synthesis using a polynucleotide synthesizer, such as OligoPilot™ (GE Healthcare), ABI 3900 DNA Synthesizer (Applied Biosystems), and the like. Enzymatic synthesis includes, but is not limited to, production of polynucleotides by enzymatic amplification, such as PCR. Polynucleotides may contain one or more analogs of typical nucleotides (e.g., modified nucleotides).

[0186] In some embodiments, a polynucleotide comprising at least 6, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, In some embodiments, the present invention relates to a region of at least 27, at least 28, at least 29, or at least 30 contiguous nucleotides that are at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical or at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary.

[0187] In various embodiments, exemplary polynucleotides comprise at least: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In various embodiments, polynucleotides comprise less than 200, 150, 100, 50, 40, 30, or 20 nucleotides. In various embodiments, exemplary polynucleotides have a length of between 6 and 200 nucleotides, between 8 and 200 nucleotides, between 8 and 150 nucleotides, between 8 and 100 nucleotides, between 8 and 75 nucleotides, between 8 and 50 nucleotides, between 8 and 40 nucleotides, between 8 and 30 nucleotides, between 15 and 100 nucleotides, between 15 and 75 nucleotides, between 15 and 50 nucleotides, between 15 and 40 nucleotides, or between 15 and 30 nucleotides.

[0188] In some embodiments, the detection of each target nucleic acid can be carried out using a primer or probe with a single label specific for each target nucleic acid. Different primers and / or probes can have the same label. By using primers or probes labeled with different detectable parts (e.g., different fluorescent reporter dyes), many target nucleic acids can be detected simultaneously in a single reaction mixture. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more different labels can be used in a single reaction mixture or multiple reaction mixtures. Each target nucleic acid can be monitored independently using such multiplexing technology. In some embodiments, a single labeled primer or probe can be used to detect multiple target nucleic acids. A melting curve can be generated to distinguish two or more target nucleic acids each using the same label, but such analysis may not be necessary.

[0189] Polynucleotide modification

[0190] In some embodiments, the methods for detecting at least one target nucleic acid described herein use one or more modified polynucleotides, such as polynucleotides comprising one or more affinity-enhanced nucleotide analogs. Modified polynucleotides useful in the methods described herein include primers for reverse transcription, PCR amplification primers, and probes. In some embodiments, the incorporation of affinity-enhanced nucleotides increases the binding affinity and specificity of the polynucleotide for its target nucleic acid compared to a polynucleotide containing only typical deoxyribonucleotides, which allows the use of shorter polynucleotides or a region of complementarity between a shorter polynucleotide and a target nucleic acid.

[0191] In some embodiments, affinity-enhanced nucleotide analogs include nucleotides containing one or more base modifications, sugar modifications, and / or backbone modifications. In some embodiments, modified bases used in affinity-enhanced nucleotide analogs include 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine, xanthine, and hypoxanthine. In some embodiments, affinity-enhanced nucleotide analogs include nucleotides with modified sugars, such as 2'-substituted sugars, such as 2'-O-alkylribose, 2'-amino-deoxyribose, 2'-fluoro-deoxyribose, 2'-fluoro-arabinose, and 2'-O-methoxyethylribose (2'MOE). In some embodiments, the modified sugar is arabinose or d-arabinose-type hexolose.

[0192] In some embodiments, affinity-enhanced nucleotide analogs include backbone modifications, such as the use of peptide nucleic acids (PNA; e.g., oligomers comprising nucleobases linked together by an amino acid backbone). Other backbone modifications include phosphorothioate linkages, phosphodiester-modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids, methylphosphonates, alkylphosphonates, phosphates, alkylthiophosphonates, phosphoamides, carbamates, carbonates, phosphotriesters, acetamidates, carboxymethyl esters, methylphosphorothioates, phosphorodithioates, p-ethoxy modifications, and combinations thereof.

[0193] In some embodiments, a polynucleotide includes at least one affinity-enhanced nucleotide analog with a modified base, at least one nucleotide (which can be the same nucleotide) with a modified sugar, and / or at least one non-naturally occurring internucleotide linkage.

[0194] In some embodiments, the affinity-enhanced nucleotide analogs contain a locked nucleic acid ("LNA") sugar, which is a bicyclic sugar. In some embodiments, the polynucleotides used in the methods described herein include one or more nucleotides having an LNA sugar. In some embodiments, the polynucleotides contain one or more regions consisting of nucleotides having an LNA sugar. In other embodiments, the polynucleotides contain nucleotides having an LNA sugar interspersed with deoxyribonucleotides. See, e.g., Frieden, M. et al. (2008) Curr. Pharm. Des. 14(11): 1138-1142.

[0195] Primers

[0196] In some embodiments, polynucleotide is a primer. Useful primers in the methods described herein are generally capable of selectively hybridizing to: genomic DNA, target RNA (genome or transcript), cDNA reverse transcribed from the target RNA, and / or amplicons amplified by genomic DNA, target RNA or cDNA (collectively referred to as "template"), and in the presence of template, polymerase and suitable buffer and reagent, they can be extended to form primer extension products. Primers generally have enough length to ensure selective hybridization to their target nucleic acid. Generally, primers with a length of at least 15 nucleotides specifically hybridize in most cases, and this length can be shortened, for example, by including modifications that enhance affinity, such as modifications discussed above. But primers may not necessarily be fully complementary to their target nucleic acid. Primers can have any degree of complementarity described above for exemplary polynucleotides. In exemplary embodiments, a primer can be 8 to 40 nucleotides in length and at least 90% complementary to its target nucleic acid; 8 to 40 nucleotides in length and at least 95% complementary to its target nucleic acid; 8 to 40 nucleotides in length and at least 99% complementary to its target nucleic acid; 8 to 30 nucleotides in length and at least 90% complementary to its target nucleic acid; 8 to 30 nucleotides in length and at least 95% complementary to its target nucleic acid; and 8 to 30 nucleotides in length and at least 99% complementary to its target nucleic acid. In embodiments where the primer has less than 100% complementarity with its target nucleic acid, making the 3' nucleotide of the primer complementary to its target nucleic acid facilitates the generation of extension products.

[0197] In some embodiments, a primer that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with an affinity that is at least 5-fold greater than that of a non-target nucleic acid under the same assay conditions. In some embodiments, a primer that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with an affinity that is at least 10-fold greater than that of a non-target nucleic acid under the same assay conditions.

[0198] In some embodiments, primer pairs are designed to produce amplicons of 50 to 1500 nucleotides in length, 50 to 1000 nucleotides in length, 50 to 750 nucleotides in length, 50 to 500 nucleotides in length, 50 to 400 nucleotides in length, 50 to 300 nucleotides in length, 50 to 200 nucleotides in length, 50 to 150 nucleotides in length, 100 to 300 nucleotides in length, 100 to 200 nucleotides in length, or 100 to 150 nucleotides in length.

[0199] In some embodiments, the primer is labeled with a detectable moiety. In some embodiments, the primer is unlabeled.

[0200] probe

[0201] In some embodiments, the polynucleotide is a probe. Probes useful in the methods described herein are generally capable of selectively hybridizing to: genomic DNA, target RNA (genomic or transcript), cDNA reverse-transcribed from target RNA, and / or amplicons that have been amplified from genomic DNA, target RNA, or cDNA (collectively, "templates"). Generally, probes of at least 15 nucleotides in length hybridize specifically in most situations, and this length can be shortened, for example, by including affinity-enhancing modifications, such as those discussed above. However, a probe need not be completely complementary to its target nucleic acid. The probe can have any degree of complementarity described above for the exemplary polynucleotides. In exemplary embodiments, the probe can be 8 to 40 nucleotides in length and at least 90% complementary to its target nucleic acid; 8 to 40 nucleotides in length and at least 95% complementary to its target nucleic acid; 8 to 40 nucleotides in length and at least 99% complementary to its target nucleic acid; 8 to 30 nucleotides in length and at least 90% complementary to its target nucleic acid; 8 to 30 nucleotides in length and at least 95% complementary to its target nucleic acid; and 8 to 30 nucleotides in length and at least 99% complementary to its target nucleic acid. In embodiments where the primer is less than 100% complementary to the target nucleic acid, any non-complementary points or regions are typically located so as not to disrupt the ability of the probe to selectively hybridize to its target nucleic acid.

[0202] In some embodiments, a probe that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with an affinity that is at least 5-fold greater than that of non-target nucleic acids under the same assay conditions. In some embodiments, a probe that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with an affinity that is at least 10-fold greater than that of non-target nucleic acids under the same assay conditions.

[0203] Polynucleotide markers

[0204] In some embodiments, primer or probe are used detectable moiety mark.Detectable moiety comprises the part of direct detection, such as fluorescent dye, and indirectly detectable part, such as in conjunction with right member.In some embodiments, when detectable moiety is in conjunction with right member, by probe and in conjunction with the detectable label of second right member combination hatch together, probe can be detected.In some embodiments, primer or probe are not labeled, such as when primer or probe are fixed on for example microarray or bead.The primer of labeling is extendable, for example, by polymerase.In some embodiments, probe is extendable.In other embodiments, probe is non-extendable.The following discussion concentrates on the probe, because these are more commonly used in the detection in the method described herein, but those skilled in the art understand that the polynucleotide labeling strategy described below is equally applicable to the labeling of primer.

[0205] In some embodiments, the probe is a FRET probe. In some embodiments, the FRET probe is labeled with a fluorescent dye (donor) at the 5' end and a quencher (acceptor) at the 3' end. A quencher is a chemical group that absorbs (i.e., suppresses) fluorescence emission from the dye when the groups are in close proximity (e.g., attached to the same probe). Therefore, in some embodiments, the emission spectrum of the dye should overlap significantly with the absorption spectrum of the quencher. In other embodiments, the dye and quencher are not located at the ends of the FRET probe.

[0206] Exemplary FRET probes include, but are not limited to, TaqMan® probes, molecular beacon probes, and scorpion probes. The TaqMan® probe is a linear probe that typically has a fluorescent dye covalently bound to one end of the DNA and a quencher molecule covalently bound elsewhere, such as the other end of the DNA. The FRET probe comprises a sequence complementary to a region of the cDNA or amplicon, such that when the FRET probe hybridizes to the cDNA or amplicon, the dye fluorescence is quenched; when the probe is digested during the cDNA or amplicon amplification process, the dye is released from the probe and a fluorescent signal is generated. In some embodiments, the amount of target nucleic acid in the sample is proportional to the amount of fluorescence measured during the amplification process.

[0207] Similar to TaqMan® probes, molecular beacons use FRET to detect PCR products by attaching a fluorescent dye and a quencher to the probe end. Unlike TaqMan® probes, molecular beacons remain intact during PCR cycling. When the molecular beacon probe is free in solution, it forms a stem-loop structure, bringing the dye and quencher into close proximity to cause fluorescence quenching. When the molecular beacon hybridizes to the target nucleic acid, the stem-loop structure is disrupted, spatially separating the dye and quencher, and the dye emits fluorescence. Molecular beacons are available, for example, from Gene Link™ (see www.genelink.com / newsite / products / mbintro.asp).

[0208] In some embodiments, scorpion probes can be used as sequence-specific primers and for PCR product detection. Similar to molecular beacons, scorpion probes form a stem-loop structure when not hybridized to a target nucleic acid. However, unlike molecular beacons, scorpion probes achieve both sequence-specific priming and PCR product detection. A fluorescent dye molecule is attached to the 5' end of the scorpion probe, and a quencher is attached elsewhere, such as to the 3' end. The 3' portion of the probe is complementary to the extension product of the PCR primer, and this complementary portion is connected to the 5' end of the probe via a non-amplifiable portion. After the scorpion primer is extended, the probe's target-specific sequence binds to its complementary sequence within the extended amplicon, thereby opening the stem-loop structure and causing the dye on the 5' end to fluoresce and generate a signal. Scorpion probes are available, for example, from Premier Biosoft International (see www.premierbiosoft.com / tech_notes / Scorpion.html).

[0209] In some embodiments, labels that can be used on FRET probes include colorimetric dyes and fluorescent dyes, such as Alexa Fluor dyes; BODIPY dyes, such as BODIPY FL, Cascade Blue, and Cascade Yellow; coumarins and their derivatives, such as 7-amino-4-methylcoumarin, aminocoumarins, and hydroxycoumarins; cyanine dyes, such as Cy3 and Cy5; eosin and erythrosine; fluorescein and its derivatives, such as fluorescein isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum Dye™; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red, tetramethylrhodamine, and rhodamine 6G; Texas Red; fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer; and TOTAB.

[0210] Specific examples of dyes include, but are not limited to, the dyes described above and the following dyes: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750; amine-reactive BODIPY dyes such as BODIPY 493 / 503, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 655, BODIPY FL, BODIPY R6G, BODIPY TMR, and BODIPY-TR; Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, 2',4',5',7'-tetrabromosulfonylfluorescein, and TET.

[0211] Examples of dye / quencher pairs (i.e., donor / acceptor pairs) include, but are not limited to, fluorescein / tetramethylrhodamine; IAEDANS / fluorescein; EDANS / dabcyl; fluorescein / fluorescein; BODIPY FL / BODIPY FL; and fluorescein / QSY 7 or QSY 9 dyes. When the donor and acceptor are the same, in some embodiments, FRET can be detected by fluorescence depolarization. Some specific examples of dye / quencher pairs (i.e., donor / acceptor pairs) include, but are not limited to, Alexa Fluor 350 / Alexa Fluor 488; Alexa Fluor 488 / Alexa Fluor 546; Alexa Fluor 488 / Alexa Fluor 555; Alexa Fluor 488 / Alexa Fluor 568; Alexa Fluor 488 / Alexa Fluor 594; Alexa Fluor 488 / Alexa Fluor 647; Alexa Fluor 546 / Alexa Fluor 568; Alexa Fluor 546 / Alexa Fluor 594; Alexa Fluor 546 / Alexa Fluor 647; Alexa Fluor 555 / Alexa Fluor 594; Alexa Fluor 555 / Alexa Fluor 647; Alexa Fluor 568 / Alexa Fluor 647; Alexa Fluor 594 / Alexa Fluor 647; Alexa Fluor 350 / QSY35; Alexa Fluor Alexa Fluor 555 / QSY 7 or QSY 9; Alexa Fluor 568 / QSY 7 or QSY 9; Alexa Fluor 568 / QSY 21; Alexa Fluor 594 / QSY 21; and Alexa Fluor 647 / QSY 21. In some cases, the same quencher can be used for multiple dyes, for example, a broad-spectrum quencher such as Iowa Black® quencher (Integrated DNA Technologies, Coralville, IA) or Black Hole Quencher™ (BHQ™; Sigma-Aldrich, St. Louis, MO).

[0212] Specific examples of fluorescently labeled ribonucleotides useful in the preparation of probes for use in certain embodiments of the methods described herein are available from Molecular Probes (Invitrogen), including Alexa Fluor 488-5-UTP, fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP. Other fluorescent ribonucleotides are available from Amersham Biosciences (GE Healthcare), such as Cy3-UTP and Cy5-UTP.

[0213] Specific examples of fluorescently labeled deoxyribonucleotides useful in preparing probes for use in the methods described herein include dinitrophenyl (DNP)-1′-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, rhodamine green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY Fluorescently labeled nucleotides are commercially available and can be purchased from, for example, Invitrogen.

[0214] As described above, exemplary detectable moieties also include members of binding pairs. Exemplary binding pairs include, but are not limited to, biotin and streptavidin, antibodies and antigens, and the like.

[0215] sample

[0216] The sample to be tested can be any sample suspected of containing at least one of the MPX clade II biomarkers described herein. In some embodiments, the sample is a biological sample collected from a subject. In other embodiments, the sample is not collected directly from a subject, such as, for example, a wastewater sample or a sample from an air filter in a building.

[0217] Exemplary biological samples include skin samples, lesion swabs, vesicular lesion fluid samples, pustular lesion fluid samples, rectal samples, and body fluid samples, such as nasal aspirates, nasal washes, nasal swabs, nasopharyngeal swabs, saliva, oropharyngeal swabs, throat swabs, bronchoalveolar lavage fluid samples, bronchial aspirates, bronchial washes, endotracheal aspirates, endotracheal washes, tracheal aspirates, nasal secretion samples, mucus samples, sputum samples, plasma samples, whole blood samples, and the like.

[0218] In some embodiments, the sample to be tested is fresh (i.e., never frozen). In other embodiments, the sample is a frozen specimen. In some embodiments, the sample is a tissue sample, such as a formalin-fixed paraffin-embedded sample. In some embodiments, the sample is a liquid cytology sample.

[0219] In some embodiments, the sample to be tested is contacted with a buffer after collection. For example, in the case of a skin sample, a lesion swab, a blister lesion fluid sample, a pustular lesion fluid sample, or a rectal sample, a buffer (including, for example, a preservative) may be added to the sample. In embodiments where the sample is a swab sample, the swab may simply be placed in the buffer. In some embodiments, the sample is immediately contacted with the buffer; in the case of a swab, the swab is immediately placed in the buffer. In some embodiments, the sample (e.g., including the swab) is contacted with the buffer within 5 minutes, 10 minutes, 30 minutes, 1 hour, or 2 hours of sample collection.

[0220] In some embodiments, less than 5 ml, less than 4 ml, less than 3 ml, less than 2 ml, less than 1 ml, or less than 0.75 ml of sample or buffered sample is used in the current method. In some embodiments, 0.1 ml to 1 ml of sample or buffered sample is used in the current method.

[0221] Subjects

[0222] Biological samples useful in the methods described herein can be collected from any subject that may be infected with one, several, or all of the above-mentioned viruses. In various embodiments, the subject can include non-human animals, such as canines, felines, equines, primates, and other non-human mammals, as well as humans.

[0223] In some embodiments, the test sample is obtained from an individual who has one or more symptoms of infection with an orthopoxvirus, such as MPX, or has been exposed to such an individual. The primary symptoms of MPX virus are fever, headache, myalgia, lymphadenopathy, and fatigue, followed by a vesicular-pustular rash that lasts about 2-3 weeks. In some such embodiments, the individual is monitored for recurrence of an orthopoxvirus, such as MPX.

[0224] In some embodiments, the methods described herein can be used for routine screening of apparently healthy individuals without risk factors. In some embodiments, the methods described herein are used to screen asymptomatic individuals, for example, during routine or preventive healthcare. In some embodiments, the methods described herein are used to screen women who are pregnant or attempting to become pregnant.

[0225] In some embodiments, the methods described herein can be used to assess the effectiveness of a treatment in an individual undergoing treatment for a disorder caused by an orthopoxvirus (eg, MPX).

[0226] Determination method

[0227] Any analytical process capable of achieving specific detection of a target nucleic acid can be used in the methods proposed herein. In some embodiments, DNA targets can be detected by direct hybridization, or more easily by amplifying a DNA template and detecting the amplicon. In some embodiments, RNA targets can be detected by direct hybridization, or more easily by reverse transcribing the target RNA to produce a cDNA complementary to the target RNA. The cDNA can be directly detected by direct hybridization or by amplifying a cDNA template.

[0228] Nucleic acid amplification provides rapid, sensitive, and specific detection of nucleic acid targets and has been used in a wide variety of assay formats to detect nucleic acid targets. Those skilled in the art, following the guidance herein, can perform the methods described herein in any number of different nucleic acid amplification-based assays, using, for example, any of the nucleic acid amplification methods discussed above. Such methods may, but need not, require thermal cycling, as is the case with isothermal amplification. Exemplary methods include, but are not limited to, isothermal amplification, real-time RT-PCR, endpoint RT-PCR, and amplification from a T7 promoter annealed to DNA using T7 polymerase, such as provided by the SenseAmp Plus™ kit available from Implen, Germany. Amplification and detection can be performed in solution or on a solid support (e.g., a biochip). Nucleic acid amplification-based assays can employ a single reaction chamber or multiple reaction chambers. Amplification can be nested or non-nested. In some embodiments, detection comprises electrochemical detection.

[0229] In some embodiments, target nucleic acids, such as MPX clade II biomarkers (as well as clade I and other orthopoxvirus biomarkers), optional host biomarkers, and / or optional controls can be detected by: (a) contacting nucleic acid from a sample with a set of primers and optional probes to detect the presence of the desired target nucleic acid, (b) subjecting the nucleic acid, primers, and optional probes to amplification conditions; (c) optionally detecting the presence of any (multiple) amplification products by real-time PCR, melting curve analysis, or a combination thereof, and (d) differentially confirming the presence of a viral pathogen in the sample, or determining the absence of a viral pathogen detectable using the set of primers, based on the detection or absence of the (multiple) amplification products, respectively. In this context, "differentially confirming" refers to the ability to determine the presence of a particular target organism and the absence of one or more other target organisms being assayed. In some embodiments, an assay is capable of determining the presence of any target organism present in a sample while excluding the presence of other target organisms (above the detection limit of the assay).

[0230] In some embodiments of amplification by polymerase chain reaction (PCR), exemplary cycles include an initial denaturation at 90°C to 100°C for 20 seconds to 5 minutes, followed by cycles of denaturation at 90°C to 100°C for 1 to 10 seconds, followed by annealing and amplification at 60°C to 75°C for 10 to 40 seconds. Further exemplary cycles include 20 seconds at 94°C, followed by up to 3 cycles of 1 second at 95°C and 35 seconds at 62°C, 20 cycles of 1 second at 95°C and 20 seconds at 62°C, and 14 cycles of 1 second at 95°C and 35 seconds at 62°C. In some embodiments, the cyclic denaturation step is omitted for the first cycle after the initial denaturation step. In some embodiments, Taq polymerase is used for amplification. In some embodiments, the cycle is carried out at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times or at least 45 times. In some embodiments, Taq with a hot start function is used. In some embodiments, from initial denaturation through final extension, detection of the target nucleic acid occurs in less than 3 hours, less than 2.5 hours, less than 2 hours, less than 1 hour or less than 30 minutes. In some embodiments, by comprising a method for real-time quantitative PCR, for example, using FRET probes, such as those mentioned above, to detect the target nucleic acid.

[0231] In some embodiments, quantification of real-time PCR assay results is accomplished by constructing a standard curve from nucleic acids of known concentrations and then extrapolating the quantitative information for target nucleic acids of unknown concentrations. In some embodiments, the nucleic acid used to generate the standard curve is DNA (e.g., an endogenous control or an exogenous control). In some embodiments, the nucleic acid used to generate the standard curve is purified double-stranded plasmid DNA or single-stranded DNA generated in vitro.

[0232] In some embodiments, in order for an assay to indicate that a given target nucleic acid is not present in a sample, the Ct value of an endogenous control (such as SAC) and / or an exogenous control (such as SPC) must be within a previously determined valid range. For example, in some embodiments, the absence of a particular target nucleic acid cannot be confirmed unless the control is detected, and detection of the control indicates that the assay was successful.

[0233] In some embodiments, a threshold Ct (or "cutoff Ct") value has been previously determined for the target nucleic acid (including an endogenous control and / or an exogenous control), below which a gene is considered detected. In some embodiments, the threshold Ct is determined using assay conditions and systems that are substantially the same as those used to test the sample (such as GeneXpert®).

[0234] Any PCR instrument available in the art can be used for real-time PCR. Typically, the instrument used for real-time PCR data collection and analysis includes a thermal cycler, an optical system for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.

[0235] In some embodiments, the number of target nucleic acids in an assay exceeds the number of labels that can be detected, for example, in a particular instrument. Thus, PCR amplification can be followed by a melting analysis to increase the number of possible reportable results. In general, target organisms requiring high sensitivity, such as viruses, can be detected by real-time PCR using TaqMan probes or molecular beacon probes or by melting analysis.

[0236] Another method for detecting target nucleic acids can involve only high-resolution melting. For example, nested multiplex PCR can be performed by first performing reverse transcription, followed by multiple first-stage PCR reactions (PCR1). Multiple simultaneous second-stage PCR reactions (PCR2) are then performed in an array to amplify sequences within the PCR1 products. Endpoint melting curve data can then be run to detect target nucleic acids and analyzed to generate results for each analyte.

[0237] Target nucleic acids can also be detected by real-time PCR but in more than one reaction chamber. Another method for detecting target nucleic acids can include digital microfluidics or electrowetting and electrochemical detection. For example, digital microfluidics or electrowetting can be used to move and transfer samples and reagents within the box. A system for such operation can include a microarray for detection, which consists of target-specific capture probes connected to gold electrodes (solid supports) that generate a voltage signal if the "target DNA / signal probe" hybridizes with the capture probe. Target nucleic acids can also be detected using a chip comprising an integrated sensor array.

[0238] Examples of other methods that can be used for the methods described herein include bead-based flow cytometry assays. See Lu J. et al. (2005) Nature 435: 834-838, which is incorporated herein by reference for this description. An example of bead-based flow cytometry assays is Luminex, Inc's xMAP® technology. See www.luminexcorp.com / technology / index.html. Another approach uses microfluidic devices and single-molecule detection. See U.S. Patent Nos. 7,402,422 and 7,351,538 to Fuchs et al. of US Genomics, Inc., each of which is incorporated herein by reference in its entirety. Yet another approach is simple gel electrophoresis and detection with labeled probes (e.g., probes labeled with radioactive or chemiluminescent markers), such as by northern blotting.

[0239] In some embodiments, the method for detecting a target nucleic acid does not include bead-based flow cytometry assays, microfluidic devices and single molecule detection, simple gel electrophoresis, use of capture probes attached to a solid support, separation of the reaction mixture into multiple reaction chambers, array-based detection, nested amplification, electrochemical detection, high-resolution melting alone, or a combination thereof.

[0240] Automated measurement method

[0241] Methods that are easy to automate are of great interest. The methods described herein can be performed in a substantially automated manner using commercially available nucleic acid amplification systems. Exemplary non-limiting nucleic acid amplification systems that can be used to implement the methods of the present invention include the GENEXPERT® system, the GENEXPERT® Infinity system, and the GENEXPERT® Xpress system (Cepheid, Sunnyvale, California). In some embodiments, the amplification system can be available at the same location as the individual to be tested, such as a healthcare provider's office, clinic, or community hospital, so that processing is not delayed by transporting the sample to another facility. Using an automated system, such as the GENEXPERT® system, determinations according to the methods described herein can be completed within 3 hours, in some embodiments within 2 hours, in some embodiments within 1 hour, in some embodiments within 45 minutes, in some embodiments within 35 minutes, and in some embodiments within 30 minutes. GENEXPERT® uses an independent disposable box. Sample extraction, amplification, and detection can all be performed in this independent sample box as described herein.

[0242] In some embodiments, after adding a sample to the cartridge, the sample is contacted with a lysis buffer, and the released nucleic acids (NAs) bind to a NA-binding substrate, such as a silica or glass substrate. The sample supernatant is then removed, and the NAs are eluted in an elution buffer, such as a Tris / EDTA buffer. The eluate can then be processed in the cartridge to detect target nucleic acids as described herein. In some embodiments, the eluate is used to reconstitute at least some of the PCR reagents present in the cartridge as lyophilized particles.

[0243] A cassette having multiple chambers may have the set of primers and optional probes described herein, or a subset thereof, disposed in one chamber. In some embodiments, the set of primers and optional probes described herein, or a subset thereof, are disposed in more than one of the multiple chambers.

[0244] In some embodiments, RT-PCR is used to amplify and analyze the presence of target nucleic acids. In some embodiments, reverse transcription uses MMLV and / or CAT-A RT enzymes and is incubated at 40°C to 50°C for 5 to 20 minutes. In some embodiments, PCR uses a Taq polymerase with a hot start function, such as AptaTaq (Roche). In some embodiments, initial denaturation is performed at 90°C to 100°C for 20 seconds to 5 minutes; cycling denaturation at 90°C to 100°C for 1 to 10 seconds; cycling annealing and amplification at 60°C to 75°C for 10 to 40 seconds; and up to 50 cycles are performed.

[0245] In some embodiments, a double denaturation approach is used to amplify low-copy number target nucleic acids. In some embodiments, the double denaturation approach includes a first denaturation step followed by the addition of primers and / or probes for detecting the target nucleic acid. Then, in some cases, all or most of the nucleic acid-containing sample (such as a DNA eluate) is subjected to a second denaturation step before a portion of the sample is aliquoted for circulation and detection of the target nucleic acid. While not wishing to be bound by any particular theory, a double denaturation approach can increase the chance that the low-copy number target nucleic acid (or its complement) will be present in the aliquot selected for circulation and detection because the second denaturation effectively doubles the amount of target nucleic acid (i.e., it separates the target nucleic acid and its complement into two separate templates) before the aliquot is selected for circulation. In some embodiments, the first denaturation step involves heating to a temperature of 90°C to 100°C for a total time of 30 seconds to 5 minutes. In some embodiments, the second denaturation step involves heating to a temperature of 90°C to 100°C for a total time of 5 seconds to 3 minutes. In some embodiments, the first and / or second denaturation steps are performed by heating aliquots of the sample separately. In some embodiments, each aliquot can be heated for the times listed above. As a non-limiting example, a first denaturation step for a NA-containing sample (such as a DNA eluate) can include heating at least one, at least two, at least three, or at least four aliquots of the sample separately (either sequentially or simultaneously) to a temperature of 90°C to 100°C for 60 seconds each. As a non-limiting example, a second denaturation step for a NA-containing sample (such as a DNA eluate) containing an enzyme, primers, and a probe can include heating at least one, at least two, at least three, or at least four aliquots of the eluate separately (either sequentially or simultaneously) to a temperature of 90°C to 100°C for 5 seconds each. In some embodiments, the aliquot is the entire NA-containing sample (such as a DNA eluate). In some embodiments, the aliquot is less than the entire NA-containing sample (such as a DNA eluate).

[0246] In some embodiments, for example, for samples with low cell content, offline centrifugation is employed. The sample, with or without the addition of buffer, is centrifuged and the supernatant removed. The pellet is then resuspended in a smaller volume of supernatant or buffer. The resuspended pellet is then analyzed as described herein.

[0247] Exemplary Automation and Systems

[0248] Many existing fully integrated nucleic acid amplification and testing systems capable of sample preparation are often complex and costly. The nucleic acid amplification and testing system provided herein enables rapid, simple, convenient, and affordable nucleic acid analysis.

[0249] System Overview

[0250] In one aspect, the present invention relates to a sample cartridge utilizing a valve body platform that enables detection of both enveloped and free target nucleic acids. In some embodiments, the valve body includes a sample processing region or lysis chamber that provides one or both of mechanical and chemical lysis. This allows a single cartridge to provide lysis for multiple different types of targets, and thus can be considered a "group assay cartridge." In some embodiments, the sample cartridge can process and detect viral targets suitable for chemical lysis.

[0251] The sample box device can be any device configured to perform one or more processing steps related to the preparation and / or analysis of a biological fluid sample according to any of the methods described herein. In some embodiments, the sample box device is configured to at least perform sample preparation. The sample box can also be further configured to perform additional processes, such as detecting a target nucleic acid in a nucleic acid amplification test (NAAT), such as a polymerase chain reaction (PCR) assay, by using a reaction vessel connected to the sample box. In some embodiments, the reaction vessel extends from the body of the box. Preparation of a fluid sample typically involves a series of processing steps, which may include chemical, electrical, mechanical, thermal, optical, or acoustic processing steps depending on the particular protocol. Such steps can be used to perform various sample preparation functions, such as cell capture, cell lysis, binding of analytes, and binding of unwanted materials.

[0252] Sample cartridges suitable for use with the present invention include one or more delivery ports through which a prepared fluid sample can be delivered to a connected reaction vessel for analysis. Figure 1Figure A illustrates an exemplary panel assay cartridge 100 suitable for sample preparation and analytical testing via PCR when received in an instrument module, according to some embodiments. The sample cartridge is connected to a reaction vessel 116 (also referred to as a "reaction tube" or "PCR tube") suitable for analyzing a fluid sample processed within the sample cartridge 100. In some embodiments, the reaction vessel extends from the cartridge body. Such a sample cartridge 100 comprises various components, including a main housing 102 with one or more chambers 108 for processing the fluid sample, which typically includes sample preparation prior to analysis. In these embodiments, the sample cartridge can be a fully integrated nucleic acid amplification and testing system that combines sample preparation, amplification, and detection. The instrument module facilitates the processing steps required for sample preparation, and the prepared sample is delivered via one of a pair of delivery ports to a fluid conduit connected to the reaction vessel 116 of the sample cartridge 100 housing. The prepared biological fluid sample is then delivered to the reaction chamber of the reaction vessel, where it undergoes nucleic acid amplification. In some embodiments, the amplification is a polymerase chain reaction. In some embodiments, while amplifying the biofluid sample, the excitation tool and optical detection tool of the module are used to detect the target nucleic acid analyte of indication being paid close attention to, such as the light emission of the presence or absence of bacteria, viruses, pathogens, toxins or other target analytes. It should be understood that such a reaction vessel can include various chambers, conduits or micropore arrays for detecting target analytes. The sample box can be equipped with an instrument for preparing the biofluid sample before being transported to the reaction vessel. Any chemical reagent required for virus or cell lysis or the instrument (e.g., reagent beads) for combining or detecting the analyte of interest can be contained in one or more chambers of the sample box, and therefore can be used for sample preparation.

[0253] Exemplary uses of reaction vessels for analyzing biological fluid samples are described in commonly assigned U.S. Patent No. 6,818,185, filed May 30, 2000, entitled "Cartridge for Conducting a Chemical Reaction," which is incorporated herein by reference in its entirety for all purposes. Examples of sample cartridges and associated modules are shown and described in the following patents: U.S. Patent No. 6,374,684, filed on August 25, 2000, entitled “Fluid Control and Processing System,” and U.S. Patent No. 8,048,386, filed on February 25, 2002, entitled “Fluid Processing and Control,” U.S. Patent Application No. 63 / 217,672, filed on July 1, 2021, entitled “Universal Assay Cartridge and Methods of Use,” U.S. Provisional Application No. 63 / 319,993, filed on March 15, 2022, entitled “Unitary Cartridge Body and Associated Components and Methods of Manufacture,” and U.S. Provisional Application No. 63 / 319,993, filed on March 15, 2022, entitled “Molecular Diagnostic Assay System", U.S. Patent No. 10,562,030; the entire contents of which are incorporated herein by reference in their entirety for all purposes. The above patents are included in the attached appendix.

[0254] Various aspects of the sample cartridge 100 may be further understood by reference to U.S. Patent No. 6,374,684 (the "'684 patent"), which describes certain aspects of the sample cartridge in greater detail. Such a sample cartridge may include a fluid control mechanism, such as a rotary fluid control valve assembly, which is fluidically connected to a chamber of the sample cartridge. The term "chamber" may be used interchangeably with terms such as "well," "tube," and the like. Rotation of the rotary fluid control valve allows fluid communication between the chamber and the valve to control the flow of a biological fluid sample deposited in the cartridge into different chambers, where various reagents may be provided as needed according to a specific protocol to prepare the biological fluid sample for analysis. To operate the rotary valve, the cartridge processing module includes a motor, such as a stepper motor, which is typically connected to a transmission system that engages with features of the valve in the sample cartridge to coordinate valve movement with syringe movement, thereby causing the fluid sample to be moved according to a desired sample preparation protocol. The '684 patent describes the fluid metering and dispensing functions of the rotary valve according to a specific sample preparation protocol.

[0255] Exemplary Assay Cartridge and Valve Assemblies

[0256] Overview

[0257] like Figure 1 As shown in Figure A, the group test box 100 includes a box body 102, which includes a plurality of chambers 108 for reagents or buffers and sample processing. The chambers are arranged around a central syringe barrel 106, which is connected to a valve body 110 (see Figure 1 B and 1C) are fluidically connected and sealed with gasket 104. The valve body 110 may include a cover 112, and the entire cartridge body may be supported on the cartridge base 101. The valve body typically contains one or more channels or cavities (chamber(s) 114), which may contain filters as described herein, which can serve to bind and elute nucleic acids. In some embodiments, the cartridge also includes one or more temperature-controlled channels or chambers, which, in certain embodiments, can function as thermal cycling chambers. A "plunger," not shown, can be operated to draw fluid into the syringe barrel 106, and rotation of the valve body 110 provides selective fluid communication between the various reagent chambers 108 and the channels, reaction chamber(s), mixing chamber, and optionally any temperature-controlled zones. Thus, the various reagent chambers 108, reaction chambers, filter(s), and temperature-controlled chambers or channels are selectively fluidically connected by rotation of the plunger, and reagent movement (e.g., chamber loading or unloading) is controlled by the "injection" action of the plunger within the valve assembly. In other embodiments, various reagent chambers, reaction chambers, filter materials, and temperature-controlled chambers or channels are selectively fluidly connected by linear advancement (e.g., by forced motion) of reagents and samples from one chamber to the next.

[0258] Exemplary valve assembly

[0259] Figure 3 illustrates the use of Figure 1Figure 1 shows the different valve assemblies in a sample cartridge (e.g., A). To better illustrate the components of the valve body, each valve assembly is shown inverted. Valve assembly A performs only mechanical lysis and is suitable for lysing recalcitrant targets (e.g., certain bacteria, spores). Valve assemblies B and C perform only chemical lysis and are suitable for lysing less recalcitrant targets (e.g., viruses, free NA, certain spores, certain bacteria, and yeast). Valve assembly D can perform both mechanical and chemical lysis on all target types. In all such cartridges, the valve assembly includes a syringe barrel 106, a valve body 110, and a valve cover 112. The additional capabilities of the sample cartridge's valve assembly depend, in part, on the characteristics of the filter, valve body, and cover, as well as the specific workflow sequence implemented by the module's instrument interface. For example, valve assembly A has a valve body 110 shaped with a circular cavity to support a polymer filter disc 116 for filtering the sample, and a cover 112 with a sonication dome feature 113 that couples to the ultrasonic horn of the cartridge receiving module to ultrasonically lyse the target. In contrast, valve assemblies B and C have a valve body 110 with a rectangular filter recess 111 that receives a glass filter 114 (e.g., a glass column) therein, which is configured to bind to nucleic acids released from the target by chemical lysis. Valve assembly D has a design more similar to valve assembly A, having a cover 112 with an ultrasonication dome 113 and a valve body with a circular cavity for supporting a disc filter, but this design uses a glass fiber filter 117. The use of glass fibers to form the filter promotes affinity binding to free nucleic acids released by chemical lysis. Therefore, this filter is suitable for both mechanical and chemical lysis. The assay cartridge of valve assembly D is further described in detail in U.S. Patent Application Publication No. 2023 / 0044516, which is incorporated herein by reference in its entirety.

[0260] Reaction Module

[0261] In certain embodiments, the cartridge 200 is configured to be inserted into a reaction module 300, e.g., Figure 4 As shown in A. Figure 4As shown in B, the module is configured to receive a box 200 therein. In some embodiments, the reaction module provides a heating plate 308 to heat a temperature-controlled chamber or channel. The module may optionally further include a fan 304 to provide cooling, wherein the temperature-controlled channel or chamber is a thermal cycling channel or chamber. An electronic circuit 302 may be provided to transmit information (e.g., optical information) to a computer for analysis. In some embodiments, the module may include an optical block 306 to provide excitation and / or detection of one or more (e.g., 1, 2, 3, 4 or more) optical signals, which represent, for example, signal DNA amplified for various PCR targets. In various embodiments, an electrical connector 312 may be provided for docking the module with a system (e.g., a system controller) or with a discrete analysis / controller unit. As Figure 4 As shown in B, a sample can be introduced into the cartridge using a pipette 310. In certain embodiments, the module also contains a controller that operates the rotation of the plunger and valve body in the syringe barrel.

[0262] Analysis System

[0263] In certain embodiments, a system (eg, a processing unit) is provided. Figure 4 C shows an illustrative, but non-limiting embodiment. System 400 includes a housing 401, which is configured to support and power a plurality of sample processing modules 300, wherein each processing module is configured to hold and operate a detachable box 100. In some embodiments, the system is configured to operate the sample processing module to perform PCR assays on one or more target nucleic acid analytes and optionally determine the level of one or more target RNA / DNA sequences in the corresponding detachable sample box. Typically, the processing of the sample in the corresponding detachable sample box includes operating the box to perform the method described herein. In certain embodiments, the system is configured to include a sample processing module. In certain embodiments, the system is configured to include at least two or more sample processing modules (e.g., at least 4, 8, 12, 16, 20, 24, 28, 32, 64, 128 or more sample processing modules). In some embodiments, the system provides a user interface that allows a user to input operating instructions and / or monitor the operation of the box to determine the presence and / or quantity of one or more nucleic acids.

[0264] Although the methods described herein are primarily described with reference to the GENEXPERT® kits from Cepheid Inc. (Sunnyvale, CA) (e.g., see Figure 1A), but it will be appreciated that, in view of the teachings provided herein, the methods can be implemented on other cartridges / microfluidic systems, including alternative cartridge designs having a valve assembly including a plurality of docking components and a cartridge body defined by the plurality of docking components to form a plurality of chambers of the cartridge, such as those described in Korean Application Nos. 102293717B1 and KR102362853B1, cartridges that utilize ultrasound to lyse cells in a biological sample, such as those described in International Application Publication No. WO2021 / 245390A1, cartridges and systems utilizing electrowetting grids for droplet operations, and electrical sensor arrays configured to detect analytes of interest, such as those described in International Application Publication No. WO2016 / 077341A2. International applications, cartridges that facilitate the movement of nucleic acids from one chamber to the next by opening a vent bag, such as those described in International Application Publication No. WO2012 / 145730A2, multiplexed assay systems that include multiple thermal cycling units that allow a single chamber to be heated, cooled, and / or compressed to mix fluids within a chamber or to propel fluids within a chamber to another chamber, such as those described in International Application Publication No. WO2015 / 138343A1, and systems for rapid nucleic acid amplification that facilitate temperature cycling for nucleic acid amplification by adjusting a flexible portion of a sample cartridge, such as those described in International Application Publication No. WO2017 / 147085A1. Such cartridges / microfluidic systems may include, for example, microfluidic systems implemented using soft lithography, micro / nanofabricated microfluidic systems implemented using hard lithography, and the like.

[0265] In one exemplary embodiment, a cartridge may include multiple cartridge bodies, such as a first body, a second body, a central syringe barrel in fluid communication with the first and second bodies, a reaction vessel, and the like. The first body may be formed with multiple, separate chambers for reagents or buffers and sample processing. In some embodiments, the first body can be used to store multiple reagents. The second body may be formed with one or more separate chambers and include pathways through which reagents or samples from the first body pass. When the first and second bodies of the cartridge are combined, a liquid flow path and, optionally, an air flow path, may be formed between the two compartments through the central syringe barrel. The liquid flow path connects to the first body to provide space for sample and reagent movement and mixing. The air flow path may connect to the vacuum control area of ​​the reaction vessel and the plunger to control the vacuum that may occur when extracted nucleic acids are transferred into the reaction vessel. Rotation of the syringe barrel, including the plunger, sequentially draws sample and reagents from the multiple chambers into the syringe barrel's interior and discharges the mixture from the interior into any of the cartridge's multiple chambers (the first or second body). Rotation of the syringe barrel including the "plunger" can draw reagents from the multiple chambers of the box into the interior space of the syringe barrel, and then discharge the mixed reagents into the nucleic acid amplification reaction container.

[0266] In another exemplary embodiment, the box includes a flow channel cover plate and a substrate, which together form a closed channel therein. In one embodiment of this configuration, the inner chamber containing the reagents required for dielectric extraction is provided separately from the outer chamber, and the upper and lower parts of the inner chamber are sealed. In addition, a double-structured flow channel cover plate-pad can be provided between the outer chamber and the substrate. A closed flow path is formed by achieving a strong connection between the substrate-flow channel cover plate-pad-outer chamber. Beads necessary for dielectric extraction and amplification are also provided in this configuration, and the beads are accommodated in a dual-chamber structure of the outer chamber-bead chamber. Even when the bead chamber is opened, the beads can be maintained by a dehumidification unit located above the bead chamber.

[0267] In other exemplary embodiments, the box may include multiple reaction chambers, and in particular, the reaction vessel may include multiple reaction chambers. In these embodiments, different types of freeze-dried primers and probes may be provided in each reaction chamber. For example, primers and probes for virus-related nucleic acids may be provided in one reaction chamber, and primers and probes for virus-related nucleic acids may be provided in a second chamber for amplification and detection, and so on. Of course, various amplification and detection processes may be carried out simultaneously in a single reaction chamber. Therefore, the amplification of each target nucleic acid as described herein may be carried out separately in separate reaction chambers or wells, or may be carried out with multiple reactions in a single reaction chamber or well.

[0268] Furthermore, it should be understood that the panel assay methods described herein (i.e., confirming multiple conditions based on the comparative levels of multiple target nucleic acids obtained from a single sample) can be further implemented in completely different systems, including: isothermal nucleic acid amplification systems, digital RT-PCR, electrochemical PCR, lateral flow test kits, electrochemical sensors, nucleic acid sequencing, CRISPR / Cas-based technologies, chemiluminescence, and nanoparticle-based colorimetric detection.

[0269] In various embodiments, signal DNA from a PCR (nucleic acid amplification) reaction is amplified for detection and / or quantification. In certain embodiments, amplification comprises any of a number of methods, including but not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), ligase detection reaction (LDR), multiplex ligation-dependent probe amplification (MLPA), post-ligation Q-replicase amplification, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), rolling circle amplification (RCA), and the like.

[0270] In illustrative but non-limiting embodiments, the amplification reaction can generate a light signal proportional to the amount of amplified target nucleic acid (e.g., signal DNA). Exemplary light signals include, but are not limited to, fluorescent signals, chemiluminescent signals, electrochemiluminescent signals, colorimetric signals, and the like. In certain embodiments, the light signal is a fluorescent light signal generated by a fluorescent indicator. In certain embodiments, the fluorescent indicator is a non-specific intercalating dye that binds to the double-stranded DNA product, while in certain other embodiments, the fluorescent indicator comprises a target sequence-specific probe (e.g., a TAQMAN® probe, a SCORPION® probe, a MOLECULAR BEACON® probe, etc.).

[0271] A single PCR reaction (nucleic acid amplification) or multiple PCR reactions (nucleic acid amplification) performed sequentially (or in separate temperature-controlled channels or chambers) can also use the same detectable label, as the DNA signals from sequentially performed PCR reactions are analyzed sequentially, and the DNA signals from simultaneous PCR reactions can be distinguished by their presence in different temperature-controlled channels or chambers. The signal generated by this amplification reaction can be distinguished from other amplification products because it is not performed simultaneously and / or because it is performed in different reaction channels / chambers. However, when multiple nucleic acid amplification reactions are performed simultaneously in the same chamber, the reaction products analyzed in each reaction are typically detected and / or quantified using different and distinguishable labels.

[0272] In certain embodiments, the amplified products (amplified nucleic acids from nucleic acid analysis) can be detected using methods well known to those skilled in the art. In certain embodiments, amplification is a direct, simple PCR amplification reaction. However, in certain embodiments, nested PCR reactions are used to amplify nucleic acids from nucleic acid analysis. In various embodiments, multiplex PCR assays are contemplated, particularly when it is desired to analyze multiple products of nucleic acid analysis in the same amplification reaction. In certain embodiments, in such multiplex amplification reactions, each probe (e.g., for each specific analyte) has its own unique dye / fluor, allowing it to be detected independently of other probes. In certain embodiments, typically, for signal generation, the probes used in the various amplification reactions utilize changes in the fluorescence of a fluorophore to detect and / or quantify the amplified products, the changes in the fluorescence of the fluorophore being due to changes in its interaction with another molecule or moiety caused by changes in the distance between the fluorophore and the interacting molecule or moiety. Alternatively, other methods for detecting polynucleotides in a sample are contemplated, including but not limited to the use of radiolabeled probes.

[0273] Assay workflow

[0274] Before the sample is subjected to the amplification reaction, it undergoes one or more sample preparation procedures. Typically, these sample preparation procedures include extraction of intracellular material, such as nucleic acids, from whole-cell samples, viruses, etc., to form a crude extract, and additional sample processing for subsequent processing, such as denaturation, purification, filtration, and desalting of contaminating (e.g., DNA-binding) proteins. Release of nucleic acids from sample cells or viruses and denaturation of DNA-binding proteins can typically be performed using chemical, physical, or electrolytic lysis methods. For example, chemical methods typically use lysis agents to disrupt and extract nucleic acids from cells, followed by treatment of the extract with a lytic salt, such as guanidine isothiocyanate or urea, to denature any contaminating and potentially interfering proteins. Typically, when chemical extraction and / or denaturation methods are used, suitable reagents may be contained in the sample preparation chamber, a separate accessible chamber, or introduced externally. Preferably, sample preparation is performed in only one step, or no more than two steps. For example, sample preparation may include heating the sample in a lysis solution without further purification prior to the amplification reaction. In some embodiments, the lysed sample may be diluted prior to the amplification reaction. One or more of these various sample preparation operations can be readily incorporated into the fluidically closed cartridge systems contemplated herein.

[0275] On the one hand, the assay sample box as described herein can carry out a special workflow, and the workflow is according to the needs of a specific group of assays, and different target analytes are cracked and detected. In some embodiments, the box is configured to be used for the chemical cracking of a variety of target organisms. In other embodiments, the box is configured to be used for the mechanical cracking of a variety of target organisms. In other other embodiments, the box is configured to be used for both mechanical cracking and chemical cracking, to realize the cracking of different types of multiple targets. Therefore, the sample box can be configured to carry out group assays by the existing workflow relevant to conventional boxes, or can operate according to the new workflow configured for a group assay.

[0276] In one aspect, a sample cartridge with a valve assembly as described herein in FIG3D is capable of performing a variety of workflows: chemical lysis of a target organism, mechanical lysis of a target organism, or both. Thus, the sample cartridge can perform existing workflows associated with traditional dedicated cartridges, or can perform entirely new workflows that perform both.

[0277] Exemplary assay workflows AC (below) can be performed using a single universal cartridge. In any of these embodiments, the filter can be formed from a glass filter to promote affinity binding of nucleic acids (NAs) to the glass fibers, and the pore size is also suitable for chemical cleavage. In any of these workflows, nucleic acid amplification can be PCR, real-time PCR, isothermal amplification (including but not limited to nucleic acid sequence-based amplification, loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, multiple displacement amplification, whole genome amplification, or recombinase polymerase amplification), or other nucleic acid amplification methods known to those skilled in the art.

[0278] In Workflow A, the sample is optionally subjected to sample treatment or chemical lysis, and the treated or lysed fluid sample is then passed through a filter where the target organisms are captured. In some embodiments, sample treatment is used to weaken cell walls, inactivate the sample, or reduce its viscosity to facilitate passage through the filter. The filter is then washed, leaving the target organisms on the filter. Next, the target organisms are mechanically lysed, for example by sonication, to release nucleic acids (NAs). In some embodiments, mechanical lysis includes packing glass beads along the filter to aid in mechanical lysis of the target. Next, the NAs are eluted from the filter, and nucleic acid amplification is performed.

[0279] In Workflow B, the sample is chemically lysed to obtain the NA target. In some embodiments, after chemical lysis, the NA is bound to the filter by the presence of a precipitating reagent and a binding reagent. Next, the filter is washed with a flushing reagent while the NA remains bound to the filter. Typically, the washing reagent has a certain amount of salt that still promotes the binding of the NA to the filter while allowing the removal of non-target substances. Next, the filter is eluted to remove the NA target. In some embodiments, the elution is performed with a pH neutral buffer or an alkaline buffer fluid. The target NA is then delivered to a connected reaction vessel for nucleic acid amplification.

[0280] In workflow C, the fluid sample undergoes sample processing and / or chemical lysis of the target organism. Next, the NAs released by chemical lysis are bound to a filter. This step can utilize both precipitation and binding reagents. Next, the filter is washed with a rinse reagent while the NAs remain bound to the filter. Typically, the wash reagent contains a certain amount of salt that still promotes NA binding to the filter while allowing the removal of non-target substances. Next, the target organisms captured in the filter are heated and / or mechanically lysed. This can utilize sonication, and glass beads can further be utilized to promote mechanical lysis of the selected target organisms. The lysed target NAs are then eluted from the filter. In some embodiments, elution is performed with a pH-neutral buffer or alkaline buffer fluid. The target NAs are then delivered to a connected reaction vessel for nucleic acid amplification. Thus, in this workflow, the lysis of multiple different target organisms is achieved, some requiring only chemical lysis (e.g., viral targets) while others requiring mechanical lysis (e.g., bacteria, spores, etc.), allowing all of these target NAs to be released from a single sample and tested using the same sample cartridge. While the above workflow describes mechanical lysis followed by chemical lysis, it will be appreciated that other workflows may be used in which chemical lysis occurs after mechanical lysis.

[0281] In some embodiments, the sample cartridge includes an identifier with information regarding the appropriate workflow required for a particular set of assays, such that an instrument module receiving the sample cartridge operates according to the specified workflow.

[0282] Exemplary assay configuration

[0283] Reagents for MPOX Clade II Biomarker Panel Assay

[0284] Figure 5An exemplary sample cartridge that can be used as part of the GENEXPERT® system is shown, with the various chambers numbered. An exemplary cartridge may include: one or more lyophilized reagents in the form of beads, as described herein; an optional lysis reagent; an alkaline reagent; an optional binding reagent; a filtration reagent; a washing reagent; and an elution reagent. The last four reagents are named according to the function they perform on nucleic acids. Thus, for example, a binding reagent facilitates the binding of nucleic acids to substrates, and a filtration reagent facilitates the filtration of nucleic acids.

[0285] In some embodiments, the lysis reagent may include a chaotropic agent, a chelating agent, a buffer, an alkaline agent, or a detergent. The chaotropic agent may be selected from a guanidine compound such as guanidine thiocyanate or guanidine hydrochloride, an alkali metal perchlorate salt such as lithium perchlorate, an alkali metal iodide, magnesium chloride, urea, thiourea, formamide, or a combination thereof. The chaotropic agent concentration may range from about 1 M to about 10 M, such as from about 2.5 M to about 7.5 M, or less than 4.5 M, less than 2 M, or less than 1 M. The chelating agent may be selected from N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-disuccinic acid (EDDS), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and a phosphonate chelating agent. The chelating agent concentration may range from about 10 mM to about 100 mM and / or comprise about 0.5% to about 5% of the lysis reagent. The buffer can be selected from Tris, phosphate buffer, PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, SSC, and MES. The concentration of the buffer can range from about 5 mM to about 100 mM, such as about 5 mM to about 50 mM. The detergent can be selected from an ionic detergent or a non-ionic detergent. In some examples, the detergent includes a detergent selected from N-lauroylsarcosine, sodium dodecyl sulfate (SDS), cetylmethylammonium bromide (CTAB), TRITON®-X-100, n-octyl-β-D-glucopyranoside, CHAPS, n-octanoylsucrose, n-octyl-β-D-maltopyranoside, n-octyl-β-D-thioglucopyranoside, PLURONIC® F-127, TWEEN® 20, and n-heptyl-β-D-glucopyranoside. The detergent may comprise about 0.1% to about 2% of the lysing agent, and / or range from about 10 mM to about 100 mM. The lysing agent may have a pH ranging from about pH 3.0 to about pH 5.5.

[0286] In some embodiments, the assays disclosed herein do not use a chaotropic agent or lysis buffer. When a chaotropic agent or lysis buffer is not used, the sample can be contacted with a buffer (or filtration reagent) including, for example, saline (containing one or more inorganic salts, such as CaCl2, MgSO4, KCl, NaHCO3, NaCl, etc.), phosphate buffer, Tris buffer, 2-amino-2-hydroxymethyl-1,3-propanediol, HEPES, PBS, citrate buffer, TES, MOPS, PIPES, cacodylate, SSC, MES, a sugar or disaccharide, or a combination thereof. For example, the buffer can be a commercially available buffer, such as Hanks' balanced salt solution available from Sigma Aldrich or TE buffer available from Fisher BioReasons.

[0287] In some embodiments, the alkaline agent can be selected from alkali metal hydroxides, such as sodium hydroxide or potassium hydroxide. The concentration of the alkaline agent can be about 0.5 N to 5 N.

[0288] Binding agents can promote the binding of nucleic acids to the filter and facilitate the removal of non-target substances. In some embodiments, the binding agent can include a binding polymer such as polyacrylic acid (PAA), polyacrylamide (PAM), polyethylene glycol (PEG), poly(sulfobetaine), or salts thereof, or combinations thereof. In some embodiments, the filtration agent and / or wash reagent can include a binding agent. For example, the binding agent, filtration agent, and / or wash reagent can include a binding polymer (e.g., PEG 200), a buffer, an inorganic salt, an antioxidant and / or chelating agent, defoamer SE15, sodium azide, a disaccharide or disaccharide derivative, a carrier protein, a detergent, or DMSO. The binding polymer can be present in an amount of at least 10% v / v, at least 20% v / v, at least 30% v / v and / or less than 60% v / v, less than 40% v / v, less than 30% v / v, less than 20% v / v or less than 10% v / v of a binding reagent, a filtering reagent and / or a washing reagent, or can fall within any range limited by any of these values, for example 10% to 60% v / v. The buffer can be selected from Tris, 2-amino-2-hydroxymethyl-1,3-propanediol, HEPES, phosphate buffer, PBS, citrate buffer, TAPS, Bicine, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, dimethyl arsenate, SSC and MES. The concentration range of the buffer can be from about 5 mM to about 100 mM, such as from about 5 mM to about 50 mM. Salts, such as NaCl, KCl, or MgCl2, can be present at a concentration of about 0.05 M to about 1 M, such as about 0.1 M to about 0.5 M. Antioxidants and / or chelating agents include agents selected from the group consisting of N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-disuccinic acid (EDDS), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and phosphonate chelating agents. In some embodiments, the antioxidant and / or chelating agent comprises EDTA. In certain embodiments, the antioxidant and / or chelating agent comprises 0.2% to about 5%, about 0.2% to about 3%, about 0.5% to about 2%, or about 0.5% of the binding reagent, filtration reagent, and / or wash reagent. In some embodiments, the concentration of the antioxidant and / or chelating agent in the binding reagent, filtration reagent, and / or washing reagent ranges from about 2 mM to about 50 mM or from about 5 mM to about 20 mM. In some embodiments, the detergent is an ionic detergent or a non-ionic detergent. The detergent can be selected from an ionic detergent or a non-ionic detergent.In some examples, the detergent includes a detergent selected from the group consisting of N-lauroylsarcosine, sodium dodecyl sulfate (SDS), cetylmethylammonium bromide (CTAB), TRITON®-X-100, n-octyl-β-D-glucopyranoside, CHAPS, n-octylsucrose, n-octyl-β-D-maltopyranoside, n-octyl-β-D-thioglucopyranoside, PLURONIC® F-127, TWEEN® 20, Brij-35, and n-heptyl-β-D-glucopyranoside. The detergent may comprise from about 0.1% to about 2% of the binding reagent, filtration reagent, and / or wash reagent, and / or the detergent may range from about 10 mM to about 100 mM. The binding reagent, filtration reagent, and / or wash reagent may have a pH ranging from about pH 6.0 to about pH 8.0 (such as from about 6.5 to about 7.5).

[0289] In some embodiments, the elution reagent may have a pH greater than about 9, greater than about 10, greater than about 11, or greater than about 12. The use of a high pH to elute nucleic acids such as DNA is particularly unique to the cartridges described herein and provides improved speed and performance for the disclosed methods. Speed ​​is provided by the rapid neutralization of acidic ammonium ions by high concentrations of hydroxide ions. Alkylamines have a pKa of approximately 10-11 and are immediately deprotonated at pH 12.7 to form a neutral free base on the solid surface, releasing cationic DNA. Another advantage of the high pH is the denaturing effect of KOH on the captured DNA or RNA. The acidic functional groups in the heterocyclic bases of the DNA or RNA are immediately deprotonated and cannot form Watson-Crick bonds. The double-stranded structure and other secondary structures are destroyed, but can be renatured when neutralized, for example, with Tris HCl. This chemical denaturation of the captured genomic DNA is advantageous for isothermal assays that do not undergo conventional thermal denaturation for PCR. The box provided herein allows the eluted DNA or RNA to be rapidly neutralized in KOH and subsequently reacted with Tris to produce a final pH of about 8.5 for downstream PCR or other nucleic acid assays. In some embodiments, the pH value of the elution reagent can be less than about 9, less than about 8.5, or less than about 8. This lower pH elution of the bound DNA or RNA may be advantageous, particularly for devices that are not conducive to rapid neutralization of KOH solutions. RNA is known to be hydrolyzed by high pH, ​​but short exposure to KOH can provide good quality RNA. In some examples, the elution reagent includes a polyanion, a polycation, ammonia, or an alkali metal hydroxide. For example, the elution reagent may include a polyanion such as carrageenan, a carrier nucleic acid, or a combination thereof.

[0290] In some cases, to reduce bubble formation in one or more chambers, the detergent Brij can be added to one or more of the reagents described herein.

[0291] It will be appreciated that various other reagents and initial volumes may be used to perform automated PCR panel assays on samples inserted into the cartridge.

[0292] Dedicated box parts

[0293] Figure 5 An exemplary cartridge suitable for performing the multi-target panel assays described herein is shown. The cartridge shown is based on the GENEXPERT® cartridge (Cepheid, Inc., Sunnyvale, CA). The cartridge 100 includes a cartridge body 102 having a plurality of chambers 108 defined therein for containing various reagents and / or buffers. The chambers are arranged around a central syringe barrel 109, which is in fluid communication with a valve body 110 via a valve syringe tube 106 extending through the syringe barrel 109. In some embodiments, the valve assembly comprises any of the valve assemblies described herein or any suitable valve assembly. In this embodiment involving a cartridge, the cartridge utilizes a valve assembly capable of chemical lysis, such as any of the valve assemblies BD in FIG. 3 . The valve body 110 is an interface within the cartridge body and is supported on a cartridge base 101. The cartridge typically contains one or more channels or cavities, which may contain a filter material (e.g., a glass filter column) as described herein, which may function to bind and elute nucleic acids. In various embodiments, the box also includes one or more temperature-controlled channels or chambers, which in certain embodiments can function as thermal cycle chambers. A "plunger" (not shown) can be operated to draw fluid into the syringe barrel 109, and the rotation of the valve body / syringe tube provides selective fluid communication between various reagent chambers and channels, (multiple) reaction chambers. Therefore, various reagent chambers, reaction chambers, (multiple) matrix materials, and channels are selectively fluidically connected by the rotation of the valve and plunger, and reagent movement (e.g., chamber loading or unloading) is operated by the "syringe" action of the plunger. The connected reaction vessel 116 provides an optical window to provide, for example, real-time detection of amplification products and base identity in sequential operation by operating the modules within the system described herein.

[0294] While the above methods are described with respect to specific chambers within a GENEXPERT® cartridge, it will be appreciated that the specific reagent / chamber assignments may vary depending on the specifics of the nucleic acid detection / quantification assay. It will also be appreciated that, in certain embodiments, variations of the GENEXPERT® cartridge are contemplated. Such variations may include, but are not limited to, more or fewer reagent chambers and / or chambers of varying sizes, two (or more) sample receiving chambers, two (or more) temperature-controlled channels or chambers, stacked cartridges (with one module providing control of both cartridges), and the like. In one aspect, the sample cartridge includes one or more features or components specifically configured for the unique requirements of a particular multi-target assay. In this embodiment, the sample cartridge utilizes certain components developed specifically for the MPX Clade II group assay.

[0295] Figure 5 A-6B shows a detailed view of a valve assembly for a multi-target panel assay cartridge according to some embodiments. In this embodiment, the valve assembly is configured for chemical lysis. The valve assembly includes a valve body 110 having a disc shape and a tubular syringe type 106 along its proximal portion. Figure 6A As shown, the disc-shaped portion has a generally flat outer upper port surface 120 with one or more ports (e.g., 142, 146) therein. The valve assembly can be rotated relative to the cartridge housing, for example, by engaging the interface feature 123 with the module's drive mechanism. The ports 142, 146 on the top flat surface allow fluid communication between the syringe barrel 106 and each chamber and / or one or more channels within the cartridge. In this embodiment, the ports 142, 146 dock with a circular gasket 126 having a plurality of ports defined therein, which dock and fluidically connect with corresponding ports on the bottom of the chambers in the cartridge body. When the valve assembly is rotated, the top ports 142, 146 dock with different pairs of ports to fluidically connect the various chambers to each other or to the channels of the reaction vessels connected to the cartridge, so that the "plunger" propels fluid between the various chambers and / or channels through the movement of the syringe barrel to facilitate processing of the fluid sample and subsequent analytical testing.

[0296] like Figure 6B As shown, the valve body 110 includes a lysis chamber 121 on its underside, which may contain a solid phase material for capturing cells, spores, viruses or microorganisms to be lysed. Suitable solid phase materials include, but are not limited to, filters, beads, fibers, membranes, filter paper, glass wool, polymers or gels. One or more internal ports facilitate the flow of fluid into and out of the lysis chamber. In this embodiment, the solid phase material is a glass filter 114, which is located in a filter groove 111 in the valve body 110 and is used to capture nucleic acids released by the chemical lysis of target organisms in the fluid sample.

[0297] like Figures 6A-6B As shown in FIG, the outer wall 122 of the valve body and the bottom cap 112 together seal the cracking chamber 121. Figure 6B As shown, the lysis chamber 121 includes a pair of processing ports to facilitate the flow of fluid samples into and out of the lysis chamber. A first fluid processing port 124 is connected to a first fluid processing channel defined within the valve body, and a second fluid processing port 125 is connected to a second fluid processing channel defined within the valve body. Through these channels, the first fluid processing channel is connected to the internal tubing and external port 142 of the syringe barrel 106, while the second fluid processing channel is connected to the second external port 146. With this configuration, fluid is drawn into the syringe barrel from one chamber by applying pressure through the syringe barrel 106 by moving a "plunger," acting like a syringe, and after rotating the valve assembly to position the respective ports to connect with the other chamber, the movement of the plunger causes the fluid to advance through the valve body and channels into the other chamber or channel to the reaction vessel. This method can perform various processing steps that are required to perform a sample preparation protocol for target analytes for a multi-target panel assay.

[0298] In one aspect, the assay described herein chemically lyses the target virus. Widely applicable lysis buffers can have elevated alkalinity (e.g., sodium hydroxide). Other high-alkalinity elution buffers (e.g., ammonia or alkali metal hydroxides) can also be used to elute nucleic acids bound to glass filters. While these buffers facilitate chemical sample preparation, their practical use in conventional cartridges can be problematic because these high-alkalinity buffers can degrade the sealing interface between valve assembly materials and cartridge components, leading to valve assembly cracking and leakage during processing. Such leakage can be detrimental to sample processing. Specialized valve assemblies can be used to withstand the elevated alkalinity of these buffers (e.g., pH values ​​greater than 10, pH values ​​greater than 11, or pH values ​​greater than 12). One difficulty in developing conventional valve assemblies is that they are manufactured by injection molding certain polymeric materials, typically polycarbonate, polyolefins (including polyethylene or polypropylene), or combinations thereof. Typical polymeric materials used in injection molding are not tolerant to these elevated alkalinity ranges, and those that do can be significantly more expensive and less suitable for injection molding small-scale microfluidic features. Therefore, specialized valve assemblies can be developed by annealing valve body assemblies formed from conventional polymers to sufficiently harden the polymer material to withstand elevated alkalinity. Annealing of polycarbonate reduces chemical attack by the mixture of NaOH and GTC in the cleavage and elution reagents. In some embodiments, after the valve assemblies are formed, they are heated to an elevated temperature (e.g., 90-100°C, approximately 100°C) for approximately one hour or longer and then allowed to cool slowly in a controlled manner for at least 30 minutes. Studies have shown that these annealed valve assemblies produce valve assemblies of substantially the same design and materials as conventional valve assemblies, except that they are resistant to high-alkalinity buffers, which are no longer usable in conventional cartridges. The alkali resistance of the annealed valve body can be tested by exposing the valve body assembly (VBA) to NaOH / GTC and then visually inspecting the VBA for cracking after approximately 30 minutes. It should be understood that such annealed cartridges can be advantageous for a variety of reasons and need not be associated with any specific assay described herein. Furthermore, it should be understood that the assays described herein can be performed using a variety of other cartridge designs, devices, and systems, and need not be associated with the specific cartridge designs described herein.

[0299] Exemplary Detection Methods, Results, and Processing of Results

[0300] In some embodiments, computer-based analysis programs are used to convert raw data generated by a detection assay into data that has predictive value to a clinician.

[0301] Melting curve analysis can be evaluated using GENEXPERT® software to determine the presence of PCR products.M ) and the melting peak height of the curve are automatically calculated by the analysis software. m falls within the effective T specified for each target nucleic acid m If the melting curve is not within the appropriate T m If the melting curve is within the range of 100,000, the melting curve is called negative. The software automatically calculates the cycle threshold (Ct), endpoint, and probe check value. Illustrative detection limit concentrations, Ct cutoff values, and methods for determining them are provided in the examples below.

[0302] Before the PCR reaction begins, the GENEXPERT® System measures the fluorescent signal from the probe to monitor bead rehydration, reaction tube filling, probe integrity, and dye stability. If the Probe Check Control (PCC) meets the validated acceptance criteria, it passes.

[0303] In some embodiments, the raw data produced by the detection assay is converted into data with predictive value to the clinician using a computer-based analysis program. The clinician can use any suitable method to access the predicted data. Therefore, in some embodiments, the present invention provides further benefits, i.e., it is unlikely that the trained clinician in genetics or molecular biology does not need to understand the raw data. The data are directly presented to the clinician in its most useful form. The clinician can then utilize information immediately to optimize the diagnosis and treatment of the experimenter.

[0304] When using the GENEXPERT® System, results are automatically interpreted and displayed in the "View Results" window. Positive targets are highlighted in red, negative targets in green, and indeterminate targets in light gray. Samples with co-infections may show positive results for multiple targets. Invalid, erroneous, or no results are highlighted in light gray.

[0305] In some embodiments, the results and interpretations are provided in Tables 3-1 to 3-2.

[0306]

[0307]

[0308] Exemplary detection methods, results, and processing of results for host biomarker targets are described in U.S. Patent Application Publication No. 2022 / 0298572, which is incorporated by reference for purposes of this description.

[0309] The present disclosure contemplates any method capable of receiving, processing, and transmitting information to and from laboratories performing assays, information providers, medical personnel, and subjects. For example, in some embodiments of the present invention, a sample is obtained from a subject and submitted to a testing service (e.g., a clinical laboratory at a medical facility, a genomic analysis company, etc.) located anywhere in the world (e.g., in a country different from the subject's country of residence or the country where the information will ultimately be used) to generate raw data. If the sample comprises a tissue or other biological sample, the subject can visit a medical center to collect the sample and send it to the testing service, or the subject can collect the sample themselves and send it directly to the testing service. If the sample comprises previously determined biological information, the information can be sent directly by the subject to the testing service (e.g., an information card containing the information can be scanned by a computer and transmitted to a computer at an analysis center using an electronic communication system). Once the sample is received by the testing service, it processes the sample and generates a set of test results specifically tailored to the diagnostic or prognostic information desired by the subject.

[0310] The test results can be prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw expression data, the prepared format can represent a diagnosis or risk assessment of the subject, with or without recommendations for specific treatment options. The test results can be displayed to the clinician by any suitable method. For example, in some embodiments, the testing service generates a report that can be printed for the clinician (e.g., at the clinician's site) or displayed to the clinician on a computer monitor.

[0311] In some embodiments, information is first analyzed at the site of treatment or at a regional facility. The raw data is then sent to a central processing facility for further analysis and / or conversion of the raw data into useful information for clinicians or patients. A central processing facility offers the advantages of privacy (all data is stored in a central facility with unified security protocols), speed, and consistency in data analysis. The central processing facility can then control the disposition of the data after treatment. For example, using electronic communication systems, the central facility can provide data to clinicians, patients, or researchers.

[0312] In some embodiments, the subject can directly access the data using an electronic communication system. The subject can choose further intervention or consultation based on the results. In some embodiments, the data is used for research purposes. For example, the data can be used to further optimize the inclusion or elimination of markers that are useful indicators of a specific state or stage of a disease, or as companion diagnostics to determine a course of therapeutic action.

[0313] Reagent test kit

[0314] Also contemplated are kits for performing the methods described herein. Such kits include one or more reagents useful for performing any of these methods. Kits typically include a package having one or more containers for containing the reagents, either as one or more separate compositions, or optionally as a mixture as reagent compatibility permits. Kits may also include other materials that may be desired from a user's perspective, such as buffers, diluents, standards, and / or any other materials useful in sample processing, washing, or any other step in performing an assay.

[0315] The kit preferably includes instructions for practicing one or more of the screening methods described herein. The instructions included in the kit may be affixed to the packaging material or included as a package insert. While instructions are typically written or printed, they are not limited thereto. Any medium capable of storing such instructions and transmitting them to an end user may be used. Such media include, but are not limited to, electronic storage media (e.g., disks, magnetic tapes, cassettes, chips), optical media (e.g., CD ROMs), etc. As used herein, the term "instructions" may include the address of an internet website providing the instructions.

[0316] In some embodiments, the kits include primer pairs for amplifying and / or detecting the above-described MPXV (e.g., MPX clade II and non-smallpox MPXV) biomarker panel targets as described above, optionally with probes specific for these targets. Such kits may additionally include primer pairs and optional probes for detecting one or more of the above-described host biomarker targets. In some embodiments, these kits may include primer pairs and optional probes for detecting one or more of the above-described controls.

[0317] In some embodiments, the kit can include any of the reagents described above provided with or in one or more GENEXPERT® cartridges. See, for example, U.S. Patent Nos. 5,958,349, 6,403,037, 6,440,725, 6,783,736, and 6,818,185, each of which is incorporated herein by reference for this description. In some embodiments, reagents for measuring and detecting MPXV (e.g., MPX clade II and non-smallpox MPXV) and for detecting host biomarkers are provided in separate cartridges within the kit.

[0318] In some embodiments, any kit described herein can include a container for a sample and / or a swab for collecting a sample. Example

[0319] Example 1: MPOX assay design to distinguish between clade II and clade I

[0320] The following method was used to obtain a primer / probe set specific for MPOX (MPX or MPOX) clade II that did not detect clade I.

[0321] Dataset and Methods

[0322] Sequence and metadata are Figure 7 Sequences and metadata used in the NextStrain analysis shown. Sequences were all from NCBI (for MPX, NCBI and GISAID datasets appear to be effectively equivalent).

[0323] Clade assignments are taken from NextStrain metadata. Their positions indicate a specific set of nucleotide differences that define a clade. Furthermore, they exclude the terminal regions of the genome from their analysis. Specifically, they describe "several regions masking the genome, including the first 1500 and last 7000 base pairs and repetitive regions of variable length." The terminal regions contain repetitive sequences. Not all sequences in the dataset have an assigned clade.

[0324] The initial dataset consisted of 2103 MPX sequences, of which 152 were assigned to clade I, 1712 to clade II, and 149 were unassigned. Of the clade II genomes, 107 were clade IIa, 1542 were clade IIb, and 62 were unassigned to a subclade.

[0325] All Clade I and Clade II genomes were formatted into a BLAST database. GenBank sequence NC_063383.1 (Clade IIb) was used as a reference sequence. This sequence was divided into 150-nucleotide segments that overlapped by 50 nucleotides. These were compared with the Clade I and Clade II datasets to identify segments that were highly conserved in Clade II and showed minimal or no similarity to Clade I.

[0326] A set of candidate segments is selected and then compared to a small set of non-MPX orthopoxvirus genomes to identify any segments that have little or no similarity to non-MPX species.

[0327] Two good candidates were identified and candidate primer / probe sets were designed. These are shown below.

[0328] In the clade II alignment, the last number in each sequence tag is the number of isolates with a unique amplicon sequence. Differences from the most common sequence are capitalized and bold.

[0329] Multiple alternative designs for each oligonucleotide are provided.

[0330] Neither amplicon has a clear match to the Clade I genome. Among the matches to the OPX subset, there are a number of indels relative to the Clade II amplicons, which are capitalized and bold.

[0331] Both amplicons had no significant matches to any other species besides orthopoxviruses.

[0332]

[0333] Sequences identified as U84503 (positions 2268_2166), OP245318 (positions 133040_133142), OX044345 (positions 133966_134068), and OX044338 (positions 133966_134067) are from the clade II genome (obtained from NCBI GenBank). Capitalized and bold letters represent mismatches in the genomic sequence compared to the coding region of the MPXV clade II gene OPG153 (deposited in NCBI GenBank as NC_063383.1). The identified sequences mpx_cld2_1_fwd, mpx_cld2_1_prb, and mpx_cld2_1_rev represent potential forward primers, probes, and reverse primers that can be used to detect clade II in the MPX assay.

[0334] The above shows no clear match to clade 1. Therefore, this amplicon is located within a larger fragment that is not present in clade 1.

[0335]

[0336] This analysis used a small subset of non-MPX OPX genomes (obtained from NCBI). Specifically, sequences identified as NC_055231.1 (positions 144914_14501), NC_008291.1 (positions 135985_136090), NC_003663.2 (positions 152956_153061), NC_006998.1 (positions 136036_136141), NC_001611.1 (positions 126867_126972), NC_003391.1 (positions 138318_138401), NC_055230.1 (positions 151168_151270), and NC_004105.1 (positions 144647_144731) were from non-MPX orthopoxvirus genomes. Capital and bold letters are mismatches in the genomic sequence compared to the coding region of gene OPG153 of MPXV clade II (the MPX clade IIb reference sequence is deposited in NCBI GenBank as NC_063383.1). Underlined dashed lines ( --- ) indicates a 3-nt deletion in the coding region of gene OPG153 of MPXV clade II compared with the OPX genome.

[0337] Amplification primers will amplify these OPX genomes, but a 3 nt insertion relative to the probe should prevent probe binding.

[0338]

[0339]

[0340] Sequences identified as OX297403 (position 156398_156506), OP324469 (position 155995_156103), MN346703 (position 157947_158053), OK573120 (position 778_672), OP215284 (position 156398_156506), ON622721 (position 156227_156333), KJ642616 (position 158008_158114), OP215279 (position 156398_156506), and OP215286 (position 156398_156506) were from the clade IIa genome (obtained from NCBI). Capitalized and bold letters indicate mismatches in the genomic sequence compared to the OPG183 intergenic region of MPXV clade II (Gene Accession No. (GenBank) NC_063383.1). The identified sequences mpx_cld2_2_fwd, mpx_cld2_2_prb, and mpx_cld2_2_rev represent potential forward primers, probes, and reverse primers that can be used to detect clade II in the MPX assay. The shaded dashed line (--) indicates a 2-nt deletion in the clade IIa genome compared to the OPG183 intergenic region of MPXV clade II.

[0341] The above shows no clear match to clade 1. Therefore, this amplicon is located within a larger fragment that is not present in clade 1.

[0342]

[0343]

[0344] Sequences identified as NC_055231.1 (positions 168806_168907), NC_004105.1 (positions 168152_168252), NC_003663.2 (positions 176827_176927), NC_003391.1 (positions 160851_160948), NC_008291.1 (positions 158375_158472), NC_006998.1 (positions 159720_159777), and NC_055230.1 (positions 174991_175061) were from a non-MPX orthopoxvirus genome. Capital and bold letters indicate mismatches in the genomic sequence compared to the OPG183 intergenic region of MPXV clade II (MPX clade IIb reference sequence; GenBank accession number NC_063383.1). --) indicates a 2-nt deletion in the OPG183 intergenic spacer of MPXV clade II compared with the OPX genome.

[0345] Mismatches in the amplification primers should prevent amplification in this genomic sample. Two 2nt insertions and deletions relative to the probe should prevent binding.

[0346] Example 2: Inclusive and Exclusive Analysis of Non-Variola Orthopoxviruses and MPOX Viruses (Clade II) Computer simulation report

[0347] The illustrative MPOX assay described in this example is an automated real-time PCR test for the qualitative detection of DNA from MPOX virus clade II and / or non-variola orthopoxviruses in lesional swab specimens (i.e., swabs from acute pustular or vesicular rashes) collected from individuals by their healthcare providers for suspected MPOX infection.

[0348] Tests on the GeneXpert Dx and GeneXpert Infinity instruments are included in laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, which meet regulatory requirements for performing high- or medium-complexity testing. Tests on the GeneXpert Xpress (hub configuration) instrument are authorized for point-of-care (POC) use, meaning use in patient care settings operated under a CLIA certificate of waiver, certificate of compliance, or certificate of certification.

[0349] Results are used to detect and identify MPOX virus (clade II) and / or non-variola orthopoxvirus DNA. MPOX virus (clade II) and / or non-variola orthopoxvirus DNA is typically detected in swabs collected from lesions during the acute phase of infection. A positive result indicates the presence of MPOX virus (clade II) and / or non-variola orthopoxvirus DNA; clinical correlation with the patient's medical history and other diagnostic information is appropriate for determining the patient's infection status.

[0350] Principle of Operation

[0351] The MPOX test is an automated in vitro test for the qualitative detection and identification of MPOX (clade II) viral DNA and non-variola orthopoxvirus DNA. In some instances, the MPOX test can be used as a diagnostic test. In other instances, the MPOX test is a non-diagnostic test. The MPOX test can be performed on the GeneXpert® instrument system. The GeneXpert instrument system automates and integrates sample preparation, nucleic acid extraction and amplification, and detection of target sequences in simple or complex samples using real-time PCR assays. The system includes an instrument, a computer, and pre-installed software for running the test and viewing the results. The system requires the use of a single-use, disposable cartridge that holds the real-time PCR reagents and carries the real-time PCR process. Because the cartridges are self-contained, cross-contamination between samples is minimized. The test can be performed at room temperature or below, such as 0-30 o C or 2-28 o C storage box.

[0352] The MPOX test includes reagents for detecting MPXV-clade II and OPXV-E9L NVAR targets in lesion swab specimens. A sample processing control (SPC), a sample adequacy control (SAC), and a probe check control (PCC) are also included in the cartridge for use with the GeneXpert instrument. The SPC is included to control for adequate sample processing and to monitor for the presence of possible inhibitor(s) in the real-time PCR reaction. The SPC also ensures that the real-time PCR reaction conditions (temperature and time) are appropriate for the amplification reaction and that the real-time PCR reagents are functional. The SAC reagent detects the presence of a single copy of the human gene and monitors whether the sample contains human DNA. The PCC verifies reagent rehydration, PCR tube filling, and confirms the presence of all reaction components in the cartridge, including monitoring probe integrity and dye stability.

[0353] An exemplary MPOX test is used with lesion swabs collected by a healthcare provider from a patient suspected of having an MPOX infection.

[0354] Collect the sample and place it into a transfer tube containing 3 ml of viral transport medium (VTM). Briefly mix the sample by quickly inverting the collection tube five times. Using the provided pipette, transfer the sample to the sample chamber of the Xpert MPOX cartridge. Load the GeneXpert cartridge onto the GeneXpert instrument system platform, which performs hands-free automated sample processing and real-time PCR for detection of viral DNA.

[0355]

[0356] This example focuses on the inclusive and exclusive analysis of non-variola orthopoxviruses and MPOX viruses (clade II).

[0357] Purpose

[0358] The first objective of this in silico analysis was to evaluate the performance of the MPOX test in detecting variants representing MPOX viruses (clade II) and orthopoxviruses (non-variola) using sequences relevant to the listed inclusive disease pathogens. The second objective was to assess the risk of cross-reactivity of MPOX viruses (clade II) and orthopoxviruses (non-variola) with non-MPOX viruses and non-orthopoxvirus organisms, as well as with all listed exclusive disease pathogens. The third objective was to analyze the potential cross-reactivity of sample processing control (SPC) and sample adequacy control (SAC) assay oligonucleotides with non-target sequences. This example covers in silico inclusion and exclusion analyses of primers and probes for non-variola orthopoxvirus and MPOX virus (clade II) assays.

[0359] Inclusive analysis

[0360] Sequence data available for computer simulation analysis

[0361] GISAID includes a subset of MPOX virus (MPXV or MPOX virus) sequences, but excludes sequences of other orthopoxviruses (OPXVs). Most, but not all, of these sequences are annotated with the MPXV clade (I, IIa, and IIb) to which they belong.

[0362] NCBI / GenBank contains sequences for MPXV and other OPXV species. Sequence annotations are contributed by the submitters and can vary widely in the detail provided. Notably, many MPXV sequences do not clearly identify the clade to which they belong.

[0363] Datasets for MPOX viruses were downloaded from NCBI and GISAID. Datasets for cowpox, camelpox, ectromelia (mousepox), and vaccinia viruses were downloaded from NCBI. These data sources contain full-length genome sequences and shorter gene fragments. For analysis, these shorter fragments, which may not overlap with the amplicons used for the assay, were removed. The genome length of orthopoxviruses is approximately 200,000 nucleotides. An artificial cutoff of 160,000 nucleotides was selected as the minimum length of sequences in the dataset. This excludes shorter sequences and includes all full-length or near-full-length genomes.

[0364] The NCBI dataset was filtered to remove repetitive sequences and those that appeared to be synthetic constructs. Repetitive sequences included those included in the RefSeq database, which are repeat sequences of other GenBank records.

[0365] Sequences from vaccinia and cowpox were examined because these viruses, particularly vaccinia, can be used as synthetic cloning vectors to introduce foreign genes into mammalian cells. Including these in the analysis dataset could have yielded misleading inclusion results, as some sequence fragments are not part of the viral genome. Furthermore, some sequences are listed in patents. These also likely represent synthetic viral constructs and were therefore not included.

[0366] The filtered dataset represents the virus isolates that were not otherwise modified. Results from the two datasets are presented separately. Table 4 provides an overview of the sources of the datasets used for each virus.

[0367]

[0368] Description of MPOX Test Oligonucleotides and Amplicons

[0369] The MPOX test includes five primer / probe sets for two target regions in the MPXV (clade II) genome, one target region in the non-smallpox OPXV genome, a sample adequacy control—the human genome (SAC), and a sample processing control (SPC). The forward primer (F), reverse primer (R), and probe (P) sequences are provided in Table 5. The associated amplicon sequences can be found in Table 6.

[0370]

[0371]

[0372] BLAST analysis of Xpert MPOX test amplicons

[0373] Procedure: For the OPX assay and both MPX assays, corresponding amplicons were defined in the MPX reference sequence (GenBank NC_063383). These were compared to all available sequences using BLASTN. Matching fragments were extracted, condensed into a set of unique sequences, and aligned.

[0374] In a few cases, due to sequencing / assembly errors, the region that should have matched the amplicon contained stretches of consecutive "N" letters. These were excluded from the analysis.

[0375] Some sequences contain consecutive "N" letters, which represent unknown bases at these positions. Letters other than A, C, G, or T represent ambiguous codes. To determine the inclusiveness of each oligonucleotide, the aligned sequences were first filtered to remove sequences containing ambiguous or unknown nucleotides. The number of mismatches for each oligonucleotide was counted in this unambiguous data set.

[0376] Tables 7, 8, and 9 show the percentage of matches with 0, 1, or more mismatches, as well as the actual counts in each category. Generally, we found that a single mismatch in an oligonucleotide match did not affect primer or probe binding. In many cases, oligonucleotides with 2 mismatches also bound, but in this analysis, we defined the inclusive percentage as the number of oligonucleotides with 0 or 1 mismatch.

[0377] result

[0378] Non-Variola Orthopoxvirus Assay Inclusivity: The expected inclusivity for the probes used in the orthopoxvirus assay was 98.9%, the reverse primer was 99.8%, and the forward primer was 100% (see Table 7). Sequences with two or more mismatches were from isolates of vaccinia, cowpox, and camelpox. Not all isolates from these species contained mismatches. Two MPX sequences (ncbi_MPX_FV537352.1_142088_142183_2) contained multiple mismatches throughout the amplicon relative to the other MPX amplicons. These indicate sequencing / assembly errors.

[0379]

[0380] MPOX Virus (Clade II) Assay #1 Inclusivity: The estimated inclusivity of the MPOX Virus Assay primers and probe can be found in Table 8. This assay is expected to have 100% inclusivity for both NCBI and GISAID MPX Clade IIa and IIb sequences. Amplicon alignment can be found in Scheme 2. This assay was designed not to detect MPX Clade I isolates. No significant matches were found to the 43 sequences in the Clade I dataset.

[0381]

[0382] MPOX Virus (Clade II) Assay #2 Inclusiveness: This assay is expected to be 100% inclusive of both NCBI and GISAID MPX Clade IIa and IIb sequences. The expected inclusiveness of the MPOX Virus Assay primers and probes can be found in Table 9.

[0383]

[0384] in conclusion

[0385] MPXV assays #1 and #2 are expected to have 100% inclusivity for both NCBI and GISAID MPXV clade IIa and IIb sequences. These assays are designed not to detect MPXV clade I isolates. The non-smallpox OPXV assay probe has 98.9% inclusivity, while the reverse primer has 99.8% and the forward primer has 100%. The analytical sensitivity for the MPOX target is 50 copies / ml or less, or 30 copies / ml or less. The analytical sensitivity for the OPX target is 100 copies / ml or less.

[0386] Exclusivity Analysis

[0387] Objective: To assess the risk of cross-reactivity of Xpert MPOX oligonucleotides and amplicons with other non-MPOX viruses and against all exclusive disease pathogens.

[0388] BLAST analysis of Xpert MPOX test amplicons

[0389] Process: For each of the 33 species listed in the EUA submission template, a representative genome was identified in the NCBI RefSeq or nucleotide databases, and the sequences were downloaded and formatted into a BLAST database. All oligonucleotides and amplicons were compared to the dataset using BLAST, and the percent homology / identity for each oligonucleotide was determined.

[0390] result

[0391] The tables (see Tables 10, 11, 12, 13, and 14) show 80% or greater identity with each listed species. Species with a best match of less than 80% identity are left blank. An explanation of these results is provided in the text following each table.

[0392] Note: Streptococcal group C and group G species are not well characterized in terms of genome sequencing. In this analysis, the genome of Streptococcus dysgalactiae subsp. equisimilis was used to represent these two groups, consistent with the literature.

[0393] In the case of a match to the human genome, the specific chromosome containing the match is listed.

[0394] Note: Four eukaryotic species contain multiple chromosomes. Sequences for all of these were downloaded and retrieved.

[0395]

[0396]

[0397] *See note in paragraph

[0355] .

[0398] A single match of the forward primer to the human genome did not produce an amplification product.

[0399] The OPX oligonucleotides are based on designs derived from CDC targeting the E9L gene. This is described in the article by Yu Li et al. (Journal of Clinical Virology 36 (2006) 194–203) pubmed.ncbi.nlm.nih.gov / 16731033 / . The OPX assay is designed to detect MPOX (clades I and II), cowpox, camelpox, mousepox, and vaccinia. It is specifically designed for No For smallpox detection, the paper demonstrated this to be the case in laboratory testing. According to the computer simulation analysis shown in the table above, the forward and reverse primers matched the smallpox genome exactly, while the probe had three mismatches. Although the probe shared 91% identity with the smallpox genome, the paper demonstrated that this did not produce a positive assay result in the laboratory when tested against a wide range of smallpox samples (see Table 2 in Yu Li et al. (Journal of Clinical Virology 36 (2006) 194–203)).

[0400] For detailed results and alignment of target orthopoxvirus species, see the OPX assay section on inclusivity analysis.

[0401]

[0402] *See note in paragraph

[0355] .

[0403]

[0404] *See note in paragraph

[0355] .

[0405] A single match between the forward primer and smallpox (see Table 12) did not produce an amplification product. The matches between the forward and reverse primers in the human genome (see Table 12) are located on different chromosomes and therefore did not produce an amplification product. A match between the reverse primer and Trichomonas vaginalis (see Table 12) of less than 100% did not produce an amplification product.

[0406] The oligonucleotide's alignment with the Orthopoxvirus genome (see Table 12) may result in amplification products when using the cowpox and mousepox genomes. The probe contains a 2-nt insertion relative to the OPX genome and a second 2-nt deletion relative to the genome. This is sufficient to prevent the probe from binding to these genomes.

[0407] Therefore, MPX Clade II Assay #2 will not detect other orthopoxvirus genomes.

[0408] This assay was designed not to detect MPX clade I isolates. Primers and probes do not display any noteworthy homology to clade I sequences. The best-scoring alignment between the MPOX (Clade II) assay #2 amplicon and the MPX clade I dataset is shown here (one example of 43 identical search results). This region is inconsistent with the MPOX (Clade II) assay #2 primers and probe, ensuring exclusivity for MPXV clade I isolates.

[0409]

[0410]

[0411] *See note in paragraph

[0355] .

[0412] Sample adequacy control (SAC) oligonucleotides amplify a segment of the human hydroxymethylcholine synthase (HMBS) gene. As shown above (see Table 13), these oligonucleotides have a 100% match in the human genome. Other human genomic chromosomes were examined for <100% matches (see Table 13), but none of them were located close enough to form an amplification product. Therefore, none of these matches posed a problem for the assay. The <100% match of the forward primer to Candida albicans (see Table 13) did not pose a problem for the assay, as the assay reverse primer and probe had less than 80% homology.

[0413]

[0414] *See note in paragraph

[0355] .

[0415] The two oligonucleotides that matched the human genome (see Table 14) were located on different chromosomes and therefore could not generate amplification products.

[0416] Determination of cross-reactivity between oligonucleotides: The MPOX assay is a multiplex, five-component assay, each of which uses two amplification primers and a probe. The entire set of 15 oligonucleotides was compared to each other to examine possible inter-oligonucleotide binding. No significant homology was confirmed with any pair of oligonucleotides in the entire set.

[0417] in conclusion

[0418] The MPOX test met the requirements of inclusivity and exclusivity. In silico cross-reactivity checks did not reveal any relevant interactions between the tested primers and probes.

[0419] Example 3: MPOX assay of samples with different concentrations of inactivated MPOX virus

[0420] The MPOX panel assay was performed using the GENEXPERT® system essentially as described above. Positive samples contained inactivated MPOX virus, human cells, and SPC template at final concentrations ranging from 3 to 500 copies / mL. Negative samples contained only human cells and SPC template. Figure 8 The Ct value for each analyte is shown. Figure 9 The endpoint fluorescence (EPF) is displayed for each analyte. Figure 10 Displays test results from GENEXPERT® software. Figure 11 Graph showing fluorescence of 50 copies / mL NATtrol MPOX virus input versus cycle output. Figure 12 A plot showing fluorescence of a negative sample versus cycle output is shown. The results demonstrate that the assay performs as expected.

[0421] sequence

[0422]

[0423]

[0424]

[0425]

[0426]

Claims

1. A set of primers and optional probe(s) for detecting and / or confirming the presence of Monkeypox (MPOX) clade II virus in a sample, the set comprising: at least one primer pair and optionally a probe that selectively hybridizes to the MPOX clade II OPG153 gene, the MPOX clade II OPG183 intergenic region, or a combination thereof, of an MPOX clade II virus, wherein the set of primers and optional probe(s) selectively hybridize to one or more conserved regions of nucleic acid from an MPOX clade II virus.

2. The panel of claim 1 , wherein each primer and optional probe has less than 90%, preferably less than 85%, sequence identity to a similar region, when present in MPOX clade 1 or a non-smallpox orthopoxvirus.

3. The panel of claim 1 or 2, wherein the primers and optional probe(s) do not selectively hybridize to, detect and / or confirm the presence of, MPOX clade I viruses or non-smallpox orthopoxviruses.

4. The panel of any one of claims 1 to 3, further comprising additional primer pairs and optional probes for detecting and / or confirming the presence of one or more non-variola orthopoxviruses, wherein the additional primer pairs and optional probes comprise at least one primer pair and optional probe that selectively hybridize to the DNA polymerase (E9L) gene of a non-variola orthopoxvirus.

5. The panel of claim 4, wherein the additional primer pairs and optional probes for identifying one or more non-variola orthopoxviruses selectively hybridize to one or more conserved regions of nucleic acid from non-variola orthopoxviruses.

6. The panel of any one of claims 1-5, further comprising additional primer pairs and optional probes for detecting and / or confirming the presence of one or more MPOX clade 1 viruses, wherein the additional primer pairs and optional probes comprise at least one primer pair and optional probe that selectively hybridize to one or more MPOX clade 1 viruses.

7. The panel of any one of claims 1-6, further comprising one or more additional primer pairs and optionally probe(s) for detecting and / or confirming the presence of one or more of herpes simplex virus (HSV), varicella zoster virus (VZV), and syphilis.

8. The set of any one of claims 4-6, wherein the set comprises: at least one primer pair and probe that selectively hybridize to the OPG153 gene of MPOX clade II, at least one primer pair and probe that selectively hybridize to the intergenic region of MPOX clade II, and At least one primer pair and probe that selectively hybridize to a DNA polymerase (E9L) gene of a non-smallpox orthopoxvirus.

9. The set of any one of claims 1 to 8, wherein When present, The primers and optional probes that selectively hybridize to the OPG153 gene of MPOX clade II comprise a sequence identical or complementary to at least 15 consecutive nucleotides of one or more of SEQ ID NOs: 1, 25, 26, 27, and 28; The primers and optional probes that selectively hybridize to the OPG183 intergenic region of MPOX clade II comprise a sequence that is identical or complementary to at least 15 consecutive nucleotides of one or more of SEQ ID NOs: 2, 29, 30, 31, and 32; The primers and optional probes that selectively hybridize to the DNA polymerase (E9L) gene of a non-smallpox orthopoxvirus comprise a sequence that is identical or complementary to at least 15 consecutive nucleotides of one or more of SEQ ID NOs: 3, 33, 34, 35, and 36.

10. The set of any one of claims 1-9, wherein at least one of the primer pair and optional probe(s) comprises a detectable label, optionally wherein at least one probe, optionally each probe, of the set comprises a fluorescent dye and a quencher molecule.

11. The panel of any one of claims 1-10, wherein the panel further comprises primer pairs that selectively hybridize to an exogenous control and / or an endogenous control, wherein the exogenous control is a sample processing control, and wherein the endogenous control is a sample adequacy control.

12. The set of any one of claims 1-11, wherein the set is contained within one or more cassettes.

13. The set of claim 12, wherein the set is contained within a box.

14. A kit for detecting and / or identifying monkeypox (MPOX) clade II in a sample, the kit comprising: A cartridge body comprising a plurality of chambers, wherein the plurality of chambers comprises: i) a sample chamber having at least one fluid outlet in fluid communication with another chamber of the plurality of chambers; and ii) optionally a lysis chamber in fluid communication with said sample chamber, optionally wherein said sample chamber and lysis chamber are one and the same; a reaction vessel fluidly connected to the plurality of chambers of the cartridge body and configured for: i) nucleic acid amplification, and ii) detection and identification of one or more amplification products by real-time PCR, melting curve analysis, or a combination thereof; a filter disposed in the fluid path between the lysis chamber and the reaction vessel, and A set of primers and / or probes according to any one of claims 1 to 12, for confirming the presence of MPOX clade II.

15. The cartridge of claim 14, wherein the cartridge is a Clinical Laboratory Improvement Amendments (CLIA) compliant cartridge.

16. A method for detecting and identifying monkeypox (MPOX) clade II in a sample, the method comprising: a) contacting nucleic acid from the sample with a set of primers and optionally probe(s) according to any one of claims 1 to 11; b) subjecting the nucleic acid, primers and optional probe(s) to amplification conditions, optionally followed by melting curve determination; c) detecting the presence of any amplification product(s) by real-time PCR, melting curve analysis, or a combination thereof, and d) confirming the presence of an MPOX clade II virus in the sample or determining the absence of an MPOX clade II virus detectable using the set of primers and / or probes based on the detection or absence of the amplification product(s), respectively.

17. The method of claim 16, wherein d) comprises differentially confirming the presence of MPOX clade II and / or non-variola orthopoxviruses in the sample based on the detection or absence of the amplification product(s), respectively, or determining that no MPOX clade II and non-variola orthopoxviruses are detectable using the provided set of primers and / or probes.

18. The method of any one of claims 16-17, wherein: a) contacting the nucleic acid from the sample with the set of primers and / or probes comprises: placing the sample in a cartridge comprising a cartridge body having a plurality of chambers in fluid communication, a reaction vessel having one or more reaction chambers and configured for nucleic acid amplification and detection of one or more amplification products, a fluid path between the plurality of chambers and the reaction vessel, and a filter in the fluid path; and if the sample comprises cells, lysing the cells in the sample with one or more lysing reagents present in at least one of the plurality of chambers; b) subjecting the nucleic acid, primers and / or probes to amplification conditions comprises amplifying the nucleic acid using primers and / or probes present in solution in a reaction vessel.

19. The method of any one of claims 16-18, wherein the sample is a skin sample, a lesion swab sample, a vesicular lesion fluid sample, a pustular lesion fluid sample, a rectal sample, a nasal aspirate sample, a nasal wash sample, a nasal swab sample, a nasopharyngeal swab sample, a saliva sample, an oropharyngeal swab sample, a throat swab sample, a bronchoalveolar lavage fluid sample, a bronchial aspirate sample, a bronchial wash sample, an endotracheal aspirate sample, an endotracheal wash sample, a tracheal aspirate sample, a nasal secretion sample, a mucus sample, a sputum sample, a plasma sample, a whole blood sample, or a combination thereof.

20. The method of any one of claims 16-19, wherein the method is performed to differentiate between highly virulent pathogens and less virulent pathogens.

21. The panel of any one of claims 1-13, the box of any one of claims 14-15, or the method of any one of claims 16-20, wherein the box facilitates detection of MPOX clade II virus in the sample within 60 minutes, within 45 minutes, or within 30 minutes of collecting the sample from the subject and / or the method comprises detecting MPOX clade II virus in the sample within 60 minutes, within 45 minutes, or within 30 minutes of collecting the sample from the subject.

22. The panel of any one of claims 1-13, the box of any one of claims 14-15, or the method of any one of claims 16-20, wherein the box facilitates detection of MPOX clade II virus in the sample within 60 minutes, 45 minutes, 30 minutes, or 20 minutes from the time the sample is placed in the box and / or the method comprises detecting MPOX clade II virus in the sample within 60 minutes, 45 minutes, 30 minutes, or 20 minutes from the time the sample is placed in the box.

23. The set, cartridge or method of claim 21 or claim 22, wherein the reaction vessel comprises up to 4 reaction chambers.

24. The set, cartridge or method of any one of claims 21-23, wherein the reaction vessel comprises a reaction chamber.

25. A system for detecting monkeypox (MPOX) clade II in a biological sample, the system comprising: a module having a receiving area for receiving the cartridge of any one of claims 14-15, wherein the module includes one or more mechanisms within the receiving area for manipulating a fluid sample within the cartridge and an instrument for docking with the reaction vessel interface; A memory having programmable instructions recorded thereon, the programmable instructions being specifically configured to operate the module according to a monkeypox clade II assay protocol to determine the nucleic acid sequence characteristics of MPOX clade II.

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