Sample lysis reagent compositions and methods of production and use thereof

Abstract

To satisfy a need for methods and reagents utilized during sample handling for viral inactivation, thereby rendering samples non-infectious and providing safer sample handling conditions.SOLUTION: Methods of utilizing sample lysis reagent compositions in handling of samples containing viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are disclosed. The reagent composition comprises a nonionic octylphenol ethoxylate surfactant and water. Kits containing the sample lysis reagent compositions are also disclosed.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS / INCORPORATION-BY-REFERENCE STATEMENT This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 200,083, filed February 12, 2021. The entire contents of the above-referenced application are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable. [Background technology]

[0003] The field of medical diagnostics utilizes many different forms of assay technology. When a patient is suspected of being infected with a microorganism (such as, but not limited to, a bacterium or a virus), an assay is performed on a biological sample from the patient to detect antigens from the microorganism or antibodies against the microorganism produced by the patient's immune system.

[0004] COVID-19 (coronavirus disease 2019) is an illness caused by infection with the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus. The virus spreads easily from person to person, primarily through infected secretions such as saliva and respiratory droplets or aerosols. Evidence supports spread by both symptomatic and asymptomatic individuals. The incubation period for the virus ranges from 2 to 14 days after exposure, with symptoms appearing within approximately 5 days of exposure in most cases.

[0005] SARS-CoV-2 nucleic acid amplification tests, such as reverse transcription-polymerase chain reaction (RT-PCR), are considered the gold standard for diagnostic testing for current infections, typically using upper respiratory specimens. While RT-PCR detects viral genetic material, antigen tests detect viral proteins (e.g., nucleocapsid). Immunoassays detecting SARS-CoV-2 nucleocapsid antigens are also being used to diagnose current infections. Due to the high infectiousness and mode of transmission of this virus, the need for additional, less complex testing solutions has been recognized. Antigen testing, as part of SARS-CoV-2 testing strategies, is used to identify symptomatic and asymptomatic individuals currently infected with SARS-CoV-2.

[0006] Antigen tests for SARS-CoV-2 are particularly desirable during the COVID-19 pandemic because their use can expand access to more rapid screening of at-risk individuals. They are useful in responding to suspected COVID-19 outbreaks, especially when PCR is not readily available, and they allow for early implementation of infection control measures. SARS-CoV-2 antigen tests are also useful for investigating outbreaks in closed or semi-closed communities and for monitoring trends in disease incidence in the community. In cases of widespread community transmission, antigen tests not only enable early detection and isolation of positive cases but also enable effective infection control and contact tracing. Summary of the Invention [Problem to be solved by the invention]

[0007] Just as there is a need for new and improved assays for various viruses and viral infections, including (but not limited to) COVID-19, there is a corresponding need for new and improved reagents and sample handling methods for use therewith, particularly those that inactivate viruses, thereby rendering samples non-infectious, and allowing for safer sample handling conditions. There is a need for methods and reagents utilized during sample handling to provide the desired results. The present disclosure is directed to such reagents, kits containing same, and methods of making and using same. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows a schematic of viral antigen sample flow for two non-limiting examples of laboratory-based viral antigen assay formats, including rt-PCR and high-throughput assays. VTM: viral transport medium; UTM: universal transport medium. Throughput ADVIA Centaur 240 tests / hour; Atellica IM 440 tests / hour. DETAILED DESCRIPTION OF THE INVENTION

[0009] Before describing in detail at least one embodiment of the present disclosure by way of exemplary language and results, it should be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. Accordingly, the language used herein is intended to have the broadest possible scope and meaning; the embodiments are meant to be illustrative, not comprehensive. It should also be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.

[0010] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and described in various general and more specific references cited and discussed throughout the specification. The nomenclatures utilized in connection with analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the experimental procedures and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0011] All patents, published patent applications, and non-patent publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any part of this application are expressly incorporated herein by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0012] All of the compositions, kits, devices, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions, kits, devices, and / or methods have been described with reference to specific embodiments, it will be apparent to those skilled in the art that changes can be made to the compositions, kits, devices, and / or methods, and in the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure, as defined by the appended claims.

[0013] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0014] The word "comprising" in the claims and / or specification When used in conjunction with other terms, the use of the terms "a" or "an" can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." Thus, the terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "plurality" refers to two or more.

[0015] Use of the term "at least one" will be understood to include one and any amount greater than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend to 100 or 1000 or more, depending on the term to which it is attached; furthermore, an amount of 100 / 1000 should not be considered limiting, as higher limits may also provide satisfactory results. Furthermore, use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. Use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is for the purpose of distinguishing between two or more items only and is not intended to imply, for example, any permutation or order or any order of importance or addition of one item to another.

[0016] The use of the word "or" in the claims is used to mean an inclusive "and / or" unless expressly indicated to refer only to alternatives or unless the alternatives are mutually exclusive. For example, the condition "A or B" is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and A and B are both true (or exist).

[0017] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. For example, the appearances of the phrases "in some embodiments" or "one example" in various places in the specification are not necessarily all referring to the same embodiment. Moreover, all references to one or more embodiments or examples should not be construed as limiting the scope of the claims.

[0018] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method used to determine the value, or the variation that exists among study subjects. For example, and not by way of limitation, when the term "about" is used, the specified value may vary by plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent from the specified value, with such variations being appropriate to practice the disclosed methods and as would be understood by one of ordinary skill in the art.

[0019] As used in this specification and claims, "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and having, such as "have" and "has"), The words "including" (and any form of "including" such as "includes" and "include"), "including" (and any form of "including" such as "contains" and "contain"), or "containing" (and any form of containing such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0020] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and, where order is important in the particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing this example, combinations containing repeats of one or more items or terms are expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. One of skill in the art will understand that typically there is no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0021] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs completely, or that the subsequently described event or circumstance occurs to a large or significant extent. For example, when referring to a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are within close proximity to each other but not 100% adjacent to each other, or that a portion of one of two items is not 100% adjacent to the other item but is within close proximity to the other item.

[0022] As used herein, the phrases "associated" and "linked" include both direct association / binding of two moieties to one another and indirect association / binding of two moieties to one another. Non-limiting examples of association / linkage include, for example, covalent binding of one moiety to another moiety by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety, either directly or by a specific binding pair member attached to both moieties, incorporation of one moiety into another moiety, such as by dissolving or synthesizing one moiety into another moiety, and coating one moiety onto another moiety.

[0023] As used herein, the term "sample" will be understood to include any type of biological sample that can be utilized in accordance with the present disclosure. Examples of fluid biological samples that can be utilized include, but are not limited to, nasal, nasopharyngeal, anterior nasal, saliva, mucus, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cyst fluid, whole blood or any fraction thereof (i.e., plasma or serum), urine, sweat, interstitial fluid, extracellular fluid, tears, bladder washings, semen, feces, pleural fluid, combinations thereof, and the like.

[0024] Certain non-limiting embodiments of the present disclosure are directed to a viral sample lysis reagent composition effective for inactivating one or more viruses in a biological sample prior to performing an assay for detecting viral antigens in the biological sample. The reagent composition includes a non-ionic octylphenol ethoxylate surfactant and water. The reagent composition may optionally include other components, such as (but not limited to) antimicrobial agents (such as, but not limited to, azides), preservatives, or other agents commonly found in lysis reagents. Alternatively, the sample lysis reagent composition may include only surfactant and water.

[0025] The virus to be inactivated in the biological sample can be the virus being assayed and / or any other virus present in the sample. For example (and not by way of limitation), the virus can be a coronavirus, such as SARS-CoV-2, influenza virus, respiratory syncytial virus (RSV), hepatitis virus, human papillomavirus (HPV), varicella-zoster virus, herpes simplex virus, Epstein-Barr virus, cytomegalovirus (CMV), rotavirus, human immunodeficiency virus (HIV), morbillivirus (measles virus), paramyxovirus (mumps virus), Ebola virus, poliovirus, norovirus, BK virus, West Nile virus, Zika virus, dengue virus, picornavirus, enterovirus, parainfluenza virus, rubivirus, and the like, and any combination thereof.

[0026] Any nonionic octylphenol ethoxylate surfactant known in the art or otherwise contemplated herein can be utilized in accordance with the present disclosure. Non-limiting examples of octylphenol ethoxylate surfactants that can be utilized include polyethylene glycol octylphenyl ether, particularly 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol.

[0027] The non-ionic surfactant can be present in the sample lysis reagent composition at any initial concentration that enables the reagent composition to function in accordance with the present disclosure (i.e., inactivate viruses and not interfere with the antigen assay). In certain non-limiting embodiments, the surfactant is present in an amount of about 0.01 wt%, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0.07 wt%, about 0.08 wt%, about 0.09 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0. ... t%, approximately 0.5wt%, approximately 0.6wt%, approximately 0.7wt%, approximately 0.8wt%, approximately 0.9wt%, approximately 1.0wt%, approximately 1.1wt%, approximately 1.2wt%, Approx. 1.3wt%, approx. 1.4wt%, approx. 1.5wt%, approx. 1.6wt%, approx. 1.7wt%, approx. 1.8wt%, approx. 1.9wt%, approx. 2wt%, approx. 3wt% , about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, or more, or a range formed by two of the above values ​​(i.e., a range of about 0.1 wt% to about 20 wt%, a range of about 0.5 wt% to about 15 wt%, a range of about 1 wt% to about 10 wt%, etc.), or any value within a range of two of the above values ​​(i.e., about 2.7 wt%).

[0028] In a specific (but non-limiting) embodiment, the nonionic surfactant is present in the sample lysis reagent composition at an initial concentration ranging from about 1 wt% to about 15 wt%. In a specific (but non-limiting) embodiment, the nonionic surfactant is present in the sample lysis reagent composition at an initial concentration ranging from about 5 wt% to about 10 wt%. In another specific (but non-limiting) embodiment, the nonionic surfactant is present in the sample lysis reagent composition at an initial concentration of about 10 wt%.

[0029] Deionized water can be present in the sample lysis reagent composition at any concentration that enables the reagent composition to function in accordance with the present disclosure. For example (and not by way of limitation), deionized water can be present at about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 81 wt%, about 82 wt%, about 83 wt%, about 84 wt%, about 85 wt%, about 86 wt%, about 87 wt%, about 88 wt%, about 89 wt%, about 90 wt%, about 91 wt%, about 92 wt%, about 93 wt%, about 94 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, about 99 wt%, or more, or any of the foregoing values. It may be present at a concentration selected from a range formed by any two of the above values ​​(i.e., a range of about 85 wt% to about 99 wt%, a range of about 90 wt% to about 95 wt%, etc.), or any value within the range between any two of the above values.

[0030] Similarly, any additional components present in the reagent composition (i.e., one or more antimicrobial agents, preservatives, bulking agents, etc.) can each be present in any concentration that enables the reagent composition formed therefrom to function in accordance with the present disclosure. For example (and not by way of limitation), each additional component can be present in an amount of about 0.001 wt%, about 0.002 wt%, about 0.003 wt%, about 0.004 wt%, about 0.005 wt%, about 0.006 wt%, about 0.007 wt%, about 0.008 wt%, about 0.009 wt%, about 0.01 wt%, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0. ...8 wt%, about 0.009 wt%, about 0.01 wt%, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0.06 wt%, about 0.07 wt%, about 0.06 wt%, about 0.06 wt%, about 0.06 wt%, about 0.06 wt%, about 0.06 wt%, about 0.06 wt%, about 0.06 wt%, about 0.06 wt%, about 0.06 wt%, about 0.06 wt%, about 0.06 wt%, about 0.06 wt%, approx. 0.04wt%, approx. 0.05wt%, approx. 0.06wt%, approx. 0.07wt%, approx. 0.08wt%, approx. 0.09wt%, approx. 0.1wt%, approx. 0.2 wt%, approx. 0.3wt%, approx. 0.4wt%, approx. 0.5wt%, approx. 0.6wt%, approx. 0.7wt%, approx. 0.8wt%, approx. 0.9wt%, approx. 1.0wt%, approx. 1 The present invention may be present at a concentration independently selected from about 0.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about 1.5 wt%, about 1.6 wt%, about 1.7 wt%, about 1.8 wt%, about 1.9 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, or more, or a range formed by two of the above values ​​(i.e., a range of about 0.001 wt% to about 10 wt%, a range of about 0.1 wt% to about 5 wt%, a range of about 2 wt% to about 60 wt%, etc.), or any value within a range between two of the above values.

[0031] The sample lysis reagent composition of the present disclosure is substantially stable for a desired period of time at a desired temperature. For example (but not limited to), the sample lysis reagent composition may be substantially stable for at least about 7 days, at least about 14 days, at least about 28 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 2 years, at least about 3 years, or longer. Furthermore, the sample lysis reagent composition can be stored at room temperature (i.e., in the range of about 18°C ​​to about 25°C) or under refrigerated or frozen conditions. If a preservative is present in the sample lysis reagent composition, the preservative used is selected based on the specific storage conditions and the desired storage period.

[0032] The sample lysis reagent composition of the present disclosure may be provided in any form that allows the reagent composition to function according to the present disclosure. For example, but not by way of limitation, the reagent composition may be provided in liquid form. Alternatively, the reagent composition may be freeze-dried or lyophilized and provided in the form of a dry reagent.

[0033] Viral antigen assays can detect any type of virus that can infect a patient (such as, but not limited to, a human or livestock) and cause a disease state, for which specific antigens are known. Examples of viruses that may be detected by viral antigen assays according to the present disclosure include, but are not limited to, coronaviruses such as SARS-CoV-2; influenza viruses; respiratory syncytial virus (RSV); hepatitis viruses; human papillomavirus (HPV); varicella-zoster virus; herpes simplex virus; Epstein-Barr virus; cytomegalovirus (CMV); rotavirus; human immunodeficiency virus (HIV); morbillivirus (measles virus); paramyxovirus (mumps virus); Ebola virus; poliovirus; norovirus; BK virus; West Nile virus; Zika virus; dengue virus; picornawi virus; and other viruses. enteroviruses; parainfluenza viruses; rubiviruses; and the like; and any combination thereof.

[0034] Specific (but non-limiting) examples of viruses that may be detected by viral antigen assays according to the present disclosure include coronaviruses (such as, but not limited to, SARS-CoV-2 virus) and influenza viruses.

[0035] SARS-CoV-2 antigen assays utilizing the disclosed sample lysis reagent composition can detect antigens of any known virus, including, but not limited to, at least a portion of the nucleocapsid (N) protein, at least a portion of the spike (S) protein, at least a portion of the matrix (M) protein, at least a portion of the envelope (E) protein, at least a portion of the ssRNA, and combinations thereof.

[0036] Certain non-limiting embodiments of the present disclosure are directed to kits that include any one or more of the sample lysis reagent compositions described or otherwise contemplated herein.

[0037] The kit may include one or more other reagents / components for use in sample handling and / or assays according to the present disclosure. For example (and not by way of limitation), the kit may further include at least one reagent composition for use in performing a viral antigen assay (such as, but not limited to, a SARS-CoV-2 or other coronavirus antigen assay, or an influenza antigen assay). For example, and not by way of limitation, the kit may further include at least one reagent that specifically binds to an antigen (such as, but not limited to, an antibody that specifically binds to a viral antigen (i.e., an anti-SARS-CoV-2 antibody or an anti-influenza antibody)), at least one reagent for use in the particular assay format being utilized, and / or at least one viral antigen-containing reagent, as well as various combinations thereof. When the reagent includes a viral antigen, the viral antigen may be the same as or different from the viral antigen being assayed. Non-limiting examples of viral antigen-containing reagents within the scope of the present disclosure include calibrators containing viral antigens, quality control solutions containing viral antigens, and the like.

[0038] Non-limiting examples of coronavirus antigen-containing reagents included within the scope of the present disclosure include calibrators containing coronavirus antigens, quality control solutions containing coronavirus antigens, etc. Non-limiting examples of SARS-CoV-2 antigen-containing reagents included within the scope of the present disclosure include calibrators containing SARS-CoV-2 antigens, quality control solutions containing SARS-CoV-2 antigens, etc. Non-limiting examples of influenza antigen-containing reagents included within the scope of the present disclosure include calibrators containing influenza antigens, quality control solutions containing influenza antigens, etc.

[0039] Alternatively and / or additionally, the kit may include one or more sample collection tubes.

[0040] In certain (but non-limiting) embodiments, the kit may include one or more sample collection tubes having any of the sample lysis reagent compositions described herein or contemplated in connection with the sample collection tubes.

[0041] The sample lysis reagent compositions present in any of the kits described herein are provided in any form that enables them to function in accordance with the present disclosure. For example, and not by way of limitation, each of the reagents may be provided in liquid form and may be provided in bulk and / or single-stranded form within the kit. Alternatively, in certain (but non-limiting) embodiments, one or more of the reagents are disposed in the kit in the form of a single aliquot of lyophilized reagent. The use of dried reagents in kits and microfluidic devices is described in detail in U.S. Pat. No. 9,244,085 (Samproni), the entire contents of which are expressly incorporated herein by reference. If one or more of the reagents are lyophilized, the kit may further include an excipient for reconstituting the reagent; alternatively, the sample itself may function as an excipient when mixed with the lyophilized reagent.

[0042] If the kit includes a sample collection tube into which the sample lysis reagent composition is disposed, and if the sample lysis reagent is in liquid form, the sample lysis reagent composition may be disposed in the sample collection tube in any volume that enables the sample lysis reagent composition to function in accordance with the present disclosure. Non-limiting examples of volumes per mL of sample collection tube volume utilized in accordance with the present disclosure include about 1 μL, about 2 μL, about 3 μL, about 4 μL, about 5 μL, about 6 μL, about 7 μL, about 8 μL, about 9 μL, about 10 μL, about 11 μL, about 12 μL, about 13 μL, about 14 μL, about 15 μL, about 16 μL, about 17 μL, about 18 μL, about 19 μL, about 20 μL, about 21 μL, about 22 μL, about 23 μL, about 24 μL, about 25 μL, about 26 μL, about 27 μL, about 28 μL, about 29 μL, about 30 μL, about 31μL, about 32μL, about 33μL, about 34μL, about 35μL, about 36μL, about 37μL, about 38μL, about 39μL, about 40μL, about 41μL, about 42μL, about 43μL, about 44μL, about 45μL, about 46μL, about 47μL, about 48μL, Approximately 49μL, approximately 50μL, approximately 51μL, approximately 52μL, approximately 53μL, approximately 54μL, approximately 55μL, approximately 56μL, approximately 57μL, approximately 58μL, approximately 59μL, approximately 60μL, approximately 61μL, approximately 62μL, approximately 63μL, approximately 64μL, approximately 65μL, approximately 66μL, Approximately 67μL, approximately 68μL, approximately 69μL, approximately 70μL, approximately 71μL, approximately 72μL, approximately 73μL, approximately 74μL, approximately 75μL, approximately 76μL, approximately 77μL, approximately 78μL, approximately 79μL, approximately 80μL, approximately 81μL, approximately 82μL, approximately 83μL, approximately 84μL , about 85 μL, about 86 μL, about 87 μL, about 88 μL, about 89 μL, about 90 μL, about 91 μL, about 92 μL, about 93 μL, about 94 μL, about 95 μL, about 96 μL, about 97 μL, about 98 μL, about 99 μL, about 100 μL, about 101 μL, about 10 Included are 2 μL, about 103 μL, about 104 μL, about 105 μL, about 106 μL, about 107 μL, about 108 μL, about 109 μL, about 110 μL, about 115 μL, about 120 μL, about 125 μL, about 130 μL, about 135 μL, about 140 μL, about 145 μL, about 150 μL, or more, or a range formed by two of the above values ​​(i.e., a range of about 1 μL to about 150 μL, a range of about 10 μL to about 100 μL, etc.), or any value within a range between two of the above values.

[0043] In certain (but non-limiting) embodiments, the sample lysis reagent composition is placed in the sample collection tube in an amount ranging from about 10 μL to about 100 μL per mL of sample collection tube volume. In certain (but non-limiting) embodiments, the sample lysis reagent composition is placed in the sample collection tube in an amount of about 50 μL per mL of sample collection tube volume.

[0044] Alternatively, if the kit includes a sample collection tube into which the sample lysis reagent composition is placed, the sample lysis reagent composition is placed in the sample collection tube in freeze-dried or lyophilized form. In this way, the sample acts as a vehicle for dissolving the lyophilized reagent; alternatively, the kit may include a separate vehicle that is placed in the sample collection tube to reconstitute the sample lysis reagent composition before adding the sample to the sample collection tube. Furthermore, in certain (but non-limiting) embodiments, using the sample lysis reagent composition in lyophilized form allows the lyophilized reagent to adhere to the sidewall of the sample collection tube.

[0045] In addition to the assay components / reagents described in detail hereinabove, any of the kits described or otherwise contemplated herein may further include other reagents for carrying out any of the particular assays described or otherwise contemplated herein. The nature of these additional reagents will depend on the particular assay format, and their identification is well within the skill of those in the art; therefore, further description thereof is not believed to be necessary. Each of the components / reagents present in the kit may be in a separate container / compartment, or the various compositions / reagents may be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the components / reagents. Furthermore, the kit may include a microfluidic device in which the components / reagents are disposed.

[0046] The relative amounts of the various components / reagents in the kit can be varied widely to provide concentrations of the components / reagents that fully optimize the reactions required to occur during the assay method and further optimize the sensitivity of the assay. Under appropriate circumstances, one or more of the components / reagents in the kit can be provided as a dry powder, such as a lyophilized powder, and the kit can further include an excipient for dissolving the dried reagent; in this way, a reagent solution having the appropriate concentration for performing a method or assay according to the present disclosure can be obtained from these components. Non-limiting examples of other reagents that can be included in the kit include wash solutions, diluents, excipients, interference solutions, positive controls, negative controls, etc. Furthermore, the kit can further include a set of written instructions describing how to use the kit. Kits of this nature can be used in any of the methods described or otherwise contemplated herein.

[0047] Certain non-limiting embodiments of the present disclosure are directed to a method for producing any of the sample lysis reagent compositions for SARS-CoV-2 antigen assays described herein or otherwise contemplated. In this method, water is added to a desired amount of nonionic surfactant until the desired surfactant concentration (as described in detail hereinabove) is achieved. The solution is then thoroughly mixed and stored at a desired temperature for a desired period of time (as described herein).

[0048] Additionally, the method of making any of the sample lysis reagent compositions may include one or more additional steps to form the reagent compositions described herein.

[0049] Certain non-limiting embodiments of the present disclosure are directed to a method of inactivating at least one virus present in a biological sample, in which any of the sample lysis reagent compositions described or otherwise contemplated herein is added to a biological sample to form a mixture, and the mixture is incubated for a desired period of time and at a temperature sufficient to substantially inactivate the at least one virus.

[0050] Any biological sample potentially containing evidence of infection by one or more viruses may be utilized in accordance with the present disclosure. For example, and without limitation, the biological sample may be nasal, nasopharyngeal, anterior nasal, saliva, mucus, sputum, cerebrospinal fluid, intestinal fluid, peritoneal fluid, cyst fluid, whole blood or any portion thereof, urine, sweat, interstitial fluid, extracellular fluid, tears, bladder washings, semen, feces, pleural fluid, and combinations thereof.

[0051] In certain (but non-limiting) embodiments, the biological sample is selected from the group consisting of nasal, nasopharyngeal, anterior nasal, saliva, mucus, sputum, and combinations thereof.

[0052] The sample lysis reagent composition is utilized to inactivate any of the viruses described herein or otherwise contemplated. In certain (but non-limiting) embodiments, the one or more viruses inactivated include (but are not limited to) coronaviruses, such as SARS-CoV-2; influenza viruses; respiratory syncytial virus (RSV); hepatitis viruses; human papillomavirus (HPV); varicella-zoster virus; herpes simplex virus; Epstein-Barr virus; cytomegalovirus (C); MV); rotavirus; human immunodeficiency virus (HIV); morbillivirus (measles virus); paramyxovirus (mumps virus); Ebola virus; poliovirus; norovirus; BK virus; West Nile virus; Zika virus; dengue virus; picornavirus; enterovirus; parainfluenza virus; rubivirus; and the like; and any combination thereof.

[0053] The sample lysis reagent composition is added to a biological sample in any volume that enables the sample lysis reagent composition to function in accordance with the present disclosure (and therefore any final concentration based on the initial concentration of the sample lysis reagent composition detailed hereinabove). Non-limiting examples of volumes per mL of biological sample volume utilized in accordance with the present disclosure include about 1 μL, about 2 μL, about 3 μL, about 4 μL, about 5 μL, about 6 μL, about 7 μL, about 8 μL, about 9 μL, about 10 μL, about 11 μL, about 12 μL, about 13 μL, about 14 μL, about 15 μL, about 16 μL, about 17 μL, about 18 μL, about 19 μL, about 20 μL, about 21 μL, about 22 μL, about 23 μL, about 24 μL, about 25 μL, about 26 μL, about 27 μL, about 28 μL, about 29 μL, about 30 μL, and about 31 μL. L, about 32μL, about 33μL, about 34μL, about 35μL, about 36μL, about 37μL, about 38μL, about 39μL, about 40μL, about 41μL, about 42μL, about 43μL, about 44μL, about 45μL, about 46μL, about 47μL, about 48μL, about 49 50μL, 51μL, 52μL, 53μL, 54μL, 55μL, 56μL, 57μL, 58μL, 59μL, 60μL, 61μL, 62μL, 63μL, 64μL, 65μL, 66μL, 6 7μL, approximately 68μL, approximately 69μL, approximately 70μL, approximately 71μL, approximately 72μL, approximately 73μL, approximately 74μL, approximately 75μL, approximately 76μL, approximately 77μL, approximately 78μL, approximately 79μL, approximately 80μL, approximately 81μL, approximately 82μL, approximately 83μL, approximately 84μL, approximately 85μL, about 86μL, about 87μL, about 88μL, about 89μL, about 90μL, about 91μL, about 92μL, about 93μL, about 94μL, about 95μL, about 96μL, about 97μL, about 98μL, about 99μL, about 100μL, about 101μL, about 102 10 μL, about 103 μL, about 104 μL, about 105 μL, about 106 μL, about 107 μL, about 108 μL, about 109 μL, about 110 μL, about 115 μL, about 120 μL, about 125 μL, about 130 μL, about 135 μL, about 140 μL, about 145 μL, about 150 μL, or more, or a range formed by two of the above values ​​(i.e., a range of about 1 μL to about 150 μL, a range of about 10 μL to about 100 μL, etc.), or any value within a range between two of the above values.

[0054] In certain (but non-limiting) embodiments, the sample lysis reagent composition is added to the biological sample in an amount ranging from about 10 μL to about 100 μL per mL of biological sample present. In certain (but non-limiting) embodiments, the sample lysis reagent composition is added to the biological sample in an amount of about 50 μL per mL of biological sample.

[0055] The sample lysis reagent composition can be present in the sample lysis reagent composition / biological sample mixture at any final concentration that enables the sample lysis reagent composition to function in accordance with the present disclosure (i.e., inactivate viruses and not interfere with antigen assays). In certain non-limiting embodiments, the surfactant is present at about 0.01 wt%, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0.07 wt%, about 0.08 wt%, about 0.09 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1.0 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about 1.6 wt%, about 1.8 wt%, about 1.9 wt%, about 2.0 wt%, about 2.1 wt%, about 2.2 wt%, about 2.3 wt%, about 2.4 wt%, about 2.5 wt%, about 2.6 wt%, about 2.7 wt%, about 2.8 wt%, about 2.9 ... The reagent composition may be present in a concentration independently selected from about 0.4 wt%, about 1.5 wt%, about 1.6 wt%, about 1.7 wt%, about 1.8 wt%, about 1.9 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, or more, or a range formed by two of the above values ​​(i.e., a range of about 0.01 wt% to about 2 wt%, a range of about 0.01 wt% to about 1 wt%, a range of about 0.05 wt% to about 1 wt%, etc.), or any value within a range of two of the above values.

[0056] As described herein above, the mixture can be incubated for any desired period of time and at any temperature sufficient to substantially inactivate the virus. For example (and not by way of limitation), the mixture can be incubated at temperatures of about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, or higher, and any range formed by any two of the above values. In a specific (but non-limiting) embodiment, the mixture is incubated at room temperature (i.e., a temperature ranging from about 18°C ​​to about 25°C).

[0057] Also, for example (and not by way of limitation), the mixture may be incubated for at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 11 minutes, at least about 12 minutes, at least about 13 minutes, at least about 14 minutes, at least about 15 minutes, at least about 16 minutes, at least about 17 minutes, at least about 18 minutes, at least about 19 minutes, at least about 20 minutes, at least about 21 minutes, at least about 22 minutes, at least about 23 minutes, at least about 24 minutes, at least about 25 minutes, at least about 26 minutes, at least about 27 minutes, at least about 28 minutes, at least about 29 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 90 minutes, at least about 120 minutes, or more, and any range formed by two of the above values ​​(i.e., such as a range of about 5 minutes to about 20 minutes). In a specific (but non-limiting) embodiment, the mixture is incubated for about 10 minutes.

[0058] One or more of the steps of the disclosed methods are performed simultaneously or in whole or in part sequentially with one or more assay steps for detecting one or more viral antigens in a biological sample.

[0059] Furthermore, the mixture containing the inactivated virus may be stored for any desired period of time and at any temperature, so long as the antigen present in the biological sample is detectable. Specific (but non-limiting) examples of storage conditions that may be utilized include up to about 4 hours at room temperature, up to about 3 days at 2-8°C, and longer if stored at -20°C or below. [Example]

[0060] Examples are provided herein below. However, it should be understood that the present disclosure is not limited in its application to the specific experiments, results, and experimental procedures disclosed herein. Rather, examples are provided merely as one of various embodiments, and are intended to be illustrative, not comprehensive. [Example]

[0061] This example describes the preparation of a sample lysis reagent composition constructed in accordance with the present disclosure.

[0062] The required amount of Triton X-100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol) was placed in a container, and then approximately 10 wt % Triton Deionized water was added until a Triton X-100 concentration was achieved. The solution was then mixed to ensure that the Triton X-100 was properly dispersed therein. For example (but not by way of limitation), the Triton X-100 and deionized water solution were mixed on a roller mixer set at 30-45 or a 3D Orbital Mixer set at 70-90 RPM for 120-240 minutes to ensure proper mixing.

[0063] The pH of the solution was then measured at room temperature (i.e., in the range of about 18°C ​​to about 25°C) to confirm that the pH was in the range of about 4.0 to about 6.0. Optionally, the solution was also filtered through a 0.2 μm filter, if desired.

[0064] The resulting sample dissolution reagent composition was then stored at room temperature or in the range of about 2°C to about 8°C. [Example]

[0065] This example discloses the determination of infectivity for sample lysis reagent composition treatment of SARS-CoV-2 culture fluid. This study was conducted to determine the extent to which Triton X-100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol) can inactivate SARS-CoV-2 without destroying the ability of viral proteins to be detected in antigen-based detection assays.

[0066] material and method: A sample lysis reagent composition prepared similarly to Example 1, except that a Triton X-100 concentration of approximately 0.5 wt% was used, was used to test its effectiveness in inactivating SARS-CoV-2 (isolate: USAWA1 / 2020). A lower initial Triton X-100 concentration was used to ensure that the cells being tested remained intact when exposed to the sample lysis reagent composition; however, because retaining intact cells is not a requirement for the SARS-CoV-2 antigen assay, higher initial surfactant concentrations can be used in the sample lysis reagent composition utilized in accordance with the methods of the present disclosure.

[0067] SARS-CoV-2 infected VeroE6 cell cultures (ZeptoMetrix, Buffalo, NY Part #0610587CF Lot #544347) were grown and frozen (-65°C or colder) for experimental procedures. The cultures were thawed and 2 × 10 6 TCID 50 The virus was diluted to 1 / mL and combined with lysis buffer at a 1:10 dilution. The lysis buffer and virus had a contact time of 1, 2, 5, and 10 minutes before setting up the infectivity plates. As a control, the buffer was combined with uninfected VeroE6 cell culture fluid at a 1:10 dilution for a 10-minute contact time to observe whether cytotoxicity could affect the infectivity test results. All samples were tested in quadruplicate and observed for 7 days by microscopic visualization of cytopathic effect (CPE).

[0068] On day 0, SARS-CoV-2 was administered at 2 × 10 6 TCID 50 / mL and combined with 0.5% Triton X-100 at a 1:10 dilution. The mixture was incubated at room temperature for 1, 2, 5, and 10 minutes. After incubation, the mixture was transferred to a 96-well plate. No lysis buffer was added to control wells containing cells only. As a negative control, 0.5% Triton was mixed with 2% MEM medium at a 1:10 dilution for 10 minutes. After incubation, the cell / reagent mixture was transferred to a 96-well plate. No lysis reagent was added to control wells containing cells only. As a positive control, VeroE6 cell culture medium was used to measure TCID 50 The assay was performed. All cultures were incubated at 37°C for 7 days. After all conditions were set, VeroE6 cells were added to the wells. All conditions in the 96-well plate were observed 3 and 7 days post-infection (dpi) and recorded on a plate map. Cytopathic effect (CPE) was scored as shown in Table 1.

[0069] [Table 1]

[0070] result: Infectious SARS-CoV-2 virus positive control was TCID 50 The assay showed visible CPE, which was completely destroyed by day 7 post-infection in wells 1-7, demonstrating proper viral infection of VeroE6 cells in the absence of any lytic reagent. The positive control was 1.05 x 10 6 TCID 50 Each negative control consisted of VeroE6 cells alone, which remained healthy on each plate throughout the course of the assay.

[0071] Uninfected VeroE6 cells were also exposed to 0.5% Triton at a 1:10 dilution as a control for cytotoxicity. VeroE6 cells treated with 0.5% Triton at a 1:10 ratio showed no signs of cytotoxicity 3 and 7 days after treatment, indicating this ratio was effective in treating SARS-CoV-2 and did not interfere with overall cell health.

[0072] SARS-CoV-2 infection can cause complete or partial destruction of the VeroE6 cell monolayer. Upon infection, many of the cells rapidly shrink, become dense, and detach from the monolayer. This phenomenon was observed in VeroE6 cells infected with SARS-CoV-2. SARS-CoV-2 treated with 0.5% Triton at a 1:10 ratio for 1, 2, and 5 minutes exhibited cytopathic effects throughout the assay. SARS-CoV-2 incubated for 1 minute resulted in the highest number of wells exhibiting CPE. As the incubation time increased, the effect decreased. SARS-CoV-2 treated with 0.5% Triton at a 1:10 dilution was not completely effective in inactivating the SARS-CoV-2 virus when exposed for 1, 2, and 5 minutes (Table 2).

[0073] Infectious SARS-CoV-2 treated with 1:10 diluted 0.5% Triton for 10 minutes showed no visible CPE, indicating that 0.5% Triton added at a 1:10 dilution is effective in inactivating the SARS-CoV-2 virus when exposed for 10 minutes or longer.

[0074] [Table 2]

[0075] Acceptance criteria were as follows: (1) the positive control infectious SARS-CoV-2 culture must show CPE, (2) all cell-only control wells must remain healthy throughout the entire 7-day assay, and (3) the effect of the 0.5% Triton reagent on healthy cells must allow reading of the infectious plate (i.e., cell death must not prevent CPE).

[0076] Conclusion: The cell-only negative control remained healthy throughout the course of the assay in all test plates. The positive control SARS-CoV-2 cultures showed infectious CPE throughout the course of the assay and therefore met the control acceptance criteria. VeroE6 cell-only wells showed no signs of CPE. SARS-CoV-2 treated with 0.5% Triton at a 1:10 dilution was not completely effective in inactivating SARS-CoV-2 virus when exposed for 1, 2, and 5 minutes. SARS-CoV-2 treated with 0.5% Triton at a 1:10 dilution was effective in inactivating SARS-CoV-2 virus when exposed for 10 minutes or longer. [Example]

[0077] This example provides one non-limiting protocol for how to use the sample lysis reagent composition to inactivate viruses in a biological sample.

[0078] If necessary, nasopharyngeal (NP) or anterior nasal (AN) swab sample tubes were thawed. The NP / AP swab sample tubes were then vortexed for 10 seconds. The caps were carefully removed from the sample tubes, and the swabs were rolled against the side of the tube at least three times and then removed. The swabs were then disposed of in a biohazardous waste collection container.

[0079] CoV2Ag Sample Lysis Reagent (prepared as described above in Example 1) was then dispensed into the NP / AP swab sample tube in an amount of 50 μL (or 2 drops) per mL of sample; for example, this required 150 μL (or 6 drops) of CoV2Ag Sample Lysis Reagent for a 3 mL sample (or 5 μL of Sample Lysis Reagent for a 100 μL sample). The mixture was then gently mixed.

[0080] The mixture was incubated at room temperature for 10 minutes to inactivate the sample, which was then optionally transferred to an appropriate sample cup or other assay device to be placed on the assay system (or for performing a manual assay).

[0081] The inactivated sample can be tested immediately after the incubation step is complete. Alternatively, the inactivated sample can be stored at room temperature for a short period of time before performing the assay. For example (and not by way of limitation), inactivated samples are stable on the system for up to 4 hours, and inactivated samples can be stored at 2-8°C for up to 3 days, or at -20°C or below for longer storage periods. Inactivated samples should not be stored in a frost-free freezer, and more than two freeze-thaw cycles should be avoided.

[0082] Figure 1 provides a schematic diagram illustrating sample flow through two types of laboratory-based assays (rt-PCR and high-throughput SARS-CoV-2 antigen assays) and includes steps of the disclosed methods (i.e., addition of a sample lysis reagent composition to the sample, mixing, and incubation to inactivate the virus). However, this schematic is for illustrative purposes only and is not intended to limit the disclosure; it will be understood that different samples, different assays, and different sample flow steps may be utilized as described herein. Additionally, the specific sample lysis reagent composition concentrations and incubation conditions listed in Figure 1 are also for illustrative purposes only and are not intended to limit the methods disclosed or otherwise contemplated herein.

[0083] Thus, in accordance with the present disclosure, there have been provided compositions, kits, and devices, and methods for making and using the same, that fully satisfy the objects and advantages set forth hereinabove. While this disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth hereinabove, it will be apparent that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that are within the spirit and broad scope of this disclosure.

Claims

1. 1. A method for inactivating at least one virus present in a biological sample, comprising: (i) adding a sample lysis reagent composition to a biological sample to form a mixture, the sample lysis reagent composition comprising a non-ionic octylphenol ethoxylate surfactant and water, the surfactant being present in the sample lysis reagent composition at an initial concentration in the range of 1 wt % to 15 wt %; (ii) incubating the mixture for a period ranging from 5 minutes to 20 minutes at a temperature sufficient to substantially inactivate at least one virus; Including, The method, wherein the sample dissolution reagent composition is added to the biological sample at a concentration ranging from 20 μL to 100 μL per mL of the biological sample.

2. The method of claim 1 , wherein the nonionic surfactant comprises polyethylene glycol octylphenyl ether.

3. The method of claim 1, wherein the nonionic surfactant comprises 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol.

4. 10. The method of claim 1, wherein the surfactant is present in the sample lysis reagent composition at an initial concentration ranging from 5 wt % to 10 wt %.

5. 2. The method of claim 1, wherein the surfactant is present in the sample lysis reagent composition at an initial concentration of 10 wt %.

6. 10. The method of claim 1, wherein the surfactant is present in the mixture at a final concentration ranging from 0.01 wt % to 2 wt %.

7. The method of claim 1, wherein the mixture is incubated at a temperature ranging from 18°C ​​to 25°C. Law.

8. The method of claim 1 , wherein the mixture is incubated for 10 minutes.

9. 2. The method of claim 1, wherein the biological sample is selected from the group consisting of nasal, nasopharyngeal, anterior nasal, saliva, mucus, sputum, cerebrospinal fluid, intestinal fluid, peritoneal fluid, cyst fluid, whole blood or any portion thereof, urine, sweat, interstitial fluid, extracellular fluid, tears, bladder washings, semen, feces, pleural fluid, and combinations thereof.

10. 10. The method of claim 1, wherein the biological sample is selected from the group consisting of nasal, nasopharyngeal, anterior nasal, saliva, mucus, sputum, and combinations thereof.

11. 10. The method of claim 1, further comprising: (iii) storing the incubated mixture at a temperature ranging from -80°C to 8°C.

12. The method of claim 1 , wherein the virus is a coronavirus.

13. 13. The method of claim 12, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

14. 2. The method of claim 1, wherein the virus is an influenza virus.

15. at least one sample collection tube having at least one sample lysis reagent composition associated therewith, the sample lysis reagent composition comprising a non-ionic octylphenol ethoxylate surfactant and water, the surfactant being present in the range of 1 wt % to 15 wt %; 2. The kit for carrying out the method of claim 1, wherein the amount of sample lysis reagent composition present in the sample collection tube is in the range of 20 μL to 100 μL per mL of volume of the sample collection tube.

16. 16. The kit of claim 15, wherein the surfactant is present in the sample dissolution reagent composition at a concentration ranging from 5 wt % to 10 wt %.

17. 16. The kit of claim 15, wherein the surfactant is present in the sample lysis reagent composition at a concentration of 10 wt%.

18. 16. The kit of claim 15, wherein the non-ionic surfactant comprises polyethylene glycol octylphenyl ether.

19. The kit of claim 15, wherein the surfactant comprises 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol.

20. 16. The kit of claim 15, wherein the nonionic octylphenol ethoxylate surfactant is lyophilized.

21. 21. The kit of claim 20, wherein the non-ionic octylphenol ethoxylate surfactant is attached to the sidewall of the sample collection tube.

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