Buffer compositions for reducing aggregation

Abstract

Compositions are provided for nucleic acid-based detection and identification of vaginal diseases, such as vulvovaginal candidiasis, trichomoniasis, and / or bacterial vaginosis. [Solution] A buffer composition is provided that includes a conjugate pair of an acid and a base, a chelating or reducing agent, a nonionic surfactant, a monovalent or divalent salt selected from the group consisting of sodium salts, potassium salts, calcium salts, magnesium salts, and combinations thereof, and, optionally, a germicidal preservative, including one or more isothiazolones.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 879,310, filed July 26, 2019, which is incorporated herein by reference in its entirety. The present disclosure relates to compositions, kits, and methods for vaginal diseases and / or sexually transmitted diseases, such as buffer compositions, assay kits containing the same, and methods of use and diagnostic testing therewith. In some embodiments, the compositions, kits, and methods achieve robust, high-performance diagnostic testing from biological samples, particularly in the presence of interfering substances. Some embodiments relate to nucleic acid-based detection and identification of vulvovaginal candidiasis-associated Candida species, trichomonas vaginalis, which causes trichomoniasis, and / or bacterial vaginosis-associated bacteria from clinical vaginal swabs sampled from women exhibiting clinical signs of vaginitis and / or vaginosis using the compositions, kits, and methods disclosed herein. In some embodiments, sexually transmitted diseases, such as chlamydia infection, gonorrhea, and trichomoniasis, are diagnosed using the nucleic acid-based compositions, kits, and methods disclosed herein. [Background technology]

[0002] Candida, a genus of yeast, is the most common cause of fungal infections worldwide. Many Candida species are harmless commensals, found as part of the host's normal microbiota and can be endosymbiotic in hosts, including humans. However, in cases of host instability or immunosuppression, Candida is known to invade and cause disease. Some Candida species, such as C. krusei and C. glabrata, are known to be associated with vulvovaginal candidiasis (VVC). Trichomonas vaginalis is an anaerobic flagellate protoparasite, which is the etiologic agent of trichomoniasis. Bacterial vaginosis (BV) is a vaginal infection caused by an alteration of the normal balance of bacteria in the vagina.

[0003] Sexually transmitted infections (STIs), such as chlamydia (CT), gonorrhea (GC), and trichomoniasis (TV), pose a significant and growing health care burden. While the majority of these infections are asymptomatic, if left untreated they can have serious consequences, such as pelvic inflammatory disease (PID), ectopic pregnancy, infertility, preterm delivery or low birth weight babies, and increased risk of STI transmission or infection (including HIV) in men and women.

[0004] Targeted treatment based on accurate diagnosis of vaginal diseases and / or sexually transmitted diseases is necessary to improve patients' quality of life and achieve better clinical outcomes. Assay platforms and methods, such as fluidic manipulation of functionalized particles for nucleic acid extraction, have been developed for diagnostic testing and therapeutic intervention of VVC, trichomoniasis, BV, CT, GC, and TV. Some such assays demonstrate sensitivity to even trace amounts of interfering substances present in the biological sample being tested. In particular, clinical vaginal swab samples may be contaminated with gels, such as commercially available personal care creams used by patients and clinical lubricants introduced during testing procedures prior to sample collection. Many interfering gels contain polyelectrolytes, such as carbomer (polyacrylic acid), which can interact with functionalized particles placed in nucleic acid extraction assays. When transferred to an assay cartridge, these interfering gels can cause aggregation of functionalized particles within the fluidic channels, clogging the fluidic channels, and / or inhibiting polymerase chain reaction (PCR) of target nucleic acids. Therefore, in the presence of interfering substances, many nucleic acid-based assays not only experience increased reporting errors (false positives and false negatives), but also increased unreportable results (unresolved results due to internal control failure, indeterminate results due to excessive noise, etc.). For example, particle aggregation can lead to loss of functionality, thereby reducing assay sensitivity and increasing the rate of unresolved results. Channel clogging can increase the rate of indeterminate results, and PCR inhibition can increase the rate of false negatives. Summary of the Invention

[0005] Standard buffers used in assays such as those described above are typically designed solely to preserve biological samples after collection for subsequent diagnostic analysis. Given the various adverse events associated with interfering substances commonly present in clinical samples, there is a pressing need to develop novel buffer compositions (and methods using same) with higher interference robustness for on-chip sample processing and the associated detection of vaginal diseases (e.g., vulvovaginal candidiasis, trichomoniasis, bacterial vaginosis, etc.) while maintaining clinical efficiency. For microfluidic assays such as those discussed in the previous paragraph, there is a particular need to prevent and / or mitigate the consequences of particle aggregation, channel clogging, PCR inhibition, etc. There is also a need for novel buffer compositions that can be adapted to various clinical workflows, leading to faster diagnoses, more effective treatments, and better patient outcomes.

[0006] Embodiments of the present disclosure relate to compositions, kits, and methods for nucleic acid-based detection and identification of vaginal diseases and / or sexually transmitted diseases, particularly from vaginal samples containing interfering substances. Some of the disclosed embodiments relate to buffer compositions, kits, and methods for preventing or reducing aggregation of surface-functionalized particles, such as those disposed in microfluidic PCR devices for nucleic acid extraction and / or purification. Some embodiments may improve the efficiency of amplification and / or detection of nucleic acids from vaginal pathogenic organisms. It will be understood by those skilled in the art that the application of the compositions, kits, and methods described herein is not limited to specific samples or specific vaginal diseases.

[0007] Buffer compositions are described herein. In some embodiments, the buffer composition comprises a conjugate pair of an acid and a base, a chelating or reducing agent, a nonionic surfactant, a monovalent or divalent salt selected from the group consisting of sodium salts, potassium salts, calcium salts, magnesium salts, and combinations thereof, and, optionally, a biocidal preservative, including one or more isothiazolones. In some embodiments, the conjugate pair comprises acetic acid and its salts. In some embodiments, the conjugate pair comprises about 50 mM to about 150 mM acetic acid. In some embodiments, the conjugate pair comprises about 90 mM to about 110 mM acetic acid. In some embodiments, the conjugate pair comprises about 350 mM to 450 mM sodium acetate. In some embodiments, the conjugate pair can be present at a concentration ranging from about 400 mM to about 600 mM. In some embodiments, the conjugate pair comprises acetic acid and sodium acetate, or Tris-HCl. In some embodiments, the acetic acid can be present at a concentration of 200 mM or less, and the sodium acetate can be present at a concentration of 300 mM or more. In some embodiments, the buffer composition may have a pH of about 4.0 to about 6.0 or about 1.0 to about 3.0. In some embodiments, the buffer composition may have a pH of about 5.0. In some embodiments, the buffer composition may have a pH of about 2.4. In some embodiments, the chelating agent may include EDTA. In some embodiments, the reducing agent includes TCEP. In some embodiments, the chelating agent or reducing agent may be present at a concentration ranging from about 1 mM to about 20 mM. In some embodiments, the chelating agent is EDTA at a concentration of about 10 mM. In some embodiments, the reducing agent is TCEP at a concentration of about 15 mM. In some embodiments, the nonionic surfactant may be selected from the group consisting of ethoxylated nonionic surfactants, proxilated nonionic surfactants, co-ethoxylated-propoxylated nonionic surfactants, and combinations thereof.In some embodiments, the nonionic surfactant may be selected from the group consisting of ethoxylated sorbitan esters of mono-fatty acids, ethoxylated octylphenols, ethoxylated secondary C1-C20 alcohols, co-ethoxylated propoxylated seed oil alcohols, and combinations thereof. In some embodiments, the nonionic surfactant may be selected from the group consisting of ethoxylated sorbitan esters of mono-fatty acids containing an average of 1-50 ethylene oxide units per surfactant, ethoxylated octylphenols containing an average of 1-20 ethylene oxide units per surfactant, ethoxylated secondary C1-C20 alcohols containing an average of 1-20 ethylene oxide units per surfactant, co-ethoxylated propoxylated seed oil alcohols containing an average of 1-20 propylene oxide units and 1-30 ethylene oxide units per surfactant, and combinations thereof. In some embodiments, the nonionic surfactant may comprise one or more ethoxylated sorbitan esters of mono-fatty acids. In some embodiments, the one or more ethoxylated sorbitan esters of mono-fatty acids may contain an average of 1 to 50 ethylene oxide units. In some embodiments, the nonionic surfactant may include one or more ethoxylated secondary C1-C20 alcohols. In some embodiments, the one or more ethoxylated secondary C1-C20 alcohols may contain an average of 1 to 20 ethylene oxide units. In some embodiments, the nonionic surfactant may include one or more ethoxylated octylphenols. In some embodiments, the one or more ethoxylated octylphenols may contain an average of 1 to 20 ethylene oxide units. In some embodiments, the nonionic surfactant may include one or more co-ethoxylated-propoxylated seed oil alcohols. In some embodiments, the one or more co-ethoxylated-propoxylated seed oil alcohols may contain an average of 1 to 20 propylene oxide units and 1 to 30 ethylene oxide units. In some embodiments, the non-ionic detergent is Tergitol™ or Triton™ detergent.In some embodiments, the nonionic surfactant is Tergitol™ 15-S-9 or Triton™ X-100. In some embodiments, the nonionic surfactant may be present at a concentration ranging from about 0.5% to 1.5% by weight of the buffer composition. In some embodiments, the nonionic surfactant may be present at a concentration ranging from about 1.0% by weight of the buffer composition. In some embodiments, the divalent salt may be a calcium salt. In some embodiments, the divalent salt may be CaCl. In some embodiments, the monovalent or divalent salt may be present at a concentration ranging from about 100 mM to about 300 mM. In some embodiments, the monovalent or divalent salt may be present at a concentration of about 200 mM. In some embodiments, the antiseptic preservative may be present at a concentration ranging from about 0.03% by weight of the buffer composition. In some embodiments, the antiseptic preservative may comprise one or more isothiazolones at about 1% to about 5% by weight. In some embodiments, the antiseptic preservative may comprise about 2% to about 4% by weight of one or more isothiazolones. In some embodiments, the antiseptic preservative may comprise about 1% to about 3% by weight of one or more isothiazolones. In some embodiments, the one or more isothiazolones include chloromethylisothiazolinone and methylisothiazolinone. In some embodiments, the chloromethylisothiazolinone and methylisothiazolinone are in a weight ratio of about 1:1 to about 5:1. In some embodiments, the one or more isothiazolones include 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one in a weight ratio of about 3:1. In some embodiments, the antiseptic preservative comprises one or more salt-free proprietary glycol and alkyl carboxylate stabilizers. In some embodiments, the buffer composition does not comprise an antiseptic preservative. In some embodiments, the buffer composition is Example Buffer I, Example Buffer II, or Example Buffer III.

[0008] The present disclosure also provides kits. In some embodiments, the kit comprises a buffer composition described above and / or elsewhere herein. In some embodiments, the kit comprises a sterile container housing the buffer composition. In some embodiments, the kit comprises a manual for diagnosing a condition associated with vaginal infection or inflammation. In some embodiments, the condition is vaginitis or vaginosis, or a sexually transmitted disease, or a combination thereof. In some embodiments, the condition is vulvovaginal candidiasis (VVC), trichomoniasis, or bacterial vaginosis (BV), or a combination thereof. In some embodiments, the condition is a sexually transmitted disease, such as chlamydia (CT), gonorrhea (GC), trichomoniasis (TV), or a combination thereof.

[0009] The present disclosure provides methods for preventing or reducing aggregation of surface-functionalized particles, comprising contacting a sample with a buffer composition described above and / or elsewhere herein. In some embodiments, the sample comprises a plurality of surface-functionalized particles, and the level of aggregation of the plurality of surface-functionalized particles in the presence of the buffer composition is reduced compared to the level of aggregation in the absence of the buffer composition. In some embodiments, the level of aggregation of the plurality of surface-functionalized particles in the presence of the buffer composition may be reduced by at least 1% compared to the level of aggregation in the absence of the buffer composition. In some embodiments, the level of aggregation of the plurality of surface-functionalized particles in the presence of the buffer composition is reduced by at least 5% compared to the level of aggregation in the absence of the buffer composition. In some embodiments, the level of aggregation of the plurality of surface-functionalized particles in the presence of the buffer composition is reduced by at least 10% compared to the level of aggregation in the absence of the buffer composition. In some embodiments, the sample is a clinical sample. In some embodiments, the sample is a vaginal sample. In some embodiments, the sample is a clinical vaginal swab. In some embodiments, the sample is collected from the vagina. In some embodiments, the sample is taken from a subject exhibiting clinical signs of vaginitis, vaginosis, a sexually transmitted disease (e.g., chlamydia (CT), gonorrhea (GC), trichomoniasis (TV)), or a combination thereof. In some embodiments, the sample is taken from a subject exhibiting clinical signs of vaginitis or vaginosis, or both. In some embodiments, the sample is taken from a subject exhibiting clinical signs of chlamydia (CT), gonorrhea (GC), trichomoniasis (TV), or a combination thereof. In some embodiments, the sample may comprise a plurality of nucleic acids. In some embodiments, the plurality of nucleic acids is from one or more vulvovaginal candidiasis (VVC)-associated Candida species, Trichomonas vaginalis that causes trichomoniasis, one or more bacterial vaginosis (BV)-associated bacteria, or a combination thereof.In some embodiments, the one or more VVC-associated Candida species can include Candida glabrata, Candida albicans, Candida tropicalis, C. dubliniensis, C. parapsilosis, Candida krusei, or a combination thereof. In some embodiments, the one or more BV-associated bacteria can include Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Atopobium vaginae, Megasphaera Type 1, Megasphaera BVAB2, or a combination thereof. In some embodiments, the method may further include amplifying and / or detecting a plurality of nucleic acids, wherein the efficiency of amplification and / or detection of the plurality of nucleic acids in the presence of the buffer composition is increased compared to the efficiency in the absence of the buffer composition. In some embodiments, the efficiency of amplification and / or detection of the plurality of nucleic acids in the presence of the buffer composition is increased by at least 1% compared to the efficiency in the absence of the buffer composition. In some embodiments, the efficiency of amplification and / or detection of the plurality of nucleic acids in the presence of the buffer composition is increased by at least 5% compared to the efficiency in the absence of the buffer composition. In some embodiments, the surface-functionalized particles may have an average diameter of less than 1 mm. In some embodiments, the surface-functionalized particles are configured for nucleic acid extraction, purification, amplification, detection, or a combination thereof. In some embodiments, aggregation is induced by interfering substances in the sample. In some embodiments, aggregation may occur in a microfluidic channel. In some embodiments, the interfering substance is selected from the group consisting of a lubricant, a gel, a cream, and combinations thereof. In some embodiments, the interfering substance may include a gel comprising one or more carbomers.In some embodiments, the interfering material may include a carbomer-free gel.

[0010] Alternative or additional embodiments described herein provide buffer compositions that include one or more features described above or anywhere else herein. Alternative or additional embodiments described herein provide kits that include one or more features described above or anywhere else herein. Alternative or additional embodiments described herein provide methods of preventing or reducing aggregation of surface-functionalized particles, including one or more features described above or anywhere else herein. [Brief explanation of the drawings]

[0011] [Figure 1] 1 shows a pie chart of the composition by geographic region of the 263 donors of clinical vaginal swab samples tested in Examples 2-9. [Figure 2] Figures 2-4 show an embodiment of the reduction in particle aggregation and prevention of microfluidic clogging in a BD MAX™ PCR cartridge achieved by use of an embodiment buffer composition disclosed herein (see Table 1 below). Figures 2A-2B show photographs of an embodiment of a BD MAX™ PCR cartridge, each of which has been utilized in diagnostic testing of clinical vaginal swab samples that do not contain interfering gel. [Figure 3] 3A-3B show photographic embodiments of BD MAX™ PCR cartridges, each of which is utilized for diagnostic testing of clinical vaginal swab samples containing McKesson carbomer-based blocking gel present at 10 μL. [Figure 4] 4A-4B show additional embodiments of photographs of BD MAX™ PCR cartridges utilized in testing additional blocking gel EZ brand (Medline Industries, Inc.) present at 10 μL (FIGS. 4A-4B). [Figure 5]Figures 5-6 show an embodiment of the improved assay performance provided by Example Buffer II compared to the comparative buffer in the presence of two representative thwarting gels: EZ Lubricating Jelly (Medline Industries, Inc.) (Figures 5A-5F) and the non-carbomer-based Surgilube® (Figures 6A-6F). An embodiment of the assay for detecting bacterial vaginosis (BV) was performed on a BD MAX™ system using either the comparative buffer or the example buffer. Three sets of scatter plots were generated: using the comparative buffer according to the standard workflow (Figures 5A, 5D, 6A, 6D), using the comparative buffer according to the optimized workflow (Figures 5B, 5E, 6B, 6E), and using the example buffer according to the optimized workflow (Figures 5C, 5F, 6C, 6F). [Figure 6] Figures 5-6 show an embodiment of the improved assay performance provided by Example Buffer II compared to the comparative buffer in the presence of two representative thwarting gels: EZ Lubricating Jelly (Medline Industries, Inc.) (Figures 5A-5F) and the non-carbomer-based Surgilube® (Figures 6A-6F). An embodiment of the assay for detecting bacterial vaginosis (BV) was performed on a BD MAX™ system using either the comparative buffer or the example buffer. Three sets of scatter plots were generated: using the comparative buffer according to the standard workflow (Figures 5A, 5D, 6A, 6D), using the comparative buffer according to the optimized workflow (Figures 5B, 5E, 6B, 6E), and using the example buffer according to the optimized workflow (Figures 5C, 5F, 6C, 6F). DETAILED DESCRIPTION OF THE INVENTION

[0012] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. All literature and similar materials cited in this application, including but not limited to patents, patent applications, papers, books, articles, and Internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines or uses a term in a manner that contradicts the use of that term in this application, this application controls. While the teachings of the present invention have been described in conjunction with various embodiments, it is not intended that the teachings of the present invention be limited to such embodiments. On the contrary, the teachings of the present invention encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0013] Provided herein are buffer compositions, kits, and methods for nucleic acid-based detection of vaginal diseases, such as vulvovaginal candidiasis (VVC), trichomoniasis, and / or bacterial vaginosis (BV). For example, buffer compositions that can prevent or reduce assay aggregation of surface-functionalized particles disposed in a microfluidic PCR cartridge for nucleic acid extraction are provided for detecting pathogenic organisms from clinical vaginal swab samples in the presence of interfering substances. In some embodiments, the buffer compositions can improve or maintain the efficiency of nucleic acid-based detection of, for example, VVC-associated Candida species, trichomoniasis-causing Trichomonas vaginalis, and / or BV-associated bacteria.

[0014] As used herein, "nucleic acid" has its plain and ordinary meaning in light of the present disclosure and refers to a polymeric compound containing nucleosides or nucleoside analogs having nitrogenous heterocyclic bases or base analogs linked by nucleic acid backbone bonds (e.g., phosphodiester bonds) to form a polynucleotide. Non-limiting examples of nucleic acids include RNA, DNA, and analogs thereof. The nucleic acid backbone can contain one or more of a variety of linkages, such as sugar-phosphodiester linkages, peptide-nucleic acid linkages, phosphorothioate or methylphosphonate linkages, or a mixture of such linkages in a single oligonucleotide. The sugar moiety in a nucleic acid is ribose or deoxyribose, or similar compounds with known substitutions. Conventional nitrogenous bases (e.g., A, G, C, T, U), known base analogs (e.g., inosine), derivatives of purine or pyrimidine bases, and "abasic" residues (i.e., lacking a nitrogenous base at one or more backbone positions) are included in the term nucleic acid. That is, nucleic acids can contain only conventional sugars, bases and linkages found in RNA and DNA, or can contain both conventional components and substitutions (e.g., conventional bases and analogs linked via a methoxy backbone, or conventional bases and one or more base analogs linked via an RNA or DNA backbone).

[0015] As used herein, a "conjugated pair" has its plain and ordinary meaning in light of this disclosure and includes one proton (H + Acids (HA) and bases (A) differ in - For example, potassium phosphate monobasic (KH2PO4) and potassium phosphate dibasic (K2HPO4) are a conjugate pair. Another example is acetic acid and sodium acetate (CH3COONa). The term "sensitivity," as used herein, has its plain and ordinary meaning in light of this disclosure, and when referring to the performance of a test method is the true positives divided by the sum of the true positives and false negatives. Those skilled in the art will understand that the terms "recall," "hit rate," and "true positive rate (TPR)" are the same as "sensitivity" when referring to the performance of a test method. The term "specificity," as used herein, has its plain and ordinary meaning in light of this disclosure, and when referring to the performance of a test method is true negatives divided by the sum of true negatives and false positives. Those skilled in the art will understand that the terms "selectivity" and "true negative rate (TPR)" are the same as "specificity" when referring to the performance of a test method. The term "accuracy" as used herein has its plain and ordinary meaning in light of this disclosure, and when referring to the performance of a test method is the sum of the true positives and true negatives divided by the sum of the true positives, false positives, true negatives, and false negatives. As used herein, "C" is a set of integers where "a" and "b" are integers. a -C b " refers to the number of carbon atoms in the compound.

[0016] Buffer Composition Some embodiments disclosed herein provide a buffer composition. In some embodiments, the buffer composition comprises a conjugate pair of an acid and a base, a chelating agent, a non-ionic surfactant, a monovalent or divalent salt, and optionally a germicidal preservative. In some embodiments, the buffer composition comprises a conjugate pair of an acid and a base, a chelating agent, a non-ionic surfactant, a monovalent or divalent salt, and optionally a germicidal preservative in any of the amounts or ranges of amounts disclosed herein, including the following:

[0017] In some embodiments, the base of the conjugate pair is a salt of an acid. In some embodiments, the acid includes acetic acid. In some embodiments, the base includes a salt of acetic acid. In some embodiments, the conjugate pair includes acetic acid and its salts. In some embodiments, the base includes sodium acetate. In some embodiments, the conjugate pair includes acetic acid and sodium acetate. In some embodiments, the buffer includes Tris-HCl. In some embodiments, the concentration of the acid (e.g., acetic acid) is at, or about, or less than, or about, or greater than, or greater than: 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM , 165mM, 170mM, 175mM, 180mM, 185mM, 190mM, 195mM, 200mM, 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 23 5mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275mM, 280mM, 285mM, 290mM, 295mM, 300mM, 305mM , 310mM, 315mM, 320mM, 325mM, 330mM, 335mM, 340mM, 345mM, 350mM, 355mM, 360mM, 365mM, 370mM, 375mM, 38 In some embodiments, the concentration of the acid (e.g., acetic acid) is 5-100 mM, 5-250 mM, 200-400 mM, 250-500 mM, 300-500 mM, 400 mM, 410 mM, 415 mM, 420 mM, 425 mM, 430 mM, 435 mM, 440 mM, 445 mM, 450 mM, 455 mM, 460 mM, 465 mM, 470 mM, 475 mM, 480 mM, 485 mM, 490 mM, 495 mM, or 500 mM, or a range between any two of these values.In some embodiments, the concentration of base (e.g., sodium acetate) is at, or about, or less than, or about, or greater than, or greater than: 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 100 mM, 150 mM, 150 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 15 ... 05mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM, 180mM, 185 mM, 190mM, 195mM, 200mM, 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275mM, 280mM, 285mM, 290mM, 295mM, 300mM, 305mM, 310mM, 315mM, 320mM, 325mM, 330mM , 335mM, 340mM, 345mM, 350mM, 355mM, 360mM, 365mM, 370mM, 375mM, 380mM, 385mM, 390mM, 395m The concentration of the base (e.g., sodium acetate) is 5 mM to 100 mM, 5 mM to 250 mM, 200 mM to 400 mM, 250 mM to 500 mM, 300 mM to 500 mM, or 400 mM to 500 mM. In some embodiments, the conjugate pair comprises about 350 mM to 450 mM sodium acetate. In some embodiments, the conjugate pair comprises about 50 mM to about 150 mM acetic acid. In some embodiments, the conjugate pair comprises about 90 mM to about 110 mM acetic acid. In some embodiments, the conjugate pair is at, about, or less than the following concentrations:or at about the following concentrations less than, greater than, or equal to: 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 1 30mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM, 180mM, 185mM, 190mM, 195mM, 200mM, 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275mM, 280mM, 285mM, 290mM, 295mM, 300mM, 305mM, 310mM, 315mM, 320mM, 325mM, 330mM, 335mM , 340mM, 345mM, 350mM, 355mM, 360mM, 365mM, 370mM, 375mM, 380mM, 385mM, 390mM, 395mM, 400mM, 405m M, 410mM, 415mM, 420mM, 425mM, 430mM, 435mM, 440mM, 445mM, 450mM, 455mM, 460mM, 465mM, 470mM, 475 mM, 480mM, 485mM, 490mM, 495mM, 500mM, 505mM, 510mM, 515mM, 520mM, 525mM, 530mM, 535mM, 540mM, 545 mM, 550mM, 555mM, 560mM, 565mM, 570mM, 575mM, 580mM, 585mM, 590mM, 595mM, 600mM, 605mM, 610mM, 61 5mM, 620mM, 625mM, 630mM, 635mM, 640mM, 645mM, 650mM, 655mM, 660mM, 665mM, 670mM, 675mM, 680mM, 6 85mM, 690mM, 695mM, 700mM, 705mM, 710mM, 715mM, 720mM, 725mM, 730mM, 735mM, 740mM, 745mM, 750mM, 755mM, 760mM, 765mM, 770mM, 775mM, 780mM, 785mM, 790mM, 795mM, 800mM, 805mM, 810mM, 815mM, 820mM,825 mM, 830 mM, 835 mM, 840 mM, 845 mM, 850 mM, 855 mM, 860 mM, 865 mM, 870 mM, 875 mM, 880 mM, 885 mM, 890 mM, 895 mM or 900 mM, or a range between any two of these values. In some embodiments, the concentration of the conjugate pair (e.g., acetic acid and sodium acetate or Tris-HCl) is 5 mM to 100 mM, 5 mM to 250 mM, 200 to 400 mM, 250 mM to 500 mM, 300 mM to 500 mM, 400 mM to 500 mM, 5 mM to 1 M, 300 mM to 700 mM, 500 mM to 900 mM, 750 mM to 1 M, or 800 mM to 1 M. In some embodiments, the conjugate pair is present at a concentration ranging from about 400 mM to about 600 mM. In some embodiments, the conjugate pair comprises acetic acid and sodium acetate. In some embodiments, the acetic acid is present at a concentration of 200 mM or less and the sodium acetate is present at a concentration of 300 mM or more. In some embodiments, the conjugate pair is Tris-HCl at a concentration of 10 mM.

[0018] In some embodiments, the pH of the buffer composition is at, or about, or less than, or about, or more than, or about, 1.0, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3 , 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or a range between any of these values. In some embodiments, the buffer composition has a pH of about 4.0 to about 6.0. In some embodiments, the buffer composition has a pH of about 5.0.

[0019] In some embodiments, chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), ethylenediamine, amino acids such as glutamic acid and histidine, organic diacids such as oxalic acid, malonic acid, succinic acid, and pharmaceutically acceptable salts thereof. In some embodiments, the chelating agent comprises EDTA. In some embodiments, the concentration of the chelating agent (e.g., EDTA) is at, or about, or less than, or about, or more than, or about, 0.2 mM, 0.5 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, or a range between any two of these values. In some embodiments, the chelating agent is present at a concentration ranging from about 1 mM to about 20 mM, 0.2 mM to 2 mM, 1 mM to 7 mM, 16 mM to 20 mM, 10 mM to 20 mM, or 4 mM to 8 mM. In some embodiments, the concentration of the chelating agent (e.g., EDTA) is about 10 mM.

[0020] In some embodiments, the nonionic surfactant is selected from the group consisting of ethoxylated nonionic surfactants, propoxylated nonionic surfactants, co-ethoxylated-propoxylated nonionic surfactants, and combinations thereof. In some embodiments, the nonionic surfactant is selected from the group consisting of ethoxylated sorbitan esters of mono-fatty acids, ethoxylated octylphenols, ethoxylated secondary C1-C20 alcohols, co-ethoxylated-propoxylated seed oil alcohols, and combinations thereof. In some embodiments, the nonionic surfactant is selected from the group consisting of ethoxylated sorbitan esters of mono-fatty acids containing an average of 1-50 ethylene oxide units per surfactant, ethoxylated octylphenol containing an average of 1-20 ethylene oxide units per surfactant, ethoxylated secondary C1-C20 alcohols containing an average of 1-20 ethylene oxide units per surfactant, co-ethoxylated propoxylated seed oil alcohols containing an average of 1-20 propylene oxide units and 1-30 ethylene oxide units per surfactant, and combinations thereof. In some embodiments, the nonionic surfactant comprises one or more ethoxylated sorbitan esters of mono-fatty acids. In some embodiments, the one or more ethoxylated sorbitan esters of mono-fatty acids contain an average of 1-50 ethylene oxide units. In some embodiments, the nonionic surfactant comprises a Tween surfactant. In some embodiments, the nonionic surfactant comprises a Tween 20 surfactant, a Tween 40 surfactant, a Tween 60 surfactant, a Tween 80 surfactant, or a combination thereof. In some embodiments, the surfactant is an ECOSURF™ surfactant (Dow Chemical Co.), such as ECOSURF™ SA-4, SA-7, SA-9, or SA-15 surfactant. In some embodiments, the nonionic surfactant comprises one or more ethoxylated secondary C1-C20 alcohols. In some embodiments, the one or more ethoxylated secondary C1-C20 alcohols contain an average of 1 to 20 ethylene oxide units.In some embodiments, the nonionic surfactant comprises a Tergitol™ surfactant (Sigma-Aldrich), such as Tergitol™ 15-S-9 surfactant. In some embodiments, the nonionic surfactant comprises one or more ethoxylated octylphenols. Some of the one or more ethoxylated octylphenols contain an average of 1 to 20 ethylene oxide units. In some embodiments, the nonionic surfactant comprises a Triton surfactant, such as Triton X-100 surfactant. In some embodiments, the nonionic surfactant comprises one or more co-ethoxylated-propoxylated seed oil alcohols. Some of the one or more co-ethoxylated-propoxylated seed oil alcohols contain an average of 1 to 20 propylene oxide units and 1 to 30 ethylene oxide units. In some embodiments, the nonionic surfactant comprises an EcoSurf™ surfactant. The nonionic surfactant is present in the buffer composition at, or about, or less than, or about, or greater than, or about, or greater than the following concentrations: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9% or 3.0%, or a range between any two of these values. In some embodiments, the nonionic surfactant is present at a concentration ranging from about 0.5% to 1.5%, 0.1% to 3.0%, 0.8% to 1.9%, 1.0% to 2.0%, 2.5% to 3%, or 0.7% to 2.6% by weight of the buffer composition, hi some embodiments, the nonionic surfactant is present at a concentration ranging from about 1.0% by weight of the buffer composition.

[0021] In some embodiments, the monovalent or divalent salt is selected from the group consisting of sodium salts, potassium salts, calcium salts, magnesium salts, or combinations thereof. In some embodiments, the divalent salt is a calcium salt. In some embodiments, the divalent salt is CaCl. In some embodiments, the monovalent or divalent salt is at, or about, or less than, or about, or greater than, or greater than the following concentrations: 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, M, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 16 5mM, 170mM, 175mM, 180mM, 185mM, 190mM, 195mM, 200mM, 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275mM, 280mM, 285mM, 290mM, 295mM, 300mM, 305mM, 310 mM, 315mM, 320mM, 325mM, 330mM, 335mM, 340mM, 345mM, 350mM, 355mM, 360mM, 365mM, 370mM, 375mM, 380mM, In some embodiments, the monovalent or divalent salt (e.g., CaCl) is present at a concentration of 5 mM to 100 mM, 5 mM to 250 mM, 200 mM to 400 mM, 250 mM to 500 mM, 300 mM to 500 mM, or 400 mM to 500 mM. In some embodiments, the monovalent or divalent salt is present at a concentration ranging from about 100 mM to about 300 mM.In some embodiments, the monovalent or divalent salt is present at a concentration of about 200 mM.

[0022] In some embodiments, the buffer composition optionally includes a biocidal preservative. In some embodiments, the biocidal preservative includes one or more isothiazolones. In some embodiments, the biocidal preservative is present at, about, less than, about, less than, or equal to, or greater than, or equal to, or greater than, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% by weight of the buffer composition, or a range between any two of these values. In some embodiments, the antiseptic preservative is present at a concentration of about 0.01% to 0.1%, 0.01% to 0.03%, 0.025% to 0.045%, 0.05% to 0.1%, 0.07% to 0.01%, or 0.04% to 0.08% by weight of the buffer composition. In some embodiments, the antiseptic preservative is present at a concentration of about 0.03% by weight of the buffer composition. In some embodiments, the antiseptic preservative is ProClin™ 300. In some embodiments, the antiseptic preservative ProClin™ 300 is present at a concentration of about 0.03% by weight of the buffer composition.In some embodiments, the biocidal preservative is present in an amount at or about the following, or less than or about the following, or more than or about the following: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1% by weight. 0.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9% or 6.0% by weight of one or more isothiazolones or a range between any two of these values. In some embodiments, the antiseptic preservative is present at a concentration of about 0.1% to 6.0%, 0.1% to 3.0%, 1.0% to 4.0%, 3% to 6%, 5% to 6%, or 2% to 5% by weight of the buffer composition. In some embodiments, the antiseptic preservative comprises about 1% to about 5% by weight of one or more isothiazolones. In some embodiments, the antiseptic preservative comprises about 2% to about 4% by weight of one or more isothiazolones. In some embodiments, the antiseptic preservative comprises about 1% to about 3% by weight of one or more isothiazolones. In some embodiments, the one or more isothiazolones comprise chloromethylisothiazolinone and methylisothiazolinone.In some embodiments, chloromethylisothiazolinone and methylisothiazolinone are in a weight ratio of, at about, or less than, or about, or greater than, or equal to, 1:1, 1.5:1, 2:1, 2.5:1 3:1, 3:5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, or 10:1, or a range between any two of these values. In some embodiments, chloromethylisothiazolinone and methylisothiazolinone are in a weight ratio of from about 1:1 to about 5:1. In some embodiments, the one or more isothiazolones comprise 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one in a weight ratio of about 3:1. In some embodiments, the antiseptic preservative comprises one or more salt-free proprietary glycol and alkyl carboxylate stabilizers. In some embodiments, the buffer composition is used to preserve samples prior to testing. In some embodiments, the buffer composition does not comprise an antiseptic preservative.

[0023] In some embodiments, the buffer composition comprises a reducing agent. In some embodiments, the reducing agent is selected from the group consisting of dithiothreitol (DTT), β-mercaptoethanol, and TCEP (tris(2-carboxyethyl)phosphine). In some embodiments, the reducing agent is TCEP (tris(2-carboxyethyl)phosphine). In some embodiments, the concentration of the reducing agent (e.g., TCEP) is at, or about, or less than, or about, or more than, or about, or more than: 0.2 mM, 0.5 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM, or a range between any two of these values. In some embodiments, the reducing agent is present at a concentration ranging from 1 mM to 50 mM, 1 mM to 30 mM, or 10 mM to 20 mM. In some embodiments, the concentration of the chelating agent (e.g., TCEP) is about 15 mM.

[0024] Exemplary Buffer Compositions The following are non-limiting exemplary buffer compositions. In some embodiments, the buffer composition comprises components of Buffer #1, #2, or #3:

[0025] [Table 1]

[0026] In some embodiments of the above Buffers #1 to #3, the acid-base pair is selected from the group consisting of acetic acid / sodium acetate and Tris-HCl, the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylenediamine, amino acids such as glutamic acid and histidine, organic diacids such as oxalic acid, malonic acid, and succinic acid, etc., the surfactant is selected from the group consisting of ethoxylated nonionic surfactants, propoxylated nonionic surfactants, co-ethoxylated-propoxylated nonionic surfactants, and combinations thereof, the monovalent or divalent salt is selected from the group consisting of sodium salts, potassium salts, calcium salts, magnesium salts, or combinations thereof, and the antiseptic preservative comprises one or more isothiazolones. In some embodiments of the above buffers #1-3, the acid-base pair is acetic acid / sodium acetate, the chelating agent is EDTA, the surfactant is Tergitol™ 15-S-9, Triton X-100™ surfactant, or Ecosurf™ surfactant, the divalent salt is CaCl2, and the antiseptic preservative is a combination of chloromethylisothiazolinone and methylisothiazolinone.In some embodiments of the above buffers #1-3, the acid-base pair is acetic acid / sodium acetate, the chelating agent is EDTA, the surfactant is Tergitol™ 15-S-9 or Triton X-100™, the divalent salt is CaCl2, and the antiseptic preservative is a combination of chloromethylisothiazolinone and methylisothiazolinone. In some embodiments of the above buffers #1-3, the acid-base pair is acetic acid / sodium acetate, the chelating agent is EDTA, the surfactant is Tergitol™ 15-S-9 or Triton X-100™, the divalent salt is CaCl2, and the antiseptic preservative is ProClin™ 300. In some embodiments of the above buffers #1-3, the acid-base pair is acetic acid / sodium acetate, the chelating agent is EDTA, the surfactant is Tergitol™ 15-S-9 or Triton X-100™, the divalent salt is CaCl2, and the antiseptic preservative is ProClin™ 300.In some embodiments of Buffers #1-3 above, the acid-base pair is acetic acid / sodium acetate, the chelating agent is EDTA, the surfactant is Tergitol™ 15-S-9, the divalent salt is CaCl2, and the antiseptic preservative is ProClin™ 300. In some embodiments of Buffers #1-3 above, the acid-base pair is acetic acid / sodium acetate, the chelating agent is EDTA, the surfactant is Triton X-100™, the divalent salt is CaCl2, and the antiseptic preservative is ProClin™ 300. In some embodiments of Buffers #1-3 above and herein, including those described herein, the buffer composition does not contain an antiseptic preservative.

[0027] In some embodiments, the buffer comprises components of Buffer #4, #5, or #6: [Table 2]

[0028] In some embodiments of Buffers #4-6, the acid-base pair is selected from the group consisting of acetic acid / sodium acetate and Tris-HCl, the surfactant is selected from the group consisting of ethoxylated nonionic surfactants, propoxylated nonionic surfactants, co-ethoxylated-propoxylated nonionic surfactants, and combinations thereof, the reducing agent is selected from β-mercaptoethanol and TCEP (tris(2-carboxyethyl)phosphine), and the monovalent or divalent salt is selected from the group consisting of sodium salts, potassium salts, calcium salts, magnesium salts, or combinations thereof. In some embodiments of Buffers #4-6, the acid-base pair is Tris-HCl acetate, the surfactant is Tergitol™ 15-S-9, Triton X-100™, Tween™ surfactant, or Ecosurf™ surfactant, the reducing agent is selected from the group consisting of β-mercaptoethanol and TCEP, and the monovalent or divalent salt is a calcium salt. In some embodiments of Buffers #4-6 above, the acid-base pair is Tris-HCl, the surfactant is Tergitol™ 15-S-9 or Triton X-100™, the reducing agent is TCEP (tris(2-carboxyethyl)phosphine), and the monovalent or divalent salt is CaCl2. In some embodiments of Buffers #4-6 above, the acid-base pair is Tris-HCl, the surfactant is Triton X-100™, the reducing agent is TCEP, and the monovalent or divalent salt is CaCl2. In some embodiments, the buffer composition comprises 10 mM Tris-HCl buffer, 15 mM TCEP, 1% Triton-X100, 50-150 mM CaCl2, pH 2.4±0.2. In some embodiments of Buffers #4-6 above and herein, including those described herein, the buffer comprises a disinfectant preservative as disclosed herein. In some embodiments, the disinfectant preservative is ProClin™ 300. In some embodiments, the antiseptic preservative is ProClin™ 300 in an amount of 0.03%. In some embodiments of Buffers #4-6, including those described above and herein, the buffer composition does not contain an antiseptic preservative.

[0029] In some embodiments, the buffer is Example Buffer I or Example Buffer II: [Table 3]

[0030] In some embodiments, the buffer is Example Buffer III: [Table 4]

[0031] kit Some embodiments disclosed herein provide kits. In some embodiments, the kit includes a buffer composition described above or elsewhere herein. In some embodiments, the kit includes a sterile container housing the buffer composition. In some embodiments, the kit further includes a microfluidic cartridge. In some embodiments, the microfluidic cartridge is configured to facilitate nucleic acid processing and detection. In some embodiments, the microfluidic cartridge is disposable. In some embodiments, the kit includes a manual for diagnosing a condition associated with vaginal infection or inflammation. In some embodiments, the condition is vaginitis or vaginosis, or a combination thereof. In some embodiments, the condition is vulvovaginal candidiasis (VVC), trichomoniasis, bacterial vaginosis (BV), or a combination thereof. In some embodiments, the condition is a sexually transmitted disease, such as chlamydia (CT), gonorrhea (GC), trichomoniasis (TV), or a combination thereof.

[0032] method Some embodiments disclosed herein provide methods of preventing or reducing aggregation of surface-functionalized particles, the methods comprising contacting a sample with a buffer composition described in the "Buffer Compositions" section above or elsewhere herein. In some embodiments, the sample comprises a plurality of surface-functionalized particles, and the level of aggregation of the plurality of surface-functionalized particles in the presence of the buffer composition is reduced compared to the level of aggregation in the absence of the buffer composition.

[0033] In some embodiments, the aggregation level of the plurality of surface-functionalized particles in the presence of the buffer composition compared to the aggregation level in the absence of the buffer composition is, or approximately, or at least approximately, or at most approximately, or at most approximately: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%. The aggregation level of the plurality of surface-functionalized particles in the presence of the buffer composition is reduced by 80%, 85%, 90%, 95%, or 100%, or a range between any two of these values. In some embodiments, the aggregation level of the plurality of surface-functionalized particles in the presence of the buffer composition is reduced by at least 1% compared to the aggregation level in the absence of the buffer composition. In some embodiments, the aggregation level of the plurality of surface-functionalized particles in the presence of the buffer composition is reduced by at least 5% compared to the aggregation level in the absence of the buffer composition. In some embodiments, the aggregation level of the plurality of surface-functionalized particles in the presence of the buffer composition is reduced by at least 10% compared to the aggregation level in the absence of the buffer composition. In some embodiments, the level of aggregation of a plurality of surface-functionalized particles in the presence of the buffer composition is reduced by 1-5%, 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 1-80%, 1-90%, 5-10%, 5-20%, 5-30%, 5-40%, 5-50%, 5-60%, 5-70%, 5-80%, 5-90%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, or 10-90% compared to the level of aggregation in the absence of the buffer composition. In some embodiments, the level of particle aggregation is measured using digital image processing, and the size of the particle aggregation is determined by measuring the diameter or two-dimensional area on the image.

[0034] In some embodiments, the sample is a clinical sample. In some embodiments, the sample is a vaginal sample. In some embodiments, the sample is a clinical vaginal swab. In some embodiments, the sample is collected from the vagina. In some embodiments, the sample is collected from a subject exhibiting clinical signs of vaginitis or vaginosis, or both. In some embodiments, the sample comprises a plurality of nucleic acids. In some embodiments, the sample comprises a plurality of nucleic acids from one or more vulvovaginal candidiasis (VVC)-associated Candida species, Trichomonas vaginalis causing trichomoniasis, one or more bacterial vaginosis (BV)-associated bacteria, or a combination thereof. In some embodiments, the one or more VVC-associated Candida species comprise Candida glabrata, Candida albicans, Candida tropicalis, C. dubliniensis, C. parapsilosis, Candida krusei, or a combination thereof. In some embodiments, the one or more BV-associated bacteria include Lactobacillus crispatus, Lactobacillus junsenii, Gardnerella vaginalis, Atopobium vaginae, Megasphaera type 1, Megasphaera BVAB2, or a combination thereof.

[0035] In some embodiments, the method further includes amplifying and / or detecting a plurality of nucleic acids, and the efficiency of the amplification and / or detection of the plurality of nucleic acids is increased in the presence of the buffer composition compared to the efficiency in the absence of the buffer composition. In some embodiments, the efficiency of the amplification and / or detection of the plurality of nucleic acids is improved by, or about, or at least, or at least about, or at most, or at most, or at most, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or a range between any two of these values, compared to the efficiency in the absence of the buffer composition. In some embodiments, the efficiency of amplification and / or detection of a plurality of nucleic acids in the presence of the buffer composition is increased by at least 1% compared to the efficiency in the absence of the buffer composition, hi some embodiments, the efficiency of amplification and / or detection of a plurality of nucleic acids in the presence of the buffer composition is increased by at least 5% compared to the efficiency in the absence of the buffer composition. In some embodiments, the efficiency of amplification and / or detection of multiple nucleic acids in the presence of the buffer composition is increased by 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1-70%, 1-80%, or 1-90%, 5-10%, 5-20%, 5-30%, 5-40%, 5-50%, 5-60%, 5-70%, 5-80%, 5-90%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, or 10-90% compared to the efficiency in the absence of the buffer composition. In some embodiments, the surface-functionalized particles comprise an average diameter of less than 1 mm. In some embodiments, the surface-functionalized particles are configured for nucleic acid extraction, purification, amplification, detection, or a combination thereof. In some embodiments, the aggregation is induced by interfering substances in the sample. In some embodiments, the aggregation occurs within a microfluidic channel. In some embodiments, the interfering substance is selected from the group consisting of lubricants, gels, creams, and combinations thereof. In some embodiments, the interfering substance comprises a gel containing one or more carbomers. In some embodiments, the interfering substance comprises a gel that does not contain a carbomer.

[0036] Also disclosed herein are methods for maintaining or improving diagnostic test performance of a sample in the presence of interfering substances. In some embodiments, the method includes transferring the sample to a buffer composition described above in the "Buffer Composition" section or elsewhere herein. In some embodiments, the method further includes collecting the sample on a swab from the subject prior to the transferring step. In some embodiments, the method further includes amplifying and detecting one or more nucleic acids in the biological sample or one or more nucleic acids extracted from the biological sample following the transferring step.

[0037] In some embodiments, the performance comprises sensitivity, specificity, recall, precision, rapidity, robustness, or a combination thereof. In some embodiments, the method reduces the incidence of non-reportable results by, or about, or at least, or at least about, or at most, or at most, or at most, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or a range between any two of these values. In some embodiments, the method reduces the incidence of non-reportable results by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, non-reportable results include indeterminate results, unresolved results, incomplete results, or a combination thereof. In some embodiments, the method maintains an accuracy rate of, or about, or at least, or at least about, or at most, or at most about: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or a range between any two of these values. In some embodiments, the method maintains an accuracy rate of at least 90%, at least 95%, at least 98%, or at least 99%. In some embodiments, the accuracy rate is measured by the concordance rate for positive results, the concordance rate for negative results, the overall concordance rate, or a combination thereof. [Example]

[0038] The following examples are provided to illustrate particular situations and settings in which the present technology may be applied, and are not intended to limit the scope of the invention and the claims contained in this disclosure.

[0039] Example 1 Diagnostic assays performed on vaginal samples The studies described in this section demonstrate the robust performance of diagnostic assays achieved by using the buffer compositions disclosed herein to detect vaginal diseases, such as vulvovaginal candidiasis (VVC), trichomoniasis, and bacterial vaginosis (BV), in clinical vaginal swab samples that may contain interfering substances. For example, the assays were performed on a BD MAX™ system as fully automated in vitro diagnostic tests for the qualitative detection of Candida species (C. glabrata and C. krusei), Trichomonas vaginalis, and / or BV-associated bacteria. The assays incorporate microfluidic-based sample processing and nucleic acid-based detection methods. More specifically, the diagnostic assays used fluidically engineered magnetic particles to extract nucleic acids, real-time polymerase chain reaction (PCR) to amplify target DNA, and fluorogenic hybridization probes to identify target organisms. Diagnostic assays such as those described below in Examples 2-9 and 11 were performed using either Example Buffer I or II. For comparison, diagnostic assays were also performed using Comparative Buffers. The compositions of Comparative Buffer, Example Buffer I, and Example Buffer II are summarized in Table 1 below. The Comparative Buffers in Table 1 represent common "gold standard" buffer compositions used in the preparation of biological samples and the diagnostic detection of vaginal or sexually transmitted diseases.

[0040] [Table 5]

[0041] The use of monobasic potassium phosphate (KH2PO4) / dibasic potassium phosphate (K2HPO4) as an acid-base pair in the comparative buffer was found to be associated with greater particle aggregation and more frequent microfluidic clogging compared to the use of acetic acid / sodium acetate (CH3COONa) in the example buffer. The inclusion of surfactants (e.g., Tergitol™ 15-S-9 or Triton X-100) and salts (e.g., CaCl2) in the example buffer was found to improve assay performance robustness by reducing interactions between magnetic particles and polyelectrolytes (e.g., carbomer) commonly present in blocking gels. The presence of ethylenediaminetetraacetic acid (EDTA) as a chelating agent in all three buffer compositions was found to be essential for sample stabilization. ProClin™ 300 preservative (Sigma-Aldrich, St. Louis, Mo.) was added to all three buffer compositions as a disinfectant for sample preservation purposes.

[0042] Vaginal swabs were collected from a total of 263 women with clinical signs of vaginitis and / or vaginosis and tested on the BD MAX (test) system. Figure 1 summarizes the composition of these sample donors by geographic region: 94 donors (36%) from Biocollection Oakland, 69 donors (26%) from PPGC Houston, and 100 donors (38%) from Biocollection Miami. This example illustrates the experimental aspects of the diagnostic assay, the results of which are described below in Examples 2 through 9. In particular, this example illustrates exemplary buffer compositions disclosed herein.

[0043] Example 2 Test repeatability Table 2 summarizes the percent concordance of assay results obtained in two tests ("first take" and "second take") of the same sample ("same swab"). The comparative buffer was used for the assay in Table 2(a), and example buffer I was used for the assay in Table 2(b). Tables 2(a)-2(b) each list the percent concordance of positive results, negative results, and overall (from left to right) for Candida spp., C. glabrata, C. krusei, Trichomonas vaginalis, and BV (from top to bottom).

[0044] [Table 6]

[0045] Both the comparative buffer and example buffer I achieved at least 90.0% agreement between two different tests ("first take" and "second take") of the "same swab" when performing diagnostic detection of various pathogenicity markers. Thus, this example provides a representative baseline for the repeatability of diagnostic assays and related methods.

[0046] Example 3 Test reproducibility Table 3 summarizes the concordance rates for assay results obtained in one test ("first take") of two samples ("Swab 1" and "Swab 2") collected from the same subject (or sample donor). The comparative buffer was used in the assay in Table 3(a), and example buffer I was used in the assay in Table 3(b). Tables 3(a)-3(b) each list the positive results, negative results, and overall (left to right) concordance rates for Candida spp., C. glabrata, C. krusei, Trichomonas vaginalis, and BV (top to bottom), respectively. Both the comparative buffer and example buffer I achieved at least 90.0% concordance between two different samples ("Swab 1" and "Swab 2") obtained from the same subject when tested for various pathogenicity markers.

[0047] [Table 7]

[0048] Thus, this example provides a representative baseline for the reproducibility of diagnostic assays and related methods.

[0049] Example 4 Baseline effects of buffers on reportable outcomes Table 4 shows the buffer-dependent variance in reported assay results for five sets of representative biomarkers: Candida spp. (Table 4(a)), C. glabrata (Table 4(b)), C. krusei (Table 4(c)), Trichomonas vaginalis (Table 4(d)), and bacterial vaginosis (BV) (Table 4(e)).

[0050] For example, as shown in Table 4(a), when the Example Buffer was used in the assay, the PCR analysis reported 45 positive results and 178 negative results for Candida, while when the Comparative Example Buffer was used, the PCR analysis reported 46 positive results and 177 negative results for Candida. Of the 223 results reported in Table 4(a), three samples reported as negative with the Comparative Example Buffer (top right) were identified as pathogenic positive with the Example Buffer, and four samples reported as positive with the Comparative Example Buffer (bottom left) were identified as pathogenic negative with the Example Buffer.

[0051] [Table 8-1] [Table 8-2]

[0052] As an example, as shown in Table 4(b), when the Example Buffer was used in the assay, the PCR analysis reported 11 positive results and 212 negative results for C. glabrata, while when the Comparative Example Buffer was used, the PCR analysis reported 13 positive results and 210 negative results for C. glabrata. Of the 223 results reported in Table 4(b), two samples (bottom left) that were reported as positive with the Comparative Example Buffer were identified as pathogenic negative with the Example Buffer.

[0053] [Table 9]

[0054] As an example, as shown in Table 4(c), when the example buffer was used in the assay, the PCR analysis reported 0 positive results and 223 negative results for C. krusei, while when the comparison buffer was used, the PCR analysis reported 0 positive results and 223 negative results for C. krusei. As an example, as shown in Table 4(d), when the Example Buffer was used in the assay, the PCR analysis reported 36 positive results and 187 negative results for T. vaginalis, while when the Comparative Buffer was used, the PCR analysis reported 36 positive results and 187 negative results for T. vaginalis. Of the 223 results reported in Table 4(d), one sample reported as negative with the Comparative Buffer (top right) was identified as pathogen-positive with the Example Buffer, and one sample reported as positive with the Comparative Buffer (bottom left) was identified as pathogen-negative with the Example Buffer. As an example, as shown in Table 4(e), when the Example Buffer was used in the assay, the PCR analysis reported 129 positive results and 104 negative results for BV, while when the Comparative Example Buffer was used, the PCR analysis reported 129 positive results and 104 negative results for C. glabrata. Of the 233 results reportable in Table 4(e), seven samples reported as negative with the Comparative Example Buffer (top right) were identified as pathogenic positive with the Example Buffer, and seven samples reported as positive with the Comparative Example Buffer (bottom left) were identified as pathogenic negative with the Example Buffer.

[0055] Table 5 summarizes the percent concordance (generally greater than 84%) between the example and comparative buffers for the assay results shown in Table 4. Specifically, the percent concordance for positive results, negative results, and overall (from left to right) was tested for Candida spp., C. glabrata, C. krusei, Trichomonas vaginalis, and BV (from top to bottom). This example represents a representative baseline for assessing the effect of assay buffers on the reportable results of diagnostic assays and related methods.

[0056] [Table 10]

[0057] Example 5 Baseline effects of buffers on non-reportable outcomes Tables 6-7 show the non-reportable rates for the Comparative Buffers (Tables 6(a)-6(d) and 7(a)-7(f)) and Example Buffer I (Tables 6(e)-6(h) and 7(g)-7(l)), respectively, in the vaginal disease assay tests. The samples tested in Table 6 did not contain any interfering gels, while the samples tested in Table 7 contained various interfering gels, such as McKesson Lubricating Jelly (Tables 7(a) and 7(g)), EZ Lubricating Jelly (Tables 7(b) and 7(h)), Surgilube® lubricant (Tables 7(c) and 7(i)), Canesten® (clotrimazole antifungal cream) (Tables 7(d) and 7(j)), and Vagisil® (benzocaine / resorcinol anti-itch cream) (Tables 7(e) and 7(k)). Each swab sample tested in Tables 6-7 was assayed for both vaginitis and vaginosis, and the non-reportable rates listed in Tables 6-7 were further divided into unresolved result rates and indeterminate result rates.

[0058] With respect to assay testing of swab samples without interfering gel, Tables 6(a) and 6(e) compare the first testing of the samples (“Swab 1, Take 1”) performed with the Comparative Buffer and Example Buffer I, respectively; Tables 6(b) and 6(f) compare the second testing of the samples (“Swab 1, Take 2”) performed with the two buffers; Tables 6(c) and 6(g) compare the first testing of the second sample (“Swab 2, Take 1”); and Tables 6(d) and 6(h) compare the overall results obtained from the testing of Tables 6(a)-6(c) and 6(e)-6(g), respectively.

[0059] [Table 11-1] [Table 11-2]

[0060] [Table 12-1] [Table 12-2]

[0061] [Table 13-1] [Table 13-2]

[0062] As shown in Table 6, Example Buffer I and Comparative Example Buffer produced comparable non-reportable rates when the assay was not obstructed by the gel. Thus, the use of Example Buffer I was demonstrated to maintain a level of reportable results for samples that did not contain interfering gel.

[0063] As shown in Table 7, Example Buffer I significantly reduced the non-reportable rates when swab samples contained interfering substances. For example, for swab samples contaminated with McKesson lubricating jelly, use of Example Buffer I reduced the unresolved, indeterminate, and total non-reportable rates per sample from 34.8%, 34.8%, and 69.6% (Table 7(a)) to 8.7%, 0.0%, and 8.7% (Table 7(g)). As another example, for swab samples contaminated with EZ lubricating jelly, use of Example Buffer I reduced the unresolved, indeterminate, and total non-reportable rates per sample from 23.1%, 53.8%, and 76.9% (Table 7(b)) to 0.0%, 0.0%, and 0.0% (Table 7(h)). Overall, the unresolved, indeterminate, and total non-reportable rates per sample decreased from 47.1%, 21.8%, and 68.9% (Table 7(f)) to 12.6%, 0.0%, and 12.6% (Table 7(l)). Thus, this example demonstrates that the use of the example buffer can significantly improve assay performance by increasing the robustness of the test to the presence of various interfering substances, while maintaining reportable rates at the level of the absence of interfering substances.

[0064] Example 6 Robustness of comparative buffers to interference The diagnostic assay was performed on 119 clinical swab samples using the comparison buffer. Of these 119 samples, only 37 (approximately 31.1%) had reportable results. For each of these 119 samples, one test ("Take 1") was performed in the absence of the interfering gel and another test ("Take 2") was performed in the presence of the interfering gel.

[0065] Table 8 shows the effect of the presence of interfering gel on reportable assay results for detecting Candida spp. (Table 8(a)), C. glabrata (Table 8(b)), C. krusei (Table 8(c)), Trichomonas vaginalis (Table 8(d)), and bacterial vaginosis (BV) (Table 8(e)). For example, as shown in Table 8(e), in the presence of interfering gel, the diagnostic assay reported 34 positive results and 37 negative results for BV, while in the absence of interfering gel, the diagnostic assay reported 38 positive results and 33 negative results for BV. Of the results reported in Table 8(d), two samples reported as positive in the presence of interfering gel (top right) were identified as negative in the absence of interfering gel, and six samples reported as negative in the presence of interfering gel (bottom left) were identified as positive in the absence of interfering gel.

[0066] [Table 14-1] [Table 14-2]

[0067] Table 9 summarizes the concordance (generally greater than 84%) between tests with and without the interfering gel for the assay results shown in Table 8. Specifically, the concordance for positive results, negative results, and overall (from left to right) was tested for Candida spp., C. glabrata, C. krusei, Trichomonas vaginalis, and BV (from top to bottom). [Table 15] This example demonstrates the limited assay performance, particularly the low reportable rate, when using the comparative buffer.

[0068] Example 7 Example buffer robustness against interference Diagnostic assays were performed on 119 clinical swab samples using Example Buffer I. Of these 119 samples, 104 (approximately 87.4%) produced reportable results, compared to approximately 31.1% for Example 6. Thus, the use of Example Buffer I significantly increased the reportability rate of the diagnostic assay compared to the use of the comparative buffer in Example 6. For each of these 119 samples, one test ("Take 1") was performed in the absence of the interfering gel and another test ("Take 2") was performed in the presence of the interfering gel.

[0069] Similar to Table 8 in Example 6, Table 10 shows the effect of the presence of blocking gel on the reportable assay results for detecting Candida (Table 10(a)), C. glabrata (Table 10(b)), C. krusei (Table 10(c)), Trichomonas vaginalis (Table 10(d)), and bacterial vaginosis (BV) (Table 10(e)) when utilizing Example Buffer I. For example, as shown in Table 10(d), in the presence of interfering gel, the diagnostic assay reported 54 positive results and 50 negative results for BV, while in the absence of interfering gel, the diagnostic assay reported 55 positive results and 49 negative results for BV. Of these results reported in Table 10(d), one sample reported as positive in the presence of interfering gel (top right) was identified as negative in the absence of interfering gel, and two samples reported as negative in the presence of interfering gel (bottom left) were identified as positive in the absence of interfering gel.

[0070] [Table 16-1] [Table 16-2]

[0071] [Table 17]

[0072] Table 11 summarizes the concordance (generally greater than 87%) between tests with and without the interfering gel for assay results such as those shown in Table 10. Specifically, the concordance for positive results, negative results, and overall (from left to right) was tested for Candida spp., C. glabrata, C. krusei, Trichomonas vaginalis, and BV (from top to bottom). The agreement shown in Table 11 is at least as good as that shown in Table 9. Thus, use of Example Buffer I (Tables 10-11) was shown to maintain the same level of precision as use of the Comparative Buffers (Tables 8-9). Thus, this example demonstrates that the use of the example buffer increases the reportability rate without affecting the reproducibility of the diagnostic assay under noisy conditions.

[0073] Example 8 Example Buffers Prevent / Reduce Microfluidic Clogging Figures 2A-2B show photographs of BD MAX™ PCR cartridges, each utilized in diagnostic testing of clinical vaginal swab samples that did not contain interfering gel. Example Buffer I was utilized in the test in Figure 2A, and Comparative Buffer was utilized in the test in Figure 2B. As indicated by the red arrow in Figure 2A, when the diagnostic assay was performed using the Comparative Buffer, the magnetic particles disposed in the nucleic acid extraction cartridge formed aggregates. This particle aggregation resulted in clogging of the microfluidics, as shown in Figure 2A. Exemplary areas of particle aggregation and microfluidic clogging in Figure 2A are labeled "A" i ","A ii ", and "A iii " and magnified at the bottom. In comparison, in Figure 2B, no such particle aggregation or microfluidic clogging is observed, and the exemplary region in Figure 2B is marked "B i "," B ii " and "B iii " and is enlarged at the bottom. Figures 3A-3B show photographs of BD MAX™ PCR cartridges, each utilized for diagnostic testing of clinical vaginal swab samples containing McKesson carbomer-based interfering gel. Figures 4A-4B show additional embodiments of photographs of BD MAX™ PCR cartridges utilized for testing of additional interfering gel EZ brand (Medline Industries, Inc.) present at 10 μL (Figures 4A-4B). As indicated by the red arrows in Figures 3A and 4A, magnetic particles disposed in a nucleic acid extraction cartridge formed aggregates when the diagnostic assay was performed using the comparative buffer. Particle aggregation then resulted in microfluidic blockages, as shown in Figures 3A and 4A. Exemplary areas of particle aggregation and microfluidic blockage in Figure 3A are labeled "A i ","A ii ", and "A iii ” and magnified at the bottom. Exemplary areas of particle aggregation and microfluidic clogging in Figure 4A are marked as “A i ","A ii ", and "A iii” and enlarged at the bottom. For comparison, see Figure 3B, e.g., “B i "," B ii "," B iii In the area marked as "" and enlarged at the bottom, no such particle aggregation or clogging of the microfluidics was observed. i "," B ii In the area marked as "" and magnified at the bottom, neither particle aggregation nor clogging of the microfluidics was observed. Thus, this example demonstrates that use of the buffer compositions disclosed herein prevents or reduces particle aggregation and microfluidic clogging in BD MAX™ PCR cartridges during diagnostic assays.

[0074] Example 9 Example: Buffers improve / maintain diagnostic accuracy Assays for detecting and diagnosing representative vaginal symptoms in samples containing the interfering gel were performed on a BD MAX™ system using Example Buffer II and the comparative buffer. The vaginosis test results in Figure 5 were obtained from vaginal swab samples containing the interfering EZ Lubricating Jelly (Medline Industries, Inc.), and the vaginitis test results in Figure 6 were obtained from vaginal swab samples containing the interfering Surgilube® lubricant, a carbomer-free gel. Scatter plots were created from PCR data obtained using the standard workflow with the comparative buffer (Figures 5A, 5D, 6A, and 6D), the optimized workflow with the comparative buffer (Figures 5B, 5E, 6B, and 6E), and the optimized workflow with the example buffer (Figures 5C, 5F, 6C, and 6F). The y-axis ("Cy5.5 EP") in Figures 5-6 represents endpoint fluorescence, which is measured as the fluorescence intensity of Cyanine Dye 5.5 (Cy5.5) at the end of the PCR amplification curve. The x-axis ("Cy5.5 Ct Score") in Figures 5A-5C and 6A-6C represents the PCR cycle threshold at which amplification products can be confidently detected after correcting for signal drift. The x-axis ("Cy5.5 SDPA") in Figures 5D-5F and 6D-6F represents the "second derivative peak horizontal axis," which was used as a criterion for assessing assay reproducibility. As shown in both Figures 5 and 6, Example Buffer II produced a narrower distribution of test results than the Comparative Buffer in both Ct score and SDPA. Thus, this example demonstrates that the use of the example buffer can significantly improve assay performance by at least improving the reproducibility of diagnostic tests under noisy conditions, especially for samples in the presence of interfering gels.

[0075] Example 10 Example Buffer III reduces non-reportable rates when samples contain interfering substances (lubricants) Similar to the tests described above for Examples 2-9, additional tests were conducted using Example Buffer III. Example Buffer III contains:

[0076] [Table 18] As reported for Example Buffers I and II, the use of Example Buffer III reduced the number of indeterminate and unresolved tests in the presence of an interfering substance (EZ Jelly, Medline Industries, Inc.) compared to the conventional buffer.

[0077] Example 11 Comparison of Example Buffer I and Comparative Buffer BD MAX™ Vaginal Panel and BD MAX™ CT / GC / TV The following example describes the results of a study examining the effect of Buffer I on the non-reportable rate (NRR), agreement, and percent detection in the BD MAX™ Vaginal Panel Assay and the BD MAX™ CT / GC / TV Assay. Buffer I was compared to the comparative buffers described in Example 1, Table 1. Laboratory and Clinical Procedures: Samples were collected from donor subjects. For each donor, four collection methods were used: 1) Self-vaginal collection without lubricant, 2) Clinician-administered vaginal collection without lubrication 3) Clinician-administered vaginal sampling using a speculum and lubricant (three different brands) 4) Clinician-administered endocervical collection using a scope + lubricant (3 different brands). Lubricants were applied on the colposcope in amounts deemed appropriate by the clinician. Three different brands of lubricants were used: Surgilube® Surgical Lubricant, Sterile and Bacteriostatic (HR® Pharmaceuticals, Inc., York PA), McKesson Lubricating Jelly (McKesson Medical-Surgical Inc., Richmond VA), and Aplicare Lubricating Jelly (Aplicare, Inc., Meriden CT). Samples were run in the BD MAX™ Vaginal Panel Assay and the BD MAX™ CT / GC / TV Assay according to the manufacturer's specifications. The results of the tests are shown below.

[0078] Table 12 shows that Example Buffer I was highly effective (-7.9) in reducing the NRR (unresolved (UNR) and indeterminate (IND) results) of vaginal samples containing lubricant when performed on the BD MAX™ Vaginal Panel, while a slight increase in NRR (5.5) was observed with Example I Buffer in clinician-collected vaginal samples without lubricant. No significant changes in NRR were observed in self-collected vaginal samples or clinician-collected cervical samples using lubricant.

[0079] [Table 19]

[0080] Similar to the BD MAX™ Vaginal Panel, Example I buffer effectively reduced the NRR (-13.1) of vaginal samples containing lubricant when run in the BD MAX™ CT / GC / TV assay, but unlike the BD MAX™ Vaginal Panel, no increase in UNR was observed in clinician-collected vaginal samples without lubricant (Table 13). No significant changes in NRR were observed in self-collected vaginal samples or clinician-collected endocervical samples with lubricant.

[0081] [Table 20] In the BD MAX™ Vaginal Panel Assay, Example I buffer provided good agreement with test results using the comparison buffer for all targets. For C. glabrata, the observed positive agreement was slightly lower due to the very low number of positive results (Table 14).

[0082] [Table 21]

[0083] In the BD MAX™ CT / GC / TV assay, Example I buffer provided good agreement with test results using the comparison buffer (Table 15). [Table 22] Analysis of PCR measurements (EndPoint, SDPA, Ct score) indicated that Example Buffer I tended to have a slight negative impact on the amplification of some targets in the BD MAX™ Vaginal Panel Assay, while tending to slightly improve the amplification of TV in the BD MAX™ CT / GC / TV Assay.

[0084] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0085] Those skilled in the art will understand that for this and other processes and methods disclosed herein, the functions performed in the processes and methods can be performed in differing order. Furthermore, the outlined steps and operations are presented by way of example only, and some of the steps and operations are optional and can be combined into fewer steps and operations or expanded into additional steps and operations without detracting from the essence of the disclosed embodiments.

[0086] With respect to the use of virtually any plural and / or singular term herein, those skilled in the art can translate from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity.

[0087] In general, those skilled in the art will understand that the terms used herein, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "comprises" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including, but not limited to," etc.). Those skilled in the art will further understand that where a specific number of introduced claim recitations is intended, such intention will be expressly set forth in the claim, and that in the absence of such recitation, no such intention exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" limits a particular claim containing such an introduced claim recitation to embodiments containing only one such restatement, even when the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or"), and the same applies to the use of definite articles used to introduce claim recitations. Also, even when a specific number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such a recitation should be interpreted to mean at least the recited number (e.g., the recitation "two occurrences" without any other modifier means at least two occurrences, or more than two occurrences).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together). When a convention similar to "at least one of A, B, or C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together). Those skilled in the art will understand that virtually any separating word and / or phrase in the specification, claims, and drawings that presents two or more alternative terms contemplates the possibility of including either one of the terms, either one of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B," or "A and B."

[0088] Furthermore, when features or aspects of the present disclosure are described in terms of a Markush group, those skilled in the art will recognize that the present disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0089] As will be understood by those skilled in the art, for any and all purposes, for example, in terms of providing a written description, all ranges disclosed herein encompass all possible subranges and combinations of subranges. Any range described can be readily recognized as fully descriptive and allowing the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range described herein can be easily broken down into a lower third, middle third, upper third, etc. Additionally, as will be understood by those skilled in the art, all terms such as "up to," "at least," etc. refer to ranges that are inclusive of the recited numbers and can subsequently be broken down into subranges as described above.

[0090] Whenever a range of values ​​is provided herein, unless otherwise specified, the range includes the starting value, the ending value, each individual value, or any range of values ​​therebetween. For example, "0.2 to 0.5" refers to 0.2, 0.3, 0.4, 0.5, 0.2 to 0.3, 0.3 to 0.4, ranges therebetween such as 0.2 to 0.4, increments therebetween such as 0.25, 0.35, 0.225, 0.335, 0.49, and increments therebetween such as 0.26 to 0.39. As another example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0091] From the foregoing, it will be understood that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications can be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. A buffering agent composition, (a) 50 mM to 150 mM acetic acid and 350 mM to 450 mM sodium acetate, 1 mM to 20 mM ethylenediaminetetraacetic acid (EDTA), 0.1% to 3.0% by mass of nonionic surfactant, A divalent salt selected from the group consisting of calcium salts, magnesium salts, and combinations thereof, in concentrations of 100 mM to 300 mM, and pH 4.0 to 6.0 The buffering composition comprising the above.

2. A buffering agent composition, (b) Tris-HCl 1 mM to 20 mM, 0.1% to 3.0% by mass of nonionic surfactant, 1 mM to 30 mM tris(2-carboxyethyl)phosphine (TCEP), A divalent salt selected from the group consisting of calcium salts, magnesium salts, and combinations thereof, with a concentration of 50 mM to 150 mM, and pH 2.1 to 3.0 The buffering composition comprising the above.

3. The buffering composition according to claim 1 or 2, wherein the buffering composition of (a) contains EDTA at a concentration of 5 mM to 15 mM, or the buffering composition of (b) contains TCEP at a concentration of 10 mM to 20 mM.

4. The buffering composition according to claim 1 or 2, wherein the buffering composition of (a) contains EDTA at a concentration of 10 mM, or the buffering composition of (b) contains TCEP at a concentration of 15 mM.

5. The buffering composition according to any one of claims 1 to 4, wherein the nonionic surfactant of (a) and (b) is selected from the group consisting of ethoxylated nonionic surfactants, propoxylated nonionic surfactants, co-ethoxylated-propoxylated nonionic surfactants, ethoxylated sorbitan esters of monofatty acids, ethoxylated octylphenol, ethoxylated secondary C1-C20 alcohols, co-ethoxylated-propoxylated seed oil alcohols, and combinations thereof.

6. The buffering composition according to any one of claims 1 to 4, wherein the nonionic surfactants of (a) and (b) are Tergitol or Triton surfactants.

7. The buffering composition according to claim 6, wherein the nonionic surfactant is Tergitol 15-S-9 or Triton X-100.

8. The buffering composition according to any one of claims 1 to 4, wherein the nonionic surfactants of (a) and (b) are selected from the group consisting of ethoxylated sorbitan esters of monofatty acids containing an average of 1 to 50 ethylene oxide units per surfactant, ethoxylated octylphenols containing an average of 1 to 20 ethylene oxide units per surfactant, ethoxylated secondary C1-C20 alcohols containing an average of 1 to 20 ethylene oxide units per surfactant, co-ethoxylated-propoxylated seed oil alcohols containing an average of 1 to 20 propylene oxide units and 1 to 30 ethylene oxide units per surfactant, and combinations thereof.

9. The buffering composition according to any one of claims 1 to 8, wherein the divalent salts of (a) and (b) are CaCl2.

10. The buffering composition according to any one of claims 1 to 9, wherein the buffering composition further comprises a bactericidal preservative, and the concentration of the bactericidal preservative is 0.01% by mass to 6% by mass.

11. The buffering composition according to claim 10, wherein the bactericidal preservative comprises one or more isothiazolones in a concentration of 0.01% to 6% by mass.

12. The buffering composition according to claim 11, wherein one or more isothiazolones include chloromethylisothiazolinone and methylisothiazolinone, wherein chloromethylisothiazolinone and methylisothiazolinone are present in a mass ratio of 1:1 to 5:

1.

13. The buffering composition according to claim 12, wherein the one or more isothiazolones comprise 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one in a mass ratio of 3:

1.

14. The buffering composition according to claim 10, wherein the bactericidal preservative comprises one or more salt-free glycols and alkyl carboxylate stabilizers.

15. The buffering agent composition according to claim 2, wherein the buffering agent composition is 5–15 mM Tris-HCl; 0.5% to 1.5% by mass of polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (Tritoon X-100); 10–20 mM tris(2-carboxyethyl)phosphine (TCEP); 50-150 mM CaCl₂; and pH 2.4 ± 0.2 The buffering composition comprising the above.

16. A buffering agent composition according to claim 1, wherein the buffering agent composition is Acetic acid in a concentration of 50 mM to 150 mM; 350 mM to 450 mM sodium acetate; EDTA from 1 mM to 20 mM; 0.1% to 3.0% by mass of nonionic surfactant; CaCl₂ at concentrations of 100 mM to 300 mM; and pH 4.0 to 6.0 The buffering composition comprising the above.

17. The buffering composition according to claim 16, wherein the nonionic surfactant comprises 1.0% by mass of an ethoxylated secondary C1-C20 alcohol or 0.5% by mass of polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (Triton X-100).

18. A buffering agent composition according to claim 1, wherein the buffering agent composition is 105 mM acetic acid; 395 mM sodium acetate; EDTA at 10 mM; 1% by mass of ethoxylated secondary C1-C20 alcohol; 200 mM CaCl₂; and pH 5.0 The buffering composition comprising the above.

19. A buffering agent composition according to claim 1, wherein the buffering agent composition is 105 mM acetic acid; 395 mM sodium acetate; 10 mM EDTA 0.5% by mass of polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (Tritoon X-100); 200 mM CaCl₂; and pH 5.0 The buffering composition comprising the above.

20. The buffering composition according to any one of claims 16 to 19, further comprising 0.01% to 1.0% by mass of chloromethylisothiazolinone and methylisothiazolinone.

21. The buffering composition according to claim 20, comprising 0.03% by mass of chloromethylisothiazolinone and methylisothiazolinone.

22. A kit for diagnosing symptoms related to vaginal infection or inflammation, A buffering composition according to any one of claims 1 to 21; and A manual for diagnosing symptoms related to vaginal infections or inflammation. The kit includes the above.

23. A method for preventing or reducing aggregation of surface-functionalized particles, comprising contacting a sample with a buffering composition according to any one of claims 1 to 21, wherein the sample comprises a plurality of surface-functionalized particles configured for nucleic acid extraction, purification, amplification, detection, or a combination thereof, and the level of aggregation of the plurality of surface-functionalized particles in the presence of the buffering composition is reduced compared to the level of aggregation in the absence of the buffering composition.

24. The sample is a vaginal sample and is taken from a subject exhibiting clinical signs of vaginitis or vaginosis, or both, and the sample comprises a plurality of nucleic acids. The method according to claim 23, further comprising amplifying and / or detecting a plurality of nucleic acids, wherein the efficiency of amplifying and / or detecting the plurality of nucleic acids is improved in the presence of the buffering composition compared to the efficiency in the absence of the buffering composition.

25. The method according to claim 23 or 24, wherein aggregation is induced by an interfering substance in the sample, and the interfering substance is selected from the group consisting of lubricants, gels, creams, and combinations thereof.

26. The method according to any one of claims 23 to 25, wherein the efficiency of amplification and / or detection of a plurality of nucleic acids is improved by at least 5% in the presence of the buffering composition compared to the efficiency in the absence of the buffering composition.

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