Methods for isolating nucleic acid from specimens in liquid based cytodiagnosis preservatives containing formaldehyde

Abstract

To provide a method for treating specimens including clinical samples preserved in a liquid-based cytodiagnosis preservative containing formaldehyde.SOLUTION: The method begins with a step of preparing a reaction mixture by mixing a specimen with a protease enzyme and 2-imidazolidone or other formaldehyde scavenger. After that, the reaction mixture is incubated at an elevated temperature for a period of time sufficient enough to reverse the chemical modification by formaldehyde to the nucleic acid which may be contained in the specimen. By this step, at least part of the chemical modification caused by reaction between formaldehyde and nucleic acid or protein is reversed. For example, chemical crosslinks may be broken. Next, there is a step for isolating the nucleic acid from the reaction mixture after the incubation step. In the final step, in vitro amplification reaction is carried out using the nucleic acid from the isolation step as a template.SELECTED DRAWING: None

Description

Technical Field

[0001] (Cross - reference to related applications) This application is a non - provisional application of U.S. Patent Provisional Application No. 61 / 946,637, filed on February 28, 2014, and the entire disclosure is incorporated by reference in its entirety for all purposes. (Field of the Invention) The present invention relates to the field of biotechnology. More specifically, the present invention relates to a method for isolating nucleic acids from samples fixed in a formaldehyde - containing liquid - based cytology preservative, and the isolated RNA is suitable for use as a template in nucleic acid amplification methods.

Background Art

[0002] Experimenters engaged in molecular analysis of nucleic acids isolated from samples fixed with formaldehyde understand that certain limitations apply to the use of this type of sample. This is because formaldehyde and other specific chemical fixatives chemically modify proteins and nucleic acids. It is known that this modification impairs the usefulness of nucleic acids in subsequent analyses. Well - known chemical modifications of DNA, RNA, and proteins cause various problems. In fact, Masuda et al. (Nucleic Acids Res., 27:4436 - 4443 (1999)) investigated the reasons why samples fixed with formalin are inadequate materials for molecular biological applications. The authors solubilized fixed tissues by treatment with proteinase K to enable RNA extraction, but showed that the extracted RNA has limited use as a template for PCR. Further studies revealed chemical addition of mono - methylol groups (-CH2OH) to all four bases, as well as evidence of adenine dimer formation by methylene bridges. Certain modifications could be reversed by raising the temperature in a buffer without formalin. However, due to the instability of RNA, the use of high - temperature conditions may become undesirable.

[0003] Initial attempts to process samples fixed with formaldehyde have been somewhat successful. For example, Khrpin et al. describe in US Patent Application Publication No. 2011 / 0196146 A1 the use of formaldehyde removal compounds containing hydrazine and hydrazide (e.g., semicarbazide; thiosemicarbazide; carbazide; thiocarbazide; N - aminoguanidine and its salts such as hydrochloride; N,N - diaminoguanidine and its salts such as dihydrochloride; acetylhydrazide; adipic acid dihydrazide; succinic acid dihydrazide; formic acid hydrazide; maleic acid dihydrazide; malonic acid dihydrazide; benzenesulfonylhydrazide; tosylhydrazide; methylsulfonylhydrazide) while isolating nucleic acids from cell materials placed in a liquid - based cytology preservative containing formaldehyde. These inventors used a hybrid capture method in which an RNA probe mixture was hybridized to the isolated nucleic acids, rather than using nucleic acid amplification as an indicator of nucleic acid integrity. Subsequently, the presence of DNA target nucleic acids was determined by antibody binding to the RNA:DNA hybrid and a subsequent signal amplification method. In fact, Khrpin et al. refer to US Patent No. 6,228,578 for teachings on nucleic acid detection, which describes the treatment of nucleic acid samples under strongly alkaline and high - temperature conditions (conditions known to hydrolyze RNA). Thus, Khrpin et al. have not addressed making nucleic acids suitable for use as templates in nucleic acid amplification reactions and have not provided sufficient disclosure to enable the detection of RNA targets from formaldehyde - fixed specimens.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Non - Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] The techniques disclosed herein address the need for rapid and efficient isolation of intact nucleic acids, such as RNA, from specimens stored in a liquid-based cytology preservative containing formaldehyde.

Means for Solving the Problems

[0007] In one aspect, the present invention relates to a method of processing a specimen comprising a clinical sample placed in a liquid-based cytology preservative containing formaldehyde. The method begins with the step of mixing the specimen with a protease enzyme and 2-imidazolidone or other formaldehyde scavenger to create a reaction mixture. Thereafter, the reaction mixture is incubated at a high temperature for a time sufficient to reverse the chemical modification of nucleic acids in the specimen by formaldehyde. This step reverses at least a portion of the chemical modification due to the reaction between formaldehyde and nucleic acids or proteins. For example, chemical cross-links can be cleaved. Next, there is a step of isolating nucleic acids from the reaction mixture after the incubation step. Finally, there is a step of performing an in vitro amplification reaction using the nucleic acids from the isolation step as a template.

[0008] In some methods, by reversal, nucleic acids are released from formaldehyde-induced cross-links to polypeptides in the specimen. In some methods, the protease liberates nucleic acids from formaldehyde-induced cross-links, and 2-imidazolidone inhibits the induction of new cross-links between nucleic acids and polypeptides in the sample.

[0009] In some methods, the sample has been placed in the liquid-based cytology preservative for 7 to 120 days prior to the performance of step (a).

[0010] In some methods, the incubation step is 30 minutes or less, or the incubation step is about 5 to 30 minutes, or the incubation step is 15 minutes or less.

[0011] In some methods, the yield of the amplified nucleic acid after step (d) is higher than the control amplification excluding either proteinase or 2-imidazolidone. In some methods, the yield of the amplified nucleic acid after step (d) is at least 10% higher than the control amplification excluding either proteinase or 2-imidazolidone. In some methods, at least 90% of the nucleic acid molecules in the sample do not contain cross-links after the incubation step.

[0012] In some methods, the final concentration of 2-imidazolidone before the incubation step is 1 to 5 or 2 to 5 times higher in molarity than the final highest concentration of formaldehyde. In some methods, the proteinase is proteinase K present at a concentration of 4.3 to 43 U / mL. In some methods, the temperature of the incubation step is about 60 to 100 °C. In some methods, the temperature of the incubation step is 85 to 95 °C. In some methods, the temperature of the incubation step is 91 to 95 °C. In some methods, the temperature of the incubation step is 90 °C.

[0013] In some methods, the proteinase and the formaldehyde scavenger are mixed with the sample simultaneously. In some methods, the proteinase is mixed with the sample before the formaldehyde scavenger. In some methods, the formaldehyde scavenger is mixed with the sample before the proteinase.

[0014] In some methods, the amplification is transcription amplification, single primer nucleic acid amplification, nucleic acid sequence-based amplification, polymerase chain reaction, strand displacement amplification, self-sustained sequence replication, or DNA ligase chain reaction. In some methods, the nucleic acid contains DNA, and in some methods, it contains RNA. In some methods, the isolated nucleic acid is DNA, and in some methods, it is RNA.

[0015] In some methods, the nucleic acid is isolated by a capture assay using a capture probe that hybridizes to the nucleic acid to be isolated and an immobilized probe. In some methods, the immobilized probe is immobilized on magnetic beads.

[0016] In some methods, the assay positivity of the amplified nucleic acid is higher than the assay positivity of the amplified nucleic acid obtained from the reaction mixture excluding either proteinase or a formaldehyde scavenger. In some methods, the assay positivity of the amplified nucleic acid is at least about 12% higher than the assay positivity of the amplified nucleic acid obtained from the reaction mixture excluding either proteinase or 2-imidazolidone. In some methods, the assay positivity of the amplified nucleic acid after step (d) is about 95% after 21 days.

[0017] In some methods, the isolated nucleic acid is a human papillomavirus (HPV) RNA target nucleic acid. In some methods, the sample is a cervical cell sample.

[0018] In another aspect, the present invention relates to a composition comprising the following components: 2-imidazolidone, proteinase K, EDTA, and a pH buffer.

[0019] In another aspect, the present invention relates to a kit for processing a sample stored in a liquid-based cytology preservative containing formaldehyde. The kit includes a first vial containing a lyophilized proteinase K enzyme. Similarly, the kit includes a second vial containing a reconstitution buffer for reconstituting the lyophilized proteinase K enzyme. The reconstitution buffer includes a certain amount of a pH buffer, a certain amount of EDTA, and a certain amount of 2-imidazolidone.

[0020] In another aspect, the present invention relates to a system for processing a nucleic acid-containing sample stored in a liquid-based cytology preservative containing formaldehyde. The components of this system include a programmable controller, a pipetting device that communicates with the programmable controller, a first holder for reaction vials, and a second holder for reagent vials. According to this aspect of the present invention, the programmable controller is configured, by software instructions, to cause the pipetting device to transfer a partial volume of liquid from the reagent vial to the reaction vial when the reagent vial contains a solution containing 2-imidazolidone, proteinase K, EDTA, and a pH buffer. In addition to these components, the reaction vial may contain a specimen stored in formaldehyde as further described below, either before or after the introduction of other components. The same or another pipetting device may be used together with the programmable controller configured to operate such a pipetting device, if desired, by software, to introduce the specimen into the reaction vial. The system may optionally include a heater for controlled-time heating of the reaction vial and its contents, under the control of a programmable controller configured by suitable software. The present invention provides, for example, the following items. (Item 1) A method for processing a specimen containing a clinical sample placed in a liquid-based cytology preservative containing formaldehyde, comprising: (a) mixing the specimen with a protease enzyme and a formaldehyde scavenger to form a reaction mixture; (b) incubating the reaction mixture at a high temperature for a time sufficient to reverse chemical modification of nucleic acids that may be contained in the specimen by formaldehyde in the liquid-based cytology preservative; (c) isolating nucleic acids from the reaction mixture after the incubating step; (d) performing an in vitro amplification reaction using the nucleic acids isolated in the isolating step as a template. (Item 2) The method according to item 1, wherein the formaldehyde scavenger is 2-imidazolidone. (Item 3) The method according to item 1 or 2, wherein the nucleic acid in the sample is released from the formaldehyde-induced cross-linking to the polypeptide in the sample by the reverse. (Item 4) The method according to item 2 or 3, wherein the protease releases the nucleic acid from the formaldehyde-induced cross-linking, and 2-imidazolidone inhibits the induction of new cross-linking between the nucleic acid and the polypeptide in the sample. (Item 5) The method according to any one of items 1 to 4, wherein the sample is placed in the liquid-based cytology preservative for 7 to 120 days before the implementation of step (a). (Item 6) The method according to any one of items 1 to 5, wherein the incubating step lasts for 30 minutes or less. (Item 7) The method according to any one of items 1 to 6, wherein the incubating step is about 5 to 30 minutes. (Item 8) The method according to any one of items 1 to 7, wherein the incubating step lasts for 15 minutes or less. (Item 9) The method according to any one of items 1 to 8, wherein the yield of the amplified nucleic acid after step (d) is higher than the control amplification excluding either protease or 2-imidazolidone. (Item 10) The method according to any one of items 1 to 9, wherein the yield of the amplified nucleic acid after step (d) is at least 10% higher than the control amplification excluding either protease or 2-imidazolidone. (Item 11) The method according to any one of items 1 to 10, wherein at least 90% of the nucleic acid molecules in the sample do not contain cross-links after the incubating step. (Item 12) The method according to any one of items 2 to 11, wherein the final concentration of 2-imidazolidone before the incubating step is 1 to 5 times higher in moles than the final highest concentration of the formaldehyde. (Item 13) The method according to item 12, wherein the final concentration of 2-imidazolidone before the incubating step is 2 to 5 times higher in moles than the final highest concentration of the formaldehyde. (Item 14) The method according to item 12 or 13, wherein the protease is proteinase K present at a concentration of 4.3 to 43 U / mL. (Item 15) The method according to any one of items 1 to 14, wherein the temperature of the incubating step is about 60 to 100 °C. (Item 16) The method according to item 15, wherein the temperature of the incubating step is 85 to 95 °C. (Item 17) The method according to item 16, wherein the temperature of the incubating step is 91 to 95 °C. (Item 18) The method according to item 15, wherein the temperature is about 90 °C. (Item 19) The method according to any one of items 1 to 18, wherein the protease and the formaldehyde scavenger are mixed with the sample simultaneously. (Item 20) The method according to any one of items 1 to 18, wherein the protease is mixed with the sample before the formaldehyde scavenger. (Item 21) The method according to item 1, wherein the formaldehyde scavenger is mixed with the sample before the protease. (Item 22) The method according to any one of items 1 to 21, wherein the amplification is transcription amplification, single primer nucleic acid amplification, nucleic acid sequence-based amplification, polymerase chain reaction, strand displacement amplification, self-sustained sequence replication, or DNA ligase chain reaction. (Item 23) The method according to any one of items 1 to 22, wherein the nucleic acid comprises DNA. (Item 24) The method according to any one of items 1 to 22, wherein the nucleic acid comprises RNA. (Item 25) The method according to item 23, wherein the isolated nucleic acid is DNA. (Item 26) The method according to item 24, wherein the isolated nucleic acid is RNA. (Item 27) The method according to any one of items 1 to 26, wherein the nucleic acid is isolated by a capture assay using a capture probe that hybridizes to the nucleic acid to be isolated and the immobilized probe. (Item 28) The method according to item 27, wherein the immobilized probe is immobilized on magnetic beads. (Item 29) The method according to any one of items 1 to 28, wherein the assay positivity of the amplified nucleic acid after step (d) is higher than the assay positivity of the amplified nucleic acid obtained from the reaction mixture excluding any one of proteinase or formaldehyde scavenger. (Item 30) The method according to item 29, wherein the assay positivity of the amplified nucleic acid after step (d) is at least about 12% higher than the assay positivity of the amplified nucleic acid obtained from the reaction mixture excluding any one of proteinase or 2-imidazolidone. (Item 31) The method according to item 29, wherein the assay positivity of the amplified nucleic acid after step (d) is about 95% after 21 days. (Item 32) The method according to item 24, wherein the isolated nucleic acid is a human papillomavirus (HPV) RNA target nucleic acid. (Item 33) The method according to item 32, wherein the sample is a cervical cell sample. (Item 34) A composition comprising: (a) 2-imidazolidone and (b) Proteinase K and (c) EDTA and, (d) a pH buffer, and a composition containing the same. (Item 35) A kit for processing a specimen stored in a preservative for liquid-based cytology containing formaldehyde, (a) a first vial containing lyophilized proteinase K enzyme, (b) a second vial containing a reconstitution buffer for reconstituting the lyophilized proteinase K enzyme, The reconstitution buffer contains a certain amount of pH buffer, a certain amount of EDTA, and a certain amount of 2-imidazolidone, and a second vial, and a kit containing the same. (Item 36) A system for processing a nucleic acid-containing sample stored in a preservative for liquid-based cytology containing formaldehyde, wherein the components of the system are a programmable controller, a pipetting device communicating with the programmable controller, a first holder for reaction vials, a second holder for reagent vials, a heating element, and is provided with When the reagent vial contains a solution containing a formaldehyde scavenger, proteinase K, EDTA, and a pH buffer by a software instruction of the programmable controller, the pipetting device moves a partial amount of the liquid from the reagent vial to the reaction vial. It is configured as follows, The programmable controller is configured to heat the reaction vial to a temperature of 65°C to 95°C by the heating element by a software instruction, and a system. (Item 37) The programmable controller is configured to heat the reaction vial to a temperature of 85°C to 95°C by the heating element by a software instruction, and the system according to Item 36. (Item 38) The system according to item 37, wherein the reagent in the reaction vial contains 2-imidazolidone as the formaldehyde scavenger. (Item 39) The system according to item 38, wherein the reagent in the reaction vial contains proteinase K at a concentration of 42 to 45 U. (Item 40) The system according to item 36, wherein the programmable controller is configured to heat the reaction vial to a temperature of 90°C to 95°C by the heating element according to software instructions. (Item 41) The system according to item 40, wherein the programmable controller is configured to heat the reaction vial to a temperature of 90°C to 95°C for 15 to 30 minutes by the heating element according to software instructions. (Item 42) The system according to item 41, wherein the reagent in the reaction vial contains proteinase K at a concentration of 42 to 45 U.

Brief Description of the Drawings

[0021]

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DETAILED DESCRIPTION OF THE INVENTION

[0022] (Definition) Unless otherwise specified, the scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art of molecular biology, based on technical literature such as Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY), or other well-known technical publications in molecular biology. Unless otherwise specified, the procedures used or contemplated in this specification are standard methods well known in the technical field of molecular biology.

[0023] All patents, published applications, and other publications cited in this specification are incorporated by reference in their entirety. If the definitions set forth in this section are contrary to or inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated by reference into this specification, the definitions set forth in this section shall control.

[0024] As used herein, "a" or "an" means "at least one" or "one or more".

[0025] The terms regarding approximation used herein throughout the specification and claims may be applied to modify any quantitative or qualitative expression that can be changed within an acceptable range without causing a change in the relevant basic function. Accordingly, a value modified by a term such as "about" or "approximately" is not limited to the exact value specified, and may include a value different from the specified value.

[0026] For clarity, "formaldehyde," which has the basic form (CH2O), is a gas. The liquid called "formalin" is actually a mixture of formaldehyde gas and water. However, as used herein, "formaldehyde" can refer to the molecule (CH2O) dissolved in an aqueous solution.

[0027] As used herein, "liquid-based cytology" refers to liquid-based gynecological specimen collection, where a sample for cervical examination is collected by conventional methods using one of the brush instruments, but instead of spreading it on a slide glass, it is transferred to a sample containing a liquid preservative, i.e., a "fixative." The preserved specimen can be used for microscopic examination or molecular analysis.

[0028] As used herein, a "specimen" is something collected as an example of a specific thing. Examples of biological specimens include any tissue or material derived from a living or dead organism that may contain an analyte such as a nucleic acid analyte. Preferred biological specimens include respiratory tissue, exudates (e.g., bronchoalveolar lavage), biopsy materials, sputum, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue, feces, urine, or other fluids, tissues, or materials. Particularly preferred biological specimens include cells collected from the external opening of the cervix such as can be obtained in relation to a Pap test.

[0029] As used herein, the term "sample" refers to a part or quantity of material for use in an examination, and this part can provide information regarding what it was collected from. A sample can be derived from any source, e.g., a biological specimen or an environmental source.

[0030] As used herein, the term "nucleic acid" refers to a polynucleotide compound containing oligonucleotides that include nucleosides or nucleoside analogs having nitrogen heterocyclic bases or base analogs covalently linked by standard phosphodiester bonds or other bonds. Examples of nucleic acids include RNA, DNA, chimeric DNA-RNA polymers, or analogs thereof.

[0031] "Nucleic acid analyte" means a polynucleotide to be detected or quantified. The genome of a specific virus would be an example of a nucleic acid analyte.

[0032] As used herein, an "assay sample" is any sample that is tested for the presence of a specific nucleic acid analyte.

[0033] As used herein, "high" temperature conditions refer to temperatures higher than room temperature. Preferably, the high temperature is in the range of 60°C to 100°C, more preferably in the range of 65°C to 95°C, sometimes in the range of 80°C to 90°C, sometimes in the range of 81 - 98°C, 85 - 98°C, 85 - 95°C, 90 - 95°C, 91 - 95°C, or 91 - 99°C, and sometimes about 90°C. The use of temperatures above 80°C, such as 85 - 98°C, 90°C, or 91 - 95°C, can result in increased assay sensitivity as further defined below. However, surprisingly, as the temperature rises above 65°C, the degree of denaturation of proteinase K increases, and the use of temperatures above 85°C or 90°C is usually not recommended due to excessive denaturation. The practice of the present invention is not dependent on identifying the mechanism of this unexpected effect by high temperature, but this result may indicate that the promotion of formaldehyde removal by 2-imidazolidone at high temperature not only compensates for but also exceeds a certain degree of decrease in proteinase K activity. Preferably, in all these cases, a reaction mixture containing a clinical sample, 2-imidazolidone, protease, EDTA, and a pH buffer in a preservative for liquid-based cytology is exposed to high temperature for 10 minutes to 30 minutes, preferably 10 minutes to 20 minutes or less, sometimes up to 15 minutes, i.e., about 15 minutes.

[0034] An "amplification product" is a polynucleotide product of an in vitro nucleic acid amplification reaction, where the target nucleic acid sequence has functioned as a template for the synthesis of copies, i.e., amplification products.

[0035] "Target" or "target nucleic acid" means a nucleic acid containing a sequence to be amplified, detected, and / or quantified. The target nucleic acid sequence to be amplified is preferably located between oligonucleotides arranged in two opposite directions and contains a part of the target nucleic acid complementary to each oligonucleotide.

[0036] "Amplification" or "nucleic acid amplification" or "in vitro nucleic acid amplification" and the like mean any known procedure for obtaining multiple copies enabling RNA and DNA equivalents of the target nucleic acid sequence, or their complements or fragments.

[0037] To aid in the understanding of some of the embodiments disclosed herein, the previously described TMA method (e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, and 5,824,518) is briefly summarized. In TMA, the target nucleic acid containing the sequence to be amplified is provided as a single-stranded nucleic acid (e.g., ssRNA or ssDNA). Any conventional method for converting double-stranded nucleic acid (e.g., dsDNA) to single-stranded nucleic acid may be used. The promoter primer specifically binds to the target nucleic acid at its target sequence, and reverse transcriptase (RT) uses the target strand as a template to extend the 3' end of the promoter primer to yield a cDNA copy, resulting in an RNA:cDNA duplex. RNase activity (e.g., RNase H of the RT enzyme) digests the RNA of the RNA:cDNA duplex, and a second primer specifically binds to the target sequence in the cDNA downstream of the promoter-primer end. Subsequently, RT synthesizes a new DNA strand by extending the 3' end of the second primer using the cDNA as a template, resulting in dsDNA containing a functional promoter sequence. RNA polymerase specific for the functional promoter initiates transcription, and about 100 to 1000 RNA transcripts (amplified copies, i.e., amplification products) complementary to the initial target strand are generated. The second primer specifically binds to the target sequence in each amplification product, and RT makes cDNA from the RNA template that is the amplification product, resulting in an RNA:cDNA duplex. RNase digests the amplification product RNA from the RNA:cDNA duplex, the target-specific sequence of the promoter primer binds to the complementary sequence in the newly synthesized DNA, and RT extends the 3' end of the promoter primer as well as the 3' end of the cDNA to yield dsDNA containing a functional promoter to which RNA polymerase binds and transcribes further amplification products complementary to the target strand. By using an autocatalytic cycle that repeatedly uses these steps during the reaction, amplification of about 1 billion-fold the initial target sequence is obtained. The amplification products can be detected using a probe that specifically binds to the sequence contained in the amplification products during amplification (real-time detection) or at the end of the reaction (endpoint detection). Detection of the signal generated by the bound probe indicates the presence of the target nucleic acid in the sample.

[0038] As used herein, "detection" of an amplification product may be achieved using any known method. For example, the amplified nucleic acid may bind to a surface that results in a detectable physical change (e.g., an electrical change). The amplified nucleic acid may be concentrated in solution phase, or in or on a matrix, and detected by detecting a label (e.g., an intercalating agent such as ethidium bromide) bound thereto. In other detection methods, a probe complementary to a sequence in the amplification product is used to detect the presence of a probe:product complex, or a probe complex is used to amplify a signal detected from the amplification product (e.g., U.S. Pat. Nos. 5,424,413, 5,451,503, and 5,849,481). In other detection methods, a signal change such as a molecular label, molecular torch, or hybridization switch probe occurs only when a labeled probe binds to the amplification product, so a probe is used that generates a signal related to the presence of the target sequence (e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 5,925,517, 6,150,097, 6,361,945, 6,534,274, 6,835,542, 6,849,412, and 8,034,554, and U.S. Patent Application Publication No. 2006 / 0194240A1). Such probes typically use a label (e.g., a fluorophore) attached to one end of the probe, and an interaction compound (e.g., a quencher) attached to another position on the probe that inhibits signal generation from the label when the probe is in one conformation ("closed") indicating that the probe is not hybridized to the amplification product, but a detectable signal is generated when the probe hybridizes to the amplification product and the conformation changes ("opens"). Detection of a signal directly or indirectly resulting from a labeled probe that specifically binds to the amplification product indicates the presence of the amplified target nucleic acid.

[0039] As used herein, a "probe" is an oligonucleotide that specifically hybridizes to a target sequence in a nucleic acid, preferably an amplified nucleic acid, under conditions that promote hybridization and form a detectable hybrid.

[0040] As used herein, the term "contacting" means bringing two or more components together. Contacting can be accomplished by mixing all components in a fluid or semi-fluid mixture. Contacting can also be achieved when one or more components are physically contacted with one or more other components on a solid surface such as a solid tissue section or substrate.

[0041] As used herein, the term "target capture" refers to the selective separation of a target nucleic acid from other components of a sample mixture such as cell fragments, organelles, proteins, lipids, carbohydrates, or other nucleic acids. A target capture system can specifically and selectively separate a given target nucleic acid from other sample components (e.g., by use of a nucleic acid sequence specific for the target nucleic acid of interest) or can non-specifically and selectively separate the target nucleic acid from other sample components by use of other characteristics of the target (e.g., its physical properties that distinguish it from other sample components that do not exhibit such physical characteristics such as hybridization to non-specific nucleic acids, binding to porous glass beads, capture and elution from a silica-packed column, etc.). Preferred nucleic acid hybridization target capture methods and compositions have been described in detail previously (U.S. Pat. Nos. 5,750,338, 6,060,246, 6,110,678, 6,534,273, and 7,993,853, and U.S. Patent Application Publication No. 2008 / 0286775 A1). Preferred embodiments of target capture form complexes with the target nucleic acid using target capture oligonucleotides in solution phase and immobilized capture probes attached to a support, and separate the captured target from other components.

[0042] As used herein, the term "target capture oligonucleotide" refers to at least one nucleic acid oligonucleotide that cross-links, i.e., binds, a target nucleic acid to an immobilized capture probe using a complementary nucleic acid sequence or binding pair members such as biotin and streptavidin. In one method, the target capture oligonucleotide binds non-specifically to the target nucleic acid and immobilizes the target nucleic acid on a solid support. In a different method, the target-specific (TS) sequence of the target capture oligonucleotide specifically binds to a sequence in the target nucleic acid. In either method, the target capture oligonucleotide includes an immobilized capture probe binding region that binds to the immobilized capture probe (e.g., by a specific binding pair interaction). In embodiments where both the TS sequence and the immobilized capture probe binding region are nucleic acid sequences, they may be covalently linked to each other or on different oligonucleotides linked by one or more linkers.

[0043] An "immobilized capture probe" provides a means for binding a target capture oligonucleotide to a solid support. The immobilized capture probe is a base sequence recognition molecule bound to a solid support and facilitates separation of the bound target polynucleotide from unbound material. Any known solid support such as a matrix and particles free in solution may be used. For example, the solid support may be nitrocellulose, nylon, glass, polyacrylate, mixed polymer, polystyrene, silane polypropylene, and preferably may be magnetically attractable particles. A particularly preferred support is a monodisperse (i.e., uniform within ± about 5% in size) magnetic sphere, which provides consistent results particularly advantageous for use in automated assays. The immobilized capture probe may be bound to the solid support directly (e.g., via a covalent bond or ionic interaction) or indirectly. General examples of useful solid supports include magnetic particles or beads.

[0044] As used herein, the term "separating" or "purifying" generally refers to removing one or more components of a mixture (e.g., a sample) from one or more other components in the mixture. Sample components generally include nucleic acids in an aqueous phase, and may include cell fragments, proteins, carbohydrates, lipids, and other compounds. Preferred embodiments separate, i.e., remove, at least 70% - 80%, more preferably about 95% of the target nucleic acid from the other components in the mixture.

[0045] "Kit" typically means a packaged combination of materials intended to be used together simultaneously. Kits according to the present invention may include instructions or other information in a "tangible" form (e.g., printed information, electronically recorded on a computer-readable medium, or otherwise recorded on a machine-readable medium such as a barcode for storing numerical values).

[0046] "Consisting essentially of" means that additional components, compositions, or method steps that do not substantially change the basic and novel features of the present invention may be included in the present invention. Any component, composition, or method step that substantially affects the basic and novel features of the present invention is outside the scope of this term.

[0047] Unless otherwise apparent from the context, "about" indicates the potential variation in the accuracy of measuring a value.

[0048] "Reversing" the modification of DNA means releasing the DNA from modifications induced by formaldehyde, particularly cross-linking to polypeptides. The reversal may be partial or complete, and as a result, the DNA may or may not be restored to the correct situation before the formaldehyde-induced modification occurs.

[0049] Detailed Description Disclosed herein are methods, systems, compositions, and kits for isolating nucleic acids from a specimen stored in a liquid-based cytology preservative containing formaldehyde. Briefly, the disclosed methods are based on contacting a sub-volume of the sample, i.e., a cell sample placed in a liquid preservative, with a combination of 2-imidazolidone and a protease enzyme. In a particularly preferred embodiment, the protease is proteinase K enzyme. Subsequently, incubating the mixture under high heat conditions inactivates free formaldehyde and can reverse at least a portion of the chemical modification of nucleic acids due to formaldehyde. This method advantageously results in nucleic acids, such as RNA, that are rapid compared to previous methods, efficiently isolated (e.g., by capture probe hybridization), reversely transcribed (if desired), and amplified in vitro.

[0050] Introduction and Overview The methods disclosed herein facilitate the detection of nucleic acid targets that may be present in a biological sample contained in a liquid-based cytology preservative containing formaldehyde. Such biological samples can be stored for a significant period of time (e.g., at least 1, 2, 7, 14, 30, 50, or 100 days, or 7 - 120 days) prior to analysis. During storage, formaldehyde may induce modification of the nucleic acids in the sample, particularly cross-linking with polypeptides present in the sample. These modifications inhibit subsequent processing of the DNA, such as the ability to hybridize (e.g., to a capture probe), or the ability to amplify. Modifications such as cross-linking can be cleaved by treatment with a protease such as proteinase K. Treatment with a protease increases the nucleic acid molecules available for capture and / or amplification, and ultimately, more amplification products can be obtained and / or the detection threshold for a particular target can be lowered. These beneficial effects of the protease are enhanced by co-treating the sample with 2-imidazolidone. 2-imidazolidone acts as a formaldehyde scavenger, i.e., it reacts with formaldehyde such that it cannot induce or at least substantially reduces the ability to induce cross-linking between nucleic acids and polypeptides. 2-imidazolidone was used as an exemplary and preferred formaldehyde scavenger in most of the subsequent descriptions, but other formaldehyde scavengers such as those described in the background art can be used instead, except where the context requires otherwise, particularly in embodiments carried out at temperatures of 85°C or higher. By removing reactive formaldehyde, 2-imidazolidone can inhibit polypeptides such as those released by the action of the protease from forming or reforming cross-links with the nucleic acids in the sample. 2-imidazolidone can also prevent the inactivation of the protease by removing formaldehyde.2-Imidazolidone has previously been reported to be a formaldehyde scavenger, but considering the long period during which the sample can be stored and cross-links can form before protease and 2-imidazolidone are supplied, it is surprising that materials with improved nucleic acid availability for capture, amplification, and subsequent processing can be obtained by potential inhibition of new cross-link formation during short-term incubation. Since some imidazolines are known nucleic acid denaturants, it is even more surprising that the mere presence of 2-imidazolidone does not impair the ability of nucleic acids to undergo capture, amplification, or other nucleic acid hybridization.

[0051] This method can be used in any form of nucleic acid, including DNA and RNA. In particular, the DNA can be genomic or cDNA. In particular, the RNA can be mRNA, rRNA, hnRNA, tRNA, or viral RNA. DNA can be the desired analyte, but RNA has more stringent requirements for sample processing due to its chemical instability, such as instability at high temperatures. Therefore, for procedures for isolating RNA, demonstration of a novel sample preparation method under the most stringent conditions was sought.

[0052] A model system for gynecological specimen collection using a formaldehyde-containing liquid-based cytology preservative was used to illustrate the nucleic acid isolation technique. In actual applications, this system involves first obtaining a swab of cervical cells, transferring the obtained cell sample to a SUREPATH (registered trademark of TriPath Imaging, Inc.) liquid-based cytology preservative, and subsequently processing the stored cells for subsequent molecular testing. The molecular testing in this case requires in vitro amplification and detection of human papillomavirus (HPV) RNA target nucleic acids.

[0053] HPV is associated with the development of cervical cancer, and the detection of expressed HPV RNA is particularly valuable as a diagnostic and monitoring assay. In fact, HPV molecular testing in combination with cytology is now recommended for cervical cancer screening and patient management. Thus, the liquid-based Pap test is considered an example of an assay system that benefits from improved recovery of amplifiable RNA by processing samples preserved in formalin or other liquid-based cytology preservatives containing formaldehyde. Furthermore, automated testing procedures based on in vitro amplification of nucleic acids followed by detection of HPV-specific amplification products would similarly provide benefits.

[0054] The APTIMA® HPV Genotype Assay is a commercially available multiplex nucleic acid test that detects E6 / E7 mRNA of 14 high-risk HPV genotypes (e.g., catalog number 303585, Gen-Probe Incorporated (San Diego, CA)). Among the genotypes detected are HPV16, HPV18, and HPV45. This assay is demonstrated for use with cervical specimens stored in a formaldehyde-free THINPREP® liquid-based cytology preservative, based on target capture using nucleic acid hybridization. The Genotype Assay has been demonstrated for use with specimens stored in SUREPATH® liquid-based cytology reagent after first mixing with specimen transport medium (STM) and then treating with a reagent containing a relatively high level of proteinase K enzyme (180 U / assay). The SUREPATH® liquid-based cytology preservative contains formaldehyde, ethanol, methanol, and isopropanol. Specimens stored in THINPREP can be processed rapidly for testing by the APTIMA® HPV Genotype Assay, whereas specimens stored in SUREPATH® require digestion with proteinase K at 65° C for 2 hours. This requirement has impaired the utility of the latter preservative. By the improvement method described below, specimens stored in SUREPATH® liquid-based cytology reagent can be processed with less enzyme reagent and with an incubation time of only 15 minutes.

[0055] Preferred reagent composition The disclosed method for preparing nucleic acids from a preservative for liquid-based cytology containing formaldehyde is based on the combined use of a protease enzyme and 2-imidazolidone. The disclosed method is further based on using the protease enzyme at a high temperature. In a preferred method, the protease enzyme is in a lyophilized form that is initially reconstituted using a buffered solution containing 2-imidazolidone. Preferably, the reconstitution buffer further contains EDTA. In one particularly preferred embodiment, the protease enzyme is proteinase K, which is known to maintain activity within a broad range of pH 4.0 to pH 12.0. However, the pH of the buffer used for reconstituting the protease enzyme preferably falls within the range of about pH 7.5 to about pH 8.5. This range allows for optimal enzyme activity while still protecting RNA from hydrolytic cleavage that occurs under strongly alkaline conditions. Even more preferably, the buffer used in the reconstitution buffer is a Tris buffer at about pH 8.0.

[0056] When mixed with any diluent and a preservative for liquid-based cytology containing formaldehyde, the final concentrations of the main reagent components are within a preferred range. The any diluent may be a buffered solution containing a surfactant that lyses cell membranes. Exemplary surfactants include anionic surfactants such as sodium dodecyl sulfate (SDS) and lithium lauryl sulfate (LLS). Other surfactants, such as nonionic surfactants, may also be useful. Advantageously, strong ionic surfactants can make these proteins better targets for proteolysis by proteinase K enzyme by denaturing the proteins. The final concentration (in molarity) of 2-imidazolidine in the reaction mixture is preferably It is selected to fall within the range of 1 to 5 times, or more preferably 2 to 5 times, the final maximum concentration of mu-aldehyde. The final concentration of 2-imidazolidone is determined in moles added relative to the volume of the reaction mixture after all reagents have been added. The final concentration of formaldehyde is the molar amount present in the preservative used in specimen preparation divided by the volume of the reaction mixture after all reagents have been added. In other words, it does not subtract the formaldehyde consumed by crosslinking induction before incubation by heating. For example, if the final formaldehyde concentration of a reaction mixture prepared by mixing 1 mL of a preservative for liquid-based cytology (e.g., containing a cell specimen), 2.9 mL of a diluent, and 0.3 mL of a reagent containing 2-imidazolidone, proteinase K enzyme, EDTA, and a buffer is about 28 mM, the final concentration of about 2-fold excess of 2-imidazolidone is about 50 mM to about 60 mM. The final concentration of the proteinase K enzyme can advantageously be reduced in the formulations of the present invention as compared to the amount used without 2-imidazolidone. The practice of the present invention is not dependent on understanding any particular mechanism of action, but proteinase K may act to release nucleic acids by digesting the amine bonds that bind proteins to methyl crosslinks and make them available for target capture in the hybridization-dependent target capture step of the APTIMA® assay. The final concentration of the proteinase K enzyme is preferably in the range of about 43 U / mL to about 4.3 U / mL, and a concentration of about 10 - 11 U / mL is particularly preferred. Thus, a reaction volume of 4.2 mL preferably contains about 40 - 50 U, or 43 U, of the proteinase K enzyme. The final concentration of EDTA is preferably in the range of 10 mM to 100 mM, more preferably in the range of 10 mM to 50 mM, and even more preferably in the range of 30 mM to 40 mM. The final concentration of the buffering agent varies depending on its structure, but is sufficient to provide sufficient buffering capacity to ensure that the RNA contained in the sample is not substantially degraded by alkaline hydrolysis.This requirement can be met by using a final buffer concentration in the range of at least 10 mM up to about 500 mM, more preferably up to about 250 mM, even more preferably up to about 100 mM, and still even more preferably up to about 50 mM. A particularly preferred final concentration range for the buffer in the reaction mixture prior to incubation at elevated temperature is 10 mM to 50 mM.

[0057] The sample may be mixed with the protease and 2-imidazolidone in any order. For example, the protease and 2-imidazolidone may be mixed with the sample simultaneously. Alternatively, the protease may first be mixed with the sample, followed by 2-imidazolidone, or vice versa.

[0058] Both the reconstitution buffer and the lyophilized protease may be components of the kit and may be mixed immediately prior to use. That is, the end user of the kit can reconstitute the lyophilized enzyme to prepare an enzyme reagent containing, for example, 2-imidazolidone, proteinase K enzyme, EDTA, and a pH buffer. Preferably, one solution of the reagent kit contains 2-imidazolidone, EDTA, and a pH buffer, but does not contain the proteinase K enzyme. The proteinase K enzyme is preferably packaged in a separate vial of the kit, where the enzyme is in the form of a lyophilizate.

[0059] Preferred target enrichment methods Before initiating the amplification reaction, it may be desirable to first concentrate or isolate the target nucleic acid using a target capture technique. In a preferred method, the nucleic acids in the reaction mixture that are incubated at a high temperature after adding a reagent containing 2-imidazolidone and proteinase K enzyme come into contact with a solid support on which an immobilized probe is disposed. According to one embodiment, target nucleic acids that are treated with a combination of 2-imidazolidone and protease enzyme under high temperature conditions in the presence of EDTA and a pH buffer hybridize specifically to the immobilized capture probe directly. In different embodiments, the "target capture probe" serves to cross-link the solid support-immobilized capture probe and the target nucleic acid to be amplified. The general features of this method are disclosed by Weisburg et al. in U.S. Patent No. 6,534,273, and this disclosure is incorporated herein by reference. Regardless of the method selected, it is clear that hybridization involving the target nucleic acid to be amplified is an essential feature of this procedure.

[0060] A variant target capture method that can be used in connection with the techniques disclosed herein and that relies on non-specific hybridization to the target to be amplified is detailed in U.S. Patent Application Publication No. 2008 / 0286775 A1, and this disclosure is incorporated herein by reference. According to the non-specific hybridization method, the capture probe contains at least one sequence that exhibits different base pairing characteristics with respect to the target nucleic acid compared to standard base pairing (i.e., G:C and A:T / U binding). The target nucleic acid purified by this non-specific hybridization method may be RNA or DNA that is at least partially single-stranded. Again, it is clear that this sequence-independent target capture method still relies on nucleic acid hybridization.

[0061] This data shows that target capture can occur despite the presence of 2-imidazolidone, and it has been reported that some imidazolines cause nucleic acid denaturation.

[0062] Preferred nucleic acid amplification methods Examples of amplification methods useful in connection with the present invention include, but are not limited to, transcription-mediated amplification (TMA), single primer nucleic acid amplification, nucleic acid sequence-based amplification (NASBA), polymerase chain reaction (PCR), strand displacement amplification (SDA), self-sustained sequence replication (3SR), DNA ligase chain reaction (LCR), and amplification methods using self-replicating polynucleotide molecules and replicating enzymes, such as MDV-1 RNA and Qβ enzyme. Methods for performing these various amplification techniques are described in U.S. Patent No. 5,399,491, U.S. Patent Application No. 11 / 213,519, European Patent Application Publication No. 0 525 882, U.S. Patent No. 4,965,188, No. 5,455,166, Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990), International Publication No. 89 / 09835, U.S. Patent No. 5,472,840, and Lizardi et al., Trends Biotechnol. 9:53-58 (1991). The disclosures of these documents describing methods for carrying out nucleic acid amplification reactions are incorporated herein by reference.

[0063] Reaction mechanism The practice of the method of the present invention does not depend on understanding the mechanism, but FIG. 6 shows a possible reaction mechanism underlying the method. Under acidic conditions, formaldehyde 2 in the sample can react with a nucleophilic functional group of either a nucleotide or a polypeptide. Subsequently, elimination of water gives rise to a reactive imine 4. Subsequently, a second nucleotide can react with the imine to form a dimer 5. Proteinase K, a serine protease, hydrolyzes and cleaves the nitrogen-methylene linker. This cleavage can regenerate the starting nucleotide and formaldehyde. 2-Imidazolidone, a formaldehyde scavenger, can react with two molecules of formaldehyde to produce an imidazolidone-methanol compound 7. This reaction effectively prevents formaldehyde from reacting again with either the released nucleotide or polypeptide.

[0064] Sensitivity By treating the sample with protease and 2-imidazolidone as described herein, more modifications, particularly cross-links, on the nucleic acids in the sample are reversed, more nucleic acids are released from the cross-linked polypeptides, the yield of the captured nucleic acids is higher, the yield of the amplified nucleic acids is higher, and improved assay sensitivity (i.e., the threshold of the target DNA required to be present for the target is lowered) can be achieved. Such improvement can be measured against a comparable control except for the absence of protease or 2-imidazolidone or both. Preferably, the improvement is shown as compared to both a control without protease and a control without 2-imidazolidone. Improvement means an improvement of sufficient magnitude beyond typical experimental variation (p < 0.05). For example, in some methods, the treatment can result in at least a 5%, 10%, 20%, or 30% improvement in the yield of the nucleic acids released from cross-links, or the captured nucleic acids, or the amplified nucleic acids. The presence of cross-links can be evaluated by assays that separate by molecular weight, such as gel electrophoresis or various forms of column chromatography. In some methods, as a result of the treatment, at least 50, 60, 70, 80, or 90% of the nucleic acid molecules are free of cross-links to the polypeptide for potential use in hybridization capture assays or amplification. In some methods, as a result of the treatment, a higher assay positivity (or lower detection threshold), meaning that some samples give positive results (the target nucleic acid is present) after treatment according to the method of the invention than a control treatment without either protease or 2-imidazolidone, is obtained.

[0065] Preferred Embodiment The following examples disclose experimental procedures conducted to demonstrate the benefits of treating specimens in a formaldehyde-containing liquid-based cytology preservative at high temperature using a combination of 2-imidazolidone and proteinase K enzyme. In a preferred embodiment, this combination is used in the presence of Tris buffer and EDTA. In all cases, the SurePath liquid-based cytology preservative served as a model liquid-based preservative containing formaldehyde. In the following description, the "demodification solution" refers to a pH-buffered solution (pH 8.0) containing EDTA (500 mM) and 2-imidazolidone (in the range of 740 mM to 750 mM). A preferred buffer for use in the demodification solution is Tris buffer. As used herein, "specimen transport medium" (STM) refers to a phosphate-buffered surfactant solution that protects released RNA by inhibiting RNase activity that may be active in the sample being tested, in addition to lysing cells. Preferred surfactants that can be used in STM include sodium dodecyl sulfate (SDS) and lithium lauryl sulfate (LLS), with LLS being slightly more preferred. When a sample of a formaldehyde-containing liquid-based cytology preservative is mixed with the demodification solution and proteinase K enzyme, it may be convenient to reconstitute the lyophilized enzyme with the demodification solution, and a partial amount of the reconstituted enzyme solution may be added to a reaction vessel containing the liquid-based cytology preservative.

[0066] Example 1 describes the procedure used to evaluate the analytical sensitivity of the experimental system with a test panel containing in vitro transcripts for each of 14 high-risk HPV genotypes. The success of the nucleic acid treatment method involving treatment with 2-imidazolidone and proteinase K under high temperature conditions was measured by detection of HPV RNA using a commercially available assay. As shown below, the results indicated that this treatment condition did not impair the amplification and detection of HPV RNA.

Example

[0067] (Example 1) Establishment of the analytical sensitivity of the HPV assay using synthetic transcripts In vitro synthesized transcripts were used as templates for amplification in a conventional TMA reaction performed using the APTIMA® HPV Gene Probe Assay for the amplification and detection of HPV RNA. The copy number of transcripts used for each different HPV type was established in a preliminary procedure using specimens stored in a THINPREP liquid-based cytology preservative (i.e., a model preservative containing no formaldehyde), APTIMA corresponded to the limit of detection (LOD) of the HPV Gene Probe Assay. The LOD is the copy level that results in a minimum positivity of at least 95% for all specimens tested. In this case, all in vitro transcripts were used at 20 - 600 copies per reaction, as needed.

[0068] Three different sample processing conditions were tested. First, in vitro transcripts were added to THINPREP® liquid-based cytology samples in STM (1 mL sample + 2.9 mL STM), and then processed according to the manufacturer's instructions for the APTIMA® HPV Gene Probe Assay. Second, in vitro transcripts were added to clinical HPV-negative residual SUREPATH liquid-based cytology preservative specimens in STM (1 mL sample + 2.9 mL STM). Aliquots of the mixture (3.9 mL each) were mixed with 100 μL of proteinase K reagent (1.8 U / μL proteinase K, sodium azide, and CaCl2 in Tris buffer (pH 8.0)) and then incubated at 65 °C for 2 hours. After the enzymatic digestion step, the mixture was processed according to the manufacturer's instructions for the APTIMA HPV Gene Probe Assay. Finally, as in the second case, in vitro transcripts were added to clinical HPV-negative residual SUREPATH liquid-based cytology preservative specimens in STM. In this case, 3.9 mL aliquots of each sample STM mixture were mixed with 0.3 mL of a proteinase K enzyme reagent containing 2-imidazolidone in Tris-EDTA buffer. This reagent had been prepared by reconstituting lyophilized proteinase K using a de-modification solution. The final mixture contained 36 mM EDTA, 36 mM Tris-HCl, approximately 53 mM 2-imidazolidone, and 43 U of proteinase K enzyme. The mixture was incubated at 90 °C for 15 minutes and then processed according to the manufacturer's instructions for the APTIMA® HPV Gene Probe Assay. After amplification and detection of HPV RNA, the frequency of positive HPV detection was compared between replicates.

[0069] Figure 1 shows the results of the analytical sensitivity evaluation performed using in vitro transcripts. The assay positivity of samples stored in the SUREPATH liquid-based cytology preservative was at least 95% for 11 out of 14 HPV genotypes, and three genotypes, HPV56, 58, and 59, yielded positivity rates of 93.3, 91.7, and 90%, respectively. These results were similar to those obtained for samples stored in the THINPREP liquid-based cytology preservative, and similar to or better than samples treated with proteinase K enzyme alone after storage in the SUREPATH liquid-based cytology preservative. This established the utility of the HPV test system and showed that the combination of 2-imidazolidone and proteinase K enzyme used under high temperature conditions did not substantially inhibit the in vitro amplification and detection reactions.

[0070] Example 2 describes the procedure used to evaluate the analytical sensitivity of the HPV assay by testing a human cell panel containing HPV. The procedure was generally the same as that described in Example 1, except that (1) an HPV-expressing cell line was used instead of in vitro transcripts, and (2) the samples were incubated for an extended period in the presence of a formaldehyde-containing preservative.

[0071] (Example 2) Establishment of the analytical sensitivity of the HPV assay using a human cell line containing HPV Human cell lines containing HPV were added to a sample pool of a SUREPATH liquid-based cytology preservative stored at 25°C for 7 days, and then treated with a combination of a de-modification solution and proteinase K at 90°C for 15 minutes or with proteinase K alone at 65°C for 2 hours, followed by testing at a half-log dilution (3 - 30 cells / 1 reaction). As in Example 1, the combination of the de-modification solution and proteinase K could be conveniently delivered as a single aliquot by reconstituting lyophilized proteinase K with the de-modification solution. Of course, there is no requirement to mix the reagents in this way. The cells used in this procedure were (1) SiHa cells (expressing HPV16), (2) HeLa cells (expressing HPV18), and (3) MS751 cells (expressing HPV45). Again, the APTIMA HPV gene probe assay was used according to the manufacturer's instructions to capture, amplify, and detect HPV nucleic acid. The positivity was compared for samples treated under two conditions.

[0072] Figures 2A - 2C show the results of the analytical sensitivity obtained using cell lines stored at 25°C for 7 days in a SUREPATH liquid-based cytology preservative. For all three HPV-positive cell lines in samples treated with the combination of the de-modification solution and proteinase K enzyme, the assay positivity was at least 95% at concentrations of 30, 10, and 30 cells / 1 reaction for SiHa, HeLa, and MS751 cells, respectively. These results were similar to or better than those obtained using samples stored at 25°C for 7 days in a SUREPATH liquid-based cytology preservative and then treated with proteinase K enzyme alone. The most dramatic difference was observed in tests using the lowest number of introduced HeLa cells. There was clearly a statistically significant advantage for sample treatment using 2-imidazolidone and high temperature in combination with proteinase K.

[0073] Example 3 describes a procedure showing a method for improving the recovery of amplifiable nucleic acids from samples stored for a long period in a preservative for liquid-based cytology containing formaldehyde by combining 2-imidazolidone and proteinase K under high-temperature conditions. As described below, the difference in RNA recovery for tests treated with proteinase K alone was most prominent over a long period of time.

[0074] (Example 3) Enhancement of recoverable mRNA from cell specimens stored in a preservative for liquid-based cytology containing formaldehyde To mimic clinical specimens, 10 residual specimen pools in SurePath preservative for liquid-based cytology, previously determined to be HPV-negative, were divided in half using the APTIMA HPV gene probe assay, and SiHa or HeLa cells were added. All tubes were stored as such at 25 °C for up to 42 days. Sub-aliquots of each pool were diluted at a SurePath:STM matrix of 1:2.9 on each test day to a final cell concentration of 30 and 100 cells per reaction. Samples were treated either with proteinase K alone at 65 °C for 2 hours or with a combination of a de-modification solution and proteinase K (this combination is delivered as a single aliquot of proteinase K reconstituted with the de-modification solution) at 90 °C for 15 minutes. Again, the APTIMA HPV gene probe assay was used according to the manufacturer's instructions to capture, amplify, and detect HPV nucleic acids.

[0075] Figures 3A - 3B show results supporting that sample treatment involving the combination of 2 - imidazolidone and proteinase K under high - temperature conditions is more advantageous than treatment with proteinase K alone. All the results during this test were valid. For 30 cells per reaction, both SiHa and HeLa cells treated with the combination of 2 - imidazolidone and proteinase K (e.g., proteinase K reconstituted in a de - modification solution) maintained 100% positivity until day 14. Beyond this storage period, the combination treatment enhanced the recovery of amplifiable RNA to a greater extent than treatment with proteinase K alone. For 100 cells per reaction, HeLa cells maintained 100% positivity until day 28, while SiHa cells remained 100% positive until day 21 (data not shown).

[0076] Example 4 describes the procedure used to show that clinical samples stored in a formaldehyde - containing liquid - based cytology preservative are treated with a combination of 2 - imidazolidone and proteinase K under high - temperature conditions and are then processed to yield a substantially constant amount of RNA regardless of the length of the period for which the clinical samples were stored.

[0077] (Example 4) Combination treatment enables efficient recovery of RNA from clinical samples over a long storage period Thirty validated HPV-positive clinical specimens in the SUREPATH liquid-based cytology preservative obtained from the reference population were evaluated in this study. A sub-volume (0.5 mL) of each specimen was added to 2.9 mL of STM and then diluted 1:10 and 1:100 in a 0.5:2.9 SP:STM matrix. The dilutions were stored at 4°C and subsequently tested by the APTIMA HPV gene probe assay at various time points over 120 days (N = 4 for each sample, total replicates per time point 120). On each day of testing, a 1 mL sub-volume of the sample was mixed with 2.9 mL of STM and 0.3 mL of reagent (containing proteinase K reconstituted in a de-modification solution to a final concentration of 143 U / mL). The mixture was incubated at 90°C for 15 minutes, processed to isolate nucleic acids by a target capture method, and tested by the APTIMA HPV gene probe assay on an automated analyzer.

[0078] Figure 4 shows results indicating that clinical specimens stored in a liquid-based cytology preservative containing formaldehyde can yield substantially constant RNA recovery rates when treated briefly with a combination of 2-imidazolidone and proteinase K under high temperature conditions. All specimens diluted 1:10 maintained a positivity of at least 97.5% after storage at 4°C for 120 days. The positivity of all 30 specimens diluted 1:100 ranged from 74.2% to 87.5% over the course of this study, and no consistent decrease in positivity was observed.

[0079] (Example 5) Temperature dependence Target modulation: Tubes containing HeLa cells infected with HPV18 were thawed at 37°C and stored in a single tube. Phosphate-buffered saline was added to the tube, and the tube was rotated in a centrifuge at 1100 rcf to form a cell pellet. The supernatant was removed by pipetting. A pool of HPV-negative SurePath® clinical specimens (NCPP) derived from the cell pellet was added to simulate SurePath clinical specimens. The tube containing HeLa cells and NCPP was inverted to break up the pellet and incubated at 25°C (concentration: 1000 cells / mL). After 0, 7, and 14 days, aliquots were removed from the tube, STM was added, and dilution was performed to a final concentration of 10 cells per reaction (NCPP:STM final ratio was 1:2.9). The tubes were processed (see next step) and tested using the APTIMA® HPV kit according to the manufacturer's instructions.

[0080] Treatment method: After incubation at 25°C and addition of STM (see previous step), the tubes were divided into three groups. Heating: 300 μL of TE (concentration in tube: 36 mM Tris, 36 mM EDTA) was added to the reaction tube. The tube was capped and placed in a 90°C water bath for 15 minutes and tested with APTIMA HPV. PK: 50 mg of proteinase K was diluted with 1 mL of Fast Express diluent. 100 μL of the PK solution was added to the reaction tube (180 units of proteinase K per tube). The tube was capped and placed in a 65°C water bath for 2 hours and tested with APTIMA HPV. Heating + PK: 50 mg of proteinase K was dissolved in 12 mL of TE. 300 μL of the TE + PK solution was added to the reaction tube (concentration in tube: 36 mM Tris, 36 mM EDTA, 45 units of PK). The tube was capped and placed in a 90°C water bath for 15 minutes and tested using the APTIMA® HPV kit according to the manufacturer's instructions. The results are shown in Table 1.

[0081]

Table 1

[0082] These data indicate that the combination treatment was most effective for the samples that had undergone the longest storage. Furthermore, the samples treated with proteinase K at high temperature had a higher recovery rate than those treated with proteinase K at low temperature or those treated only at high temperature.

[0083] (Example 6) Formaldehyde scavenger, proteinase K, and high temperature As substantially described in Example 5 above, the targets were prepared. Briefly, the HeLa cell line (HPV-18+), SiHa cell line (HPV-16+), MS751 cell line (HPV-45+), and Trichomonas cell line (Trichomonas +) were incubated in SurePath solution for 7 days. After 7 days, aliquots of each SurePath cell sample were subsequently mixed under the conditions shown in Table 2.

[0084] [Table 2] * HeLa and SiHa cells were incubated in the presence of 180 U proteinase K and in the presence of 45 U proteinase K. ** 1× and 2× refer to the molar concentration of 2-imidazolidone in the solution. 1× means that the molar concentration is approximately equal to that of formaldehyde in the solution, and 2× means twice the concentration of formaldehyde.

[0085] Subsequently, the combined solution was incubated either for 15 minutes or 2 hours and at either 65 °C or 90 °C. The incubation conditions are shown in Table 3.

[0086] [Table 3]

[0087] After incubation, the samples were assayed to determine the nucleic acid recovery rates by various treatments. In the first assay, 3 cells per reaction of HeLa cells under Conditions 1 - 4 and 10 cells per reaction of SiHa cells under Conditions 1 - 4 (see Table 3) were assayed using the APTIMA HPV Kit (Catalog No. 303585, Gen-Probe Incorporated) usually according to the manufacturer's instructions. In the second assay, 0.05 cells per reaction of Trichomonas cells under Conditions 1, 2 & 4 (see Table 3) were assayed using the APTIMA Vaginal Trichomonas Assay (Catalog No. 303563, Gen-Probe Incorporated) usually according to the manufacturer's instructions. In the third assay, 3 cells per reaction of each of HeLa, SiHa, and MS751 cells under Conditions 1, 2 & 5 (see Table 3) were incubated with 45 U of Proteinase K each and assayed using the APTIMA HPV Kit (Catalog No. 303585, Gen-Probe Incorporated) usually according to the manufacturer's instructions. In the fourth assay, 3 cells per reaction of each of HeLa, SiHa, and MS751 cells under Conditions 1, 2 & 5 (see Table 3) were incubated with 45 U of Proteinase K each and assayed using the APTIMA HPV Genotyping Kit (Catalog No. 303234, Gen-Probe Incorporated) usually according to the manufacturer's instructions. The results are shown in Tables 4 - 7.

[0088]

Table 4

[0089]

Table 5

[0090]

Table 6

[0091]

Table 7

[0092] These data indicate that when specimens stored in a liquid-based cytology preservative containing formaldehyde are treated for a short time with a combination of a formaldehyde scavenger, proteinase K, and high temperature, a substantially constant RNA recovery rate can be achieved. These data further indicate that formulations containing proteinase K are useful at high temperatures where proteinase K is known to be denatured and inactivated and is further known to destroy RNA, and that they provide excellent nucleic acid recovery rates as compared to the recovery rates at low temperatures. These data further indicate formulations capable of recovering nucleic acids from formalin-containing solutions using low concentrations of protease.

[0093] (Example 7) The following assay was performed to measure the RNA recovery rate from samples treated with SurePath® reagent for 7 days, where the samples were treated with proteinase K for 15 minutes at various high temperatures. Samples were prepared as substantially described in Example 6. Briefly, the HeLa cell line (HPV-18+) and the SiHa cell line (HPV-16+) were incubated in Surepath® at 25° C. for 7 days. After 7 days, aliquots of each SurePath® cell sample were subsequently mixed under Condition 3 shown in Table 2 above. Next, as shown in Table 8, the mixed solution was incubated at various temperatures for 15 minutes and assayed using an HPV detection kit (Catalog No. 303585, Gen-Probe Incorporated).

[0094] [Table 8]

[0095] The following assays were performed to measure the RNA recovery rate from samples treated with SurePath® reagent for 7 days. These samples were treated with proteinase K at 90°C for various short incubation times. Samples were prepared as substantially described in Example 6. Briefly, the HeLa cell line (HPV-18+) and the SiHa cell line (HPV-16+) were incubated in Surepath® at 25°C for 7 days. After 7 days, aliquots of each SurePath® cell sample were subsequently mixed under condition 3 shown in Table 2 above. Next, as shown in Table 9, the mixed solution was incubated at 90°C for various minutes and subsequently assayed using an HPV detection kit (Catalog No. 303585, Gen-Probe Incorporated).

[0096]

Table 9

[0097] The following assays were performed to measure the RNA recovery rate from samples treated with SurePath® reagent for 7 days. These samples were treated with proteinase K at various concentrations at 90°C for an incubation time of 15 minutes. Samples were prepared as substantially described in Example 6. Briefly, the HeLa cell line (HPV-18+) and the SiHa cell line (HPV-16+) were incubated in Surepath® at 2°C for 7 days. After 7 days, aliquots of each SurePath cell sample were subsequently mixed under conditions substantially similar to condition 3 shown in Table 2 above, except that the proteinase K concentration was as listed in Table C. Next, as shown in Table 10, the mixed solution was incubated at 90°C for 15 minutes and subsequently assayed using an HPV detection kit (Catalog No. 303585, Gen-Probe Incorporated).

[0098]

Table 10

[0099] The following assay was performed to measure the RNA recovery rate from samples treated with SurePath reagent for 7 days, where the samples were treated with 2-imidazolidone at various concentrations at 90° C. for an incubation time of 15 minutes. Samples were prepared as substantially described in Example 6. Briefly, the HeLa cell line (HPV-18+) and SiHa cell line (HPV-16+) were incubated in Surepath at 25° C. for 7 days. After 7 days, aliquots of each SurePath cell sample were subsequently mixed under conditions substantially similar to Condition 3 shown in Table 2 above, except that the 2-imidazolidone concentration was as listed in Table D. Next, as shown in Table 11, the mixed solution was incubated at 90° C. for 15 minutes and subsequently assayed using an HPV detection kit (Catalog No. 303585, Gen-Probe Incorporated).

[0100] [Table 11]

[0101] In all assays, the number of replicates was 40.

[0102] (Example 8) Workflow including the treatment of specimens stored in a liquid-based cytology preservative containing formaldehyde Example 8 describes a typical workflow in clinical sample processing. A clinical sample obtained using a swab device is introduced into a vial containing a preservative for liquid-based cytology containing formaldehyde, and the lid of the vial is tightly closed. SUREPATH preservative for liquid-based cytology may be used as the preservative for liquid-based cytology. The cell material dispersed in the liquid contents of the vial is transferred to a clinical laboratory for tests such as molecular analysis of nucleic acids. In the clinical laboratory, a partial volume of the vial is mixed with a partial volume of a diluent such as a buffered surfactant solution. A phosphate-buffered surfactant solution is an example of a preferred diluent. The surfactant used for this application is preferably an anionic surfactant such as sodium dodecyl sulfate (SDS) or lithium lauryl sulfate (LLS). This mixture is further mixed with 2-imidazolidone and a protease. The protease used for this purpose may be proteinase K enzyme. In a simplified method, lyophilized proteinase K is reconstituted in a solution containing a pH buffer, EDTA, and 2-imidazolidone. The pH buffer may be a Tris buffer, and the reconstituted enzyme solution may have a pH of about 8.0. The final mixture containing the diluted clinical sample, 2-imidazolidone, and protease enzyme is then heated to a high temperature for 5 to 30 minutes. The mixture is preferably heated to about 90°C for about 15 minutes. The nucleic acids in the sample become suitable for use as templates in purification and in vitro amplification reactions. For example, RNA is purified by being captured on a solid support, for example, using sequence-specific hybridization to immobilized nucleic acid strands, and is subsequently amplified in a nucleic acid amplification reaction. The nucleic acid amplification reaction may be a transcription-mediated amplification (TMA) reaction. The amplification product is contacted with a sequence-specific hybridization probe to determine the presence or absence of a specific target sequence. The specific target sequence may be an HPV target sequence. This workflow is shown in FIG. 5.

[0103] The present invention has been described with reference to many of its specific examples and embodiments. Of course, from an overview of the above detailed description, many different embodiments of the present invention itself are suggested to those skilled in the art. Therefore, the true scope of the present invention is defined by referring to the appended claims. Unless otherwise apparent from the context, any embodiment, aspect, step, or feature of the present invention can be used with any other.

Claims

【Claim 1】 The composition described in the specification.

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