Methods for filtering clinical specimens

Abstract

To solve a problem in which: it is known that a clinical sample, for example, spittle or the like having high viscosity and food residues or the like may inhibit an immunochromato inspection or genetic screening thereafter; and it is desired that an infection risk is readily reduced and a component having high viscosity or a solid matter contained in the clinical sample, which is an inhibition factor for the inspection, is reduced.SOLUTION: A sample treatment method includes (i) a step of bringing a sample into contact with a part of a filter medium in a container containing a fluid sample, (ii) a step of recovering a part of the sample from a part of the filter medium not in contact with the sample in step (i) by using a tool having a water absorption action, and (iii) a step of subjecting the recovered sample to an inspection.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a method for filtering clinical specimens simply and reducing the risk of infection.

Background Art

[0002] In the field of clinical examinations, it is important to process specimens simply and reduce the risk of infection to the operator. However, it is known that, among clinical specimens, for example, highly viscous substances such as saliva and solid substances such as food residues may inhibit immunochromatographic tests and gene tests to be performed later.

[0003] When collecting saliva, a plastic test tube or the like is used. The saliva collected in this way is separated from food residues and exfoliated mucosa by centrifugation before analysis, and the supernatant is used (Patent Document 1). Some saliva collection kits, such as Salivette (manufactured by Sarstedt Co., Ltd.), can separate mucus and food residues, but even in this case, centrifugation is necessary. Also, in gene testing, when contaminants are contained in saliva, it is necessary to remove them by centrifugation (Non-Patent Document 1). Furthermore, centrifugation requires a dedicated device and generates aerosols, so there is concern about the risk of infection to the operator.

[0004] Filtration is a simple method for removing food residues and the like without using a device such as a centrifuge. There is filtration by gravity using a simple filter paper or strainer, and filtration by manually applying pressure using a filter attached to a syringe. However, such operations require transferring the specimen to another container, and there is concern about contamination and the risk of infection to the operator.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006] [Non-Patent Document 1] Xpert Xpress SARS-CoV-2 "Cephied" Package Insert [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a specimen processing method characterized by filtering clinical specimens in a simple manner and with reduced risk of infection, thereby reducing viscous components and solid matter such as food residue that may interfere with subsequent tests. [Means for solving the problem]

[0008] The inventors of this invention have diligently studied and conducted research to solve the above problems, and as a result have completed the present invention. That is, the present invention encompasses the following aspects. <1> A sample processing method comprising the following steps (i) to (iii). (i) The process of bringing a liquid sample into contact with a portion of the filter material in a container containing the liquid sample. (ii) A step of recovering a portion of the sample from the part of the filter material in (i) that has not come into contact with the sample, using an absorbent instrument. (iii) The process of subjecting the collected samples to testing. <2> The filter media is selected from at least one of the following: filter paper, filter cloth, sponge, or strainer. <1> Methods used. <3> The absorbent device described above is selected from at least one of a syringe, a pipette, or a cotton swab. <1> or <2> Methods used. <4> The aforementioned liquid sample is saliva. <1> from <3> One of the methods described below. <5> The aforementioned test detects a target selected from at least one of the following: proteins, nucleic acids, viruses, microorganisms, animal cells, plant cells, or extracellular vesicles. <1> from <4> One of the following methods <6> The aforementioned tests are selected from either immunochromatographic testing or genetic testing. <1> from <5> One of the following methods <7> <1> from <6> A kit for carrying out one of the methods described in each of the following.

[0009] The present invention will be described in detail below.

[0010] In the present invention, a liquid sample is a biological solution containing the object to be tested, and examples include saliva, sputum, nasal secretions, urine, tears, sweat, feces, and blood, which contain highly viscous parts or solid matter. The solution may have undergone appropriate pretreatment; for example, a fecal emulsion obtained by dispersing feces in an appropriate solution, or nasopharyngeal swabs, nasal swabs, and pharyngeal swabs obtained by swabbing a biological sample and then dispersing it in an appropriate solution, are also examples. The solution may be subjected to appropriate treatments such as freezing and thawing, heating, drug addition, and dilution.

[0011] In this invention, the container can be any shape that can hold a liquid sample, but it is preferable that it has a structure that can be opened and closed with a lid or the like. Examples include test tubes, centrifuge tubes, and test tubes. The inside of the container is a situation in which the operator can handle the sample without directly touching it, and in a narrow sense it refers to the inside of the test tubes and the like that exemplified, but it is also acceptable for part of the filter material or water-absorbing instrument to be outside the container.

[0012] In the present invention, the filter material is a material capable of filtering out highly viscous portions and solids contained in a liquid sample, and examples include filter paper, filter cloth, sponge, and strainer. The shape and material can be appropriately selected depending on the container and the object to be tested, but a preferred filter material is a cellulose analytical filter paper folded into a pleat, corresponding to Type 1 as specified in JIS P 3801. The step of bringing a part of the filter material into contact with the sample can also be appropriately selected depending on the container and the filter material, but if the aforementioned analytical filter paper is folded into a pleat, it is preferable from the viewpoint of filtration speed to bring the pleated tip into contact with the sample.

[0013] In the present invention, the portion that has not been contacted with the sample refers to the other portions except for the contacted portion, because the contacted portion has adhered thereto viscous components and solids contained in the liquid sample. For example, if the filter paper for analysis described above is folded in a zigzag manner, the back side as viewed from the tip portion can be exemplified.

[0014] In the present invention, the instrument having a water absorption function may be any instrument as long as it can collect a required amount of the sample from the portion of the filter medium that has not been contacted with the sample. In order to collect the required amount of the sample in a short time, examples include a syringe and a pipette that can absorb water by negative pressure, and further, a micropipette using a disposable tip can be exemplified as a preferred example. Also, a cotton swab that can absorb water by capillary action can be exemplified as a preferred example, and filter paper, sponge, etc. can also be used.

[0015] In the present invention, the examination of the collected sample may include examinations targeting proteins, nucleic acids, viruses, microorganisms, animal cells, plant cells, and extracellular vesicles. Also, examples of the examination method include an immunochromatographic examination in which the inhibition of development by solids is known, and a gene examination in which nucleic acids and viscous polysaccharides contained in solids cause inhibition.

Effects of the Invention

[0016] According to the present invention, there is provided a sample treatment method characterized by simply reducing the risk of infection and filtering clinical samples to reduce viscous components and solids that cause inhibition to subsequent examinations. Therefore, stable examinations can be carried out by the sample treatment method of the present invention.

Brief Description of the Drawings

[0017] [Figure 1] It is a diagram showing the precipitate after centrifugation of Evaluation Sample 1. [Figure 2] Left: Evaluation Sample 3, Right: It is a diagram showing the precipitate after centrifugation of Evaluation Sample 4. [Figure 3] It is a diagram showing the precipitates after centrifugation of Evaluation Samples 6, 7, 8, and 9 from the left.

Best Mode for Carrying Out the Invention

[0018] The present invention will be described in detail below. However, the present invention can be implemented in different forms and is not limited only to the examples of the embodiments and examples shown below.

Examples

[0019] Hereinafter, the present invention will be described in detail with reference to examples and reference examples when saliva is used as the liquid specimen, but the present invention is not limited by these examples.

[0020] (Example 1) Evaluation by Saliva Saliva from healthy individuals was collected, and specimens with many precipitates were collected visually and pooled to obtain a pooled specimen, which was designated as evaluation specimen 1. 500 μL of the uniformly suspended evaluation specimen 1 was dispensed into a 1.5 mL capacity microtube and centrifuged at 12,000 rpm for 1 minute. As a result, obvious precipitation occurred at the bottom of the microtube (see Figure 1). This supernatant was dispensed into another microtube and designated as evaluation specimen 2.

[0021] Next, evaluation specimen 1 was placed in a 70 mL container (manufactured by Biomedical Sciences), and a cell strainer with a pore size of 40 μm (manufactured by Corning) was placed so that the bottom surface contacted the specimen. A micropipette was applied to the upper surface of the cell strainer, and the specimen was collected by suction to obtain evaluation specimen 3.

[0022] Subsequently, evaluation specimen 1 was placed in a 50 mL tube (manufactured by Corning), and a cellulose-based analytical filter paper (manufactured by Advantec) corresponding to one type specified in JIS P 3801, which was folded in a zigzag pattern, was placed so that the tip contacted the specimen. A micropipette was applied to the upper surface of the filter paper, and the specimen was collected by suction to obtain evaluation specimen 4. Further, a cotton swab (manufactured by Nippon Cotton Swab Co., Ltd.) was applied to the upper surface of the filter paper, and it was waited until the cotton swab was sufficiently wetted to obtain evaluation specimen 5.

[0023] 500 μL of each of evaluation sample 3 and evaluation sample 4 were dispensed into 1.5 mL microcentrifuge tubes and centrifuged at 12,000 rpm for 1 minute. As a result, it was found that the amount of precipitate at the bottom of the microcentrifuge tubes was reduced compared to evaluation sample 1, indicating that the precipitate could be removed (see Figure 2).

[0024] (Example 2) Evaluation by genetic testing The inhibitory effects of evaluation samples 1 to 5, prepared in Example 1, on genetic testing were evaluated using an internal control to assess the inhibitory effect derived from the samples.

[0025] The primers and intercalator fluorescent dye standard nucleic acid (INAF) probes used in this embodiment, described below, were prepared by the method described in Japanese Patent Application Publication No. 2016-131498.

[0026] 24.0 μL of reagent solution with the following composition was dispensed into 0.5 mL PCR tubes (Individual Dome CapPCRTube, manufactured by SSI) and evaporated in a vacuum freeze-dryer (Virtis advantage Plus, manufactured by Central Science Trading Co., Ltd.) at 25°C for 16 hours at 100 torr, 50°C for 1.5 hours, and then at 25°C until the internal temperature stabilized. The dried solution was used as the reaction reagent, sealed tightly, and stored at 4°C with a desiccant.

[0027] Reagent solution composition: Concentration is the final concentration of the reaction solution (in 30 μL). 60 mM Tris-HCl (pH 8.35) 0.39mM each dATP, dCTP, dGTP, dTTP 2.1mM each ATP, CTP, UTP, GTP 3.2mM ITP 150 mM Trehalose 11.9U AMV reverse transcriptase 200U T7 RNA polymerase 25nM INAF probe (sequence number 1 labeled with a fluorescent dye) 0.1 μM First primer (SEQ ID NO: 2) 0.1 μM Second primer (SEQ ID NO: 3) 500 Copies ICDNA (SEQ ID NO: 4) 12.2% (w / w) 2-hydroxypropyl-γ-cyclodextrin Next, an extraction reagent with the following composition was prepared.

[0028] Composition of the extraction reagent: 20.0 mM magnesium chloride 65.0 mM potassium chloride 11.0% DMSO 0.05% (v / v)Tween20 1.9% (w / w) sodium cholate 54 mM Tris-HCl (pH 8.65) 4mM Tris(2-carboxyethyl)phosphine Hydrochloride 50 μL each of evaluation samples 1 to 4 was added to 990 μL of extraction reagent and suspended. For evaluation sample 5, a cotton swab was immersed directly in 990 μL of extraction reagent. Next, 30 μL of the extraction reagent was added to the dried reaction reagent after being heated at 52°C for 60 seconds and then stirred. Subsequently, the reaction tube was reacted at 46°C using an automated gene testing device TRCReady-80 (Tosoh Corporation), while the fluorescence intensity of the reaction solution was measured over time for 15 minutes. Detection was determined when the fluorescence intensity ratio of the reaction solution (the fluorescence intensity value at a predetermined time divided by the background fluorescence intensity ratio) exceeded 1.2. For evaluation samples 1 to 5, the time required to obtain a detection determination was defined as the detection time. If no detection determination was obtained after 15 minutes, it was marked as ND (Not Detected). The results are shown in Table 1.

[0029] [Table 1]

[0030] In evaluation sample 1, the result was ND (not detected), indicating inhibition of the amplification of the internal control. On the other hand, in evaluation sample 2, the precipitate was removed, and it was confirmed that there was almost no inhibition. In evaluation sample 3, the precipitate decreased, indicating a reduction in inhibition. In evaluation samples 4 and 5, the precipitate was removed, and it was found that there was almost no inhibition, similar to evaluation sample 2.

[0031] (Example 3) Evaluation of filter paper material using saliva Saliva samples were collected from healthy individuals, and samples with a large amount of precipitate were selected by visual inspection and pooled as evaluation sample 6. Next, evaluation sample 6 was placed in a 50 mL tube (Corning), and a quarter-folded filter paper (Asaka Filter Paper Co., Ltd.) was placed inside so that its tip was in contact with the sample. The sample was collected by aspiration using a micropipette placed on the top surface of the filter paper and used as evaluation sample. The above procedure was performed on three types of filter paper: cellulose (No. 5108), polyester (No. 55K), and rayon (No. 750), and these were evaluated as evaluation samples 7, 8, and 9, respectively.

[0032] 300 μL of each evaluation sample (6 to 8) was dispensed into 1.5 mL microcentrifuge tubes and centrifuged at 12,000 rpm for 1 minute. As a result, it was found that the amount of precipitate at the bottom of the microcentrifuge tubes was reduced compared to evaluation sample 6, indicating that the precipitate could be removed (Figure 3). Almost no precipitate was observed in the cellulose tube, followed by the rayon tube, which showed the least amount of precipitate.

[0033] (Example 4) Evaluation by genetic testing The inhibitory effects of evaluation samples 6 to 9, prepared in Example 3, on genetic testing were evaluated using an internal control for evaluating the inhibitory effect derived from the samples, in the same manner as in Example 2. The results are shown in Table 2.

[0034] [Table 2]

[0035] In evaluation sample 6, the result was ND (not detected), indicating inhibition of the amplification of the internal control. On the other hand, in evaluation samples 7, 8, and 9, the precipitate was removed, and it was confirmed that the inhibition was reduced.

[0036] (Reference example 1) Confirmation of adsorption of the novel coronavirus by filter paper. We used inactivated coronavirus (manufactured by Zeptometrix) to check whether the virus would adsorb onto the filter paper.

[0037] Inactivated SARS-CoV-2 was diluted in PBS buffer (137 mmol / L NaCl, 8.1 mmol / L Na2HPO4, 2.68 mmol / L KCl, 1.47 mmol / L KH2PO4, pH 7.4) to a concentration of 100 cp / μL. 50 μL of this solution was used as the pre-filtration sample. 300 μL of the further adjusted 100 cp / μL SARS-CoV-2 solution was placed in a 50 mL tube (Corning), and a cellulose analytical filter paper (Advantec), corresponding to type 1 as specified in JIS P 3801, was folded and placed in the tube so that the tip of the filter paper was in contact with the solution. Subsequently, 50 μL of the solution was collected from the top of the filter paper using a micropipette and filtered to obtain the sample.

[0038] Next, RNA purification was performed from each sample using the QIAamp Viral RNA Mini Kit (Qiagen) as shown below. 50 μL and 90 μL of PBS buffer from each sample were added to 560 μL of Buffer AVL containing 5.6 μg of carrier RNA, and after pulse vortexing for 15 seconds, the mixture was incubated at room temperature for 10 minutes. The tubes were spun down and the solution adhering to the inside of the caps was collected. 560 μL of ethanol was added to the samples, pulse vortexing for 15 seconds, and then the tubes were spun down and the solution adhering to the inside of the caps was collected.

[0039] 630 μL of sample was applied to a QIAamp Mini column, the lid was closed, and the column was centrifuged at 6,000 xg for 1 minute, after which the filtrate was discarded. The remaining sample was then applied to the column again, the lid was closed, and the column was centrifuged at 6,000 xg for 1 minute, after which the filtrate was discarded. 500 μL of Buffer AW1 was added to the column, and the column was centrifuged at 6,000 xg for 1 minute, after which the filtrate was discarded.

[0040] 500 μL of Buffer AW2 was added, and the filtrate was discarded after centrifugation at 20,000xg for 3 minutes. A QIAamp Mini column was set in a new 1.5 mL microcentrifuge tube, 60 μL of Buffer AVE was added, the lid was closed, and the tube was incubated for 1 minute. The RNA sample was collected by centrifugation at 20,000xg for 1 minute.

[0041] Next, 5 μL of the recovered RNA sample was subjected to real-time PCR using One Step PrimeScript 3 RT-qPCR Mix (TAKARA). The reagents were prepared on a 96-well PCR plate (applied biosystems) according to the reaction composition shown in Table 3 and sealed with an 8-cap strip (applied biosystems). Then, using Quant Studio 5 (applied biosystems), the amplification reaction was performed for 45 cycles (5 seconds at 95°C, 30°C) after heating at 25°C for 2 minutes, heating at 52°C for 5 minutes, heating at 95°C for 10 seconds, and the copy number was calculated from the Ct value. The results are shown in Table 4.

[0042] [Table 3]

[0043] [Table 4]

[0044] From the results in Table 4, it was concluded that the amount of coronavirus did not change much before and after filtration, and therefore it did not adsorb onto the filter paper, and filtration using the method described herein would not affect the test results.

Claims

1. A method for processing samples for genetic testing, comprising the following steps (i) to (iii). (i) The process of bringing the sample into contact with a part of the strainer, which is a filter material, in a container containing saliva. (ii) A step of recovering a portion of the sample from the part of the strainer, which is the filter material of (i), that has not come into contact with the sample, using an absorbent instrument. (iii) The process of subjecting the nucleic acid, which is the test subject, contained in the collected sample to genetic testing.

2. The method according to claim 1, wherein the absorbent instrument is selected from at least one of a pipette or a cotton swab.

3. (i) A strainer, which is a filter material for filtering the sample contained in the container for saliva, (ii) A device selected from at least one of a pipette or cotton swab, which has a water-absorbing function, for collecting a portion of the sample from the part of the strainer, which is the filter material of (i), that is not in contact with the sample. A kit for carrying out the method according to any one of claims 1 to 2.

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