JP2023108764A - Filtration method for clinical sample - Google Patents

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

The present invention relates to a method for filtering clinical specimens that is simple and reduces the risk of infection.

In the field of clinical testing, it is important to process specimens simply and with reduced risk of operator infection. However, it is known that highly viscous clinical samples such as saliva and solids such as food residue may interfere with subsequent immunochromatographic tests and genetic tests.

When collecting saliva, a plastic test tube or the like is used. The saliva thus collected is centrifuged to separate food debris and exfoliated mucosa before analysis, and the supernatant is used (Patent Document 1). Among saliva collection kits, there are kits such as Salivette (manufactured by Sarstedt KK) that can separate mucus and food residue, but even in this case centrifugation is required. Also in genetic testing, when saliva contains contaminants, they need to be removed by centrifugation (Non-Patent Document 1). Furthermore, since centrifugation requires a special device and generates aerosols, there is concern about the risk of operator infection.

Filtration is a convenient method for removing food residue without using a device such as a centrifuge. Filtration by gravity using a simple filter paper or strainer, and filtration by manually applying pressure using a filter attached to a syringe exist. However, such an operation requires the sample to be transferred to another container, which raises concerns about contamination and the risk of infection to the operator.

JP 2015-125029 A

Xpert Xpress SARS-CoV-2 “Cefied” package insert

The object of the present invention is to filter clinical specimens simply and with reduced risk of infection, thereby reducing solids such as highly viscous components and food residues that can interfere with subsequent examinations. It is an object of the present invention to provide a sample processing method for

The present inventors have completed the present invention as a result of earnest investigations to solve the above problems. That is, the present invention includes the following aspects.
<1> A specimen processing method including the following steps (i) to (iii).
(i) step of contacting the specimen with a part of the filter medium in a container containing the liquid specimen (ii) from the part of the filter medium in (i) where the specimen is not in contact, using an instrument with a water-absorbing action; The method according to <1>, wherein the step <2> of collecting a part of the specimen (iii) subjecting the collected specimen to an examination, the filter medium is selected from at least one of filter paper, filter cloth, sponge, and strainer.
<3> The method according to <1> or <2>, wherein the water-absorbing device is selected from at least one of a syringe, a pipette, and a cotton swab.
<4> The method according to any one of <1> to <3>, wherein the liquid specimen is saliva.
<5> Any one of <1> to <4>, wherein the test detects a target selected from at least one of proteins, nucleic acids, viruses, microorganisms, animal cells, plant cells, or extracellular vesicles Method <6> The method <7> according to any one of <1> to <5>, wherein the test described above is selected from immunochromatographic test or genetic test <7><1> to <6> A kit for performing any one of the methods described.

The present invention will be described in detail below.

In the present invention, a liquid sample is a solution derived from a living body including a test target, and examples thereof include saliva, sputum, nasal discharge, urine, tears, sweat, stool, and blood containing highly viscous parts and solids. The solution may be one that has undergone an appropriate pretreatment, for example, a fecal emulsion in which feces are dispersed in an appropriate solution, or a nasopharyngeal swab, nasal swab, or pharynx that is obtained by scraping a living body and then dispersing it in an appropriate solution. A wiping liquid can also be exemplified. The solution may be subjected to appropriate treatments exemplified by freezing/thawing, heating, addition of chemicals, dilution, and the like.

In the present invention, the container may have any shape as long as it can hold a liquid specimen, but preferably has a structure that can be opened and closed with a lid or the like. For example, spitz tubes, centrifuge tubes, test tubes and the like can be exemplified. The term “inside the container” refers to a situation in which the operator can handle the specimen without directly touching it. But it's okay.

In the present invention, the filter medium is one that can filter highly viscous parts and solids contained in liquid specimens, and examples thereof include filter paper, filter cloth, sponge, and strainer. The shape and material can be appropriately selected according to the container and the object to be inspected, but a preferable filter medium is a cellulose analysis filter paper corresponding to type 1 defined in JIS P 3801, which is folded in pleats. . The step of contacting a part of the filter medium in the present invention can also be appropriately selected according to the container and the filter medium. It is also preferable from the point of filtration speed to let it come into contact with.

In the present invention, the part that is not in contact with the specimen refers to the part other than the part that is in contact with the highly viscous part or solid matter contained in the liquid specimen. If the filter paper is pleated, the back side can be exemplified when viewed from the tip.

In the present invention, the device having a water-absorbing action may be any device as long as it can collect the required amount of sample from the portion of the filter medium that is not in contact with the sample. Syringes and pipettes that can absorb water under negative pressure can be exemplified, and micropipettes using disposable tips can be exemplified as preferable examples, in order to collect the required amount of specimen in a short period of time. A cotton swab capable of absorbing water by capillary action can also be exemplified as a preferable example, and filter paper, sponge and the like can also be used.

In the present invention, the inspection of collected specimens can be exemplified by inspections targeting proteins, nucleic acids, viruses, microorganisms, animal cells, plant cells, and extracellular vesicles. Examples of testing methods include immunochromatographic testing in which solid substances are known to inhibit development, and genetic testing in which nucleic acids and viscous polysaccharides contained in solid substances cause inhibition.

According to the present invention, a specimen processing method characterized by filtering a clinical specimen simply and with a reduced risk of infection, thereby reducing highly viscous components and solids that may hinder subsequent examinations. can provide. Therefore, the sample processing method of the present invention enables stable testing.

FIG. 2 is a diagram showing a precipitate after centrifugation of evaluation sample 1; Left: Fig. 3 shows the sediment after centrifugation of evaluation specimen 3, right: evaluation specimen 4. Figs. FIG. 10 shows the precipitates after centrifugation of evaluation specimens 6, 7, 8, and 9 from the left.

The present invention will be described in detail below. However, the present invention can be embodied in different forms, and is not limited to the embodiments and examples shown below.

The present invention will be described in detail below with reference to examples and reference examples using saliva as a liquid sample, but the present invention is not limited to these examples.

(Example 1)
Evaluation by Saliva Saliva from healthy subjects was collected, and samples with a large amount of sediment visually observed were collected and used as a pool sample. The uniformly suspended evaluation sample 1 was dispensed into 500 μL and 1.5 mL microtubes, and centrifuged at 12,000 rpm for 1 minute. As a result, clear sedimentation occurred at the bottom of the microtube (see Figure 1). This supernatant was dispensed into another microtube and used as an evaluation specimen 2 .

Next, the evaluation sample 1 was placed in a 70 mL container (manufactured by Biomedical Science), and a cell strainer with a pore size of 40 μm (manufactured by Corning) was placed so that the bottom surface was in contact with the sample. A micropipette was placed on the upper surface of the cell strainer and a sample was collected by aspiration to obtain an evaluation sample 3.

Subsequently, the evaluation specimen 1 is put in a 50 mL tube (manufactured by Corning), and the tip of the cellulose analysis filter paper (manufactured by Advantech) corresponding to the type 1 specified in JIS P 3801 is folded and brought into contact with the specimen. I put it in. A micropipette was applied to the upper surface of the filter paper and a sample was collected by aspiration to obtain an evaluation sample 4 . Furthermore, a cotton swab (manufactured by Nippon Cotton Swab Co., Ltd.) was applied to the upper surface of the filter paper, and the cotton swab was allowed to absorb water sufficiently.

500 μL of each of the evaluation sample 3 and the evaluation sample 4 was dispensed into each 1.5 mL microtube, 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 microtube was reduced compared to that of the evaluation sample 1, and that the precipitate could be removed (see FIG. 2).

(Example 2)
Evaluation by Genetic Test The inhibitory effect of the test specimens 1 to 5 prepared in Example 1 on the genetic test was evaluated using an internal control for evaluating the inhibitory effect derived from the specimen.

The primers and intercalating fluorescent dye standard nucleic acid (INAF) probes used in this example described later were prepared by the method described in JP-A-2016-131498.

24.0 μL of the reagent solution having the following composition was dispensed into 0.5 mL PCR tubes (Individual Dome Cap PCR Tube, manufactured by SSI) and dried at 25° C. in a vacuum freeze dryer (Virtis advantage Plus, manufactured by Central Scientific Trading Co., Ltd.). Evaporation drying was carried out at 100 torr for 16 hours, 50° C. for 1.5 hours, and 25° C. until the temperature inside the chamber stabilized. The dried product was used as a reaction reagent, and was stored at 4° C. together with a desiccant after sealing tightly.

Composition of reagent solution: Final concentration of reaction solution (in 30 μL) 60 mM Tris-HCl (pH 8.35)
0.39 mM each dATP, dCTP, dGTP, dTTP
2.1 mM each ATP, CTP, UTP, GTP
3.2 mM ITPs
150 mM trehalose 11.9 U AMV reverse transcriptase 200 U T7 RNA polymerase 25 nM INAF probe (SEQ ID NO: 1 labeled with 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 having the following composition was prepared.

Composition of the extraction reagent:
20.0 mM magnesium chloride 65.0 mM potassium chloride 11.0% DMSO
0.05% (v/v) Tween 20
1.9% (w/w) sodium cholate 54 mM Tris-HCl (pH 8.65)
4 mM Tris(2-carboxyethyl) phosphine Hydrochloride
50 μL of the evaluation samples 1 to 4 were added to 990 μL of the extraction reagent and suspended. A cotton swab of the evaluation specimen 5 was immersed in 990 μL of the extraction reagent as it was. Next, after heating the extraction reagent at 52 ° C. for 60 seconds, 30 μL was added to the dried reaction reagent, and after stirring, the reaction tube was subsequently subjected to an automatic genetic test device TRCReady-80 (manufactured by Tosoh Corporation). °C, and 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 value obtained by dividing the fluorescence intensity value for a predetermined time by the background fluorescence intensity ratio) exceeded 1.2. The detection time was defined as the time required to obtain the determination of detection for the evaluation specimens 1 to 5. If no determination of detection is obtained after 15 minutes, N.E. D. and
Table 1 shows the results.

In the evaluation specimen 1, N.C. D. , and inhibition of the amplification of the internal control was observed. On the other hand, it was confirmed that the precipitate was removed in the evaluation sample 2 and there was almost no inhibition. It was found that the evaluation sample 3 had less precipitate and less inhibition. It was found that the precipitates were removed in the evaluation specimens 4 and 5, which is similar to the evaluation specimen 2, and that there was almost no inhibition.

(Example 3)
Evaluation of Filter Paper Material Using Saliva Saliva from healthy subjects was collected, and samples with a large amount of sediment visually observed were collected and used as a pool sample. Subsequently, the evaluation sample 6 was placed in a 50 mL tube (manufactured by Corning Inc.), and a piece of filter paper (manufactured by Azumi Filter Paper Co., Ltd.) folded in four was placed so that the tip thereof was in contact with the sample. A micropipette was placed on the upper surface of the filter paper and aspirated to recover the sample, which was used as an evaluation sample. The above operation was performed on three kinds of filter papers made of cellulose (No. 5108), polyester (No. 55K), and rayon (No. 750), and evaluation specimens 7, 8 and 9 were obtained, respectively.

300 μL of each of the evaluation samples 6 to 8 was dispensed into each 1.5 mL microtube, 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 microtube was reduced compared to that of the evaluation sample 6, and that the precipitate could be removed (Fig. 3). Almost no sedimentation was observed in the case of cellulose, followed by little sedimentation in the case of rayon.

(Example 4)
Evaluation by genetic test The inhibitory effect of the test specimens 6 to 9 prepared in Example 3 on the genetic test is evaluated using an internal control for evaluating the inhibitory effect derived from the specimen in the same manner as in Example 2. bottom. Table 2 shows the results.

In the evaluation sample 6, N.C. D. , and inhibition of the amplification of the internal control was observed. On the other hand, in evaluation specimens 7, 8 and 9, it was confirmed that the precipitate was removed and the inhibition was reduced.

(Reference example 1)
Confirmation of Adsorption of Novel Coronavirus by Filter Paper Using inactivated novel coronavirus (manufactured by Zeptometrix), it was confirmed whether the virus was adsorbed to the filter paper.

Dilute the inactivated novel coronavirus to 100 cp/μL 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) bottom. A portion of 50 μL of this was used as a pre-filtration sample. Furthermore, 300 μL of the adjusted 100 cp / μL novel coronavirus is put in a 50 mL tube (manufactured by Corning), and a cellulose analysis filter paper (manufactured by Advantech) corresponding to type 1 specified in JIS P 3801 is pleated. It was placed in a tube so that the tip of the filter paper was in contact with the solution. Subsequently, 50 μL of the solution was recovered from the upper surface of the filter paper using a micropipette and used as a filtered sample.

Next, RNA was purified from each sample using QIAamp Viral RNA Mini Kit (manufactured by Qiagen) as shown below. 50 μL of each sample and 90 μL of PBS buffer were added to 560 μL of Buffer AVL containing 5.6 μg of carrier RNA and incubated for 10 minutes at room temperature after pulse vortexing for 15 seconds. The tube was spun down to collect the solution adhering to the inside of the lid, 560 μL of ethanol was passed over the sample, pulse vortexed for 15 seconds, and then the tube was spun down to collect the solution adhering to the inside of the lid.

630 μL of sample was applied to the QIAamp Mini column, the lid was closed and centrifuged at 6,000×g for 1 minute to discard the filtrate. The remaining sample was applied to the column again, the lid was closed, the column was centrifuged at 6,000 xg for 1 minute, and the filtrate was discarded. 500 μL of Buffer AW1 was added to the column, centrifuged at 6,000×g for 1 minute, and the filtrate was discarded.

500 μL of Buffer AW2 was added, centrifuged at 20,000×g for 3 minutes and the filtrate discarded. Centrifuge at 20,000 xg for 3 minutes and discard the filtrate. A QIAamp Mini column was set in a new 1.5 mL microcentrifuge tube, 60 μL of Buffer AVE was added, and the lid was closed and incubated for 1 minute. RNA samples were collected by centrifugation at 20,000×g for 1 minute.

Next, 5 μL of the collected RNA sample was subjected to real-time PCR using One Step PrimeScript 3 RT-qPCR Mix (manufactured by TAKARA). Reagents with the reaction composition shown in Table 3 were prepared on a 96-well PCR plate (manufactured by applied biosystems) and sealed with an 8 cap strip (manufactured by applied biosystems). Next, using Quant Studio 5 (manufactured by applied biosystems), after heating at 25 ° C. for 2 minutes, heating at 52 ° C. for 5 minutes, heating at 95 ° C. for 10 seconds, 45 cycles (95 ° C. for 5 seconds, 60 ° C. and 30°C), and the copy number was calculated from the Ct value. Table 4 shows the results.

From the results in Table 4, there is almost no change in the amount of the new coronavirus before and after filtration, so it is considered that it does not adsorb to the filter paper and that filtering by the method described in this specification does not affect the test results. was taken.

Claims (7)

A sample processing method including the following steps (i) to (iii).
(i) step of contacting the specimen with a part of the filter medium in a container containing the liquid specimen (ii) from the part of the filter medium in (i) where the specimen is not in contact, using an instrument with a water-absorbing action; Step (iii) of collecting a portion of the sample and subjecting the collected sample to testing 2. The method of claim 1, wherein the filter medium is selected from at least one of filter paper, filter cloth, sponge, or strainer. 3. A method according to claim 1 or claim 2, wherein said absorbent device is selected from at least one of a syringe, a pipette, or a swab. 4. The method according to any one of claims 1 to 3, wherein the liquid specimen is saliva. 5. The test according to any one of claims 1 to 4, wherein the test detects a target selected from at least one of proteins, nucleic acids, viruses, microorganisms, animal cells, plant cells, or extracellular vesicles. Method. 6. The method according to any one of claims 1 to 5, wherein said test is selected from immunochromatographic test or genetic test. A kit for carrying out the method according to any one of claims 1 to 6.