JP7588815B2 - RNA filter and RNA filtering method - Google Patents

Description

The present invention relates to an RNA filter and related technologies that provide a permeable component that removes DNA from a sample by filtration prior to amplification by the RT-PCR method.

In the diagnosis of infectious diseases, pathogen testing for the causative microorganisms and viruses is extremely important. Pathogens are generally identified using methods such as microscopy, culture tests, immunological tests, and genetic tests. Among these, genetic tests are particularly superior in terms of their simplicity and sensitivity, and various genetic tests have become widespread in recent years.

Viruses are even smaller than bacteria, and cannot be easily observed even under a microscope, and culture tests may require host cells. For this reason, testing for viruses is considered to be highly difficult, and genetic testing is widely used.

The DNA (deoxyribonucleic acid) amplification reaction that is primarily used in genetic testing, i.e. PCR (polymerase chain reaction), can be performed relatively easily on DNA viruses. This is because DNA genes exist inside DNA viruses and can be directly subjected to PCR.

However, there are viruses (RNA viruses) that have RNA (ribonucleic acid) as their genes rather than DNA genes, and RNA viruses cannot be used for gene amplification as is.

Therefore, in the case of RNA viruses, a reverse transcription reaction is carried out prior to PCR to convert the RNA into DNA. The converted DNA is then subjected to PCR to amplify the genes and perform detection.

There are a variety of RNA viruses, including rotavirus, which is a double-stranded RNA virus; influenza virus, Sendai virus, respiratory syncytial virus, mumps virus, and Ebola virus, which are single-stranded negative-strand RNA viruses; and norovirus, Zika virus, dengue virus, poliovirus, rhinovirus, coxsackievirus, Japanese encephalitis virus, hepatitis C virus, and coronavirus, which are single-stranded positive-strand RNA viruses. These tests are extremely important in diagnosing each infectious disease.

Genetic testing is also extremely important for the new coronavirus (SARS-CoV-2), which emerged in Wuhan, China in 2019 and has been rapidly spreading worldwide in 2020, and the importance of widespread use of PCR testing has been advocated. This new coronavirus is also an RNA virus, and its detection method is introduced by the National Institute of Infectious Diseases in Non-Patent Document 1: "Pathogen Detection Manual 2019-nCoV." In particular, the one-step method, which allows reverse transcription and gene amplification reactions to be performed consecutively, i.e., the real-time one-step RT-PCR method, is considered to be very effective.

However, the implementation of this real-time one-step RT-PCR method requires a cumbersome pretreatment to purify the RNA, known as "RNA extraction using the QIAamp Viral RNA Mini Kit." Furthermore, this process requires biosafety level 2 safety measures to prevent secondary infection, so it can only be carried out in facilities equipped with sufficient infection control equipment and must be carried out by technicians with a certain level of experience. Furthermore, there have been reported cases of contamination between samples during the cumbersome operations, making the accuracy of the test a major issue.

The inventors have developed a simple PCR technique to eliminate such cumbersome operations and have proposed it to the market.

That is, as shown in Patent Document 1 (JP Patent Publication 2016-52267), one method is to use silica particles with a certain range of particle size as a carrier for capturing genes, adsorb genes in a sample in the presence of a chaotropic agent, wash with purified water, and then perform PCR directly.

Furthermore, Patent Document 2 (JP Patent Publication No. 2018-93750) discloses a technology that incorporates this principle in an all-in-one form into a nucleic acid amplification reaction device.

By using this technology, the examiner can pre-treat the sample by collecting it in an extraction solution containing a chaotropic agent, and then simply drop a specified amount of the pre-treated sample into a nucleic acid reaction device, allowing the capture of nucleic acid, washing, amplification reaction, and detection to be carried out fully automatically within the dedicated equipment. This makes it possible to eliminate the complicated nucleic acid purification process (drying and extraction operations) that was previously required, which not only shortens time and simplifies the process, but also has a significant effect on improving safety and preventing contamination.

However, this technique is difficult to implement when the target is an RNA virus. Samples typically contain human body fluids such as nasopharyngeal swabs, sputum, saliva, or blood taken from the affected area. This means that the samples contain large amounts of contaminants, particularly human genomic DNA and RNase (an RNA-degrading enzyme), which significantly reduces sensitivity.

The presence of these contaminants can cause non-specific reactions with primers and probes, such as DNA other than the RNA being detected, inhibiting reverse transcription of the target RNA and amplification of the target DNA after transcription, and/or making detection impossible.

It is considered that the presence of DNA competitively reduces the efficiency of capturing the target RNA, and that a significant decrease in sensitivity occurs due to the degradation of the target RNA itself caused by RNase present in the sample.
JP 2016-52267 A JP 2018-93750 A "Pathogen Detection Manual 2019-nCoV" National Institute of Infectious Diseases

The present invention aims to provide an RNA filter that can remove contaminants such as DNA from samples and extract only components that contain RNA but not DNA.

The RNA filter according to the first invention comprises an extract to which a sample containing a mixture of DNA and RNA is applied, a container for storing the extract, silica particles that come into contact with the extract and bind to DNA but not RNA, and a membrane filter that comes into contact with the extract and has a pore size smaller than the particle size of the silica particles.

In this configuration, once the DNA present in the sample binds to the silica particles, it is not subsequently separated from the silica particles; in other words, it is bound to the silica particles. Because the pore size of the membrane filter is smaller than the particle size of the silica particles, the DNA cannot pass through the membrane filter and is left behind on the membrane filter.

On the other hand, RNA present in the sample does not bind to the silica particles and can move freely, and is separated from DNA along with other components that pass through the membrane filter.

As a result, DNA, a contaminant that inhibits amplification, is removed from the sample, allowing subsequent amplification to be carried out smoothly and with high sensitivity.

In addition to the first aspect of the invention, the RNA filter preferably includes a drip nozzle that ejects the components of the extract that have passed through the membrane filter out of the container.

This configuration allows only the permeated components from which DNA has been removed to be easily discharged outside the container through the drip nozzle.

Preferably, in addition to the first invention, the RNA filter further comprises an extraction solution containing a chaotropic agent.

In addition to the first aspect of the invention, preferably, the RNA filter has silica particles with a particle size of 0.5 to 10 μm.

These configurations allow for smooth removal of DNA from samples.

Preferably, in addition to the first invention, the RNA filter has silica particles supported on a membrane filter.

This configuration allows the silica particles to be held in a stable position.

Preferably, in addition to the first invention, the RNA filter has a membrane filter capable of adsorbing proteins contained in the sample.

Preferably, in addition to the first invention, the RNA filter has a membrane filter that is a nitrocellulose membrane or a nylon membrane.

These configurations allow RNase contained in the sample to be adsorbed onto the membrane filter and removed.

As described above, according to the present invention, DNA contained in the sample is bound to the silica particles and left on the membrane filter. On the other hand, RNA contained in the sample is not bound to the silica particles and is separated from the DNA along with other components that pass through the membrane filter. As a result, DNA and RNA are separated, and the subsequent amplification can be carried out smoothly and with good sensitivity.

(Summary of the Invention)
Before describing specific embodiments of the present invention, the present invention will be outlined below. The present invention relates to a pretreatment prior to a gene amplification reaction.

To pre-treat samples containing RNA-containing pathogens, we provide an RNA filter that separates impurities (DNA, RNase, etc.) present in the sample and reduces the effect of reaction inhibition caused by the impurities.

Using this filter not only makes it easy to pretreat samples for real-time one-step RT-PCR, but also significantly reduces the risk of secondary infection and contamination.

Specifically, the sample is mixed with an extraction solution (containing silica particles that can selectively bind DNA in the presence of a chaotropic agent), the DNA that causes non-specific reactions is bound to the silica particles and restrained there, and then passed through a membrane filter with a pore size smaller than the particle size of the silica particles. Since the DNA is bound to the silica particles and cannot pass through the membrane filter, the DNA is separated as a result. In other words, the permeated components that pass through the membrane filter do not contain DNA, and only RNA and other components are extracted as permeated components.

By subjecting the obtained permeated component to RT-PCR, it is possible to detect RNA viruses without being affected by impurities in the sample. Furthermore, by using a material with high protein adsorption capacity, such as nitrocellulose, for the filter used for filtration removal, it is possible to adsorb RNase in the sample and prevent the progression of decomposition of the target RNA.

Detecting microorganisms contained in samples is extremely important in the diagnosis and treatment of infectious diseases, but various impurities in the specimen can cause reaction inhibition during nucleic acid detection, which can lead to incorrect measurement results. For this reason, purification of the nucleic acids in the sample is commonly performed as a pretreatment process, and is sold as a variety of reagents and kits. However, nucleic acid purification has issues such as being very complicated and time-consuming, requiring skilled technicians and specialized equipment, and being expensive. In addition, there is a risk of secondary infection in the measurement operator himself, and a risk of contamination such as false positives due to droplets being mixed between samples.

In the present invention, as a pretreatment method for RNA detection, silica particles that can selectively bind to DNA but not to RNA are added to an extraction solution containing a chaotropic agent.

A sample is added to this extraction solution, the DNA in the sample is bound to the silica particles, and the silica particles are then removed by filtration, essentially removing the DNA and obtaining a filtrate containing the target substance, RNA, which is then used in real-time one-step RT-PCR.

The "specimen" here refers to bodily fluids such as blood, or nasopharyngeal swabs, sputum, saliva, etc. from the site of a viral infection, but it can be anything that can be subsequently subjected to PCR.

(Embodiment 1)
Hereinafter, an embodiment of the present invention will be described with reference to the drawings. Here, Fig. 1 is an explanatory diagram of the operation of an RNA filter in one embodiment of the present invention, Fig. 2 is an enlarged cross-sectional view of the inside of the cap, and Fig. 3 is a perspective view showing the state in which the permeated component is applied to a nucleic acid amplification reaction device.

As shown in FIG. 1, the RNA filter of this embodiment has a container body 1, a lid 2, and a cap 6 as its main elements.

As shown in Figures 1(a) to 1(c), the extract 3 is stored in the bottom 1d of the container body 1, a flange 1a is formed on the upper part of the container body 1, and a threaded portion 1c is provided on the circumferential surface above the flange 1a.

Before use, the lid 2, which has a screw portion (not shown) inside that screws into the screw portion 1c, is attached, and the opening 1b of the container body 1 is in a closed state.

Extraction solution 3 is a solution containing silica particles 4 (see Figure 2), and the solution contains 5.2 [M] guanidine thiocyanate as a chaotropic agent.

It is sufficient for the silica particles 4 to bind to DNA but not to RNA, and for example, amorphous silica particles can be suitably used.

Here, silicon dioxide (catalog number: SI014PB, particle size 1 [μm], manufactured by Kojundo Chemical Laboratory Co., Ltd.) is used as the silica particles 4, but any material can be selected as long as it does not bind to RNA and binds to DNA, and is not limited to this example.

When testing, as shown in FIG. 1(b), the lid 2 is rotated to expose the opening 1b of the container body 1. Next, the tip of the swab 5 is brought into contact with a virally infected area, etc., and then the swab 5 is inserted into the bottom 1d of the container body 1 and gently rotated left and right. This brings the tip of the swab 5 into contact with the extraction liquid 3 in the bottom 1d, and the specimen is applied to the extraction liquid 3.

The swab 5 can be made of various materials such as polyester or sponge, and is not particularly limited. Furthermore, an instrument other than the swab 5 may be used as long as it can be brought into contact with both the specimen and the extraction liquid 3 and the specimen can be applied to the extraction liquid 3.

When application is complete, remove the swab 5 from the container body 1, and screw the cap 6 onto the threaded portion 1c with the drip nozzle 6a of the cap 6 facing upward, as shown in Figure 1(c).

Next, as shown in FIG. 1(d), turn the container body 1 upside down so that the drip nozzle 6a of the cap 6 faces downward.

As shown enlarged in FIG. 2, a seat 6b with a larger diameter than the drip nozzle 6a is formed directly above the drip nozzle 6a of the cap 6. A membrane filter 7 with approximately the same diameter as the seat 6b is attached to the seat 6b.

The pore size of the membrane filter 7 is set smaller than the particle size of the silica particles 4, and the membrane filter 7 filters out the silica particles 4 and the DNA bound to them, removing them from the permeated component 8.

As described above, the silica particles 4 may be contained in the extraction liquid 3 beforehand. The silica particles 4 may also be supported on the membrane filter 7. In either case, it is sufficient that the DNA is bound to the silica particles 4 and is bound to the silica particles 4 before filtration through the membrane filter 7 is performed.

If the particle size of the silica particles 4 is 1 μm, it is desirable to set the pore size of the membrane filter 7 to approximately 0.45 μm to 1 μm. Of course, if the particle size of the silica particles 4 selected is different, the pore size of the membrane filter 7 can be selected accordingly.

Furthermore, it is preferable to use a material with high protein adsorption capacity for the membrane filter 7 so that it can adsorb and remove various proteins present as impurities in the specimen, particularly RNase, an enzyme that degrades target RNA. Specific materials include nitrocellulose membrane and nylon membrane, with nitrocellulose membrane being particularly preferred.

In this embodiment, a nitrocellulose filter (manufactured by Whatman) with a pore size of 0.45 μm is cut to a diameter of 10 mm and is welded and attached to the seat 6b of the drip nozzle 6a.

Of course, the present invention is not limited to these specific examples, and it should be understood that the scope of protection of the present invention also includes compositions, materials, dimensions, etc. that provide similar effects.

As shown in FIG. 3, in this embodiment, a nuclear amplification reaction device 10 equipped with a reaction tube 11 is used (see Patent Document 2), and the permeated components (from which DNA has been removed) that permeate the membrane filter 7 are dripped onto the sample drip section 12 of the device 10 and are then subjected to the subsequent RT-PCR.

Example 1
(Verification of the effect of including silica particles in the extraction solution)
In the above embodiment, the concentration of the silica particles 4 in the extraction liquid 3 is changed to 0, 10, 20, and 50 mg/mL, and the effect is confirmed.

In this example, silica particles 4 (manufactured by Kojundo Chemical Laboratory Co., Ltd., catalog number: SI014PB, particle size 1 [μm]) are used.

In this example, a normal wet-strengthened filter paper (LS-100, manufactured by Azumi Filter Paper, pore size 0.5 μm) is used as the membrane filter 7.

The novel coronavirus detection reagent is produced on the premise that it will be applied to the nucleic acid amplification reaction device 10 of Patent Document 2 (JP Patent Publication No. 2018-93750) shown in Figure 3.

(Production of nucleic acid amplification reaction devices for detecting the new coronavirus)
A solution containing 0.6% agarose, 0.4 mM dNTP, 2.5 mM Mn(OAc), 0.54 μM NIID 2019-nCOV N F2 (forward primer), 0.18 μM NIID 2019-nCOV N R2 (reverse primer), and 0.1 μM Q Probe (trademark) is filled into the reaction tube 11 of the nucleic acid amplification reaction device 10.

The primer pair used was prepared by outsourcing to Nihon Gene Research Institute Co., Ltd., using the sequences described in the "Pathogen Detection Manual 2019-nCoV."

Q Probe (trademark) uses the sequence shown in Table 1, and the "C" at the 3' end is labeled with BODYPY FL fluorescent dye. This probe has the property of specifically quenching when it binds to a complementary sequence.

The membrane of the filter support portion is coated with 80 μg of crystalline silica (S5631, particle size 0.5 to 10 μm, manufactured by Sigma-Aldrich Japan Co., Ltd.) for capturing RNA.

3 U of DNA polymerase for one-step RT-PCR was added to the reagent application area and then dried. Purified water was used as the washing solution, and 1.2 mL was added to the washing pot and sealed.

The container body 1 to which the silica particles 4 of each of the above concentrations have been added is inverted about five times to disperse the silica particles 4 inside, and then the measurement sample is added. The nasopharynx of a healthy subject is swabbed and the extraction liquid 3 is applied, and synthetic RNA (Nihon Gene Research Institute, Ltd.) is added to the extraction liquid to make 400 copies/μL as a positive control to prepare the sample.

Four drops (approximately 120 μL) of the filtered permeated component 8 are dropped onto the sample dropping section 12 of the nucleic acid amplification reaction device 10 from the dropping nozzle 6a, and the device is inserted into the insertion port of the applicant's fully automatic measuring device (not shown), and the start button is pressed to start the measurement.

In the fully automated measuring device, washing is performed according to the set parameters, and then the RNA is transferred into a reaction tube, where a reverse transcription reaction is carried out at 51°C for 10 minutes using temperature control from the thermal cycle module. Then, a PCR reaction is carried out by repeating 45 cycles of 100°C for 10 seconds and 56°C for 25 seconds. These operations are carried out automatically in the fully automated measuring device, and the measurement results are displayed on the monitor screen (not shown) of the fully automated measuring device.

(Measurement results)
FIG. 4 is a graph showing the relationship between the amount of silica particles and the relative fluorescence value in one embodiment of the present invention, and FIG. 5 is a graph showing the relationship between the amount of silica particles and the melting curve in the same embodiment.

As shown by the solid line in Figure 4, when no silica particles 4 are present in the extract 3, almost no reaction occurs. In contrast, as the amount of silica particles added is increased to 10 (dotted dash line), 20 (coarse dash line), and 50 (fine dash line) [mg], the reactivity (quenching rate) improves. Note that in Figure 4, the greater the descent of the line, the higher the reactivity.

The thermal melting curve analysis shown in Figure 5 clearly shows that the amplification product with a peak at 66°C increases as the amount of silica particles 4 added increases. Note that in Figure 5, the higher the apex of the peak, the higher the reactivity.

This reactivity is equivalent to that when no swab specimen components are present, and it is believed that adding 50 mg/mL of silica particles can almost completely eliminate the effects of contaminants in the swab specimen components.

Example 2
In this example, based on the results of Example 1, a comparison is made between the case where silica particles 4 at a concentration of 50 mg/mL are added to the extraction liquid 3 and the control method (where no silica particles 4 are present).

Prepare samples of the extract 3 containing the wipe components with and without silica particles, and add positive control synthetic RNA to these samples to adjust the concentrations to 0 (no addition), 5, 40, and 400 [copies/μL]. These samples are dropped into the nucleic acid amplification reaction device 10 and reacted in the same manner as in Example 1.

(Table 2) shows the results of this example. With the control method, only 400 copies/μL could be detected, whereas with this method, 5 copies/μL was positive in one of two measurements, and the actual sensitivity is considered to have improved significantly by about 50 to 80 times.

This phenomenon is thought to be the result of silica particles 4 in the extract binding to inhibitors and being removed by filtration in a situation where reverse transcription and PCR reactions for the original target substance were significantly inhibited by DNA present in the sample.

Example 3
In this example, unlike Example 2, the membrane filters attached to the cap 6 of the container body 1 are filter paper LS-100, a hydrophilic nylon membrane 0.45 μm (manufactured by Merck Millipore), and a nitrocellulose membrane 0.45 μm (manufactured by Merck Millipore).

400 copies/μL of positive control synthetic RNA is added to an extraction solution containing silica particles 4 at a concentration of 50 mg/mL, and four drops of each are dropped onto the nucleic acid amplification reaction device 10 using a cap 6 to which three types of membrane filters 7 have been attached, which have been prepared after the extraction operation, and measurement is performed.

(result)
Fig. 6 is a graph showing the relationship between the material of the membrane filter and the relative fluorescence value in one embodiment of the present invention, and Fig. 7 is a graph showing the relationship between the material of the membrane filter and the melting curve in the same embodiment. The views are the same as Figs. 4 and 5, respectively.

As shown in Figure 6, the nylon membrane (dashed line) and nitrocellulose membrane (dashed line) have a clearly larger drop in the amplification curve compared to LS-100 filter paper (solid line), indicating a larger quenching rate, and as shown in Figure 7, the peak of the thermal melting curve analysis derived from the amplified product is higher and has a larger change. This result indicates that nylon membrane, and especially nitrocellulose membrane, are more suitable materials for the membrane filter 7 than LS-100 filter paper.

This phenomenon is believed to occur because impurities (particularly proteins) in the specimen are more effectively adsorbed and removed by the membrane filter 7 made of a nylon membrane or nitrocellulose membrane than by filter paper. Among the proteins in the specimen, the presence of RNase, an RNA-degrading enzyme, is particularly important and is thought to gradually degrade the target substance, RNA. Using a membrane filter 7 made of a preferred material is believed to have the effect of protecting the target RNA from degradation by more effectively removing this RNase.

(Detection of the new coronavirus using this method)
Based on the above results, the silica concentration in the extract is set to 50 [mg/mL], and a nitrocellulose membrane is used as the filter for the drip nozzle to prepare the container body 1 and cap 6. A nucleic acid amplification reaction device 10 is also prepared in the same manner as in Example 1. These are used to measure actual clinical samples. Nipro Sponge Swab Type R is used to collect the samples.

As a control method, the general-purpose transport medium UTM for transporting viruses and the attached swab will be used, and 120 μL of the sample suspended in UTM medium after swab collection will be used for RT-PCR measurement. RT-PCR will be performed in accordance with the Pathogen Detection Manual 2019-nCoV from the National Institute of Infectious Diseases. Tests will be performed on suspected novel coronavirus infections (116 cases).

(result)
Table 3 shows the results of the experiment.
The results of the measurement according to the National Institute of Infectious Diseases' pathogen detection manual were positive in 30 cases and negative in 86 cases, while the measurement by this method was positive in 29 cases and negative in 87 cases. The discrepant samples were low concentration samples with 8 copies/test by the National Institute of Infectious Diseases' method.

The obtained sensitivity was 96.6%, the specificity was 100%, and the overall agreement rate was 99.1%, which can be said to be extremely good results.

Therefore, it was shown that when measurements are performed using the RNA filter of the present invention, the effects of impurities in the sample can be avoided and good measurement results can be obtained.

According to the present invention, it is possible for anyone to easily detect target RNA in a short time using real-time one-step RT-PCR, without the time-consuming and complicated procedures such as centrifugation required in conventional methods. It will also be understood that no special equipment or skilled technicians are required, making this method useful for rapid testing closer to the bedside in medical settings.

FIG. 1 is an explanatory diagram of the operation of an RNA filter according to one embodiment of the present invention. FIG. 1 is an enlarged cross-sectional view of the inside of a cap according to an embodiment of the present invention. FIG. 1 is a perspective view showing a state in which a permeation component is applied to a nucleic acid amplification reaction device according to an embodiment of the present invention. Graph showing the relationship between the amount of silica particles and the relative fluorescence value in one embodiment of the present invention A graph showing the relationship between the amount of silica particles and the melting curve in one embodiment of the present invention. A graph showing the relationship between the material of the membrane filter and the relative fluorescence value in one embodiment of the present invention. A graph showing the relationship between the material of the membrane filter and the melting curve in one embodiment of the present invention.

1 Container body 1a Flange 1b Opening 1c Screw part 1d Bottom part 2 Lid 3 Extraction liquid 4 Silica particles 5 Swab 6 Cap 6a Dropping nozzle 6b Seat 7 Membrane filter 8 Permeation component 10 Nucleic acid amplification reaction device 11 Reaction tube 12 Sample dropping part

Claims (7)

A sample containing a mixture of DNA and RNA is applied , and an extraction solution containing a chaotropic agent is provided .
A container for storing the extract;
amorphous silica particles, which are fused silica or fused silica , that are in contact with the extract and bind to the DNA but not to the RNA;
An RNA filter comprising a membrane filter that is in contact with the extraction liquid and has a pore size smaller than the particle size of the silica particles. The RNA filter according to claim 1, further comprising a drip nozzle for ejecting the components of the extract that have permeated the membrane filter out of the container. 3. The RNA filter according to claim 1, wherein the silica particles have a particle size of 0.5 to 10 μm. 4. The RNA filter according to claim 1, wherein the silica particles are supported on the membrane filter. 5. The RNA filter according to claim 1, wherein the membrane filter has a capability of adsorbing proteins contained in the sample. 6. The RNA filter according to claim 1, wherein the membrane filter is a nitrocellulose membrane or a nylon membrane. 7. An RNA filtering method using the RNA filter according to claim 1 to provide a permeated component from the sample from which the DNA has been removed.

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