JP7585843B2 - Oligonucleotide probes, test solutions and detection methods - Google Patents

Description

The present invention relates to an oligonucleotide probe, a test solution, and a detection method.

The PCR method is mainly used to test for infection with the novel coronavirus (SARS-CoV-2). The National Institute of Infectious Diseases has published the PCR-based Pathogen Detection Manual 2019-nCoV (see Non-Patent Document 1). The manual describes how to use samples such as nasal swabs, pharyngeal swabs, nasal discharge, nasal wash, sputum, and saliva, and how to carry out pretreatment such as RNA extraction and purification (isolation) before carrying out the PCR method.

As one of the PCR methods, a real-time PCR method (hydrolysis probe method) using a TaqMan probe (registered trademark) as a hydrolysis probe is described. In the hydrolysis probe method, the amount of nucleic acid amplified by the PCR method is detected from the fluorescence intensity emitted from the probe that is hydrolyzed during amplification. Since the amount of amplified nucleic acid is detected in real time, the test result of the presence or absence of nucleic acid can be determined in a short time.

Among specimens, sputum and saliva are extremely viscous compared to nasal swabs, throat swabs, nasal mucus, and nasal washings, making it difficult to collect them directly with a pipette and perform testing. Even if the specimen is diluted with a buffer solution, it is difficult to collect a uniform specimen containing the virus because the specimen does not disperse uniformly in the diluent. For this reason, the manual recommends adding dithiothreitol (DTT) to the buffer solution as a reagent to break down proteins, reduce viscosity, and homogenize the specimen.

National Institute of Infectious Diseases, Pathogen Detection Manual 2019-nCoV Ver. 2.9.1, [online], March 19, 2020, [searched October 9, 2020], Internet https://www.niid.go.jp/niid/images/lab-manual/2019-nCoV20200319.pdf

However, because the RNA purification process, which is one of the pretreatment steps for PCR, is time-consuming, PCR is sometimes performed without purifying the RNA.

However, if DTT is added to the sample as recommended in the manual, and PCR is performed without purifying the RNA, the PCR will be performed with DTT contained in the sample. This results in strong fluorescence being observed from the start of PCR, before nucleic acid amplification, which causes a problem in that the fluorescence amplification due to nucleic acid amplification cannot be accurately detected. As a result, it becomes impossible to detect the presence or absence of the target nucleic acid, and ultimately the target virus.

The present invention aims to provide an oligonucleotide probe, a test solution, and a detection method capable of detecting nucleic acids in a sample containing a thiol reducing agent.

The first aspect of the present invention is an oligonucleotide probe for testing a sample containing a thiol reducing agent, which has a reporter portion and a quencher portion. The reporter portion has an excitation spectrum with a peak wavelength of 540 nm or more. The quencher portion is a non-azo dye capable of absorbing fluorescence from the reporter portion.

A second aspect of the present invention is a method for detecting nucleic acids comprising the steps of preparing a specimen treatment solution containing a specimen and a thiol reducing agent, and performing a hydrolysis probe method on the specimen treatment solution using an oligonucleotide probe. The peak wavelength of the excitation spectrum of the reporter part is 540 nm or longer. The quencher part is a non-azo dye capable of absorbing fluorescence from the reporter part.

According to the first and second aspects of the present invention, even when a PCR method is performed on a sample to which a thiol reducing agent has been added, it is possible to detect nucleic acids in the sample.

FIG. 1 shows a fluorescence amplification curve graph (vertical axis is relative fluorescence unit, horizontal axis is PCR cycle number) when a hydrolysis probe method was performed on a sample to which DTT was not added, using the probe of Comparative Example 1. FIG. 2 shows a graph of a fluorescence amplification curve when the hydrolysis probe method was carried out using the probe of Example 1 for a sample to which DTT was not added. FIG. 3 shows a fluorescence amplification curve graph when the hydrolysis probe method was carried out using the probe of Comparative Example 1 for a sample treatment solution containing added DTT. FIG. 4 shows a fluorescence amplification curve graph when the hydrolysis probe method was carried out using the probe of Example 1 for a sample treatment solution containing added DTT.

1. Oligonucleotide Probe The oligonucleotide probe according to the first aspect of the present invention (hereinafter also referred to as "the probe of the present invention") has an oligonucleotide portion, a reporter portion, and a quencher portion.

The oligonucleotide portion is a portion for hybridizing to the nucleic acid to be detected, and is a polynucleotide in which multiple nucleotides are polymerized in a linear chain. The number of nucleotides is, for example, 5 or more, preferably 10 or more, and, for example, 30 or less, preferably 20 or less.

The base sequence of the oligonucleotide portion is determined appropriately depending on the nucleic acid region to be hybridized, and a known base sequence can be used.

For example, if the probe is for detecting the new coronavirus (SARS-CoV-2), the base sequences published by the CDC (Centers for Disease Control and Prevention) and the National Institute of Infectious Diseases can be used. Specific examples include the base sequences shown in the table below.

The reporter portion is a functional group that emits fluorescence when released from the quencher portion by hydrolysis of the probe, thereby detecting the amplification of the target nucleic acid. The reporter portion is bound to the 5' end (or 3' end) of the oligonucleotide portion.

The reporter part is a fluorescent dye having an excitation spectrum with a peak wavelength of 540 nm or more. Such fluorescent dyes include known dyes, and for example, fluorescent dyes commercially available from Funakoshi Co., Ltd., Molecular Probes Co., Ltd., etc. can be used. Specifically, Alexa Fluor 555(555nm,565nm), HiLyte Plus 555(552nm,567nm), DyLight 549(550nm,568nm), HiLyte Fluor 555(553nm,568nm), Cy3(550nm,570nm), DyLight 547(557nm) m,570nm), Rhodamine(550nm,570nm), TRITC(550nm,570nm), DY-548(558nm,572nm), DY-554(551nm,572nm), DY-555(547nm,572nm), Alexa Fluor 546(556nm,573nm), DY-556(548nm,573nm), NorthernLights 557(557nm,574nm), B- PE(545 nm,578nm), R-PE(566nm,578nm), DY-560(559nm,578nm), TAMRA(555nm,580nm), MFP555(560nm,585nm), Spectrum Orange(559nm,588nm), ATTO 565(563nm,592nm) , Cy3.5(581nm,596nm), ROX(X-Rhodamine,Rhodamine Red X)(587nm,599nm), DY-590(580nm,599nm), 5-ROX(573nm,602nm), Spectrum Red(587nm,612nm), Texas Red(596nm,615nm), DyLight 594(593nm,618nm), Alexa Fluor 594(590nm) ,619nm), HiLyte Fluor TR(591nm,622nm), ATTO 590(594nm,624nm), MFP590(597nm,624nm), DY-610(610nm,630nm), ATTO 610(615nm,634nm), DY-615(621nm,641 nm), C-PC(C-Phycocyanin)(616nm,647nm), ATTO 620(619nm,643nm), Phycocyanin(620nm,650nm), ATTO 633(629nm,657nm), DY-630(636nm,657nm), DY-632(637nm,657nm), MFP631 (633nm,658nm), DyLight 633(638nm,658nm), NorthernLights 637(637nm,658nm), DY-631(637nm,658nm), DY-634(635nm,658nm), APC(Allophycocyanin)(650nm,660nm), APC -XL(650nm,662nm), Alexa Fluor 647(650nm,665nm), Cy5(643nm,667nm), Oyster 645(650nm,669nm), DY-635(647nm,671nm), DY-636(645nm,671nm), DY-647(653nm,672nm), DyLight 647(652nm,673nm), HiLyte Flu or 647(653nm,673nm), DyLight 649(646nm,674nm), HiLyte Plus 647(649nm,674nm), Oyster 650(655nm,674nm), DY-648(653nm,674nm), DY-650(653nm,674nm), DY-652(654nm,675nm), DY-649(655nm,676nm), DY-651(656nm,678nm), Oyster 656(662nm,679nm), ATTO 655(663nm,684nm), Cy5.5(675nm,694nm), DY-677(673nm,694nm), DY-678 (674nm,698nm), Fluor 680(678nm,699nm), DY-675(674nm,699nm), DY-676(674nm,699nm), IRDye700DX(689nm,700nm), DY-681(691nm,708nm), DY-680(690nm,709nm), DY- 682(690nm,709nm), DyLight 680(682nm,715nm), Alexa Fluor 700 (702 nm, 723 nm), DY-700 (707 nm, 730 nm), DY-701 (706 nm, 731 nm), PREX710 (710 nm, 740 nm), DY-730 (732 nm, 758 nm), DY-732 (736 nm, 759 nm), 34(736nm,759nm), DY-731(736nm,760nm), DY-752(748nm,772nm), DY-750(747nm,776nm), DyLight 750(752nm,778nm), HiLyte Fluor 750(754nm,778nm), DY- 749(752nm,778nm), HiLyte Plus 750(751nm,779nm), Examples include DY-751 (751 nm, 779 nm), DyLight 800 (770 nm, 794 nm), IRDye800CW (774 nm, 800 nm), DY-780 (782 nm, 800 nm), DY-781 (783 nm, 800 nm), DY-782 (784 nm, 800 nm), DY-776 (771 nm, 801 nm), DY-777 (771 nm, 801 nm), IRDye800 (778 nm, 806 nm), and derivatives thereof. The dye names above include registered trademarks. In addition, within the parentheses of the fluorescent dyes above, the first number indicates the peak wavelength of the excitation spectrum, and the second number indicates the peak wavelength of the fluorescence spectrum. The peak wavelengths of each fluorescent dye in the present invention are the numerical values (catalog values) indicated by the supplier.

Preferably, from the viewpoint of versatility and excellent fluorescence intensity, ROX (compound (1) below), Cy5 (compound (2) below), and derivatives thereof are used, and more preferably, ROX and its derivatives are used.

The peak wavelength of the excitation spectrum (absorption spectrum) of the reporter part is 540 nm or more, preferably 560 nm or more, more preferably 580 nm or more, and is, for example, 800 nm or less, preferably 700 nm or less, more preferably 625 nm or less. When the peak wavelength of the excitation spectrum is equal to or greater than the above lower limit, the fluorescence intensity can be detected using excitation light in the medium to high wavelength range.

The peak wavelength of the fluorescence spectrum (emission spectrum) of the reporter part is located on the longer wavelength side than the peak wavelength of the excitation spectrum, and is, for example, 560 nm or more, preferably 580 nm or more, more preferably 590 nm or more, and is, for example, 820 nm or less, preferably 720 nm or less, more preferably 630 nm or less. By having the peak wavelength of the fluorescence spectrum in the above range, overlap between the fluorescence spectrum of the reporter part and the fluorescence spectrum of the quencher part can be avoided, and the two spectra can be reliably distinguished, so that the fluorescence intensity of only the reporter part can be detected with high accuracy.

The quencher portion is a functional group that quenches the fluorescence from the reporter portion until the reporter portion is released by hydrolysis of the probe. The quencher portion is attached to the 3' end (or 5' end) of the oligonucleotide portion.

The quencher part absorbs the fluorescence from the reporter part. Specifically, the excitation spectrum of the quencher part is located on the longer wavelength side than the fluorescence spectrum of the reporter part and overlaps with the fluorescence spectrum of the reporter part. Preferably, the wavelength region at full width at half maximum of the excitation spectrum of the quencher part overlaps at least partially with the wavelength region at full width at half maximum of the fluorescence spectrum of the reporter part. This allows the quencher part to more reliably cause the phenomenon of quenching the fluorescence from the reporter part (FRET phenomenon: fluorescence resonance energy transfer phenomenon) when both the reporter part and the quencher part are bound to the oligonucleotide part.

The quencher part is a non-azo dye. In other words, the quencher part does not have an azo group (-N=N-). This makes it possible to avoid reduction of the azo group in the quencher part by a thiol reducing agent and suppress decomposition of the quencher part. This makes it possible to suppress the emission of light from the reporter part before the start of PCR and its detection.

The quencher portion is preferably a fluorescent dye having an excitation spectrum with a peak wavelength of 560 nm or greater.

The quencher portion may be a known dye, for example, a fluorescent dye commercially available from Funakoshi Co., Ltd., Molecular Probes Co., Ltd., or the like. Specifically, among the fluorescent dyes listed in the reporter portion above, a non-azo fluorescent dye with an excitation spectrum of 560 nm or more may be used. Preferred examples include Cy5.5 (compound 3 below) and its derivatives.

The peak wavelength of the excitation spectrum of the quencher part is, for example, 560 nm or more, preferably 600 nm or more, more preferably 640 nm or more, and for example, 850 nm or less, preferably 800 nm or less, more preferably 750 nm or less. When the peak wavelength of the excitation spectrum is in the above range, the peak of the excitation spectrum of the quencher part and the peak of the fluorescence spectrum of the reporter part tend to overlap, so that the FRET phenomenon can be more reliably generated.

The difference between the peak wavelength of the excitation spectrum of the quencher part and the peak wavelength of the excitation spectrum of the reporter part is, for example, 20 nm or more, preferably 30 nm or more, and is, for example, 100 nm or less, preferably 90 nm or less. By having the peak wavelength of the fluorescence spectrum in the above range, the FRET phenomenon can be more reliably generated.

The peak wavelength of the fluorescence spectrum of the quencher part is, for example, 600 nm or more, preferably 630 nm or more, more preferably 650 nm or more, and is, for example, 900 nm or less, preferably 820 nm or less, more preferably 740 nm or less. By having the peak wavelength of the fluorescence spectrum in the above range, overlap between the fluorescence spectrum of the reporter part and the fluorescence spectrum of the quencher part can be avoided and the two spectra can be reliably distinguished, so that the fluorescence intensity of only the reporter part can be detected with high accuracy.

The difference between the peak wavelength of the fluorescence spectrum of the quencher part and the peak wavelength of the fluorescence spectrum of the reporter part is, for example, 20 nm or more, preferably 50 nm or more, and for example, 150 nm or less, preferably 100 nm or less. When the peak wavelength of the fluorescence spectrum is in the above range, overlap between the fluorescence spectrum of the reporter part and the emission spectrum of the quencher part is suppressed, and they can be distinguished and discriminated, so that the amplification of the fluorescence intensity by the reporter part can be detected more accurately.

Preferred combinations of reporter and quencher moieties include, for example, a combination of ROX and Cy5.5, and a combination of Cy5 and Cy5.5.

The probe of the present invention is prepared by chemically modifying the 5' or 3' end of the oligonucleotide part with the fluorescent dye constituting the reporter part by esterification or the like, and by chemically modifying the 3' or 5' end of the oligonucleotide part with the non-azo dye constituting the quencher part by esterification or the like, using a known method.

In addition to the above-mentioned sites, the probe of the present invention may also have an MGB (Minor Groove Binder) or the like, and may also have multiple reporter sites.

The probe of the present invention is used in a test for detecting a target nucleic acid from a specimen containing a thiol reducing agent, i.e., a treatment solution in which a thiol reducing agent has been added to a specimen. Specifically, the probe of the present invention is contained in a test solution described below, and is used in a detection method described below.

2. Test solution The test solution is a test reaction solution for PCR, and contains, for example, the probe of the present invention, a PCR primer, a DNA polymerase, and physiological saline. When performing reverse transcription PCR, the reaction solution contains the probe of the present invention, a reverse transcriptase, a reverse transcription reaction primer, a PCR primer, a DNA polymerase, a dNTP mix, and physiological saline.

Reverse transcriptase is an enzyme that generates single-stranded complementary DNA (cDNA) using viral RNA as a template. Specific examples include RNA-dependent DNA polymerases derived from RNA viruses such as avian myeloblastosis virus, Moloney murine leukemia virus, and human immunodeficiency virus, as well as mutants of these viruses.

Examples of reverse transcription primers include primers specific to the sequence of the target RNA, oligo(dT) primers, random primers, etc.

The PCR primers include a pair of primers (forward and reverse primers) specific to the sequence of the cDNA generated by the reverse transcription reaction. The PCR primers may be the same as the reverse transcription primers.

The DNA polymerase is, for example, a thermostable DNA polymerase derived from a thermophilic bacterium, and specific examples thereof include Taq DNA polymerase, Tth DNA polymerase, KOD DNA polymerase, Pfu DNA polymerase, and mutants thereof.

The dNTP mix is a mixture of dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dCTP (deoxycytidine triphosphate) and dTTP (thymidine triphosphate).

Physiological saline solutions include buffer solutions such as phosphate buffer, Tris buffer, borate buffer, and Good's buffer (such as HEPES).

The probe, reverse transcriptase, reverse transcription primer, PCR primer, DNA polymerase, dNTP mix, and physiological saline of the present invention can be mixed in an appropriate ratio according to conventional methods.

The test solution preferably further contains an inhibition suppressor. This allows the nucleic acid of the sample to be amplified without purifying the nucleic acid (RNA or DNA). In other words, the PCR method can be reliably performed on samples containing impurities.

Inhibition suppressors are substances that suppress the role of substances that inhibit the amplification of nucleic acids. These are disclosed, for example, in Japanese Patent No. 4735645 and Japanese Patent Laid-Open No. 2000-93176.

In addition to the inhibitor, the test solution may contain PCR buffers such as Tris-HCl, potassium salt ions, and magnesium ions.

In addition to the probe of the present invention, the test solution may contain other oligonucleotide probes that hybridize to other nucleic acid regions different from the nucleic acid region to which the probe of the present invention hybridizes, oligonucleotide probes for internal standards, etc. In this case, the test solution may further contain PCR primers (for example, forward primers and reverse primers) corresponding to each of the other oligonucleotide probes and the oligonucleotide probe for internal standards.

The test solution may contain the above components together in one solution, or may be appropriately separated and stored in separate solutions (e.g., three solutions), which are then mixed at the time of testing to form one solution, which is then mixed with the specimen.

3. Detection Method The detection method of the second aspect is a method for detecting (testing) the presence or absence of a target nucleic acid in a sample, and includes a preparation step and a PCR step.

(1) In the preparation step, a specimen treatment solution containing a specimen and a thiol reducing agent is prepared.
Specifically, a thiol reducing agent is added to the sample. This dilutes the sample and decomposes the proteins in the sample, reducing the viscosity, making it easier to collect with a pipette and easier to process. In addition, it reduces the uneven distribution of nucleic acids in the sample and allows them to exist uniformly, allowing the target nucleic acid to be collected reliably.

The specimen may be any of nasal swabs, pharyngeal swabs, nasal discharge, nasal washings, sputum, saliva, etc. In particular, this detection method is suitable for using highly viscous specimens such as sputum and saliva.

The thiol reducing agent may be any agent having a thiol group, and examples thereof include dithiothreitol (DTT), dithioerythritol, β-mercaptoethanol, 3-mercapto-1,2-propanediol, 1,2-ethanethiol, thioglycolic acid, ammonium thioglycolate, cysteine, and glutathione, with dithiothreitol being preferred. By contacting the thiol reducing agent with the specimen, the S-S bonds of the proteins contained in the specimen are cleaved, reducing the viscosity of the specimen and allowing the nucleic acids inside the virus to be uniformly present in the specimen treatment solution.

The thiol reducing agent may be added directly to the sample, or may be mixed with a buffer solution to form a diluent and then added to the sample. Specifically, this can be done in accordance with the PCR-based pathogen detection manual 2019-nCoV provided by the National Institute of Infectious Diseases.

The specimen treatment solution to which a diluted thiol reducing agent has been added may be centrifuged to collect the supernatant, and the PCR process may be carried out using this supernatant as the specimen treatment solution. Stirring, ice cooling, etc. may also be carried out as necessary.

In the testing method of the present invention, an extraction step is preferably carried out between the preparation step and the PCR step.

The extraction process is a pretreatment process in which nucleic acids are extracted from nucleic acid encapsulation bodies. Generally, nucleic acids contained in a specimen are encapsulated within a membrane (envelope, cell membrane, etc.) or cell wall as nucleic acid encapsulation bodies (viruses, fungi, bacteria, cells, etc.), so it is necessary to extract the nucleic acids from the membrane and bring them into contact with the PCR test solution.

In the extraction process, for example, a commercially available or publicly known RNA extraction reagent may be added to the sample treatment solution. Examples of RNA extraction reagents include solutions containing phenol and guanidine thiocyanate, specifically, ISOGEN, TRI reagent, TRIZOL, etc. In addition, examples of alkaline treatment agents include those containing a reducing agent and a chelating agent that chelates divalent metal ions, specifically, those disclosed in Japanese Patent No. 4735645.

In the extraction step, heating is preferably performed. Heating conditions are, for example, 40°C or higher, preferably 60°C or higher, and, for example, less than 100°C, preferably 96°C or lower. Heating time is, for example, 30 seconds or more, preferably 1 minute or more, and, for example, 10 minutes or less, preferably 5 minutes or less. This activates the extraction reagent, and allows nucleic acid to be extracted more reliably.

In addition, in this detection method, preferably, the PCR step is carried out after the extraction step without purifying the specimen treatment solution (i.e., isolating only the nucleic acid from the specimen treatment solution). Therefore, the specimen treatment solution to be subjected to the PCR step described below contains, in addition to the target nucleic acid, a thiol reducing agent, specimen-derived impurities, etc.

(2) PCR step In the PCR step, the hydrolysis probe method is carried out on the specimen treatment solution obtained in the preparation step. The hydrolysis probe method is a method for carrying out real-time PCR using a specific probe. In this step, the probe of the present invention is used as the probe.

The real-time PCR method may be either DNA-PCR or reverse transcription PCR (RT-PCR), and is appropriately determined depending on the type of test subject. For example, when testing for infection with a virus such as the new coronavirus, reverse transcription PCR is performed because the virus has RNA. In reverse transcription PCR, PCR is performed by synthesizing cDNA from RNA using reverse transcriptase and amplifying this cDNA.

In real-time PCR, several dozen cycles (e.g., 20 to 60 cycles) are carried out, each cycle consisting of (i) a thermal denaturation step in which double-stranded DNA is converted into single strands, (ii) an annealing step in which a PCR primer and the probe of the present invention are hybridized to the single-stranded DNA, and (iii) an extension step in which DNA is extended from the PCR primer by DNA polymerase to convert it into double strands. During the extension step, the probe of the present invention is hydrolyzed, and the reporter part and quencher part are released, so the FRET phenomenon disappears and the fluorescence from the reporter part can be detected. Each time the number of PCR cycles increases, the total number of released reporter parts, and therefore the fluorescence intensity from the reporter part, amplifies, and this amplification is measured by a fluorescence analyzer.

The reaction temperature, time, and other conditions for each step of the PCR method (reverse transcription, denaturation step, annealing step, and extension step) can be set according to standard procedures. For example, a commercially available real-time PCR analyzer can be used, following the settings built into the device.

The PCR cycle is repeated several dozen times, and if an amplification of the fluorescence intensity (amplification curve) in the fluorescent region emitted by the target reporter is detected, the virus or other target being tested is present in the sample, and the result is determined to be positive. On the other hand, if no amplification of the fluorescence intensity is detected, the virus or other target being tested is not present in the sample, and the result is determined to be negative.

The oligonucleotide probe of the first aspect of the present invention has a reporter portion and a quencher portion, the peak wavelength of the excitation spectrum of the reporter portion is 540 nm or longer, and the quencher portion is a non-azo dye capable of absorbing fluorescence from the reporter portion.

Conventional oligonucleotide probes have the dyes shown below (BHQ1-3; compounds having the structure shown below) in the quencher portion. However, if a thiol reducing agent (e.g., DTT) is added to a sample to prepare a sample treatment solution that reduces the viscosity of the sample, and then a reaction solution containing a conventional oligonucleotide probe is added to the sample treatment solution without purifying it, the azo group of BHQ is reduced and BHQ is decomposed before the probe is hydrolyzed (i.e., before DNA amplification). As a result, the FRET phenomenon caused by the quencher portion does not occur, and strong fluorescence from the reporter portion is detected even before DNA amplification, making it difficult to measure the increase in fluorescence intensity caused by DNA amplification that occurs with each PCR cycle.

In contrast, the detection method of the second embodiment using the oligonucleotide probe of the first embodiment has a quencher portion that is a non-azo dye capable of absorbing fluorescence from the reporter portion, and therefore can avoid reduction and decomposition by a thiol reducing agent. Therefore, the FRET phenomenon can be reliably caused before DNA amplification, and as a result, the increase in fluorescence intensity due to DNA amplification can be accurately measured.

The detection targets of the detection method of the present invention are not limited as long as they have DNA or RNA, and examples include viruses such as the novel coronavirus (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), influenza virus, human immunodeficiency virus (HIV), norovirus, rotavirus, and hepatitis C virus, as well as fungi, bacteria, and cells.

4. Aspects It will be appreciated by those skilled in the art that the exemplary embodiments described above are illustrative of the following aspects.

(1) The oligonucleotide probe according to one embodiment is an oligonucleotide probe for testing a sample containing a thiol reducing agent, and has a reporter part and a quencher part, the peak wavelength of the excitation spectrum of the reporter part is 540 nm or more, and the quencher part may be a non-azo dye capable of absorbing fluorescence from the reporter part.

(2) In the oligonucleotide probe described in 1, the quencher portion may be a fluorescent dye having an excitation spectrum with a peak wavelength of 560 nm or more.

(3) In the oligonucleotide probe described in 1 or 2, the peak wavelength of the fluorescence spectrum of the reporter portion may be 560 nm or more and 700 nm or less, and the peak wavelength of the excitation spectrum of the quencher portion may be 600 nm or more and 800 nm or less.

(4) The test solution according to one embodiment may contain the oligonucleotide probe described in any one of 1 to 3.

(5) In one embodiment of the detection method, the method includes the steps of preparing a specimen treatment solution containing a specimen and a thiol reducing agent, and performing a hydrolysis probe method on the specimen treatment solution using an oligonucleotide probe, the oligonucleotide probe having a reporter portion and a quencher portion, the peak wavelength of the excitation spectrum of the reporter portion being 540 nm or longer, and the quencher portion may be a non-azo dye capable of absorbing fluorescence from the reporter portion.

The present invention will now be described in detail with reference to examples and comparative examples, but the scope of the present invention is not limited thereto.

<Detection Reagent Kit Including the Probe of Comparative Example 1>
A 2019 novel coronavirus detection reagent kit (manufactured by Shimadzu Corporation, P/N: 241-09560-91) was used. This kit consists of an RNA extraction reagent as a pretreatment liquid and an RT-PCR reaction liquid as a test liquid. The RNA extraction reagent is an alkaline treatment agent containing a reducing agent and a divalent chelating agent. The test liquid contains PCR components in accordance with the US CDC's "2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" and an inhibition regulator ("Ampdirect Plus" registered trademark, manufactured by Shimadzu Corporation). Specifically, the test liquid contains the components in the table below (probe, forward primer and reverse primer of the comparative example), reverse transcriptase, Taq DNA polymerase, dNTP mix, phosphate buffered saline, inhibition regulator, PCR buffer (Tris-HCl, KCl, MgCl ₂ ), etc.

The compounds used for terminal modification of functional groups are shown in the following structures (left: ROX, right: BHQ2).

<Detection Reagent Kit Including the Probe of Example 1>
For the N1 probe, an oligonucleotide probe (oligonucleotide probe of the present invention) designed as shown in the table below was manufactured. That is, the 3'-terminal functional group of the N1 probe was changed from BHQ2 to Cy5.5. The components contained in the test solution were the same as those in Comparative Example 1, except that the N1 probe was changed to the oligonucleotide probe of the present invention, and a detection reagent kit was prepared that included a test solution containing the probe of Example 1.

The compound (Cy5.5) used for terminal modification of the functional group has the following structure:

<Evaluation>
(1) Detection results when DTT was not added to the sample 1,000 copies of the novel coronavirus (SARS-CoV-2) were prepared as a sample. PCR was performed on this sample using the 2019 novel coronavirus detection reagent kit containing the probe of Example 1 or Comparative Example 1 according to the manual.

Specifically, first, an RNA extraction reagent was added to the novel coronavirus, and the mixture was subjected to a thermal activation treatment at 95°C for 5 minutes (extraction step). Next, as the PCR step, a test solution containing the probe of Example 1 or Comparative Example 1 was added to the mixture, and real-time PCR was performed using a real-time PCR device (BIO-RAD's "CFX96 Touch Deep well") (PCR step). The real-time PCR was performed using the temperature and time settings built into the device. That is, after heating at 42°C for 10 minutes and 95°C for 1 minute, 45 cycles were performed, with 95°C for 5 seconds and 60°C for 15 seconds as one cycle. The measurement results at this time are shown in Figures 1 and 2.

(2) Detection results when DTT was added to the sample 1,000 copies of the new coronavirus (SARS-CoV-2) were prepared, and 80 mM dithiothreitol (DTT) solution was added to this virus to obtain a sample treatment solution (preparation step).

Except for using this sample treatment solution, the extraction process and PCR process were carried out in the same manner as described above using the 2019 novel coronavirus detection reagent kit containing the probe of Example 1 or Comparative Example 1. The measurement results at this time are shown in Figures 3 and 4.

(3) Discussion As can be seen from Figs. 2 and 4, in the test solution containing the probe of Example 1, an amplification curve of the fluorescence intensity of the N1 probe was observed from 30 cycles onwards, whether DTT was added or not. Therefore, the target nucleic acid, and therefore the virus, could be detected regardless of the presence or absence of DTT.

On the other hand, from Fig. 1, it was found that in the test solution containing the probe of Comparative Example 1, when DTT was not added, an amplification curve of fluorescence intensity was observed, and therefore the virus could be detected. However, from Fig. 3, when DTT was added, an amplification curve of fluorescence intensity was not observed, and accurate detection was not possible. This is because, when the raw data of the fluorescence intensity before data analysis (before the baseline was taken into consideration) was observed, the baseline was set very high, i.e., a strong fluorescence intensity was already detected at the time of the 0th cycle, and therefore the increase in fluorescence intensity due to nucleic acid amplification could not be accurately measured.

Claims (5)

1. An oligonucleotide probe for testing an analyte containing a thiol reducing agent, comprising:
having a reporter portion and a quencher portion,
the peak wavelength of the excitation spectrum of the reporter moiety is 540 nm or longer;
the reporter has a peak wavelength of a fluorescence spectrum of 560 nm or more and 630 nm or less;
the quencher moiety is a non-azo dye capable of absorbing fluorescence from the reporter moiety;
An oligonucleotide probe , wherein the peak wavelength of the fluorescence spectrum of the quencher portion is 650 nm or more and 900 nm or less . The oligonucleotide probe according to claim 1, wherein the quencher portion is a fluorescent dye having an excitation spectrum with a peak wavelength of 560 nm or more. The oligonucleotide probe according to claim 1 or 2, wherein the peak wavelength of the excitation spectrum of the quencher portion is 600 nm or more and 800 nm or less. A test solution containing the oligonucleotide probe according to any one of claims 1 to 3. providing a specimen treatment solution containing a specimen and a thiol reducing agent;
and performing a hydrolysis probe method on the sample treatment solution using an oligonucleotide probe,
The oligonucleotide probe has a reporter portion and a quencher portion,
the peak wavelength of the excitation spectrum of the reporter moiety is 540 nm or longer;
The detection method, wherein the quencher moiety is a non-azo dye capable of absorbing fluorescence from the reporter moiety.

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