RNA VIRUS DETECTION METHOD

Abstract

Provided is a method for detecting an FNA virus in a biological sample, the method comprising: (1) a step for preparing a sample solution containing a biological sample, a protease, a nucleic acid that does not become a target of nucleic acid amplification, and at least one additive selected from the group consisting of chaotropic reagents and surfactants; (2) a step for preparing a nucleic acid amplification reaction solution containing the sample solution prepared in step (1) and containing a polypeptide having reverse transcriptase activity and DMA polymerase activity or a polypeptide having reverse transcription activity and a polypeptide having DNA polymerase activity; and (3) a step for amplifying a nucleic acid of the RNA virus in the reaction solution prepared in step (2).

Claims

1. A method for detecting an RNA virus in a biological sample, the method comprising: (1) a step of preparing a sample solution containing the biological sample and at least one additive selected from the group consisting of a proteolytic enzyme, a nucleic acid that is not a target of nucleic acid amplification, a chaotropic reagent and a surfactant, (2) a step of preparing a nucleic acid amplification reaction solution containing the sample solution prepared in step (1), and a polypeptide having reverse transcription activity and a polypeptide having DNA polymerase activity, or a polypeptide having reverse transcription activity and DNA polymerase activity, and (3) a step of amplifying a nucleic acid of the RNA virus in the reaction solution prepared in step (2).

2. The method according to claim 1, wherein the proteolytic enzyme is proteinase K, thermolysin, or pronase.

3. The method according to claim 1, wherein the chaotropic reagent is guanidine or a salt thereof, urea, iodine or a salt thereof, or a combination thereof.

4. The method according to claim 1, wherein the biological sample is at least one selected from the group consisting of an oral scraping, pharyngeal swab, nasal swab, nasopharyngeal swab, nasal aspirate, sputum, bronchial lavage fluid, alveolar lavage fluid, rectal swab, saliva, blood, urine, and a stool suspension.

5. The method according to claim 1, wherein the RNA virus is at least one selected from the group consisting of influenza virus, RS virus, metapneumovirus, parainfluenza virus, SARS coronavirus, MERS coronavirus, measles virus, norovirus, rotavirus, sapovirus, and HIV.

6. A method for preparing a sample solution for nucleic acid amplification, the method comprising a step of preparing a mixture containing a biological sample and at least one additive selected from the group consisting of a proteolytic enzyme, a nucleic acid that is not a target of the nucleic acid amplification, a chaotropic reagent and a surfactant.

7. A composition for the method for detecting an RNA virus in a biological sample according to claim 1, comprising: (1) a sample solution containing at least one additive selected from the group consisting of a proteolytic enzyme, a nucleic acid that is not a target of nucleic acid amplification, a chaotropic reagent and a surfactant, and a biological sample, and (2) a polypeptide having reverse transcriptase activity and a polypeptide having DNA polymerase activity, or a polypeptide having reverse transcriptase activity and DNA polymerase activity.

8. A kit for the method for detecting an RNA virus in a biological sample according to claim 1, comprising: (1) at least one additive selected from the group consisting of a proteolytic enzyme, a nucleic acid that is not a target of nucleic acid amplification, a chaotropic reagent and a surfactant, and (2) a polypeptide having reverse transcriptase activity and a polypeptide having DNA polymerase activity, or a polypeptide having reverse transcriptase activity and DNA polymerase activity.

Description

EXAMPLE 1

Experiment for Additives—1

[0062] The detection method of the present invention was demonstrated. First, an RNA positive control having the nucleotide sequence shown in SEQ ID NO: 4, called “N2 set”, which is one of RNA positive controls used for sensitivity tests of primer pairs and nucleic acid probes for detection as described in the National Institute of Infectious Diseases Manual “Pathogen Detection Manual 2019-nCoV Ver. 2.9,1”, was prepared by a conventional method. Next, a commercially available sputum sample (manufactured by NOVA Biologics) was diluted 3-fold with physiological saline to prepare a stock suspension of the sputum sample. The stock suspension was further diluted 20-fold with physiological saline, and then mixed with the RNA positive control to prepare a biological sample suspension. A mixture that did not contain the sputum sample was also prepared. Then, Proteinase K (manufactured by TAKARA BIO INC., about 400 U/ml) as a proteolytic enzyme, guanidine thiocyanate (CAS registration number 593-34-0) (manufactured by TOKYO CHEMICAL INDUSTRY CO., LTD.) as a chaotropic reagent, or sodium dodecyl sulfate (manufactured by TOKYO CHEMICAL INDUSTRY CO., LTD.) as a surfactant was used as the additive to be mixed with the biological sample. In addition, a control was prepared by adding nothing to the biological sample suspension.

[0063] To the biological sample suspension, a final concentration of 3.5 U/ml (175 μg/ml), 7 U/ml (350 μg/ml), 35 U/ml (1750 μg/ml) or 70 U/ml (3500 μg/ml) of Proteinase K, a final concentration of 209 μmol/ml of guanidine thiocyanate, and a final concentration of 0.01% (w/v) of sodium dodecyl sulfate were added; and kept warm at 55° C. for 5 to 10 minutes and then heated at 95° C. for 5 minutes, or directly heated at 95° C. for 5 minutes without keep warm. To a mixture thus obtained, the RNA positive control was added so as to become 2.5×10{circumflex over ( )}5 copies/reaction, and then subjected to RT-PCR. As RT-PCR reagents. One Step PrimeScript (trademark) III RT-qPCR Mix (manufactured by TAKARA BIO INC.) was used. Specifically, 5 μl of a mixture as obtained above was mixed with 25 μl of 2×RT-qPCR Mix; NIID_2019-nCoV_N_F primer (final concentration: 0.5 μM) and NIID_2019-nCoV_N_R primer (final concentration: 0.7 μM) described in the National Institute of Infectious Diseases Manual, having the nucleotide sequences of SEQ ID NOs: 1 and 2; NIID_2019-nCoV_N_P probe (final concentration: 0.2 μM, 5′ FAM labeled, 3′ BHQ1 labeled) having the nucleotide sequence of SEQ ID NO: 3; and RNase Free H.sub.2O to prepare a reaction mixture having a final volume of 50 μl.

[0064] As a thermal cycler, Thermal Cycler Dice (registered trademark) Real Time System III (Cy5) with PC (manufactured by TAKARA BIO INC.) was used. PCR was performed under conditions of 52° C. for 5 minutes and 95° C. for 10 seconds, followed by 45 cycles of 95° C. for 5 seconds and 60° C. for 30 seconds. Results are shown in Table 1.

TABLE-US-00001 TABLE 1 Ct Keep warm/heat value Sputum treatment RNA suspension Additive 95° C./ positive supernatant ProK SDS GT 55° C. 5 min control None (PBP) — — — — — 23.6 ∘ 3.5 U/ml — — — ∘ 28.8 ∘ 3.5 U/ml — — ∘/5 min ∘ 28.2 ∘ 3.5 U/ml — — ∘/10 min ∘ 28.7 ∘ 7.0 U/ml — — — ∘ 26.9 ∘ 7.0 U/ml — — ∘/5 min ∘ 26.4 ∘ 7.0 U/ml — — ∘/10 min ∘ 26.7 ∘ 35 U/ml — — — ∘ 28.6 ∘ 35 U/ml — — ∘/5 min ∘ 26.9 ∘ 35 U/ml — — ∘/10 min ∘ 26.5 ∘ 70 U/ml — — — ∘ 26.7 ∘ 70 U/ml — — ∘/5 min ∘ 25.3 ∘ 70 U/ml — — ∘/10 min ∘ 24.8 ∘ 3.5 U/ml 0.01% — — ∘ 24.0 ∘ 3.5 U/ml 0.01% — ∘/5 min ∘ 23.9 ∘ 3.5 U/ml 0.01% — ∘/10 min ∘ 23.8 ∘ 3.5 U/ml — 2.5% — ∘ 23.9 ∘ 3.5 U/ml — 2.5% ∘/5 min ∘ 23.9 ∘ 3.5 U/ml — 2.5% ∘/10 min ∘ 23.9

[0065] As seen from Table 1, when a high concentration 35 to 70 U/ml (175 μg/ml to 3500 μg/ml) of Proteinase K (“ProK” in the table) was added, a Ct value was almost the same as that when the biological sample suspension was not added, and thus the influence of the biological sample suspension was eliminated. When a combination of 3.5 U/ml of Proteinase K and guanidine thiocyanate (“GT” in the table) or a combination of 3.5 U/ml of Proteinase K and 0.01% of sodium dodecyl sulfate (“SDS” in the table) was added, a Ct value was almost the same as that when the biological sample suspension was not added, and thus the influence of the biological sample suspension was eliminated. Therefore, it was found that a high concentration of Proteinase K or a combination of Proteinase K with guanidine thiocyanate and/or sodium dodecyl sulfate is effective as the additive of the present invention. Even when the keep warm treatment of the sample solution at 55° C. was omitted, it was possible to detect the RNA virus in the sample.

EXAMPLE 2

Experiment for Additives—2

[0066] The detection method of the present invention was demonstrated by using an inactivated virus. In this Example, NATtrol (trademark) Influenza A/B Positive Control (manufactured by ZeptoMetrix) was used as the inactivated RNA virus. The inactivated RNA virus being undiluted was mixed with, or the inactivated RNA virus was diluted 10 to 20-fold with physiological saline and then mixed with the sputum sample prepared in Example 1 to prepare a biological sample suspension. A mixture that did not contain the sputum sample was also prepared.

[0067] Proteinase K as a proteolytic enzyme, guanidine thiocyanate as a chaotropic reagent, or sodium dodecyl sulfate as a surfactant was used as the additive.

[0068] The experiment was carried out as follows. Specifically, to the biological sample suspension, a final concentration of 3.5 U/ml or 70 U/ml of Proteinase K, a final concentration of 2.5% (w/v) of guanidine thiocyanate, or a final concentration of 0.01% (w/v) of sodium dodecyl sulfate was added in combination. A mixture was kept warm at room temperature or 55° C. for 5 minutes, heated at 95° C. for 5 minutes, and then subjected to evaluation by RT-qPCR. As RT-PCR reagents. One Step PrimeScript (trademark) III RT-qPCR Mix (manufactured by TAKARA BIO INC.) was used. Specifically, 5 μl of the biological sample suspension containing the additive(s) was mixed with 25 μl of 2×RT-qPCR Mix, IAmg-F01 and IAmg-R01 primers (final concentration: 0.2 μM) having the nucleotide sequences of SEQ ID NOs: 5 and 6, IAmg-PR01 probe for influenza virus A detection (final concentration: 0.2 μM, 5′ FAM labeled, 3′ BHQ1 labeled) having the nucleotide sequence of SEQ ID NO: 7, and RNase Free H.sub.2O to prepare a reaction mixture having a final volume of 50 μl.

[0069] As a thermal cycler, Thermal Cycler Dice (registered trademark) Real Time System III (Cy5) with PC (manufactured by TAKARA BIO INC.) was used. PCR was performed under conditions of 52° C. for 5 minutes and 35° C. for 10 seconds, followed by 45 cycles of 95° C. for 5 seconds and 60° C. for 30 seconds. Amplification results were evaluated by comparing Ct values with a Ct value of a control to which no additive was added. Results are shown in Table 2.

TABLE-US-00002 TABLE 2 ProK 70 U/ml 3.5 U/ml 3.5 U/ml SDS — ∘ — GT — — ∘ Keep warm/heat 55° C./5 min — — treatment 95° C./5 min 95° C./5 min 95° C./5 min Biological Stock 10-fold 32.6 36.2 32.5 sample suspension diluted suspension inactivated virus Undiluted 29.9 32.3 29.4 inactivated virus (1/10) 10-fold 32.6 32.7 32.4 diluted inactivated virus Undiluted 29.4 29.0 28.8 inactivated virus (1/20) 10-fold 32.8 32.4 32.5 diluted inactivated virus Undiluted 29.6 28.9 28.8 inactivated virus None (PBS) 10-fold 33.2 32.2 32.3 diluted inactivated virus Undiluted 29.2 28.8 28.9 inactivated virus

[0070] As seen from Table 2, when high concentration 70 U/ml of Proteinase K (“ProK” in the table) was added to the stock suspension to 20-fold dilution of the biological sample, a Ct value was the same as that when the biological sample suspension was not added, and thus the influence of the biological sample suspension was eliminated. When a combination of 3.5 U/ml of Proteinase K and guanidine thiocyanate (“GT” in the table) was added, a Ct value was the same as that when high concentration 70 U/ml of Proteinase K was added. Further, when a combination of 3.5 U/ml of Proteinase K and 0.01% of sodium dodecyl sulfate (“SDS” in the table) was added to a 10- to 20-fold dilution of the biological sample suspension, a Ct value was the same as that when high concentration 70 U/ml of Proteinase K was added. Therefore, it was found that a high concentration of Proteinase K or a combination of Proteinase K with guanidine thiocyanate and/or sodium dodecyl sulfate is effective as the additive of the present invention even when detecting an RNA from a biological sample spiked with an inactivated virus, which is close to an actual sample.

EXAMPLE 3

Experiment for Biological Samples

[0071] The detection method of the present invention was demonstrated by using an inactivated virus. As the inactivated RNA virus, a commercially available inactivated RNA virus, NATtrol (trademark) SARS_CORONA Positive Control (manufactured by ZeptoMetrix) was used, and diluted 1.2-fold and 12-fold with physiological saline. As the biological sample, a commercially available saliva (manufactured by LEE BIOSOLUTIONS) was used with being undiluted or diluted with a virus transport medium (product name: virus transport medium (VTM), manufactured by Sugiyama-gen). The biological sample was mixed with the inactivated virus dilution to prepare a biological sample suspension. As the additive to be mixed with the biological sample suspension. Proteinase K as a proteolytic enzyme was used.

[0072] The experiment was carried out as follows. Specifically, a final concentration of 70 U/ml or 17.5 U/ml of Proteinase K was added to the biological sample suspension. A mixture was left at room temperature for 5 minutes, held at 55° C. for 5 minutes, heated at 95° C. for 5 minutes, and then subjected to evaluation by RT-qPCR. The same RT-qPCR reagents as used in Example 1 were used. Specifically, 10 μl of the biological sample suspension containing the additive was mixed with 2×RT-qPCR Mix, PCR forward Primer N1_SARS_COV_F and R primers (final concentration: 0.2 μM) having the nucleotide sequences of SEQ ID NOs: 8 and 9, PCR Forward Primer N2_SARS COV_F and R primers (final concentration: 0.2 μM) having the nucleotide sequences of SEQ ID NOs: 11 and 12, N1_SARS COV_P probe for SARS_CORONA detection (final concentration: 0.2 μM, 5′ Cy5 labeled, 3′ BHQ3 labeled) having the nucleotide sequence of SEQ ID NO: 10, N2_SARS COV_P probe for SARS_CORONA detection (final concentration: 0.2 μM, 5′ Cy5 labeled, 3′ BHQ3 labeled) having the nucleotide sequence of SEQ ID NO: 13, and RNase Free H.sub.2O to prepare a reaction mixture having a final volume of 50 μl.

[0073] A thermal cycler and PCR conditions were the same as Example 1. Amplification results were evaluated by comparing Ct values with a Ct value of a control to which no additive was added. Results are shown in Table 3.

TABLE-US-00003 TABLE 3 Treatment with ProteinaseK ProK Biological added Natrol sample amount RT-PCR Biological added added (unit Copies/ reaction sample amount amount conc.) reaction Ct value Saliva 1 μl 39 μl 10 μl 500 35.4   (70 U/ml) 50 38.2 10 μl 500 35.9 (17.5 U/ml) 50 38.8 Saliva + 19 μl 10 μl 500 35.3 VTM (saliva) + (70.0 U/ml) 50 38.4 20 μl 10 μl 500 35.3 (VTM) (17.5 U/ml) 50 39.1

[0074] As seen from Table 3, nucleic acid amplification was observed in all the biological sample suspensions obtained by adding the inactivated virus to the undiluted saliva or the mixture of saliva and VTM. Both when 70 U/ml of Proteinase K (“ProK” in the table) was added as the additive and when 17.5 U/ml of Proteinase K was added as the additive, the same Ct value was obtained. Therefore, it was found that the present invention is effective even when detecting an RNA from a biological sample such as saliva spiked with an inactivated virus.

EXAMPLE 4

Experiment for Uracil-N-Glucosidase (UNG) Treatment

[0075] The detection method of the present invention was demonstrated by using the same biological sample suspension and the same additive as Example 3.

[0076] In this Example, treatment of the biological sample suspension before RT-PCR was performed in the same way as in Example 3. Evaluation by RT-qPCR was also performed in the same way as in Example 3 except that one Step PrimeScript (trademark) III RT-qPCR Mix, with UNG (manufactured by TAKARA BIO INC.) was used as RT-PCR reagents instead of One Step FrimeScript (trademark) III RT-qPCR Mix (manufactured by TAKARA BIO INC.). A thermal cycler and PCR conditions were the same as Example 1. Amplification results were evaluated by comparing Ct values with a Ct value of a control to which no additive was added. Results are shown in Table 4.

TABLE-US-00004 TABLE 4 Treatment with ProteinaseK Biological Inactivated virus in sample ProK Natrol RT-PCR reaction Bioligical (Added (Final (Added Copies/ Ct sample amount) conc.) amount) reaction value Saliva 39 μl 10 μl 1 μl 500 36.1   (70 U/ml) 50 39.1 10 μl 500 36.5 (17.5 U/ml) 50 38.7 Saliva + 19 μl 10 μl 500 36.5 VTM (saliva) +   (70 U/ml) 50 39.0 20 μl 10 μl 500 36.0 (VTM) (17.5 U/ml) 50 39.0

[0077] As seen from Table 4, nucleic acid amplification was observed in ail the biological sample suspensions obtained by adding the inactivated virus to the undiluted saliva or the mixture of saliva and VTM. Both when 70 U/ml of Proteinase K (“ProK” in the table) was added as the additive and when 17.5 U/ml of Proteinase K was added as the additive, the same Ct value was obtained. Therefore, it was found that the present invention is effective even in RT-qPCR containing uracil-N-glucosidase and dUTP for preventing contamination by amplified products.

INDUSTRIAL APPLICABILITY

[0078] A nucleic acid derived from an RNA virus contained in a sample can be detected without requiring isolation of the nucleic acid by using the detection method of the present invention. Thus the present invention greatly contributes to the field of clinical diagnosis.

SEQUENCE LISTING FREE TEXT

[0079] SEQ ID NO: 1: PCR Forward Primer NIID_2019-nCOV_N_F2 [0080] SEQ ID NO: 2: PCR Reverse Primer NIID_2019-nCOV_N_R2 [0081] SEQ ID NO: 3: Probe NIID_2019-nCOV_N_P2. 5′-end is labeled FAM and 3′-end is labeled BHQ1 [0082] SEQ ID NO: 4: RNA positive control sequence for 2019-nCoV N2 set [0083] SEQ ID NO: 5: PCR Forward Primer IAmg-F01 [0084] SEQ ID NO: 6: PCR Reverse Primer IAmg-R01 [0085] SEQ ID NO: 7: Probe IAmg-PB1, 5′-end is labeled FAM and 3′-end is labeled BHQ1 [0086] SEQ ID NO: 8 PCR Forward Primer N1_SARS COV_F [0087] SEQ ID NO: 9 PCR Reverse Primer N1_SARS_COV_R [0088] SEQ ID NO: 10 N1_SARS COV_P. 5′-end is labeled Cy5 and 3′-end is labeled BHQ3 [0089] SEQ ID NO: 11 PCR Forward Primer N2_SARS COVF [0090] SEQ ID NO: 12 PCR Reverse Primer N2.SARS GOVJR [0091] SEQ ID NO: 13 N2_SARS COV_p. 5′-end is labeled Cy5 and 3′-end is labeled BHQ3