PRETREATMENT METHOD, PRETREATMENT SOLUTION, AND KIT FOR DETECTING NUCLEIC ACID OF VIRUS, AND USE THEREOF

Abstract

The present invention relates to the field of viral nucleic acid detection. In particular, the present invention provides a pretreatment method for viral nucleic acid detection. The method includes mixing a pretreatment solution containing a sample with a nucleic acid releasing agent and a qPCR amplification reagent, wherein the pretreatment solution includes Tris-HCl, EDTA-2Na, sodium chloride, a ribonuclease (RNase) inhibitor, and an antibiotic; and the pretreatment solution has a pH of 6.5-8.0.

Claims

1. A pretreatment method for viral nucleic acid detection, the method comprising: mixing a sample preserved in a pretreatment solution with a nucleic acid releasing agent and a qPCR amplification reagent, wherein the pretreatment solution comprises Tris-HCl, EDTA-2Na, sodium chloride, a ribonuclease (RNase) inhibitor, and an antibiotic; and the pretreatment solution has a pH of 6.5-8.0.

2. The method according to claim 1, wherein in the pretreatment solution, the Tris-HCl is present at a concentration of 10 mM to 200 mM, the EDTA-2Na is present at a concentration of 8 mM to 50 mM, the sodium chloride is present at a concentration of 0.5% to 2% (w/v), the RNase inhibitor is present at a concentration of 2 U/mL to 800 U/mL, and the antibiotic is present at a concentration of 0.005% to 0.05%.

3. The method according to claim 1, wherein in the pretreatment solution, the Tris-HCl is present at a concentration of 80 mM to 120 mM, the EDTA-2Na is present at a concentration of 10 mM to 15 mM, the sodium chloride is present at a concentration of 0.8% to 1% (w/v), and the RNase inhibitor is present at a concentration of 10 U/mL to 30 U/mL.

4. The method according to claim 1, wherein the RNase inhibitor is diethyl pyrocarbonate (DEPC), a RNase protein inhibitor (RNasin), a ribonucleoside vanadyl complex, or SDS; and/or, the antibiotic is a Proclin antibiotic or NaN.sub.3.

5. The method according to claim 1, wherein the antibiotic is Proclin 300 or Proclin 950.

6. The method according to claim 1, wherein a reaction solution prepared by the method can be used to directly perform a qPCR amplification test.

7. The method according to claim 1, wherein the virus is a DNA virus or an RNA virus.

8. The method according to claim 1, wherein the virus is coronavirus, respiratory syncytial virus, or enterovirus.

9. The method according to claim 1, wherein the virus is SARS-CoV-2.

10. The method according to claim 1, wherein the sample is derived from human or animal blood, feces, urine, oral epithelial cells, exfoliated cells, buccal swabs, or throat swabs.

11. A pretreatment solution for viral nucleic acid detection, the pretreatment solution comprising: Tris-HCl, EDTA-2Na, sodium chloride, a ribonuclease (RNase) inhibitor, and an antibiotic, wherein the pretreatment solution has a pH of 6.5-8.0.

12. The pretreatment solution according to claim 11, wherein in the pretreatment solution, the Tris-HCl is present at a concentration of 10 mM to 200 mM, the EDTA-2Na is present at a concentration of 8 mM to 50 mM, the sodium chloride is present at a concentration of 0.5% to 2% (w/v), the RNase inhibitor is present at a concentration of 2 U/mL to 800 U/mL, and the antibiotic is present at a concentration of 0.005% to 0.05%.

13. The pretreatment solution according to claim 11, wherein in the pretreatment solution, the Tris-HCl is present at a concentration of 80 mM to 120 mM, the EDTA-2Na is present at a concentration of 10 mM to 15 mM, the sodium chloride is present at a concentration of 0.8% to 1% (w/v), and the RNase inhibitor is present at a concentration of 10 U/mL to 30 U/mL.

14. The pretreatment solution according to claim 11, wherein the RNase inhibitor is diethyl pyrocarbonate (DEPC), a RNase protein inhibitor (RNasin), a ribonucleoside vanadyl complex, or SDS; and/or, the antibiotic is a Proclin antibiotic or NaN.sub.3.

15. The pretreatment solution according to claim 11, wherein the antibiotic is Proclin 300 or Proclin 950.

16. The pretreatment solution according to claim 11, wherein the virus is a DNA virus or an RNA virus.

17. The pretreatment solution according to claim 11, wherein the virus is coronavirus, respiratory syncytial virus, or enterovirus.

18. The pretreatment solution according to claim 11, wherein the virus is SARS-CoV-2.

19. A kit for viral nucleic acid detection based on the one-step method, the kit comprising the pretreatment solution defined according to claim 11.

20. The kit according to claim 19, wherein the kit further comprises a sample releasing agent and a qPCR amplification reagent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 is a graph showing the results of “one-step method” testing of the SARS-CoV-2 nucleic acid in the samples gradient-diluted by using the pretreatment method of the present invention and after being preserved at room temperature for 72 hr.

DETAILED DESCRIPTION

[0053] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented therefrom. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, but not to limit the present invention.

Example 1. Pretreatment and Rapid Detection of Respiratory Syncytial Virus (RSV) Throat Swab Samples with the Present Invention

[0054] In order to evaluate the virus pretreatment solution of the present invention, comparative analysis was performed on the virus pretreatment solution of the present invention (Tris-HCl at a concentration of 100 mM, EDTA-2Na at a concentration of 10 mM, sodium chloride at a concentration of 0.9% (w/v), RNasin at a concentration of 20 U/mL, and Proclin 950 at a concentration of 0.04% (v/v)), a saline solution, and commercially available virus pretreatment solutions. The comparison method was employed by performing dilution (1:9, v/v) pretreatment on clinically diagnosed positive RSV throat swab samples, and direct amplification of the samples at 0 hr, 24 hr, 48 hr, and 72 hr, respectively at room temperature (25° C.). The detection efficiency of real-time quantitative PCR (real-time qPCR) under the room temperature pretreatment condition was compared by Ct values to evaluate the effects of different pretreatment solutions on virus pretreatment. The qPCR amplification test was employed by using the EFNART “one-step method” technique, such that a real-time qPCR amplification test was directly performed in a PCR amplification tube at a ratio of a pretreatment solution with the sample:a nucleic acid releasing agent:a PCR amplification reagent of 10:10:30.

[0055] Real-time qPCR amplification testing procedures are as shown in Table 1, and the results are as shown in Table 2.

TABLE-US-00001 TABLE 1 Number Step Temperature Time of Cycles Reverse transcription 60° C. 30 min 1 Pre-denaturation 95° C.  1 min 1 Denaturation 95° C. 15 sec 40-45 Annealing, extension, and 60° C. 30 sec fluorescence collection

TABLE-US-00002 TABLE 2 Ct Value by Detection in Ct Value by Detection in Original Sample 10-fold Diluted Sample Pretreatment Time 0 hr 24 hr 48 hr 72 hr 0 hr 24 hr 48 hr 72 hr Pretreatment solution 30.69 31.05 30.98 31.24 33.56 33.98 33.78 34.01 of the present invention Saline solution 31.04 32.15 33.24 34.65 34.12 35.26 36.42 36.98 Hank's pretreatment 35.12 38.20 No Ct No Ct No Ct No Ct No Ct No Ct solution Guanidine No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct hydrochloride pretreatment solution

[0056] The aforementioned results show that with the virus pretreatment method of the present invention, pretreatment at room temperature for 24 hr, the gradient-diluted RSV sample can be well pretreatment, and can be directly used for EFNART detection, and with the pretreatment method based on saline solution matrix, after pretreatment for a long time, the Ct values is delayed, and the nucleic acid of the virus is degraded to a certain extent, which affects the amplification efficiency. In contrast, the Hank's pretreatment solution and the guanidine salt pretreatment solution, which are commonly used and commercially available, cannot provide an effective way of performing virus pretreatment and a PCR amplification test in this scheme.

Example 2. Nucleic Acid Pretreatment and Rapid Detection SARS-CoV-2 Samples after Purification with the Present Invention

[0057] In order to evaluate the virus pretreatment solution of the present invention, comparative analysis was performed on the virus pretreatment solution of the present invention (Tris-HCl at a concentration of 100 mM, EDTA-2Na at a concentration of 10 mM, sodium chloride at a concentration of 0.9% (w/v), RNasin at a concentration of 20 U/mL, and Proclin 300 at a concentration of 0.01% (v/v)), a saline solution, and commercially available virus pretreatment solutions. The comparison method was employed by performing dilution (1:9, v/v) pretreatment on clinically diagnosed positive SARS-CoV-2 nucleic acid, and direct amplification of the sample at 0 hr, 24 hr, 48 hr, and 72 hr, respectively at room temperature (25° C.). The detection efficiency of real-time qPCR under the room temperature pretreatment condition was compared by Ct values to evaluate the effects of different pretreatment solutions on virus pretreatment. The qPCR amplification test was employed by using the EFNART “one-step method” technique, such that a real-time qPCR amplification test was directly performed in a PCR amplification tube at a ratio of a pretreatment solution with the nucleic acid:a nucleic acid releasing agent:a PCR amplification reagent of 10:10:30. The results are as shown in Table 3. The SARS-CoV-2 nucleic acid gradient-diluted by using the virus pretreatment method of the present invention was preserved at room temperature for 72 hr. The results of the “one-step method” testing are as shown in FIG. 1.

TABLE-US-00003 TABLE 3 Ct Value by Detection in Ct Value by Detection in Original Sample 10-fold Diluted Sample Pretreatment Time 0 hr 24 hr 48 hr 72 hr 0 hr 24 hr 48 hr 72 hr Pretreatment solution 28.59 28.68 29.01 28.97 32.01 31.89 32.14 32.20 of the present invention Saline solution 28.95 29.48 29.98 30.54 33.01 3.21 33.14 34.28 Hank's pretreatment 31.02 32.35 33.62 No Ct No Ct No Ct No Ct No Ct solution Guanidine No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct hydrochloride pretreatment solution

[0058] The aforementioned results show that with the virus pretreatment method of the present invention, pretreatment at room temperature for 24 hr, the gradient-diluted SARS-CoV-2 nucleic acid can be well pretreated, and can be directly used for EFNART detection, and with the pretreatment method based on saline solution matrix, after pretreatment for a long time, the Ct values is delayed, and the nucleic acid of the virus is degraded to a certain extent, which affects the amplification efficiency. In contrast, the Hank's pretreatment solution and the guanidine salt pretreatment solution, which are commonly used and commercially available, cannot provide an effective way of performing virus pretreatment and a PCR amplification test in this scheme.

Example 3. Pretreatment and Rapid Detection of Enterovirus (EV) Universal Throat Swab Samples with the Present Invention

[0059] In order to evaluate the virus pretreatment solution of the present invention, comparative analysis was performed on the virus pretreatment solution of the present invention (Tris-HCl at a concentration of 100 mM, EDTA-2Na at a concentration of 10 mM, sodium chloride at a concentration of 0.9% (w/v), SDS at a concentration of 0.1%, and Proclin 950 at a concentration of 0.04% (v/v)), a saline solution, and commercially available virus pretreatment solutions. The comparison method was employed by performing dilution (1:9, v/v) pretreatment on clinically diagnosed positive EV universal throat swab samples, and direct amplification of the samples at 0 hr, 24 hr, 48 hr, and 72 hr, respectively at room temperature (25° C.). The detection efficiency of real-time qPCR under the room temperature pretreatment condition was compared by Ct values to evaluate the effects of different pretreatment solutions on virus pretreatment. The qPCR amplification test was employed by using the EFNART “one-step method” technique, such that a real-time qPCR amplification test was directly performed in a PCR amplification tube at a ratio of a pretreatment solution with the sample:a nucleic acid releasing agent:a PCR amplification reagent of 10:10:30. The results are as shown in Table 4:

TABLE-US-00004 TABLE 4 Ct Value by Detection in Ct Value by Detection in Original Sample 10-fold Diluted Sample Pretreatment Time 0 hr 24 hr 48 hr 72 hr 0 hr 24 hr 48 hr 72 hr Pretreatment solution 26.12 26.32 26.25 26.34 29.65 29.78 29.82 29.16 of the present invention Saline solution 27.32 27.15 28.64 29.01 30.98 31.65 32.27 32.59 Hank's pretreatment 30.12 30.54 33.28 33.42 No Ct No Ct No Ct No Ct solution Guanidine No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct hydrochloride pretreatment solution

[0060] The aforementioned results show that with the virus pretreatment method of the present invention, pretreatment at room temperature for 24 hr, the gradient-diluted EV samples can be well pretreated, and can be directly used for EFNART detection, and with the pretreatment method based on saline solution matrix, after pretreatment for a long time, the Ct values is delayed, and the nucleic acid of the virus is degraded to a certain extent, which affects the amplification efficiency. In contrast, the Hank's pretreatment solution and the guanidine salt pretreatment solution, which are commonly used and commercially available, cannot provide an effective way of performing virus pretreatment and a PCR amplification test in this scheme.

Example 4. Pretreatment Capacity of Virus Pretreatment Solution of the Present Invention for RNA Virus

[0061] Compared with DNA viruses, pretreatment and detection of RNA viruses are more susceptible to environmental factors due to higher requirements. A pretreatment process involving RNase contained in consumables has a particularly vital impact on detection of RNA viruses. In order to evaluate the pretreatment effect of the virus pretreatment solution of the present invention (Tris-HCl at a concentration of 100 mM, EDTA-2Na at a concentration of 10 mM, sodium chloride at a concentration of 0.9% (w/v), Rnasin at a concentration of 20 U/mL, and Proclin 950 at a concentration of 0.04% (v/v)) on RNA viruses, nucleic acid extracted from an SARS-CoV-2 sample pretreated with the virus pretreatment solution of the present invention was divided into two parts (A/B), at the same time, nucleic acid (C) extracted from an SARS-CoV-2 sample with the same concentration that was pretreated in the saline solution was prepared, 0.25 μg/mL RNase A was respectively added into solution A and solution C, the three solutions containing the SARS-CoV-2 nucleic acid were pretreated at room temperature (25° C.) for 24 hr, and then the RNA virus was pretreated and detected with the pretreatment solution of the present invention by adopting the EFNART “one-step method” technique. Detection was conducted by directly performing a real-time qPCR amplification test in a PCR amplification tube at a ratio of a pretreatment solution with the nucleic acid:a nucleic acid releasing agent:a PCR amplification reagent of 10:10:30. The results are as shown in Table 5.

TABLE-US-00005 TABLE 5 SARS-CoV-2 Nucleic Acid Pretreatment Condition Ct Value by Detection Pretreated nucleic acid sample A FAM HEX ROX (pretreatment solution of the present 30.8 26.8 29.6 invention containing 0.25 g/mL Rnase A) Pretreated nucleic acid sample B FAM HEX ROX (pretreatment solution of the present 30.9 27.1 30.2 invention without 0.25 g/mL Rnase A) Pretreated nucleic acid sample C FAM HEX ROX (pretreatment with saline solution No Ct 27.1 No Ct containing 0.25 g/mL Rnase A) Pretreated nucleic acid sample D FAM HEX ROX (pretreatment with saline solution 32.2 29.4 32.9 without 0.25 g/mL Rnase A)

[0062] It is proved by the experiments that in a case where pretreated nucleic acid sample D is taken as a reference, the addition of 0.25 μg/mL RNase A in the present invention has no influence on nucleic acid detection effect of SARS-CoV-2, and effective components in the present invention can digest RNase, reduce the influence of RNase on experimental detection, and can ensure the efficiency of direct detection of RNA viruses. It can be seen from pretreated nucleic acid sample C, the addition of 0.25 μg/mL RNase A included in the experimental conditions can digest and degrade the RNA in the experiment, thereby greatly affecting direct detection of RNA viruses and leading to risk of missed detection.

Example 5. Pretreatment and Rapid Detection of Hepatitis B Virus (HBV) Serum Sample by Using Virus Pretreatment Solution of the Present Invention

[0063] In order to evaluate the effect of the virus pretreatment solution of the present invention on DNA virus preservation and amplification detection, comparative analysis was performed on the virus pretreatment solution of the present invention (Tris-HCl at a concentration of 100 mM, EDTA-2Na at a concentration of 10 mM, sodium chloride at a concentration of 0.9% (w/v), RNasin at a concentration of 20 U/mL, and Proclin 950 at a concentration of 0.04% (v/v)), a saline solution, and commercially available virus pretreatment solutions. The comparison method was employed by performing dilution (1:9, v/v) pretreatment on clinically diagnosed positive HBV serum samples, and direct amplification of the samples at 0 hr, 24 hr, 48 hr, and 72 hr, respectively at room temperature (25° C.). The detection efficiency of real-time qPCR under the room temperature pretreatment condition was compared by Ct values to evaluate the effects of different pretreatment solutions on virus pretreatment. The qPCR amplification test was employed by using the EFNART “one-step method” technique, such that a real-time qPCR amplification test was directly performed in a PCR amplification tube at a ratio of a pretreatment solution with the DNA virus:a nucleic acid releasing agent:a PCR amplification reagent of 10:10:30. The results are as shown in Table 6.

TABLE-US-00006 TABLE 6 Ct Value by Detection in HBV Serum Sample Pretreatment Time 0 hr 24 hr 48 hr 72 hr Pretreatment solution 32.23 31.98 32.24 32.67 of the present invention Saline solution 33.43 33.18 33.54 33.87 Hank’s pretreatment 33.74 33.49 33.85 34.18 solution Guanidine hydrochloride No Ct No Ct No Ct No Ct pretreatment solution

[0064] The aforementioned results show that although the original design of the virus pretreatment method of the present invention was aimed at virus preservation matrix for RNA viruses for performing amplification based on the “one-step method”, the virus pretreatment method can also be used for preservation and detection of DNA viruses by performing amplification based on the one-step method.

Example 6. Verification of Effectiveness of Various Components of Virus Pretreatment Solution of the Present Invention

[0065] In order to verify the effectiveness of various components in the present invention, after relevant components in the present invention were adjusted and reduced, the sample was preserved and was subjected to pretreatment and a comparison experiment. Separately optimized components included an RNase inhibitor, an antibiotic, EDTA-2Na, etc. The adjusted concentrations of various components are as shown in Table 7. qPCR amplification testing was directly performed after RSV was preserved with the pretreatment solutions at room temperature for 24 hr that were prepared at different concentrations, and the detection efficiency of real-time qPCR under the room temperature pretreatment condition was compared by Ct values to evaluate the effects of different pretreatment solutions on virus pretreatment. The qPCR amplification test was employed by using the EFNART “one-step method” technique, such that a real-time qPCR amplification test was directly performed in a PCR amplification tube at a ratio of a pretreatment solution with the sample:a nucleic acid releasing agent:a PCR amplification reagent of 10:10:30.

TABLE-US-00007 TABLE 7 Detection Effects of Separately Optimized components in the Present Invention on Pretreatment Antibiotic Solution pH Value RNase inhibitor (Proclin 950) EDTA-2Na Adjustment Concentration Ct value Concentration Ct value Concentration Ct value pH value Ct value 0 No Ct 0 No Ct 0 38.2 4 38.54  2 U/mL 31.54 0.01% 31.48 10 mM 31.4 6.5 31.64 40 U/mL 32.4 0.04% 32.4 50 mM 33.4 8 31.81 8000 U/mL  35.42   1% 37.62 200 mM  No Ct 12 No Ct

[0066] Based on the aforementioned experiments, the chemical components in the present invention are essential components, and at the concentrations in the present invention, the pretreatment and detection of RNA viruses by employing one-step RT-PCR are optimal.