OA20829A - Pretreatment method, pretreatment solution, kit for virus nucleic acid detection, and use thereof. - Google Patents

Abstract

The present invention belongs to the field of virus nucleic acid detection. Specifically, the present invention provides a pretreatment method for virus nucleic acid detection. The method comprises mixing a pretreatment solution containing a sample with a nucleic acid releasing agent and a qPCR amplification reagent. The pretreatment solution comprises Tris-HCl, EDTA-2Na, sodium chloride, a ribonuclease inhibitor and an antibiotic; and the pretreatment solution has a pH of 6.5-8.0.

Description

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

TECHNICAL FÏELD

[0001 ] The present invention relates to the field of nucleic acid détection of viral samples, and partlcularly to a virus sample pretreatment method and a pretreatment solution for pretreatment of DNA/RNA virus.

BACKGROUND

[0002] Currently, there are mainly two types of conventional virus pretreatment solutions: a Hank's matrix-based pretreatment solution and a guanidine salt-based pretreatment solution. Hank's solution is a common balanced sait solution (BSS) for virus transport. Due to various factors, the Hank's solution can preserve respiratory viruses (such as influenza virus, SARS-CoV-2, etc.) for only a few hours. After a few hours, the morphology of the virus will be affected, which may affect the efficiency of viral nucleic acid détection. After an extended time, bacteria and fungi will easily grow, resulting în a decrease in the pH value of the Hank's solution and accelerated dégradation of the virus. Therefore, the Hank's solution is difficult to be used for long-term préservation and nucleic acid détection of viruses. The concentration of guanidine hydrochloride or Rnasin în the guanidine salt-based virus pretreatment solution is relatively high (3 M to 5 M), and a high concentration of a guanidine sait is suitable for virus inactivation at room température. However, the high concentration of the guanidine sait greatly inhibits nucleic acid extraction or purification (such as based on magnetic beads), and thus it needs to be washed multiple times or used in a spin column-based method.

[0003] The extraction free nucleic acid release and amplification technology (EFNART), simply referred to as the “one-step” technique, refers to directly performing a nucleic acid amplification test for a sample by directly combining a sample nucleic acid releasing agent with a strong alkaline property and a highly compatible amplification System in a circumstance where no nucleic acid extraction or purification of the sample is required. The “one-step method” will greatly reduce time for viral nucleic acid détection, particularly for RNA viruses. The method is expected to save time by 60% or more and improve détection efficiency by 50% in comparison with a traditional amplification method performed after nucleic acid extraction.

[0004] In the event of a major épidémie (for example, the SARS-CoV-2 épidémie that occurred from 2019 to 2020), détection based on the one-step method is preferred because it can save détection time and improve efficiency, and will make a huge contribution to épidémie control.

[0005] However, existing common virus pretreatment solutions, such as the Hank's pretreatment solution and the guanidine salt-based pretreatment solution, cannot be well used for subséquent détection based on the one-step method. Thus, it is difficult to apply the existing common virus pretreatment solutions to scénarios in which rapid détection and screening of virus samples are required.

[0006] Therefore, there is a need in the art for a sample pretreatment solution that can be adéquate for use in one-step method-based viral nucleic acid détection.

SUMMARY

[0007] In vîew of this, in a first aspect, the present invention provides a pretreatment method for viral nucleic acid détection, the method including: mîxing a sample preserved in a pretreatment solution with a nucleic acid releasing agent and a qPCR amplification reagent;

[0008] wherein the pretreatment solution contaîns: Tris-HCl, EDTA-2Na, sodium chloride, a ribonucléase (RNase) inhibitor, and an antibiotic; and

[0009] the pretreatment solution has a pH of 6.5 to 8.0.

[0010] In the present invention, the Tris-HCl may be present at a concentration of about 10 mM to about 200 mM, preferably at a concentration of about 80 mM to about 120 mM, and most preferably at a concentration of about 100 mM.

[0011] In the present invention, the EDTA-2Na may be present at a concentration of about 8 mM to about 50 mM, preferably at a concentration of about 10 mM to about 15 mM, and most preferably at a concentration of about 10 mM.

[0012] In the présent invention, the sodium chloride may be présent at a concentration of about 0.5% (w/v) to about 2% (w/v), preferably at a concentration of about 0.8% (w/v) to about 1 % (w/v), and most preferably at a concentration of about 0.9% (w/v).

[0013] In the présent invention, the term “ribonucléase (RNase) inhibitor” refers to a Chemical substance that can inhibit and thus inactivate RNase, including, but îs not limited to, diethyl pyrocarbonate (DEPC), a RNase proteîn inhibitor (RNasîn), ribonucleoside vanadyl complexes, SDS, etc.

[0014] In the présent invention, the RNase inhibitor may be présent at a concentration of about 2 U/mL to about 800 U/mL, for example, about 40 U/mL, about 50 U/mL, and about 100 U/mL; preferably at a concentration of about 10 U/mL to about 30 U/mL; and most preferably at a concentration of 20 U/mL.

[0015] In the présent invention, the pH value ranges from 6.5 to 8.0, preferably from 7.0 to 8.0, and is most preferably 7.5.

[0016] The antibiotic includes, but is not limited to, Proclin antîbiotics (such as Proclin 300 and Proclin 950) and NaNs.

[0017] For example, in a case where Proclin 300 is used as the antibiotic, the concentration of Proclin 300 may be about 0.01% (v/v). For another example, in a case where Proclin 950 is used as the antibiotic, the concentration of Proclin 950 may be about 0.04% (v/v), but the présent invention is not limited thereto.

[0018] In a spécifie embodiment, the pretreatment solution contains 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), the RNase inhibitor at a concentration of 20 U/mL, and Proclin 950 at a concentration of 0.04% (v/v),

[0019] and the aforementioned pretreatment solution is adjusted to a pH value of 7.5.

[0020] As used herein, the term “sample” refers to a sample that may contain a virus. The sample may be derived from human or animal blood, feces, urine, oral épithélial cells, exfoliated cells, buccal swabs, throat swabs, etc.

[0021] The term “pretreatment” mentioned in the present invention refers to treatment prior to performing a test for a sample, particularly treatment prior to nucleic acid détection of the sample (based on the one-step method).

[0022] The “pretreatment solution” mentioned in the present invention refers to a lîquid for pretreating a virus sample.

[0023] In the present invention, the virus may be a DNA virus or an RNA virus.

[0024] In a preferred embodiment, the virus is an RNA virus.

[0025] In a more preferred embodiment, the virus is coronavirus (such as SARS-CoV-2), respiratory syncytial virus, or enterovîrus.

[0026] A nucleic acid détection reaction solution prepared by the pretreatment method of the present invention can be used to directly perform qPCR, without requiring an extraction or purification process, thereby improving détection efficiency and reducing détection time. The antibiotic and the RNase inhibitor employed în the present invention can be used for pretreatment of DNA vîruses or RNA vîruses, and can prevent the activity of a preserved virus from being affected by various microorganisms, such as bacteria, growing at room température. The RNA virus is more easily degraded due to the ubiquitous RNase. Damage to RNA that is caused by sam pies (various sample types such as oral épithélial cells, exfoliated cells, throat swabs, etc.) or sampling consumables in a medical sampling process is avoided while the method of the present invention is used, which is very conducive to longterm préservation and détection of the RNA virus.

[0027] It should be noted that in a case where the sample treated with the pretreatment solution of the present invention is not preserved but directly (i.e., 0 hr after treatment) subjected to EFNART-based détection, as confirmed in the examples below, components of the pretreatment solution of the present invention hâve an effect of enhancing RT-PCT.

[0028] In a second aspect, the present invention provides a method of detecting viral nucleic acid in a sample, including: directly performing détection by using a reaction solution prepared by the aforementioned method to perform qPCR amplification.

[0029] A sample releasing agent refers to a Chemical agent that can release nucleic acid in a sample, such as a Chemical agent with strong acidity or strong basicity. An exemplary sample releasing agent may include one or more of components such as 0.01-0.5 mmol/L surfactin, 100-200 mmol/L potassium chloride, 50-200 mmol/L lithium chloride, triethanolamine dodecyl sulfate with a mass/volume ratio of 0.1-1%, ethyl phenyl polyethylene glycol (NP-40) with a volume/volume ratio of 0.1-1%, sodium dodecyl sulfonate with a mass/volume ratio of 0.01-2%, éthanol with a volume/volume ratio of 0.051%, etc., but the present invention is not limited thereto.

[0030] A qPCR amplification reagent refers to a reagent for a real-time quantitative nucleic acid amplification test. It can be understood by those skilled in the art that the qPCR amplification reagent usually contains DNA polymerase, dNTP, a PCR buffer solution, etc. For example, when RNA détection is to be performed, reverse transcriptase may also be further included. It is not difficult to understand that those skilled in the art can détermine the components and concentrations of PCR reaction reagents according to spécifie needs (for example, the type and the content of a virus, etc.).

[0031] The term “one-step method” mentîoned in the present invention refers to the extraction free nucleic acid release and amplification technology (EFNART). The EFNART refers to directly performing a nucleic acid amplification test for a sample by directly combïning a sample nucleic acid releasing agent with strong basicity and a highly compatible amplification system in a circumstance where no nucleic acid extraction or purification ofthe sample îs required.

[0032] When détection of viral nucleic acid in the sample is performed based on the onestep method, the sample in the préservation solution or pretreatment solution, the sample releasing agent, and the qPCR reaction solution are directly amplîfied after being mixed. Mixing may be performed at a general ratio in the art. In an exemplary embodiment, the sample in the préservation solution or the pretreatment solution, the sample releasing agent, and the qPCR reaction solution may be present in the ratio of about 5:5:40, about 10:10:30, or about 5:15:30 (v/v), but the present invention is not limited thereto.

[0033] In a third aspect, the present invention provides a pretreatment solution for viral nucleic acid détection, including:

[0034] Trîs-HCl, EDTA-2Na, sodium chloride, an RNase inhibitor, and an antibiotic; and

[0035] the pretreatment solution has a pH of 6.5 to 8.0.

[0036] In the présent invention, Tris-HCl may be présent at a concentration of about 10 mM to about 200 mM, preferably at a concentration of about 80 mM to about 120 mM, and most preferably at a concentration of about 100 mM.

[0037] In the présent invention, EDTA-2Na may be présent at a concentration of about 8 mM to about 50 mM, preferably at a concentration of about 10 mM to about 15 mM, and most preferably at a concentration of about 10 mM.

[0038] In the présent invention, sodium chloride may be présent at a concentration of about 0.5% (w/v) to about 2% (w/v), preferably at a concentration of about 0.8% (w/v) to about 1% (w/v), and most preferably at a concentration of about 0.9% (w/v).

[0039] In the présent invention, the RNase inhibitor may be présent at a concentration of about 2 U/mL to about 800 U/mL, preferably at a concentration of about 10 U/mL to about 30 U/mL, and most preferably at a concentration of 20 U/mL.

[0040] In the présent invention, the pH value ranges from 6.5 to 8.0, preferably from 7.0 to 8.0, and îs most preferably 7.5.

[0041] The antibiotic includes, but is not limited to, Proclin antibiotics (such as Proclin 300 and Proclin 950) and NaNj.

[0042] For example, in a case where Prociin 300 is used as the antibiotic, the concentration of Proclin 300 may be about 0.01% (v/v). For another example, in a case where Proclin 950 is used as the antibiotic, the concentration of Proclin 950 may be about 0.04% (v/v), but the présent invention is not limited thereto.

[0043] In a spécifie embodiment, the pretreatment solution inlcudes 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), the RNase inhibitor at a concentration of 20 U/mL, and Proclin 950 at a concentration of 0.04% (v/v);

[0044] and the aforementioned pretreatment solution îs adjusted to a pH value of 7.5.

[0045] In some spécifie embodiments, the virus may be a DNA virus or an RNA virus.

[0046] In a preferred embodiment, the virus is an RNA virus.

[0047] In a more preferred embodiment, the virus is coronavîrus (such as SARS-CoV-2), respîratory syncytial virus, and enterovirus.

[0048] In a fourth aspect, the present invention provides use of the pretreatment solution in préparation of a one-step method-based nucleic acid amplification détection kit for virus détection.

[0049] In a fifth aspect, the present invention provides a kit for viral nucleic acid détection based on one-step method, the kit including the aforementioned pretreatment solution.

[0050] Further, the kit further includes a sample releasing agent and a qPCR amplification reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0051 ] FIG. 1 is a graph showing the results of “one-step method” testing of the SARSCoV-2 nucleic acid in the samples gradient-diluted by using the pretreatment method of the present invention and after being preserved at room température for 72 hr.

DETAILED DESCRIPTION

[0052] The present invention will be described in detail below in conjunction with spécifie 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 spécifie embodiments and exampies are used to illustrate the present invention, but not to limit the present invention.

Example 1. Pretreatment and rapid détection of respîratory syncytial virus (RSV) thorat swab samples with the present invention

[0053] 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 ofthe samples at 0 hr, 24 hr, 48 hr, and 72 hr, respectively at room température (25°C). The détection efficiency of real-time quantitative PCR (real-time qPCR) under the room température 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.

[0054] Real-time qPCR amplification testing procedures are as shown in Table 1, and the results are as shown in Table 2.

Table 1

Step	Température	Time	Number of Cycles
Reverse transcription	60°C	30 min	1
Pre-denaturation	95°C	1 min	1
Dénaturation	95°C	15 sec	40-45
Annealing, extension, and fluorescence collection	60°C	30 sec

Table 2

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

[0055] The aforementioned results show that with the virus pretreatment method of the present invention, pretreatment at room température for 24 hr, the gradient-dîluted RSV sample can be well pretreatment, and can be directly used for EFNART détection, 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 sait pretreatment solution, which are commonly used and commercîally 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 détection SARS-CoV-2 samples after purification with the present invention

[0056] 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 commercîally available virus pretreatment solutions. The comparison method was employed by performing dilution (1:9, v/v) pretreatment on clinîcally 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 température (25°C). The détection efficiency of real-time qPCR under the room température 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-dîluted by using the virus pretreatment method of the present invention was preserved at room température for 72 hr. The results of the “one-step method” testîng are as shown in FIG. 1.

Table 3

	Ct Value by Détection in Original Sample	Ct Value by Détection in 10-fold Diluted Sample
Pretreatment Time	Ohr	24 hr	48 hr	72 hr	0 hr	24 hr	48 hr	72 hr
Pretreatment solution of the présent invention	28.59	28.68	29.01	28.97	32.01	31.89	32.14	32.20
Saline solution	28.95	29.48	29.98	30.54	33.01	3.21	33.14	34.28
Hank’s pretreatment solution	31.02	32.35	33.62	No Ct	NoCt	No Ct	No Ct	No Ct
Guanidine hydrochloride pretreatment solution	No Ct	No Ct	No Ct	No Ct	No Ct	NoCt	No Ct	No Ct

[0057] The aforementioned results show that with the virus pretreatment method of the présent invention, pretreatment at room température for 24 hr, the gradient-diluted SARSCoV-2 nucleic acid can be well pretreated, and can be directly used for EFNART détection,, 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 sait pretreatment solution, which are commonly used and commercial!y avaîlabié, cannot provide an effective way of performîng virus pretreatment and a PCR amplification test in this scheme.

Example 3. Pretreatment and rapid détection of Enterovirus (EV) universal throat swab samples with the présent invention

[0058] In order to evaluate the virus pretreatment solution of the présent invention, comparative analysis was performed on the virus pretreatment solution of the présent invention (Tris-HCl at a concentraion of 100 mM, EDTA-2Na at a concentraion of 10 mM, sodium chloride at a concentraion of 0.9% (w/v), SDS at a concentraion of 0.1%, and Proclin 950 at a concentraion of 0.04% (v/v)), a saline solution, and commercially availabié virus pretreatment solutions. The comparison method was employed by performîng 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, respectîvely at room température (25 °C). The détection efficiency of real-time qPCR under the room température 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 4

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

[0059] The aforementioned results show that with the virus pretreatment method of the present invention, pretreatment ai room température for 24 hr, the gradient-diluted EV samples can be well pretreated, and can be directly used for EFNART détection, 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 ts degraded to a certain extent, which affects the amplification efficiency. In contrast, the Hank's pretreatment solution and the guanidine sait 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

[0060] Compared with DNA viruses, pretreatment and détection 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 détection of RNA viruses. In order to evaluate the pretreatment effect of the virus pretreatment solution of the present invention (Tris-HCl at a concentraion of 100 mM, EDTA-2Na at a concentraion of 10 mM, sodium chloride at a concentraion of 0.9% (w/v), Rnasin at a concentraion of 20 Ό/mL, and Proclin 950 at a concentraion 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 pg/mL RNase A was respectively added into solution A and solution C, the three solutions containing the SARSCoV-2 nucleic acid were pretreated at room température (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. Détection 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 5

SARS-CoV-2 Nucleic Acid Pretreatment Condition	Ct Value by Détection
Pretreated nucleic acid sample A (pretreatment solution of the présent invention containing 0.25 g/mL Rnase A)	FAM 30.8	HEX 26.8	ROX 29.6
Pretreated nucleic acid sample B (pretreatment solution ofthe présent invention without0.25 g/mL Rnase A)	FAM 30.9	HEX 27.1	ROX 30.2
Pretreated nucleic acid sample C (pretreatment with saline solution containing 0.25 g/mL Rnase A)	FAM No Ct	HEX 27.1	ROX No Ct
Pretreated nucleic acid sample D (pretreatment with saline solution without 0.25 g/mL Rnase A)	FAM 32.2	HEX 29.4	ROX 32.9

[0061] 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 pg/mL RNase A in the présent invention has no influence on nucleic acid détection effect of SARS-CoV-2, and effective components in the présent invention can digest RNase, reduce the influence of RNase on experimental détection, and can ensure the efficiency of direct détection of RNA viruses. It can be seen from pretreated nucleic acid sample C, the addition of 0.25 pg/mL RNase A included in the experimental conditions can digest and dégradé the RNA in the experiment, thereby greatly affecting direct détection of RNA viruses and leading to risk of missed détection.

Example 5. Pretreatment and rapid détection of hepatitis B virus (HBV) sérum sample by using virus pretreatment solution of the présent invention

[0062] In order to evaluate the effect of the virus pretreatment solution of the présent invention on DNA virus préservation and amplification détection, comparative analysis was performed on the virus pretreatment solution of the présent invention (Tris-HCI 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 sérum samples, and direct amplification ofthe samples at 0 hr, 24 hr, 48 hr, and 72 hr, respectîvely at room température (25°C). The détection efficiency of real-time qPCR under the room température pretreatment condition was compared by Ct values to evaluate the effects of different pretreatment solutions on virus pretreatment. The qPCR amplification test was empîoyed by usîng the EFNART “one-step method” technique, such that a real-time qPCR amplification test was directly performed în 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 6

	Ct Value by Détection in HBV Sérum Sample
Pretreatment Time	Ohr	24 hr	48 hr	72 hr
Pretreatment solution of the présent invention	32.23	31.98	32.24	32.67
Saline solution	33.43	33.18	33.54	33.87
Hank's pretreatment solution	33.74	33.49	33.85	34.18
Guanidine hydrochlorîde pretreatment solution	No Ct	No Ct	No Ct	No Ct

[0063] The aforementioned results show that although the original design of the virus pretreatment method of the présent invention was aimed at virus préservation matrix for RNA viruses for performing amplification based on the “one-step method”, the virus pretreatment method can also be used for préservation and détection of DNA viruses by performing amplification based on the one-step method.

Example 6. Vérification of effectiveness of various components of virus pretreatment solution of the présent invention

[0064] In order to verify the effectiveness of various components in the présent invention, after relevant components in the présent 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 température for 24 hr that were prepared at different concentrations, and the détection efficiency of real-time qPCR under the room température 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 7

Détection Effects of Separately Optimized components in the Present Invention on Pretreatment
RNase inhibitor	Antibiotîc (Proclin 950)	EDTA-2Na	Solution pH Value 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

[0065] Based on the aforementioned experiments, the Chemical components in the present invention are essentîal components, and at the concentrations in the present invention, the pretreatment and détection of RNA viruses by employing one-step RT-PCR are optimal.

Claims (10)

1. A pretreatment method for viral nucleic acid détection, 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 ribonucléase (RNase) inhibitor, and an antibiotic;

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%;

the ribonucléase (RNase) inhibitor is a RNase protein inhibitor (RNasin), the antibiotic is Proclin 300 or Proclin 950;

and, the pretreatment solution has a pH of 6.5-8.0.

2. The method according to claim 1, wherein the RNase protein inhibitor is replaced with SDS at a concentration of 0.1%.

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 a reaction solution prepared by the method can be used to directly perform a qPCR amplification test.

5. The method according to any one of daims 1-4, wherein the virus is a DNA virus or an RNA virus.

6. A pretreatment solution for viral nucleic acid détection, the pretreatment solution comprising: Tris-HCl, EDTA-2Na, sodium chloride, a ribonucléase (RNase) inhibitor, and an antibiotic, in the pretreatment solution, the Tris-HCl îs present at a concentration of 10 mM to 200 mM, the EDTA-2Na îs 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 îs present at a concentration of 0.005% to 0.05%;

the ribonucléase (RNase) inhibitor is a RNase protein inhibitor (RNasin), the antibiotic is Proclin 300 or Proclin 950;

and, the pretreatment solution has a pH of 6.5-8.0.

7. The pretreatment solution according to claim 6, wherein the RNase protein inhibitor is replaced with SDS at a concentration of 0.1%.

8. The pretreatment solution according to claim 6, wherein in the pretreatment solution, the Tris-HCl îs 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.

9. The pretreatment solution according to any one of claims 6-8, wherein the virus is a DNA virus or an RNA virus.

10. A kit for viral nucleic acid détection based on the one-step method, the kit comprising the pretreatment solution defïned according to any one of claims 6-9.