CN115948616B

CN115948616B - Quantitative detection method for coxsackievirus B1 nucleic acid

Info

Publication number: CN115948616B
Application number: CN202211623140.5A
Authority: CN
Inventors: 黄芳; 杨立生; 王倩; 刘冰; 付文锟; 施炜
Original assignee: Hangzhou Yangshengtang Biopharmaceutical Co ltd
Current assignee: Hangzhou Yangshengtang Biopharmaceutical Co ltd
Priority date: 2022-12-16
Filing date: 2022-12-16
Publication date: 2024-02-06
Anticipated expiration: 2042-12-16
Also published as: CN115948616A

Abstract

The invention relates to the field of virus detection, in particular to a primer and a probe for detecting coxsackievirus B1 (CVB 1) and a method for detecting CVB1 by using the primer and the probe. Furthermore, the present invention relates to a stabilizer for CVB1 nucleic acid extraction and a method for extracting CVB1 nucleic acid using the same.

Description

Quantitative detection method for coxsackievirus B1 nucleic acid

Technical Field

The invention relates to the field of virus detection, in particular to a primer and a probe for detecting coxsackievirus B1 (CVB 1) and a method for detecting CVB1 by using the primer and the probe.

Furthermore, the present invention relates to a stabilizer for CVB1 nucleic acid extraction and a method for extracting CVB1 nucleic acid using the same.

Background

Coxsackie virus type B1 (CVB 1) is a common human pathogen belonging to the genus Enterovirus (Enterovirus) of the family Picornaraadae, a non-enveloped single-stranded sense strand RNA virus with a genome length of about 7500nt. CVB1 mainly infects infants, and clinical symptoms are shown as fever, cold, mild upper respiratory symptoms, diarrhea, gastrointestinal diseases and the like, and can also cause severe diseases including aseptic meningitis, viral myocarditis, dilated cardiomyopathy, hepatitis and pancreatitis, and can sometimes cause death. No effective prophylactic or therapeutic drugs are currently available on the market.

The most commonly used quantitative detection methods of viruses at present mainly comprise the following three categories: (1) Detection techniques based on viral infectivity, e.g. viral plaque formation assay, half tissue culture infectious dose TCID ₅₀ The method is a traditional classical method, but is time-consuming and labor-consuming, is easily influenced by cell sensitivity and culture conditions for detection, and has larger fluctuation of detection results; (2) Methods for direct counting of viral particles, such as flow cytometry or transmission electron microscopy. The method has higher quantitative accuracy for determining the shape of the virus and the total number of virus particles, but has higher cost, low detection flux and depends on expensive equipment; (3) Viral nucleic acid and viral protein detection techniques such as quantitative polymerase chain reaction (qPCR), immunoblotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), and hemagglutination assays. Methods for detecting viral antigens or viral genes are generally relatively rapid and have good specificity and reproducibility. In particular, the qPCR-based nucleic acid quantitative method has the advantages of rapid detection, high sensitivity and large detection flux, and has been widely appliedThe method is used for quantitative detection of clinical specimens or virus biological products.

At present, the enterovirus nucleic acid detection method is reported to be a general detection method designed based on a conserved region of a non-coding region of a 5' UTR, and focuses on qualitative detection of a sample, but does not carry out systematic optimization on a quantitative system. Commercial typing detection kit and literature report typing detection mainly surround detection of hand-foot-and-mouth disease related pathogens such as EV71, CVA16, CVA6 and the like, and few quantitative detection methods for nucleic acid of coxsackievirus B group 1 (CVB 1) are reported. In the currently reported CVB1 virus nucleic acid quantification method, the optimization of an amplification system and an amplification program and the investigation of method performance are mainly performed, but the researches on sample pretreatment, accurate quantification of a standard substance and the like are lacking. In addition, because the genome of the CVB1 virus is RNA, in the nucleic acid extraction link, the virus nucleic acid is easily degraded by polluted RNase, thereby affecting the recovery rate of the virus nucleic acid and the quality of an amplified template, and finally affecting the accurate quantification of the virus nucleic acid. In addition, the detection accuracy of the nucleic acid quantitative detection method also depends on the accurate quantitative assignment of the standard substance/quality control substance. The standard assignment methods commonly used at present are as follows: (1) Quantitative assignment was performed using an ultraviolet spectrophotometer method. The quantitative assignment of the ultraviolet spectrophotometer to nucleic acid can be influenced by impurities, DNA content and the like, the sensitivity and assignment accuracy are limited, and absolute quantitative assignment cannot be carried out; (2) A virus infection titer detection method was used as a quality control/standard assignment method. Since the virus infection titer represents the virus content capable of infecting under certain conditions, the virus titer may be affected by the titer detection conditions, including cell type, culture conditions, and the infectivity of the strain, etc., and the virus nucleic acid copy number is a physical content, including the nucleic acid inside the virus particle and the free virus nucleic acid, there is no fixed correspondence between the infection titer and the virus nucleic acid.

In conclusion, developing a CVB1 specific quantitative detection method has important significance for early diagnosis and timely intervention of CVB1 related diseases and development of related vaccines or therapeutic drugs.

Disclosure of Invention

Through a great deal of research, the inventor of the application develops a primer and a probe set for detecting CVB1, and establishes a nucleic acid quantitative detection method of CVB1 based on the primer and the probe set, wherein the method has high accuracy and sensitivity and good specificity, and can be used for specifically and quantitatively detecting CVB1 in various serotypes of Coxsackie viruses.

In addition, the inventor optimizes the virus nucleic acid extraction treatment process, can obviously improve the recovery rate and stability of virus nucleic acid and eliminates the interference of free nucleic acid on the detection result.

Primer and probe set

Thus, in one aspect, the present application provides primers and probe sets for detecting CVB1 comprising a first primer having the sequence shown in SEQ ID NO.1, a second primer having the sequence shown in SEQ ID NO.2, and a probe having the sequence shown in SEQ ID NO. 3.

In certain embodiments, the probes are labeled with a reporter group (e.g., a fluorescent group) and a quencher group (e.g., a fluorescence quencher group).

One skilled in the art will readily appreciate that to facilitate detection of the presence or amount of a target sequence (e.g., a CVB1 nucleic acid sequence) using the probe, the probe emits a different signal if hybridized to the sequence it is targeting than if not hybridized to the sequence it is targeting. In certain embodiments, when the probe does not hybridize to the sequence to which it is targeted, the quenching group is located at a position that is capable of absorbing or quenching the signal of the reporter group (e.g., the quenching group is located adjacent to the reporter group), thereby absorbing or quenching the signal emitted by the reporter group. In this case, the probe does not emit a signal. Further, when the probe hybridizes to the sequence to which it is targeted, the quencher is positioned at a location that is incapable of absorbing or quenching the signal of the reporter (e.g., the quencher is positioned at a location remote from the reporter), thereby incapable of absorbing or quenching the signal emitted by the reporter. In this case, the probe emits a signal.

In certain embodiments, the probe has a reporter group (e.g., a fluorescent group) attached to the 5 'end and a quencher group (e.g., a fluorescence quencher group) attached to the 3' end.

In certain embodiments, the 5 'end of the probe is attached to a FAM fluorescent group and/or the 3' end of the probe is attached to a BHQ1 fluorescence quenching group.

In another aspect, the present application provides a method of detecting CVB1 comprising using the primer and probe set described above.

In certain embodiments, the method comprises the steps of:

(1) Providing the primer and the probe set and a sample to be tested;

(2) Contacting the primer and the probe set with a sample to be detected, and carrying out RT-qPCR reaction; and, a step of, in the first embodiment,

(3) And judging the result.

It will be readily appreciated that the source of the sample to be tested is not limited and may be from, but is not limited to, blood samples, urine samples, respiratory tract secretion samples (e.g., nasal secretions), fecal samples, and the like.

In certain embodiments, the test sample in step (1) is subjected to a pretreatment comprising steps of RNA extraction and/or purification.

In certain embodiments, the test sample is obtained by pretreatment of a viral sample, and the pretreatment comprises the steps of:

(i) Lysing the virus; and, a step of, in the first embodiment,

(ii) Purifying RNA released by virus lysis.

In certain embodiments, in step (i), the method lyses the virus in the presence of a virus stabilizer, wherein the virus stabilizer is a His buffer containing Tween-80.

In certain embodiments, the Tween-80 is at a concentration ofTo 1.5%o (v/v) (e.g.)>To 1.25%o (v/v),>to 1.0%o (v/v),>to 1.5%o (v/v),>to 1.25%>To 1.0 per mill (v/v)).

In certain embodiments, the His buffer further contains NaCl.

In certain embodiments, the concentration of NaCl is 0.3 to 0.8M (e.g., 0.3 to 0.5M, 0.5M).

In certain embodiments, the His buffer has a pH of 6.0-6.5 (e.g., 6.2-6.5,6.25-6.5,6.0-6.4,6.0-6.25 or 6.25).

In certain embodiments, the concentration of His in the His buffer is 10-50mM (e.g., 10-30mM,10-20mM,20-50mM,20-30mM,20 mM).

In certain embodiments, prior to step (i), the pretreatment further comprises the step of removing free nucleic acid from the virus sample.

In certain embodiments, the pretreatment removes free nucleic acids from the virus sample by enzymatic hydrolysis.

In certain embodiments, the pretreatment removes free nucleic acids from the virus sample by using nucleases (e.g., DNase and/or RNase).

In certain embodiments, in step (3), the method judges by the results of an RT-qPCR reaction: whether the sample to be tested contains nucleic acid derived from CVB1 or whether the sample to be tested contains CVB1.

In certain embodiments, in step (3), the method further comprises determining the content of CVB1 derived nucleic acid in the test sample from the results of the RT-qPCR reaction; or, the content of CVB1 in the sample from which the sample to be tested is derived.

In certain embodiments, in step (3), the method determines the content of CVB1 derived nucleic acid in the test sample or the content of CVB1 in the sample from which the test sample is derived by comparing the Ct value of the test sample with the Ct values of a series of quantified standards.

In certain embodiments, the standard is purified RNA of CVB1 or purified CVB1.

In certain embodiments, the standard is purified CVB1. As will be readily appreciated by those of skill in the art, in such embodiments, the standard is subjected to a pretreatment comprising RNA extraction and/or purification steps to release RNA for detection of Ct values; when the sample to be detected is obtained through pretreatment of a virus sample, the pretreatment of the standard substance is the same as the pretreatment of the virus sample.

In certain embodiments, in the method, the Ct values of the test sample and the standard are obtained by detection under the same conditions.

In certain embodiments, the standard is quantified by ddPCR.

In another aspect, the present application also provides a kit comprising a primer and a probe set as described above.

In certain embodiments, the kit further comprises one or more of the following:

(a) A virus stabilizer, wherein the virus stabilizer is His buffer solution containing Tween-80;

(b) Nucleases (e.g., DNase and/or RNase);

(c) Reagents required for RT-qPCR.

In certain embodiments, the His buffer further contains NaCl.

In another aspect, the present application also provides the use of a primer set or a probe set or a kit as described above for preparing a detection reagent for detecting CVB1.

Virus stabilizer

In another aspect, the present application also provides a virus stabilizer which is a His buffer containing Tween-80.

In certain embodiments, the Tween-80 is at a concentration ofTo 1.5%o (v/v) (e.g.)>To 1.25 per mill (v/v),/>to 1.0%o (v/v),>to 1.5%o (v/v),>to 1.25%>To 1.0 per mill (v/v)).

In certain embodiments, the His buffer further contains NaCl.

In another aspect, the present application also provides a method for extracting CVB1 viral nucleic acid, which comprises using the viral stabilizer described above.

In certain embodiments, the method comprises the steps of:

(i) Lysing the virus in the presence of the virus stabilizing agent; and, a step of, in the first embodiment,

(ii) Purifying RNA released by virus lysis.

In another aspect, the present application also provides the use of a viral stabilizer as described above in the preparation of a reagent for extracting a CVB1 nucleic acid.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the virology, biochemistry, immunology laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

When used herein, the terms "for example," such as, "" including, "" comprising, "or variations thereof, are not to be construed as limiting terms, but rather as meaning" but not limited to "or" not limited to.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Advantageous effects of the invention

The primer and the probe set provided by the application can be effectively used for CVB1 detection, and the CVB1 detection method based on the primer and the probe set has the advantages of high accuracy, high sensitivity and good specificity, and can be used for specifically and quantitatively detecting CVB1 in various serotypes of coxsackie viruses. In addition, the method can further quantify CVB1 in a sample, and the quantification range is wide (for example, 9.87×10 ¹ ～9.87×10 ⁷ The copies/. Mu.L), the detection limit is low (for example, 10 copies/. Mu.L), the amplification efficiency of the standard substance is stable, the requirement of 90-110% is met, and the quantitative CV of the sample is less than or equal to 15%.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

Fig. 1: the standard in example 4 was quantitatively fitted to the curve.

Fig. 2: effect of nuclease pretreatment on the quantitative effect of viruses in example 6.

Fig. 3: method specificity study in example 7, wherein "PC" represents positive control and "NC" represents negative control.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Example 1: design and synthesis of primers and probes

By comparing CVB1 strain sequences in GenBank database, a region which is relatively conserved in VP1 coding region and is specific to CVB1 strain is selected, a primer and a probe are designed, the nucleotide sequence of a forward primer is SEQ.ID NO.1, the nucleotide sequence of a reverse primer is SEQ.ID NO.2, the nucleotide sequence of the probe is SEQ.ID NO.3 (specific sequence is shown in Table 1), the reporting fluorescent dye FAM is marked at the 5 'end of the probe, the quenching fluorescent dye TAMRA is marked at the 3' end of the probe, and the primer and the probe are both consigned to Shanghai Bailigo company for synthesis.

TABLE 1 primer and probe sequences

Example 2: nucleic acid extraction and RT-qPCR amplification method

1) Nucleic acid extraction: the virus stabilizer (20 mM His buffer, pH=6.25, containingTween80,0.5M NaCl) was used to dilute CVB1 virus samples in a certain proportion. CVB1 virus sample nucleic acid was extracted using a magnetic bead method virus DNA/RNA extraction kit (CRITIC, cat# NVDRE-5010) and a full-automatic nucleic acid extraction and purification instrument (GenMagbio, model: DOF-9648), with the extraction procedure set forth in the kit instructions. The purified nucleic acid product should be used in time, otherwise, should be frozen at less than or equal to-60 ℃.

2) RT-qPCR amplification

Using a Hycells Fast-RT one-step reaction system (and cardiology, cat# HX 106-100), a one-step RT-qPCR reaction was performed according to the instructions. Fluorescent quantitative RT-qPCR reaction systems (25. Mu.L/well) were formulated as shown in Table 2. The real-time fluorescence quantitative PCR instrument of the Rockwell LightCycler 480II is used for nucleic acid amplification and quantitative detection, and the amplification program parameters are as follows: reverse transcription is carried out for 15min at 50-55 ℃ and reaction is carried out for 15min at 95 ℃; denaturation at 94℃for 15s, annealing/extension at 50-55℃for 45s, 40-45 cycles (FAM fluorescence signal was collected at the end of each cycle).

TABLE 2 preparation of RT-qPCR reaction System

Reagent(s)	Dosage of
		5×Fastcell RT-qPCR buffer	5μL
Fastcell RT-qPCR enzyme system	2.5μL
		Forward primer SEQ ID NO.1	0.25μL(10mM)
Reverse primer SEQ ID NO.2	0.25μL(10mM)
		Probe SEQ ID NO.3	0.25μL(10mM)
Purification of RNA	10μL
		DEPC treated water	6.75μL

3) Processing of data

The data processing was performed using the LightCycler 480II device with its own software. The sample type is named (Unknow, standard, negative control, etc.), and the standard is assigned. The selection assay method is Abs Quant/2nd Derivative Max assay and High Confidence, and Filter Comb is 485-533 (FAM channel). And (3) automatically calculating by using an instrument to obtain a drawn amplification curve, a standard fitting curve, a sample quantitative result and the like.

Example 3: preparation and quantitative assignment of standards

Preparation of a standard: HEK293 suspension cells (QuaCell) were cultured using HEK293 CD medium (QuaCell), CVB1 virus was inoculated at moi=0.01, and virus cultures were harvested 48h after inoculation. The virus culture was repeatedly freeze-thawed 3 times to disrupt cells to release virus. Host nucleic acids were digested with Benzonase nuclease (Merck), clarified, filtered, concentrated by ultrafiltration using 100KD membrane pack (pal), purified by two-step chromatography with Capto Core 400 and Capto Q ImpRes, and finally replaced in 20mM His buffer (containing 0.5m nacl, ph=6.25). The purified CVB1 sample was subjected to sub-packaging (0.5 mL/piece), and the sub-packaged standard was stored at-80℃in a frozen state.

Quantitative assignment of standard: nucleic acid of CVB1 standard was extracted as described in example 2, and nucleic acid was quantified by ddPCR (microdroplet digital PCR) method using the primers and probes of example 1, which was commissioned by microorganism company. The quantitative result was 9.87×10 ⁷ copies/μL。

Example 4: linearity of the method of the present application

Select 9.87×10 ⁷ CVB1 standard (prepared in example 3) at a concentration of cobies/. Mu.L was diluted in a 10-fold gradient and subjected to nucleic acid extraction and amplification in a total of 7 gradients by the method described in example 2 to establish a quantitative standard curve, and the results are shown in Table 3 and FIG. 1. The 7 gradient standard amplification curves conform to a typical S-type amplification curve, the Ct value and the nucleic acid concentration are fitted to be linear, and R ² More than 0.99, the amplification efficiency is 90-100%, so the quantitative linear range of the method is at least 9.87×10 ¹ copies/. Mu.L to 9.87X10 ⁷ copies/μL。

TABLE 3 quantitative standard curve and linear analysis

Example 5: screening of viral stabilizers

The standards were subjected to gradient dilution using different virus dilutions, respectively, for nucleic acid extraction and amplification detection as described in example 2. The effect of the dilution of the standard with different virus dilutions was examined, and as shown in table 4, the results showed that when a His buffer containing Tween80 and NaCl (hereinafter, collectively referred to as a virus stabilizer) was used as the dilution, the adsorption loss of virus in pretreatment and nucleic acid extraction was reduced, and the recovery rate of virus nucleic acid was effectively improved, thereby improving the amplification efficiency.

TABLE 4 Effect of different viral dilutions on amplification efficiency

Note that: in groups 3-6, the concentration of NaCl was 0.5M; his buffer concentration was 20mM, pH 6.25; the concentration of Tween80 was calculated as v/v.

In order to examine the applicability of the virus stabilizer in different types of samples, blood, nasal secretion and urine samples of cynomolgus monkeys were selected for examination, respectively.

1) Optimization group: the virus stabilizer (20 mM His buffer, pH=6.25, containingTween80,0.5M NaCl) to 4.94X10 s ³ The concentration of cobies/. Mu.L was mixed with an equal volume of blank matrix sample (blood, nasal secretions, urine), and finally RNase inhibitor (final concentration 1U/. Mu.L) was added and mixed by shaking.

2) Control group: dilution of disease with 1 XPBSToxic standard to 4.94×10 ³ The concentration of cobies/. Mu.L was mixed with an equal volume of blank matrix sample (blood, nasal secretions, urine), and finally RNase inhibitor (final concentration 1U/. Mu.L) was added and mixed by shaking.

Nucleic acid extraction, RT-qPCR amplification was performed as described in example 2. The results are shown in Table 5, and the extraction recovery rates of different types of samples are significantly improved after the addition of the virus stabilizers.

TABLE 5 extraction recovery of different types of blank samples

Example 6: effect of nuclease pretreatment on the quantitative effects of viruses

Complete viral particles containing genomic nucleic acid are key active ingredients of CVB1 related vaccines or therapeutic drugs, and therefore accurate quantification of genomic nucleic acid in complete viral particles in a sample is very important. In each type of CVB1 virus sample, free viral nucleic acid may be present, or some processing may release viral nucleic acid from the viral capsid, which may interfere with nucleic acid quantification of the whole viral particle. By adding nuclease to the sample, free viral nucleic acid can be cleaved off, while viral nucleic acid in the whole viral particle cannot be cleaved off due to the protection of the viral capsid.

To verify the potential interference of free nucleic acids, this example used Benzonase nuclease (Merck) to pre-treat different types of CVB1 samples.

1) Digestion group: adding Benzonase nuclease (final concentration 100 IU/mL) and MgCl into cell culture to be detected, freeze-drying and freezing preparation samples ₂ (final concentration 2 mM), shaking and mixing; digestion was incubated in an incubator (Mei Mo Erte, model 110 plus) at 25℃for 2h.

2) Non-digestible group: adding MgCl only into cell culture to be detected, freeze-drying and freezing preparation samples ₂ (final concentration 2 mM), shaking and mixing; incubator at 25℃ (Mei Mo Erte model 110 plus)Digestion was performed for 2h.

Nucleic acid extraction and quantitative detection were performed by the methods described in examples 2 and 4. As a result, as shown in FIG. 2, the detection value was decreased in a certain proportion after the CVB1 sample was pretreated with nuclease, indicating that there was a certain proportion of free nucleic acid in the sample. In particular, for freeze-dried CVB1 virus samples, the nucleic acid quantification results are greatly reduced after nuclease is added, which suggests that the freeze-drying process can damage the integrity of virus particles to release virus nucleic acid and the virus nucleic acid is digested and removed by nuclease. Therefore, the nuclease pretreatment can effectively remove the interference of free viral nucleic acid in the sample, so that the detection result can more accurately reflect the content of genome nucleic acid in the complete viral particles.

Example 7: specificity of the methods of the present application

Samples of CVA6, CVB6 and CVB1 virus cultures were selected for nucleic acid extraction and nucleic acid amplification as described in example 2. The results are shown in FIG. 3, which shows that CVA6 and CVB6 virus samples have no typical S-type amplification curve, and that CVB1 virus samples have typical S-type amplification curves. The result shows that the primer and the probe provided by the application can specifically detect CVB1 virus.

Example 8: detection limit of the method

The virus stabilizer (20 mM His buffer, pH=6.25, containingTween80,0.5M NaCl) was used to dilute CVB1 standards, virus samples were prepared at 30, 10 and 3.3 copies/. Mu.L concentrations, and nucleic acid extraction and amplification were performed as described in example 2. The results show that the method of the present application is capable of stably detecting 30, 10 copies/. Mu.L virus samples, whereas 3.3 copies/. Mu.L has no typical S-type amplification curve. Thus, the detection limit of the system is about 10 copies/. Mu.L.

Example 9: repeatability investigation of the method of the present application

The virus standards of 7 dilution gradients were quantitatively detected, and the detection was repeated 6 times in total, and nucleic acid extraction and quantitative detection were performed in the methods of example 2 and example 4. The results are shown in Table 6, and CV of the standard Ct is less than 5% and CV of the quantitative value is less than 10%, which show that the repeatability of the method is good.

Table 6 repeatability investigation of the method of the present application

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. A method of detecting CVB1 for non-diagnostic purposes comprising the steps of:

(1) Providing a primer and a probe set and a sample to be tested; the primer and the probe group comprise a first primer with a sequence shown as SEQ ID NO.1, a second primer with a sequence shown as SEQ ID NO.2 and a probe with a sequence shown as SEQ ID NO. 3;

(3) Judging results;

wherein the sample to be tested is obtained by pretreatment of a virus sample, and the pretreatment comprises the following steps:

(i) Lysing the virus; and, a step of, in the first embodiment,

(ii) Purifying RNA released by virus lysis;

wherein in step (i), the method lyses the virus in the presence of a virus stabilizing agent, wherein the virus stabilizing agent is a His buffer containing only Tween-80 and NaCl.

2. The method of claim 1, wherein the concentration of Tween-80 isTo 1.0%v/v)。

3. The method of claim 1, wherein the concentration of Tween-80 isTo 1.0 per mill (v/v).

4. The method of claim 1, wherein the concentration of NaCl is from 0.3 to 0.5M.

5. The method of claim 1, wherein the His buffer has a pH of 6.0-6.5.

6. The method of claim 1, wherein the His buffer has a pH of 6.2-6.5.

7. The method of claim 1, wherein the His buffer has a pH of 6.25-6.5.

8. The method of claim 1, wherein the His buffer has a pH of 6.0-6.4.

9. The method of claim 1, wherein the His buffer has a pH of 6.0-6.25.

10. The method of claim 1, wherein the His buffer has a pH of 6.25.

11. The method of claim 1, wherein the concentration of His in the His buffer is 10-50mM.

12. The method of claim 1, wherein the concentration of His in the His buffer is 10-30mM.

13. The method of claim 1, wherein the concentration of His in the His buffer is 10-20mM.

14. The method of claim 1, wherein the concentration of His in the His buffer is 20-50mM.

15. The method of claim 1, wherein the concentration of His in the His buffer is 20-30mM.

16. The method of claim 1, wherein the concentration of His in the His buffer is 20mM.

17. The method of claim 1, wherein the probe is labeled with a reporter group and a quencher group.

18. The method of claim 17, wherein the probe has a reporter group attached to the 5 'end and a quencher group attached to the 3' end.

19. The method of claim 17 or 18, wherein the reporter group is a fluorescent group and/or the quencher group is a fluorescence quencher group.

20. The method of claim 1, wherein the 5 'end of the probe is attached to a FAM fluorescent group and/or the 3' end of the probe is attached to a BHQ1 fluorescence quenching group.

21. The method of claim 1, wherein prior to step (i), the pretreatment further comprises the step of removing free nucleic acid from the virus sample.

22. The method of claim 21, wherein the pretreatment removes free nucleic acids from the virus sample by enzymatic hydrolysis.

23. The method of claim 22, wherein the pretreatment removes free nucleic acids from the virus sample by using nucleases.

24. The method of claim 23, wherein the pretreatment removes free nucleic acids from the virus sample by using DNase and/or RNase.

25. The method of claim 1, wherein in step (3), the method judges by the result of an RT-qPCR reaction: whether the sample to be tested contains nucleic acid derived from CVB1 or whether the sample to be tested contains CVB1.

26. The method of claim 25, further comprising, in step (3), determining the content of CVB1 derived nucleic acid in the test sample from the result of the RT-qPCR reaction; or, the content of CVB1 in the sample from which the sample to be tested is derived.

27. The method of claim 26, wherein in step (3), the method determines the content of CVB1 derived nucleic acid in the test sample or the content of CVB1 in the sample from which the test sample is derived by comparing the Ct value of the test sample with the Ct values of a series of quantified standards.

28. The method of claim 27, wherein the standard is purified RNA of CVB1 or purified CVB1.

29. The method of claim 27, wherein the standard is purified CVB1.

30. The method of claim 27, wherein the standard is quantified by ddPCR.

31. A kit comprising a primer and a probe set, and a virus stabilizer;

wherein the primer and probe set are as defined in any one of claims 1, 17-20;

the virus stabilizer is His buffer solution only containing Tween-80 and NaCl.

32. The kit of claim 31, wherein the kit further comprises a nuclease and/or reagents required for RT-qPCR.

33. The kit of claim 31, wherein the concentration of Tween-80 isTo 1.0 per mill (v/v).

34. The kit of claim 31, wherein the concentration of Tween-80 isTo 1.0 per mill (v/v).

35. The kit of claim 31, wherein the concentration of NaCl is from 0.3 to 0.5M.

36. The kit of claim 31, wherein the His buffer has a pH of 6.0-6.5.

37. The kit of claim 31, wherein the His buffer has a pH of 6.2-6.5.

38. The kit of claim 31, wherein the His buffer has a pH of 6.25-6.5.

39. The kit of claim 31, wherein the His buffer has a pH of 6.0-6.4.

40. The kit of claim 31, wherein the His buffer has a pH of 6.0-6.25.

41. The kit of claim 31, wherein the His buffer has a pH of 6.25.

42. The kit of claim 31, wherein the concentration of His in the His buffer is 10-50mM.

43. The kit of claim 31, wherein the concentration of His in the His buffer is 10-30mM.

44. The kit of claim 31, wherein the concentration of His in the His buffer is 10-20mM.

45. The kit of claim 31, wherein the concentration of His in the His buffer is 20-50mM.

46. The kit of claim 31, wherein the concentration of His in the His buffer is 20-30mM.

47. The kit of claim 31, wherein the concentration of His in the His buffer is 20mM.

48. Use of a kit according to any one of claims 31 to 47 in the preparation of a detection reagent for the detection of CVB1.

49. A virus stabilizer which is a His buffer containing only Tween-80 and NaCl;

wherein the concentration of Tween-80 isTo 1.5 per mill (v/v).

50. The virus stabilizing agent of claim 49, wherein the concentration of Tween-80 isTo 1.0 per mill (v/v).

51. The virus stabilizing agent of claim 49, wherein the concentration of NaCl is 0.3 to 0.5M.

52. The viral stabilizing agent of claim 49, wherein the His buffer has a pH of 6.0-6.5.

53. The viral stabilizing agent of claim 49, wherein the His buffer has a pH of 6.2-6.5.

54. The viral stabilizing agent of claim 49, wherein the His buffer has a pH of 6.25-6.5.

55. The viral stabilizing agent of claim 49, wherein the His buffer has a pH of 6.0-6.4.

56. The viral stabilizing agent of claim 49, wherein the His buffer has a pH of 6.0-6.25.

57. The viral stabilizing agent of claim 49, wherein the His buffer has a pH of 6.25.

58. The viral stabilizing agent of claim 49, wherein the concentration of His in the His buffer is 10-50mM.

59. The viral stabilizing agent of claim 49, wherein the concentration of His in the His buffer is 10-30mM.

60. The viral stabilizer of claim 49, wherein the concentration of His in the His buffer is 10-20mM.

61. The viral stabilizer of claim 49, wherein the concentration of His in the His buffer is 20-50mM.

62. The viral stabilizer of claim 49, wherein the concentration of His in the His buffer is 20-30mM.

63. The viral stabilizer of claim 49, wherein the concentration of His in the His buffer is 20mM.

64. A method of CVB1 viral nucleic acid extraction comprising using the viral stabilizer of any one of claims 49-63.

65. The method of claim 64, wherein the method comprises the steps of:

(ii) Purifying RNA released by virus lysis.

66. Use of a viral stabilizing agent according to any one of claims 49-63 in the preparation of a reagent for extracting CVB1 nucleic acid.