CN115948616A - Nucleic acid quantitative detection method for coxsackievirus B1 - Google Patents

Nucleic acid quantitative detection method for coxsackievirus B1 Download PDF

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CN115948616A
CN115948616A CN202211623140.5A CN202211623140A CN115948616A CN 115948616 A CN115948616 A CN 115948616A CN 202211623140 A CN202211623140 A CN 202211623140A CN 115948616 A CN115948616 A CN 115948616A
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sample
cvb1
virus
nucleic acid
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CN115948616B (en
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黄芳
杨立生
王倩
刘冰
付文锟
施炜
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Hangzhou Yangshengtang Biopharmaceutical Co ltd
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Abstract

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

Description

Nucleic acid quantitative detection method for coxsackievirus B1
Technical Field
The invention relates to the field of virus detection, in particular to a primer and a probe for detecting coxsackievirus B1 type (CVB 1) and a method for detecting the CVB1 by using the primer and the probe.
In addition, the present invention relates to a stabilizer for CVB1 nucleic acid extraction and a method for extracting CVB1 nucleic acid using the same.
Background
Coxsackievirus type B1 (CVB 1) is a common human pathogen, belonging to the Enterovirus (Enterovirus) of picornaviridae (picornadiaceae), a non-enveloped single-stranded positive sense strand RNA virus with a genome length of about 7500nt. CVB1 mainly infects infants, clinical symptoms are fever, cold, mild upper respiratory symptoms, diarrhea, gastrointestinal diseases, etc., and serious diseases such as aseptic meningitis, viral myocarditis, dilated cardiomyopathy, hepatitis and pancreatitis can be caused, sometimes leading to death. No effective prophylactic or therapeutic drug is currently available on the market.
The most commonly used methods for quantitative detection of viruses at present mainly include the following three major categories: (1) Techniques for detecting 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 large fluctuation of detection results; (2) Methods for directly counting 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 and low detection flux and depends on expensive equipment; (3) Techniques for detecting viral nucleic acids and viral proteins, such as quantitative real-time polymerase chain reaction (qPCR), immunoblotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), hemagglutination assay, and the like. Methods for detecting viral antigens or viral genes are generally rapid and highly specific and reproducible. Particularly, the nucleic acid quantitative method based on qPCR has the advantages of rapid detection, high sensitivity and large detection flux, and is widely applied to the quantitative detection of clinical specimens or virus biological products.
At present, enterovirus nucleic acid detection methods are reported to be general detection methods designed based on 5' UTR non-coding region conserved regions, and are focused on sample qualitative detection, but do not systematically optimize quantitative systems. The commercial typing detection kit and the literature report typing detection mainly surround the detection of hand-foot-and-mouth disease related pathogens such as EV71, CVA16, CVA6 and the like, and the nucleic acid quantitative detection method for coxsackie virus B group 1 type (CVB 1) is rarely reported. The currently reported methods for quantifying the nucleic acid of the CVB1 virus mainly aim at optimizing an amplification system and an amplification program and investigating method performance, but lack research on sample pretreatment, accurate quantification of a standard substance and the like. In addition, as the genome of the CVB1 virus is RNA, in the nucleic acid extraction link, the viral nucleic acid is easily polluted and degraded by RNA enzyme, so that the recovery rate of the viral nucleic acid and the quality of an amplification template are influenced, and the accurate quantification of the viral nucleic acid is finally influenced. 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 following standard assignment methods are commonly used at present: (1) Quantitative assignment was performed using uv spectrophotometer method. The quantitative assignment of the ultraviolet spectrophotometer to nucleic acid is possibly influenced by impurities, DNA content and the like, the sensitivity and the assignment accuracy are limited, and absolute quantitative assignment cannot be carried out; (2) The virus infection titer detection method is used as the quality control product/standard product assignment method. Because the virus infection titer represents the virus content with the infection ability under certain conditions, and may be influenced by titer detection conditions, including cell types, culture conditions, virus strains with strong and weak infectivity, and the like, and the virus nucleic acid copy number is a physical content, including nucleic acid in virus particles and free virus nucleic acid, there is no fixed corresponding relationship between the infection titer and the virus nucleic acid.
In conclusion, the development of a CVB1 specific quantitative detection method has important significance for the early diagnosis and timely intervention of CVB 1-related diseases and the development of related vaccines or therapeutic drugs.
Disclosure of Invention
Through a large amount of researches, the inventor of the application develops a primer and a probe group for detecting CVB1, establishes a nucleic acid quantitative detection method of the CVB1 based on the primer and the probe group, has high accuracy and sensitivity and good specificity, and can specifically and quantitatively detect the CVB1 in various serotypes of Coxsackie virus.
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 a primer and probe set for detecting CVB1, comprising a first primer having a sequence shown in SEQ ID No.1, a second primer having a sequence shown in SEQ ID No.2, and a probe having a sequence shown in SEQ ID No. 3.
In certain embodiments, the probe is labeled with a reporter (e.g., a fluorophore) and a quencher (e.g., a fluorescence quencher).
It will be readily understood by those skilled in the art that to facilitate the detection of the presence or amount of a target sequence (e.g., a CVB1 nucleic acid sequence) using the probe, the signal emitted by the probe when hybridized to the sequence it targets will be different from the signal emitted when not hybridized to the sequence it targets. In certain embodiments, the quencher is positioned such that it absorbs or quenches the signal from the reporter (e.g., the quencher is positioned adjacent to the reporter) when the probe is not hybridized to the sequence to which it is targeted, thereby absorbing or quenching the signal from the reporter. 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 located at a position that is unable to absorb or quench the signal from the reporter (e.g., the quencher is located at a position remote from the reporter), and thus unable to absorb or quench the signal from the reporter. In this case, the probe emits a signal.
In certain embodiments, the probe has a reporter group (e.g., a fluorophore) attached to the 5 'end and a quencher group (e.g., a fluorescence quencher) attached to the 3' end.
In certain embodiments, the 5 'end of the probe is attached to a FAM fluorophore, 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 the use of a primer and probe set as described above.
In certain embodiments, the method comprises the steps of:
(1) Providing the primer and the probe group and a sample to be detected;
(2) Contacting the primer and the probe group with a sample to be detected, and carrying out RT-qPCR reaction; and (c) and (d),
(3) And (5) judging the result.
It will be readily appreciated that the sample to be tested is not limited in origin, and may be derived from, but not limited to, a blood sample, a urine sample, a respiratory secretions sample (e.g., nasal secretions), a fecal sample, and the like.
In certain embodiments, the sample to be tested in step (1) is subjected to a pretreatment comprising a step 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) Splitting the virus; and the combination of (a) and (b),
(ii) The RNA released by viral lysis is purified.
In certain embodiments, in step (i), the method lyses the virus in the presence of a viral stabilizing agent, wherein the viral stabilizing agent is a His buffer containing Tween-80.
In certain embodiments, the Tween-80 is present at a concentration of
Figure BDA0004003285170000041
To 1.5% o (v/v) (e.g., based on the total weight of the blood ‰)>
Figure BDA0004003285170000043
To 1.25 ‰ (v/v), -or ∑ based>
Figure BDA0004003285170000042
To 1.0 ‰ (v/v),. Or>
Figure BDA0004003285170000045
To 1.5 ‰ (v/v),. Or>
Figure BDA0004003285170000046
To 1.25% o (v/v) or +>
Figure BDA0004003285170000044
To 1.0% o (v/v)).
In certain embodiments, the His buffer further comprises NaCl.
In certain embodiments, the concentration of NaCl is from 0.3 to 0.8M (e.g., 0.3 to 0.5M, 0.5M).
In certain embodiments, the pH of the His buffer is 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 a step of removing free nucleic acids from the viral sample.
In certain embodiments, the pretreatment removes free nucleic acids from the viral sample by enzymatic digestion.
In certain embodiments, the pretreatment removes free nucleic acids in the viral sample by using a nuclease (e.g., DNase and/or RNase).
In certain embodiments, in step (3), the method determines by the results of the RT-qPCR reaction: whether the sample to be tested contains a nucleic acid derived from CVB1 or whether the sample from which the sample to be tested is derived contains CVB1.
In certain embodiments, in step (3), the method further comprises determining the content of the nucleic acid derived from CVB1 in the test sample by the result of 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 the nucleic acid derived from CVB1 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 understood by those skilled in the art, in such embodiments, the standard is subjected to a pre-treatment comprising RNA extraction and/or purification steps to release RNA for detection of Ct values; and when the sample to be detected is obtained by preprocessing a virus sample, the preprocessing of the standard substance is the same as the preprocessing of the virus sample.
In certain embodiments, in the methods, the Ct values of the test sample and the standard are determined 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 probe set as described above.
In certain embodiments, the kit further comprises one or more of the following:
(a) 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 Tween-80 concentration is
Figure BDA0004003285170000051
To 1.5% o (v/v) (e.g., based on the total weight of the blood ‰)>
Figure BDA0004003285170000053
To 1.25 ‰ (v/v), -or ∑ based>
Figure BDA0004003285170000052
To 1.0 ‰(v/v)，/>
Figure BDA0004003285170000055
To 1.5 ‰ (v/v),. Or>
Figure BDA0004003285170000056
To 1.25% o (v/v) or +>
Figure BDA0004003285170000054
To 1.0% o (v/v)).
In certain embodiments, the His buffer further comprises NaCl.
In certain embodiments, the concentration of NaCl is from 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 another aspect, the present application also provides the use of the primer and probe set or the kit as described above in the preparation of 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 present at a concentration of
Figure BDA0004003285170000061
To 1.5% o (v/v) (e.g., based on the total weight of the blood ‰)>
Figure BDA0004003285170000062
To 1.25 ‰ (v/v),. Or>
Figure BDA0004003285170000063
To 1.0 ‰ (v/v),. Or>
Figure BDA0004003285170000064
To 1.5 ‰ (v/v),. Or>
Figure BDA0004003285170000065
To 1.25% o (v/v) or +>
Figure BDA0004003285170000066
To 1.0% o (v/v)).
In certain embodiments, the His buffer further comprises NaCl.
In certain embodiments, the concentration of NaCl is from 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 another aspect, the present application also provides a CVB1 virus nucleic acid extraction method, which includes using the virus stabilizer as described above.
In certain embodiments, the method comprises the steps of:
(i) Lysing the virus in the presence of the virus stabilizing agent; and the combination of (a) and (b),
(ii) Purifying RNA released by viral lysis.
In another aspect, the present application also provides the use of a virus stabilizer as described above in the preparation of a reagent for extracting CVB1 nucleic acid.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, virological, biochemical, immunological laboratory procedures used herein are all routine procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
When the terms "for example," "such as," "including," "containing," or variants thereof are used herein, these terms are not to be considered limiting terms, but rather are to be construed to mean "without limitation" or "without limitation".
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, the method for detecting the CVB1 based on the primer and the probe set is high in accuracy and sensitivity and good in specificity, and the CVB1 can be specifically and quantitatively detected in multiple serotypes of Coxsackie viruses. In addition, the method can further quantify CVB1 in the sample, and the quantitative range is wide (for example, 9.87 multiplied by 10) 1 ～9.87×10 7 copies/mu L), low detection limit (for example, 10 copies/mu L), stable amplification efficiency of the standard product, meeting the requirement of 90-110 percent, and quantitative CV of the sample being less than or equal to 15 percent.
In addition, the inventor of the application also 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.
Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit 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 accompanying drawings and the following detailed description of the preferred embodiments.
Drawings
FIG. 1: the standard was fit to the curve quantitatively in example 4.
FIG. 2 is a schematic diagram: effect of nuclease pretreatment on virus quantification in example 6.
FIG. 3: example 7 for a process-specific study, wherein "PC" indicates a positive control and "NC" indicates a 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 a GenBank database, selecting a region with relatively conserved VP1 coding region and specificity of the CVB1 strain, designing a primer and a probe, wherein the nucleotide sequence of the forward primer is SEQ.ID NO.1, the nucleotide sequence of the reverse primer is SEQ.ID NO.2, the nucleotide sequence of the probe is SEQ.ID NO.3 (the specific sequence is shown in Table 1), the 5 'end of the probe is marked with a report fluorescent dye FAM, the 3' end of the probe is marked with a quenching fluorescent dye TAMRA, and the primer and the probe are both synthesized by Shanghai Baili Geji company.
TABLE 1 primer and Probe sequences
Figure BDA0004003285170000081
Example 2: nucleic acid extraction and RT-qPCR amplification method
1) Nucleic acid extraction: viral stabilizers (20 mM His buffer, pH =6.25, containing
Figure BDA0004003285170000082
Tween80,0.5m NaCl) diluted the CVB1 virus samples in a certain ratio. The CVB1 virus sample nucleic acid was extracted using a paramagnetic particle method virus DNA/RNA extraction kit (Yiruicheng, cat # NVDRE-5010) and a fully automatic nucleic acid extraction and purification instrument (GenMagbio, model: DOF-9648), and the extraction procedure was set with reference to the kit instructions. The purified nucleic acid product should be used in time, otherwise should be frozen at-60 deg.C or below.
2) RT-qPCR amplification
The one-step RT-qPCR reaction was carried out using the Hycells Fast-RT one-step reaction system (and Heart medical treatment, cat. HX 106-100) according to the instructions. A fluorescent quantitative RT-qPCR reaction system (25. Mu.L/well) was prepared as shown in Table 2. The amplification and quantitative detection of nucleic acid are carried out by using a Roche LightCycler 480II real-time fluorescent quantitative PCR instrument, and the amplification program parameters are as follows: reverse transcription is carried out for 15min at the temperature of 50-55 ℃, and reaction is carried out for 15min at the temperature of 95 ℃; denaturation at 94 ℃ for 15s, annealing/extension at 50-55 ℃ for 45s, and cycles of 40-45 (FAM fluorescence signal was collected at the end of each cycle).
TABLE 2RT-qPCR reaction System formulation
Reagent 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
And the LightCycler 480II equipment is used for carrying out data processing by self-contained software. Sample types were named (Unknow, standard, negative control, etc.) and standards assigned. The selective analysis method is Abs Quant/2nd Derivative Max analysis and High Confidence, and the Filter Comb is selected to be 485-533 (FAM channel). And (4) automatically calculating by the 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 standard product
Preparation of a standard substance: HEK293 suspension cells (QuaCell) were cultured using HEK293 CD medium (QuaCell), inoculated with CVB1 virus at MOI =0.01, and virus cultures harvested 48h after inoculation. The virus culture was freeze-thawed 3 times repeatedly to disrupt the cells and release the virus. Host nucleic acids were digested with Benzonase nuclease (Merck), clarified filtered, concentrated by ultrafiltration using a 100KD membrane module (PALL), purified by two-step chromatography using Capto Core 400 and Capto Q imprres, and finally replaced in 20mM His buffer (containing 0.5m nacl, ph = 6.25). Purified CVB1 samples were aliquoted (0.5 mL/aliquot) and the aliquoted standards were stored frozen at-80 ℃.
Quantitative assignment of the standard: nucleic acid of CVB1 standard was extracted by the method described in example 2, and the primers and probes of example 1 were used to quantify the nucleic acid of the standard by ddPCR (digital PCR) method. The quantitative result was 9.87X 10 7 copies/μL。
Example 4: linearity of the claimed method
Selection 9.87X 10 7 A CVB1 standard (prepared in example 3) was diluted with 10-fold gradient and subjected to nucleic acid extraction and amplification as described in example 2 for 7 gradients to establish a quantitative standard curve, and the results are shown in Table 3 and FIG. 1. The amplification curves of the 7 gradient standards conform to a typical S-shaped amplification curve, and the Ct value and the nucleic acid concentration are fitted to be linear, R 2 More than 0.99, and the amplification efficiency is 90-100%, so that the quantitative linear range of the method is at least 9.87 multiplied by 10 1 copies/. Mu.L to 9.87X 10 7 copies/μL。
TABLE 3 quantitative Standard Curve and Linear analysis
Figure BDA0004003285170000101
Example 5: screening for viral stabilizers
The standards were diluted in different virus dilutions in a gradient to perform nucleic acid extraction and amplification assays as described in example 2. The standard was diluted with different virus dilutions and the effect on amplification efficiency was examined, as shown in table 4, the results showed that when using His buffer containing Tween80 and NaCl (hereinafter collectively referred to as virus stabilizer) as the diluent, the adsorption loss of virus in pretreatment and nucleic acid extraction was reduced, and the recovery rate of virus nucleic acid was effectively increased, thereby increasing amplification efficiency.
TABLE 4 Effect of different virus dilutions on amplification efficiency
Figure BDA0004003285170000102
Note: in groups 3-6, the concentration of NaCl was 0.5M; the concentration of the His buffer solution is 20mM, and the pH value is 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 specimens of the cynomolgus monkey are respectively selected for examination.
1) And (3) optimizing group: viral stabilizers (20 mM His buffer, pH =6.25, containing
Figure BDA0004003285170000112
Tween80,0.5M NaCl) diluted virus standard to 4.94X 10 3 Mixing copies/μ L with blank matrix sample (blood, nasal secretion, urine) of equal volume, adding RNase inhibitor (final concentration of 1U/μ L), and shaking for mixing.
2) Control group: viral standards were diluted to 4.94X 10 using 1 XPBS 3 The samples with copies/mu L concentration are mixed with blank matrix samples (blood, nasal secretion, urine) with equal volume, and finally RNase inhibitor (with the final concentration of 1U/mu L) is added and mixed evenly 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 remarkably improved after the virus stabilizer is added.
TABLE 5 recovery of different types of blank samples
Figure BDA0004003285170000111
Example 6: effect of nuclease Pre-treatment on the quantification Effect of viruses
Complete virus particles containing genomic nucleic acid are key active ingredients of CVB 1-related vaccines or therapeutic drugs, and accurate quantification of genomic nucleic acid in complete virus particles in a sample is therefore very important. In each type of CVB1 virus sample, free viral nucleic acid may be present or released from the viral capsid by some process treatment, which may interfere with the nucleic acid quantification of the intact viral particles. Free viral nucleic acid can be removed by enzyme digestion by adding nuclease to the sample, while viral nucleic acid in the intact viral particles cannot be removed by enzyme digestion because of the protection of the viral capsid.
To verify potential interference with 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 is 100 IU/mL) and MgCl into the cell culture, freeze-dried and frozen preparation samples to be detected 2 (final concentration is 2 mM), shaking and mixing uniformly; digestion was incubated in an incubator (Meyer, model 110 plus) at 25 ℃ for 2h.
2) Nondigestible group: adding only MgCl into the cell culture, freeze-dried and frozen preparation samples to be detected 2 (final concentration is 2 mM), shaking and mixing uniformly; digestion was incubated in an incubator (Meyer, model 110 plus) at 25 ℃ for 2h.
Nucleic acid extraction and quantitative detection were carried out by the methods of examples 2 and 4. As shown in FIG. 2, the detection value was decreased by a certain ratio after pretreatment of the CVB1 sample with nuclease, indicating that a certain ratio of free nucleic acids was present in the sample. Particularly, for a CVB1 virus sample subjected to freeze-drying treatment, the nucleic acid quantitative result is greatly reduced after nuclease is added, and the suggestion that the freeze-drying process can damage the integrity of virus particles to release virus nucleic acid which is digested and removed by the nuclease is provided. Therefore, the nuclease pretreatment can effectively remove the interference of free virus nucleic acid in the sample, so that the detection result can more accurately reflect the content of the genome nucleic acid in the complete virus 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 the CVA6 and CVB6 virus samples have no typical S-type amplification curve, and the CVB1 virus sample has a typical S-type amplification curve. The result shows that the primers and the probes provided by the application can specifically detect the CVB1.
Example 8: detection limit of the method of the present application
Viral stabilizers (20 mM His buffer, pH =6.25, containing
Figure BDA0004003285170000121
Tween80,0.5m naci) diluted CVB1 standards, virus samples were prepared at 30, 10 and 3.3copies/μ L concentrations for nucleic acid extraction and amplification as described in example 2. The results show that the method can stably detect 30 and 10 copies/. Mu.L virus samples, and 3.3 copies/. Mu.L does not have a typical S-shaped amplification curve. Therefore, the detection limit of the system is about 10 copies/. Mu.L.
Example 9: repeatability inspection of the method of the present application
The virus standard samples with 7 dilution gradients were quantitatively detected, and the detection was repeated 6 times in total, and the nucleic acid extraction and quantitative detection were performed by the methods in examples 2 and 4. The results are shown in Table 6, in which the CV of Ct for the standard is less than 5% and the CV for the quantitative value is less than 10%, indicating that the method of the present application has good reproducibility.
TABLE 6 reproducibility examination of the methods of the present application
Figure BDA0004003285170000131
While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

Claims (14)

1. The primer and probe set for detecting CVB1 comprises 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.
2. The primer and probe set of claim 1, wherein the probe is labeled with a reporter group (e.g., a fluorescent group) and a quencher group (e.g., a fluorescence quencher group);
preferably, 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;
preferably, the 5 'end of the probe is connected with FAM fluorescent group, and/or the 3' end of the probe is connected with BHQ1 fluorescence quenching group.
3. A method of detecting CVB1 comprising using the primer and probe set of claim 1 or 2.
4. The method of claim 3, comprising the steps of:
(1) Providing the primer and probe set of claim 1 or 2, and a sample to be tested;
(2) Contacting the primer and the probe group with a sample to be detected, and carrying out RT-qPCR reaction; and the combination of (a) and (b),
(3) And (5) judging the result.
5. The method of claim 4, wherein the sample to be tested in step (1) is subjected to a pretreatment comprising a step of RNA extraction and/or purification.
6. The method of claim 5, wherein the test sample is obtained by pretreatment of a viral sample, and wherein the pretreatment comprises the steps of:
(i) Splitting the virus; and the combination of (a) and (b),
(ii) Purifying RNA released by virus cracking;
preferably, in step (i), the method lyses the virus in the presence of a viral stabilizing agent, wherein the viral stabilizing agent is a His buffer containing Tween-80;
preferably, the concentration of the Tween-80 is
Figure FDA0004003285160000021
To 1.0% o (v/v) (e.g.; v/v)>
Figure FDA0004003285160000022
To 1.0 ‰ (v/v));
preferably, the His buffer further contains NaCl;
preferably, the concentration of the NaCl is 0.3-0.5M;
preferably, the His buffer has a pH of 6.0 to 6.5 (e.g., 6.2 to 6.5,6.25 to 6.5,6.0 to 6.4,6.0 to 6.25, or 6.25);
preferably, the concentration of His in the His buffer is 10-50mM (e.g., 10-30mM,10-20mM,20-50mM,20-30mM, 20mM).
7. The method of claim 6, wherein, prior to step (i), the pretreatment further comprises the step of removing free nucleic acids from the viral sample;
preferably, the pretreatment removes free nucleic acids in the virus sample by enzymatic digestion;
preferably, the pretreatment removes free nucleic acids in the viral sample by using nucleases (e.g., DNase and/or RNase).
8. The method of any one of claims 4 to 7, wherein, in step (3), the method judges by the result of the RT-qPCR reaction: whether the sample to be tested contains a nucleic acid derived from CVB1 or whether the sample from which the sample to be tested is derived contains CVB1.
9. The method of claim 8, wherein in step (3), the method further comprises judging the content of the nucleic acid derived from CVB1 in the sample to be tested by the result of RT-qPCR reaction; or, the content of CVB1 in the sample from which the sample to be tested is derived;
preferably, in step (3), the method determines the content of the nucleic acid derived from CVB1 in the sample to be tested or the content of CVB1 in the sample from which the sample to be tested is derived by comparing the Ct value of the sample to be tested with the Ct values of a series of quantified standards;
preferably, the standard is purified RNA of CVB1 or purified CVB1;
preferably, the standard is purified CVB1;
preferably, the standard is quantified by ddPCR.
10. A kit comprising the primer and probe set of claim 1 or 2;
preferably, the kit further comprises one or more of:
(a) The virus stabilizer is His buffer solution containing Tween-80;
(b) Nucleases (e.g., DNase and/or RNase);
(c) Reagents required for RT-qPCR;
preferably, the concentration of the Tween-80 is
Figure FDA0004003285160000031
To 1.0 ‰ (v/v) (e.g., based on ‰)>
Figure FDA0004003285160000032
To 1.0 ‰ (v/v));
preferably, the His buffer further contains NaCl;
preferably, the concentration of the NaCl is 0.3-0.5M;
preferably, the pH of the His buffer is 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);
preferably, the concentration of His in the His buffer is 10-50mM (e.g., 10-30mM,10-20mM,20-50mM,20-30mM, 20mM).
11. Use of the primer and probe set of claim 1 or 2, or the kit of claim 10, for the preparation of a detection reagent for the detection of CVB1.
12. A virus stabilizer which is His buffer solution containing Tween-80;
preferably, the concentration of the Tween-80 is
Figure FDA0004003285160000033
To 1.5% o (v/v) (e.g., based on the total weight of the blood ‰)>
Figure FDA0004003285160000034
To 1.0% o (v/v));
preferably, the His buffer further contains NaCl;
preferably, the concentration of NaCl is 0.3-0.5M;
preferably, the His buffer has a pH of 6.0 to 6.5 (e.g., 6.2 to 6.5,6.25 to 6.5,6.0 to 6.4,6.0 to 6.25, or 6.25);
preferably, the concentration of His in the His buffer is 10-50mM (e.g., 10-30mM,10-20mM,20-50mM,20-30mM, 20mM).
13. A CVB1 virus nucleic acid extraction method comprising using the virus stabilizer of claim 12;
preferably, the method comprises the steps of:
(i) Lysing the virus in the presence of the virus stabilizing agent; and (c) and (d),
(ii) Purifying RNA released by viral lysis.
14. Use of a virus stabilizer according to claim 12 for the preparation of a reagent for extracting CVB1 nucleic acid.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102534051A (en) * 2012-02-28 2012-07-04 浙江大学 Kit for detecting enterovirus
CN108473964A (en) * 2015-11-19 2018-08-31 诺华股份有限公司 Buffer solution for stablizing slow virus preparation
CN112593011A (en) * 2020-12-25 2021-04-02 中山大学 Primer and probe for detecting coxsackie virus B group
CN112626067A (en) * 2021-02-23 2021-04-09 苏州易迈吉生物医药科技有限公司 Method and kit for rapidly extracting virus RNA
CN113797333A (en) * 2020-06-12 2021-12-17 上海君实生物医药科技股份有限公司 Novel pharmaceutical composition of coronavirus antibody and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102534051A (en) * 2012-02-28 2012-07-04 浙江大学 Kit for detecting enterovirus
CN108473964A (en) * 2015-11-19 2018-08-31 诺华股份有限公司 Buffer solution for stablizing slow virus preparation
CN113797333A (en) * 2020-06-12 2021-12-17 上海君实生物医药科技股份有限公司 Novel pharmaceutical composition of coronavirus antibody and application thereof
CN112593011A (en) * 2020-12-25 2021-04-02 中山大学 Primer and probe for detecting coxsackie virus B group
CN112626067A (en) * 2021-02-23 2021-04-09 苏州易迈吉生物医药科技有限公司 Method and kit for rapidly extracting virus RNA

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SUPRIYA GUPTA等: "Development of a multidose formulation for a humanized monoclonal antibody using experimental design techniques", AAPS PHARMSCI, vol. 5, no. 2, pages 2 *
葛霞琴;崔大伟;谢国良;陈理华;王祥;郑书发;余斐;秦志梅;范剑;陈凌晓;王帅;毛佳燕;陈瑜;: "双重荧光定量RT-PCR法检测肠道病毒方法的建立及应用", 临床检验杂志, no. 06, pages 421 - 423 *

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