阅读说明：本技术 一种猪繁殖与呼吸综合征病毒美洲经典株核酸标准物质及其制备方法 (Porcine reproductive and respiratory syndrome virus American classical strain nucleic acid standard substance and preparation method thereof ) 是由 原霖 王传彬 杨林 宋晓晖 董浩 徐波 蒋菲 刘玉良 徐琦 胡冬梅 亢文华 于 2018-06-19 设计创作，主要内容包括：本发明公开了一种猪繁殖与呼吸综合征病毒美洲经典株核酸标准物质及其制备方法。该方法包括如下步骤：制备猪繁殖与呼吸综合征病毒美洲经典株的病毒液,灭活,得到灭活的病毒液；然后将灭活的病毒液和保护剂混合,冻干或真空干燥；保护剂中含有甘露醇、BSA、海藻糖、蔗糖和甘氨酸；最后通过均匀性评估、稳定性评估和定值,得到猪繁殖与呼吸综合征病毒美洲经典株核酸标准物质。猪繁殖与呼吸综合征病毒美洲经典株核酸标准物质不仅可用于评价检测试剂和控制检测质量,还为检测研究、医药研究、应用研究提供了核酸标准物质的需求,同时为检验检疫机构技术指导、服务出口企业提供技术上的支持。本发明具有重大的应用价值。(The invention discloses a porcine reproductive and respiratory syndrome virus American classical strain nucleic acid standard substance and a preparation method thereof. The method comprises the following steps: preparing virus liquid of the porcine reproductive and respiratory syndrome virus American classical strain, and inactivating to obtain inactivated virus liquid; then mixing the inactivated virus solution and a protective agent, and freeze-drying or vacuum-drying; the protective agent contains mannitol, BSA, trehalose, sucrose and glycine; and finally, obtaining the American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus through uniformity evaluation, stability evaluation and definite value. The American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus can be used for evaluating a detection reagent and controlling the detection quality, provides the requirements of the nucleic acid standard substance for detection research, medical research and application research, and provides technical support for technical guidance of inspection and quarantine organizations and service export enterprises. The invention has great application value.)

1. A method for preparing a porcine reproductive and respiratory syndrome virus American classical strain nucleic acid standard substance comprises the following steps:

(1) preparing virus liquid of the American classical strain of the porcine reproductive and respiratory syndrome virus;

(2) inactivating the virus liquid prepared in the step (1) to obtain inactivated virus liquid;

(3) and (3) taking the inactivated virus liquid obtained in the step (2), and storing to obtain the American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus.

2. The method of claim 1, wherein: in the step (2), the inactivation is heat inactivation or chemical inactivation.

3. The method of claim 1 or 2, wherein: in the step (3), the storage mode is A1) or A2):

A1) mixing the inactivated virus solution and a protective agent, and then freeze-drying or vacuum-drying; the protective agent contains mannitol, BSA, trehalose, sucrose and glycine;

A2) inactivated virus fluid, rnase inhibitor, TE buffer and BSA were mixed.

4. The method of claim 3, wherein: in A1), the "mixing the inactivated virus solution and the protective agent" is that 1 volume part of the inactivated virus solution is mixed with 0.5-1.5 volume parts of the protective agent.

5. The method of claim 3 or 4, wherein:

the freeze-drying parameters are as follows: freezing at-20 deg.C to-40 deg.C for 4-10 h;

the parameters of the vacuum drying are as follows: drying at 4-20 deg.C for 2-6 h under 0.025-0.1 mbar.

6. The method of any of claims 1 to 5, wherein: the method further comprises the step (4): and (4) carrying out virus inactivation test, mycoplasma test and virus test on the porcine reproductive and respiratory syndrome virus American classical strain nucleic acid standard substance obtained in the step (3).

7. The method of any of claims 1 to 6, wherein: the method further comprises step (5): and (4) carrying out uniformity evaluation and stability evaluation on the American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus obtained in the step (3).

8. The method of claim 7, wherein: the method further comprises step (6): carrying out value determination on the American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus with high uniformity and stability; the fixed value is expressed as a standard value ± an extension uncertainty;

respectively detecting more than 2 uniform and stable American classical strain nucleic acid standard substances of the porcine reproductive and respiratory syndrome virus by using a digital PCR method in a plurality of independent laboratories to obtain characteristic values; then, carrying out statistical processing on each characteristic value to determine a standard value;

extended uncertainties were calculated using statistical methods, including uncertainties introduced in uniformity evaluations, uncertainties introduced in stability evaluations, and uncertainties introduced in fixed values.

9. A porcine reproductive and respiratory syndrome virus american classical strain nucleic acid standard prepared by the method of any one of claims 1 to 8.

10. A protective agent as claimed in claim 3 or 4.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a porcine reproductive and respiratory syndrome virus American classical strain nucleic acid standard substance and a preparation method thereof.

Background

The WHO published international hepatitis C nucleic acid standard substances in 1997, and 16 similar standard substances have been published so far. The research and development of nucleic acid standard substances in China are relatively late, and only 5 nucleic acid standard substances which can be inquired on a national standard substance resource sharing platform relate to microorganisms and transgenic plants. In recent years, the development of animal pathogenic nucleic acid standard substances is also reported, and the animal pathogenic nucleic acid standard substances mainly comprise avian influenza, newcastle disease, Asian I type foot-and-mouth disease virus nucleic acid and the like.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious infectious disease characterized by reproductive disorders in pregnant sows, particularly dyspnea in suckling piglets, caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The PRRSV is divided into two genotypes of European type and American type, the American type is divided into two gene subtypes of classical American strain and highly pathogenic American strain, and the three subtype strains have certain differences in gene sequences. The main epidemic in China is the American strain, and the European strain is occasionally discovered. PRRS was first outbreak in the united states in 1987 and subsequently appeared in succession in canada, europe and asia, and is now prevalent worldwide, causing enormous economic losses to the aquaculture industry each year. Our country first developed PRRS in 1995 and then rapidly spread. The Ministry of agriculture in China releases a 'category of disease species of animal epidemic diseases of two and three classes', and lists PRRS-NA as animal epidemic diseases of two classes. The porcine reproductive and respiratory syndrome diagnostic method (GB/T18090-2008) provides for detecting PRRSV-NA by an RT-PCR method, but no corresponding nucleic acid standard substance is available at present, and a detection reagent cannot be evaluated and the detection quality cannot be controlled.

Disclosure of Invention

The invention aims to provide a American classical strain nucleic acid standard substance of porcine reproductive and respiratory syndrome virus.

The invention firstly provides a method for preparing a nucleic acid standard substance of a porcine reproductive and respiratory syndrome virus American classical strain, which comprises the following steps:

(1) preparing virus liquid of the American classical strain of the porcine reproductive and respiratory syndrome virus;

(2) inactivating the virus liquid prepared in the step (1) to obtain inactivated virus liquid;

(3) and (3) taking the inactivated virus liquid obtained in the step (2), and storing to obtain the American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus.

The step (1) can comprise the following steps in sequence:

(1-1) culturing Marc-145 cells or PAM cells;

(1-2) inoculating the American classical strain of porcine reproductive and respiratory syndrome virus, and culturing;

(1-3) repeated freezing and thawing;

(1-4) centrifuging and collecting supernatant;

and (4) collecting the supernatant in the step (1-4), namely the virus liquid of the porcine reproductive and respiratory syndrome virus American classical strain.

In the step (1-1), the Marc-145 cells can be specifically products of ATCC.

In the step (1-1), the culture may be specifically at 37 ℃ with 5% CO₂And (5) culturing.

In the step (1-1), the initial cell concentration of the cultured culture system may be 2X 10⁴-2×10⁶CELLS/mL (e.g., 2X 10)⁴-2×10⁵CELLS/mL、2×10⁵-2×10⁶CELLS/mL、2×10⁴CELLS/mL、2×10⁵CELLS/mL or 2X 10⁶CELLS/mL)。

In the step (1-1), the initial cell concentration of the cultured culture system may be 2X 10⁵CELLS/mL。

In the step (1-2), the culturing time can be 24h-72h (such as 24h-48h, 48h-72h, 24h, 48h or 72 h).

In the step (1-2), the initial concentration of the inoculation is 10⁵TCID₅₀/mL。

In step (1-2), the culturing is carried out until the CPE reaches 70% -80% (e.g., 70% -75%, 75% -80%, 70%, 75% or 80%).

In the step (1-3), the number of times of repeated freeze-thawing may be 2-4 times (e.g., 2 times, 3 times, or 4 times).

In the step (1-3), the number of times of repeated freeze-thawing may be specifically 3 times.

In the step (1-4), the centrifugation can be 1000r/min centrifugation.

In the step (2), the inactivation may be heat inactivation or chemical inactivation.

The heat inactivation can be carried out at 50-60 deg.C (such as 50-56 deg.C, 56-60 deg.C, 50 deg.C, 56 deg.C or 60 deg.C) for 0.5-4h (such as 0.5-2h, 2-4h, 0.5h, 2h or 4 h).

The heat inactivation condition can be specifically 56 ℃ for 4 h.

In the step (3), the storage mode can be A1) or A2):

A1) mixing the inactivated virus solution and a protective agent, and then freeze-drying or vacuum-drying; the protective agent contains mannitol, BSA, trehalose, sucrose and glycine;

A2) inactivated virus fluid, rnase inhibitor, TE buffer and BSA were mixed.

The protective agent may contain 15-25% (15-25mg/100mL) (e.g., 15-20mg/100mL, 20-25mg/100mL, 15mg/100mL, 20mg/100mL or 25mg/100mL) mannitol, 4-6% (4-6mg/100mL) (e.g., 4-5mg/100mL, 5-6mg/100mL, 4mg/100mL, 5mg/100mL or 6mg/100mL) BSA, 1-3% (1-3mg/100mL) (e.g., 1-2mg/100mL, 2-3mg/100mL, 1mg/100mL, 2mg/100mL or 3mg/100mL) trehalose, 3-5% (3-5mg/100mL) (e.g., 3-4mg/100 mL), 4-5mg/100mL, 3mg/100mL, 4mg/100mL, or 5mg/100mL) sucrose and 1-3% (1-3mg/100mL) (e.g., 1-2mg/100mL, 2-3mg/100mL, 1mg/100mL, 2mg/100mL, or 3mg/100mL) glycine.

The protective agent may be a mixture comprising 15-25% (15-25mg/100mL) (e.g., 15-20mg/100mL, 20-25mg/100mL, 15mg/100mL, 20mg/100mL, or 25mg/100mL) mannitol, 4-6% (4-6mg/100mL) BSA, 1-3% (1-3mg/100mL) (e.g., 1-2mg/100mL, 2-3mg/100mL, 1mg/100mL, 2mg/100mL, or 3mg/100mL) trehalose, 3-5% (3-5mg/100mL) (e.g., 3-4mg/100mL, 4-5mg/100mL, 3mg/100mL, 4mg/100mL, or 5mg/100mL) sucrose, and 1-3% (1-3mg/100mL) (e.g., 1-2mg/100mL, 2-3mg/100mL, 1mg/100mL, 2mg/100mL, or 3mg/100mL) glycine.

The protective agent can be specifically an aqueous solution containing 20% (20mg/100mL) mannitol, 5% (5mg/100mL) BSA, 2% (2mg/100mL) trehalose, 4% (4mg/100mL) sucrose and 2% (2mg/100mL) glycine.

The TE buffer may be Tris-HCl buffer of pH7.5-8.5 (e.g., pH7.5-8.0, pH8.0-8.5, pH7.5, pH 8.0-8.0 or pH8.5), 8-12mM (e.g., 8-10mM, 10-12mM, 8mM, 10mM or 12mM) containing 0.5-1.5mM (e.g., 0.5-1.0mM, 1.0-1.5mM, 0.5mM, 1.0mM or 1.5mM) EDTA.

The TE buffer may be specifically Tris-HCl buffer solution containing 1mM EDTA, pH8.0 and 10 mM.

The "mixing the inactivated virus solution and the protective agent" may be a mixture of 1 part by volume of the inactivated virus solution and 0.5 to 1.5 parts by volume (e.g., 0.5 to 1.0 part by volume, 0.5 part by volume, 1.0 part by volume, or 1.5 parts by volume) of the protective agent.

The "mixing the inactivated virus solution and the protective agent" may specifically be mixing 1 part by volume of the inactivated virus solution and 1 part by volume of the protective agent.

The parameters of the lyophilization may be: freezing at-20 deg.C to-40 deg.C (such as-20 deg.C to-30 deg.C, -30 deg.C to-40 deg.C, -20 deg.C, -30 deg.C or-40 deg.C) for 4h to 10h (such as 4h to 8h, 8h to 10h, 4h, 8h or 10 h).

The parameters of the vacuum drying can be: drying at 0.025 mbar-0.1 mbar (such as 0.025 mbar-0.05 mbar, 0.05 mbar-0.1 mbar, 0.025mbar, 0.05mbar or 0.1mbar) at 4 deg.C-20 deg.C (such as 4 deg.C-10 deg.C, 4 deg.C, or 20 deg.C) for 2 h-6 h (such as 2 h-4 h, 4 h-6 h, 2h, 4h or 6 h).

The parameter of the lyophilization may be in particular a1) or a2) or a 3): a1) freezing at-40 deg.C for 10 h; a2) freezing at-30 deg.C for 8 h; a3) freezing at-20 deg.C for 4 h.

The parameters of the vacuum drying can be b1) or b 2): b1) drying at 0.025mbar at 4 deg.C for 6 h; b2) drying at 20 deg.C for 2h under 0.1 mbar.

The A2) ratio of inactivated virus solution, RNase inhibitor, TE buffer and BSA may be 1mL of inactivated virus solution, 150-250U (e.g., 150-200U, 200-250U, 150U, 200U or 250U) RNase inhibitor, 0.5-1.5mL (e.g., 0.5-1.0mL, 1.0-1.5mL, 0.5mL, 1.0mL or 1.5mL) of TE buffer and 0.03-0.07g (e.g., 0.03-0.05g, 0.05-0.07g, 0.03g, 0.05g or 0.07g) of BSA.

In A2), the ratio of the inactivated virus solution, the RNase inhibitor, the TE buffer solution and the BSA can be 1mL of the inactivated virus solution, 200URNA enzyme inhibitor, 1mL of the TE buffer solution and 0.05g of the BSA.

The method of any of the above may further comprise step (4): and (4) carrying out virus inactivation test, mycoplasma test and virus test on the porcine reproductive and respiratory syndrome virus American classical strain nucleic acid standard substance obtained in the step (3).

The virus inactivation assay may use a cytopathic assay.

The mycoplasma test may use a mycoplasma detection kit.

The virus assay may use a fluorescent PCR detection kit.

The mycoplasma detection kit and the fluorescence PCR detection kit can be products of Beijing Shijiheng animal epidemic prevention technology GmbH.

The method of any of the above may further comprise step (5): and (4) carrying out uniformity evaluation and stability evaluation on the American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus obtained in the step (3).

The step of performing uniformity evaluation may be: sampling the American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus obtained in the step (3), extracting nucleic acid from the sample, detecting by using a digital PCR method, and evaluating the uniformity of the result.

The sampling method for uniformity evaluation may be to extract 10-30 (e.g., 30) nucleic acid standard substances of the American classical strain of porcine reproductive and respiratory syndrome virus. The number of detections per sample may specifically be 3.

The uniformity evaluation may be a uniformity evaluation using a one-factor analysis of variance method according to a sampling method and the number of detections.

The step of performing stability assessment may be: sampling the American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus obtained in the step (3), extracting nucleic acid from the sample, detecting by using a digital PCR method, calculating an average value, and evaluating the stability.

The stability assessment may be a long term stability assessment or a short term stability assessment.

The sampling method for long-term stability assessment may be: storing the nucleic acid standard substance at-20 deg.C for N1 months, N2 months, N3 months, N4 months or N5 months (N1-N5 are natural numbers and N1-N5 are 0-14), extracting not less than 2 bottles of nucleic acid standard substance of porcine reproductive and respiratory syndrome virus American classical strain, wherein the detection frequency of each sample can be 1.

The sampling method for short-term stability assessment may be: storing the nucleic acid standard substance at 4 deg.C, 25 deg.C or 37 deg.C for 2 months, 7 days or 1 day, and extracting no less than 2 bottles of nucleic acid standard substance of porcine reproductive and respiratory syndrome virus American classical strain, wherein the detection frequency of each sample can be 1.

The stability assessment may be a stability assessment using regression or T-test, depending on the sampling method and the number of detections.

Any one of the above-mentioned nucleic acid extractions can be performed using a fully-automatic nucleic acid extractor.

The method of any of the above may further comprise step (6): carrying out value determination on the American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus with high uniformity and stability; the fixed value is expressed as a standard value ± an extension uncertainty;

respectively detecting more than 2 uniform and stable American classical strain nucleic acid standard substances of the porcine reproductive and respiratory syndrome virus by using a digital PCR method in a plurality of independent laboratories to obtain characteristic values; then, carrying out statistical processing on each characteristic value to determine a standard value;

extended uncertainties were calculated using statistical methods, including uncertainties introduced in uniformity evaluations, uncertainties introduced in stability evaluations, and uncertainties introduced in fixed values.

The number of laboratory collaborations is fixed, the minimum number of participating laboratories is usually 6-8. When the same method is adopted, the number of independent constant value groups is generally not less than 8. For statistical processing, 4-6 independent repetitions of the measurement data are required.

The invention provides a method for preparing a nucleic acid standard substance of a porcine reproductive and respiratory syndrome virus American classical strain, which comprises the following steps:

(1) preparing virus liquid of the American classical strain of the porcine reproductive and respiratory syndrome virus;

(2) inactivating the virus liquid prepared in the step (1) to obtain inactivated virus liquid;

(3) and (3) adding a protective agent into the inactivated virus liquid obtained in the step (2) for preservation, performing virus inactivation inspection and mycoplasma inspection and virus inspection, performing uniformity evaluation and stability evaluation, performing fixed value determination on standard substances, and calculating the extension uncertainty to obtain the American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus.

The porcine reproductive and respiratory syndrome virus American classical strain nucleic acid standard substance prepared by any one of the methods also belongs to the protection scope of the invention.

The invention also protects any one of the protective agents.

The application of any protective agent in the preservation of the American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus also belongs to the protection scope of the invention.

Any of the porcine reproductive and respiratory syndrome virus American classical strains can be a porcine reproductive and respiratory syndrome virus VR2332 strain.

The American classical strain nucleic acid standard substance of the porcine reproductive and respiratory syndrome virus prepared by the method can be used for evaluating detection reagents and controlling laboratory quality, provides the requirements of the nucleic acid standard substance for scientific research, vaccine research and application research, and provides technical support for technical guidance of inspection and quarantine organizations and service export enterprises. The invention has great application value.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.