阅读说明：本技术 一种冻干型新城疫病毒核酸标准物质的制备方法及其检测方法 (Preparation method and detection method of freeze-dried Newcastle disease virus nucleic acid standard substance ) 是由 冯小宇 高晓龙 梅力 王英超 韦海涛 宋彦军 沈光年 王林 于 2019-09-26 设计创作，主要内容包括：本发明提供了一种冻干型新城疫病毒核酸标准物质的制备方法及其检测方法,包括如下步骤：S1、新城疫病毒液的制备；S2、将制备的新城疫病毒液热灭活,得到灭活的病毒液；S3、取步骤S2得到的灭活病毒液加入保护剂,冻干,得到新城疫病毒核酸标准物质。其检测方法以推进新城疫病毒核酸分子检测标准化及新城疫病毒体外诊断试剂的量值溯源和检测结果一致化。新城疫病毒核酸标准物质不仅可为兽医实验室开展量值溯源、能力验证、质量控制、方法评价、试剂选择等工作提供技术支撑,还能提高全国不同类型兽医检测实验室的检测能力和技术水平,更为动物疫病净化和畜产品质量安全评价、身份验证、国内监管、进出口检验、企业自控和国际贸易互认提供有力保障。(The invention provides a preparation method and a detection method of a freeze-dried Newcastle disease virus nucleic acid standard substance, which comprises the following steps: s1, preparing Newcastle disease virus liquid; s2, performing heat inactivation on the prepared Newcastle disease virus liquid to obtain an inactivated virus liquid; s3, adding a protective agent into the inactivated virus liquid obtained in the step S2, and freeze-drying to obtain the Newcastle disease virus nucleic acid standard substance. The detection method promotes the standardization of the detection of the newcastle disease virus nucleic acid molecules and the quantity value traceability and detection result consistency of the newcastle disease virus in-vitro diagnostic reagent. The Newcastle disease virus nucleic acid standard substance can provide technical support for veterinary laboratories to carry out work such as quantity value traceability, capability verification, quality control, method evaluation, reagent selection and the like, can also improve the detection capability and technical level of veterinary detection laboratories of different types nationwide, and provides powerful guarantee for animal epidemic disease purification and animal product quality safety evaluation, identity verification, domestic supervision, import and export inspection, enterprise automatic control and international trade mutual recognition.)

1. A preparation method of a freeze-dried Newcastle disease virus nucleic acid standard substance is characterized by comprising the following steps:

s1, preparing Newcastle disease virus liquid;

s2, performing heat inactivation on the Newcastle disease virus solution prepared in the step S1 to obtain an inactivated virus solution;

s3, adding a protective agent into the inactivated virus liquid obtained in the step S2, and freeze-drying; obtaining the nucleic acid standard substance of the Newcastle disease virus.

2. The method of claim 1, wherein the heat inactivation is a treatment at 65 ℃ for 2h in step S2.

3. The method according to claim 1, wherein in step S3, the protective agent contains gelatin and sucrose.

4. The preparation method according to claim 1, wherein the protective agent is an aqueous solution containing 5% by mass of gelatin and 25% by mass of sucrose, and is sterilized before use.

5. The method according to claim 1, wherein in step S3, the ratio of the inactivated virus solution to the protective agent is 1: 0.5 to 1.5.

6. The preparation method according to claim 1, wherein the freeze-drying condition is-10 ℃ to-40 ℃ for 4h to 10 h.

7. The method according to claim 1, further comprising performing a virus inactivation test on the lyophilized Newcastle disease virus nucleic acid standard substance,

and/or mycoplasma testing;

and/or other viral tests.

8. The method according to claim 7, further comprising performing uniformity evaluation and/or stability evaluation on the Newcastle disease virus nucleic acid standard substance.

9. The method of claim 7, wherein the step of evaluating the uniformity is: sampling the newcastle disease virus nucleic acid standard substance, extracting nucleic acid from the sample, detecting by using a digital PCR method, and evaluating the uniformity of the result;

the stability assessment short-term stability assessment and long-term stability assessment, the sampling method of the short-term stability assessment may be: storing the nucleic acid standard substance at 4 deg.C, 25 deg.C or 60 deg.C for1 week, 2 weeks or 4 weeks, randomly extracting 3 bottles of nucleic acid standard substance of Newcastle disease virus under each condition, and detecting each sample for 3 times;

the sampling method for evaluating the long-term stability comprises the following steps: storing the nucleic acid standard substance at-20 ℃ for N1 months, N2 months, N3 months, N4 months or N5 months, wherein N1-N5 are natural numbers and are not less than 0 and not more than 14 of N1-N5, extracting 3 bottles of the Newcastle disease virus nucleic acid standard substance, and detecting each sample for 3 times;

the primer pair adopted by the digital PCR method is shown as SEQ ID NO.1 and SEQ ID NO.2, the probe is shown as SEQ ID NO.3, the concentration of the primer pair adopted by an amplification system is 900nmol/L and the concentration of the probe is 250 nmol/; the amplification program is 15min at 45 ℃; 2min at 95 ℃; 15s at 95 ℃, 45s at 55 ℃ and 40 cycles; 10min at 98 ℃; 60min at 12 ℃.

10. The method according to claim 7, further comprising subjecting the newcastle disease virus nucleic acid standard substance to a constant value; the constant values are expressed as standard values ± uncertainty; respectively detecting more than 2 uniform and stable Newcastle disease virus nucleic acid standard substances 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;

the uncertainty was calculated using statistical methods, including uncertainty arising in the homogeneity assessment, uncertainty arising in the stability assessment, and uncertainty arising in the fixed value.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method and a detection method of a freeze-dried Newcastle disease virus nucleic acid standard substance.

Background

Newcastle Disease (ND) is an acute, febrile, septic and highly contagious infectious disease caused by Newcastle Disease Virus (NDV), mainly comprises the cell injury of digestive tract, respiratory tract and central nervous system, has the main clinical symptoms of mucosal and serosal hemorrhage, diarrhea, respiratory altitude difficulty and nervous disorder, has the morbidity and mortality rate of 100 percent, and causes serious threat and huge economic loss to the poultry industry. In 1926, java island in indonesia was the first outbreak of the disease, which occurred in the same year in new city, uk, and subsequently in asia areas, middle east, etc. The world animal health Organization (OIE) ranks ND as an animal epidemic disease of class A, and once the occurrence of the disease needs to be reported to OIE, China also ranks ND as an animal epidemic disease of class A. NDV mainly infects birds such as chickens, pheasants and turkeys, and waterfowls can also be toxic, but waterfowls generally do not transmit viruses to poultry. Occasionally, humans may also become infected, which can lead to conjunctivitis if workers are exposed to large amounts of NDV for extended periods of time.

NDV has only 1 serotype, so there is some cross-protection between different strains. NDV can be classified into Class I and Class II according to the genome length and F gene sequence of Newcastle disease virus. Class I NDV can be further divided into 10 genotypes (1-10), mostly of low virulent strains, which mainly infect wild waterfowl and spread gradually to terrestrial birds. Class II NDV has a long prevalence time, and poultry mainly infects hosts and can be divided into at least 18 genotypes, and most of the genotypes are strong toxicity except that the genotypes I and II are weak toxicity. Class ii is the dominant strain of NDV epidemics, of which the major current epidemics are genotype vi and vii.

Currently, the commonly used NDV live vaccines mainly comprise a plurality of strains, namely a strain I (moderate virulence), a strain II, a strain III (weak virulence), a strain IV (LaSota strain), a strain V4 and the like, wherein the LaSota weak virulence live vaccine is widely applied and has a good well-known immune effect.

At present, no standard substance aiming at detection methods of serum or nucleic acid of the Newcastle disease virus exists at home and abroad, and all levels of detection mechanisms lack corresponding standard substances of positive serum, antigen, nucleic acid and the like. At home and abroad, no mechanism or company produces NDV related standard substances, and because the software and hardware conditions of various veterinary detection laboratories are greatly different, the types of diagnostic reagents are various, and the loss of the standard substances causes the lack of consistency and comparability in NDV detection, thereby restricting the effective prevention and control of ND. Therefore, relevant standard substances for ND detection are developed, so that the detection capability and the technical level of different veterinary detection laboratories in China can be improved, and powerful guarantee is provided for animal epidemic disease purification and animal product quality safety.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method and a detection method of a freeze-dried newcastle disease virus nucleic acid standard substance, which are used for promoting the standardization of newcastle disease virus nucleic acid molecule detection and the quantity value tracing of a newcastle disease virus in-vitro diagnostic reagent and the consistency of detection results.

In order to achieve the purpose, the invention adopts the following technical scheme that:

a preparation method of freeze-dried Newcastle disease virus nucleic acid standard substance comprises the following steps:

s1, preparing Newcastle disease virus liquid;

s2, performing heat inactivation on the Newcastle disease virus solution prepared in the step S1 to obtain an inactivated virus solution;

s3, adding a protective agent into the inactivated virus liquid obtained in the step S2, and freeze-drying to obtain the Newcastle disease virus nucleic acid standard substance.

In the preparation method described above, preferably, in step S1, the preparation of the newcastle disease virus solution includes the following steps:

s101, selecting SPF chick embryos and marking inoculation positions;

s102, inoculating a Newcastle disease virus LaSota strain, and culturing;

s103, harvesting allantoic fluid; namely the Newcastle disease virus liquid.

Further, the culture conditions are 37 ℃, the relative humidity is 55%, and the culture time is 48-72 h.

The preparation method as described above, preferably, in step S2, the heat inactivation is treatment at 65 ℃ for 2 h.

In the above-described production method, preferably, in step S3, the protective agent contains gelatin and sucrose.

Furthermore, the protective agent is an aqueous solution containing 5% of gelatin and 25% of sucrose by mass, and high-temperature and high-pressure sterilization (116 ℃) is required for 20min before use.

The preparation method as described above, preferably, in step S3, the inactivated virus solution and the protective agent are mixed in a volume ratio of 1: 0.5-1.5.

The preparation method as described above, preferably, in step S3, the lyophilization conditions are-10 ℃ to-40 ℃ for 4h to 10 h.

The preparation method as described above, preferably, it further comprises performing virus inactivation test on the freeze-dried Newcastle disease virus nucleic acid standard substance obtained by the preparation method as described above,

and/or mycoplasma testing;

and/or other viral tests.

Wherein the virus inactivation test can use chicken embryo infection experiment test;

the mycoplasma test uses a mycoplasma culture format test;

the other virus tests can be tested by using a fluorescent quantitative PCR method, and the other virus tests comprise the tests on H9N2 subtype avian influenza virus (H9N2), avian Infectious Bronchitis Virus (IBV), avian infectious laryngotracheitis virus (AILTV), avian Egg Drop Syndrome Virus (EDSV), avian adenovirus type 4 (FAdV-4) and avian Infectious Bursal Disease Virus (IBDV).

The preparation method further comprises the step of evaluating the uniformity and/or stability of the Newcastle disease virus nucleic acid standard substance.

Further, the uniformity evaluation step is: sampling the newcastle disease virus nucleic acid standard substance, extracting nucleic acid from the sample, detecting by using a digital PCR method, and evaluating the uniformity of the result.

Preferably, the sampling is to extract 10-30 (e.g., 15) newcastle disease virus nucleic acid standard substances; the number of detections per sample may specifically be 3.

Preferably, the primer pairs adopted by the digital PCR method are shown as SEQ ID NO.1 and SEQ ID NO.2, the probes are shown as SEQ ID NO.3, the concentrations of the primer pairs adopted by an amplification system are 900nmol/L respectively, and the probes are 250 nmol/; the amplification program is 15min at 45 ℃; 2min at 95 ℃; 15s at 95 ℃, 45s at 55 ℃ and 40 cycles; 10min at 98 ℃; 60min at 12 ℃.

Preferably, 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 preparation method as described above, preferably, the stability assessment short-term stability assessment and long-term stability assessment, and the sampling method of the short-term stability assessment may be: storing the nucleic acid standard substance at 4 deg.C, 25 deg.C or 60 deg.C for1 week, 2 weeks or 4 weeks, randomly extracting 3 bottles of nucleic acid standard substance of Newcastle disease virus under each condition, and detecting each sample for 3 times;

the sampling method for the 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 more than or equal to 0 and less than or equal to 14), extracting 3 bottles of nucleic acid standard substance of Newcastle disease virus, wherein the detection frequency of each sample can be 3 times.

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

The preparation method as described above, preferably, the preparation method further comprises the steps of performing valuing on the newcastle disease virus nucleic acid standard substance; the constant values are expressed as standard values ± uncertainty; respectively detecting more than 2 uniform and stable Newcastle disease virus nucleic acid standard substances 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;

the uncertainty was calculated using statistical methods, including uncertainty arising in the homogeneity assessment, uncertainty arising in the stability assessment, and uncertainty arising in the fixed value.

The number of laboratory collaborations is fixed, the minimum number of participating laboratories is usually 6-8.

The newcastle disease virus adopted by the invention is specifically a newcastle disease virus LaSota strain. At present, the research on the standard substance for detecting the pathogenic microorganisms of domestic animals is still in the initial stage, and only standard serum, standard antigen and strain standard substance aiming at a few pathogens exist. The nucleic acid standard substance of the Newcastle disease virus prepared by the method provided by the invention is used for detection, on one hand, the nucleic acid standard substance can be used for evaluating detection reagents and controlling laboratory quality, on the other hand, the efficiency of epidemic disease monitoring and diagnosis can be greatly improved, and the nucleic acid standard substance has very important significance for improving the quality safety detection capability of animals and animal products, ensuring the health and safety of consumers, promoting the healthy development of animal husbandry and improving the international competitiveness of products. Therefore, the invention has great application value.

The invention has the beneficial effects that:

the invention provides a Newcastle disease virus nucleic acid standard substance. The standard substance is the key for verifying whether the detection method and the detection reagent are reliable or not and is an important factor of a laboratory quality certification system, and the use of the standard substance can bring reliable detection guarantee for an animal epidemic disease detection laboratory, so that accurate data can be obtained, and a technical guarantee is provided for accurate epidemic disease prevention and control.

According to the invention, the Newcastle disease virus LaSota strain is used, and through a series of tests such as preparation, inactivation, freeze-drying and the like of virus liquid, a Newcastle disease virus nucleic acid standard substance easy to store and transport is finally obtained, so that a key standard reagent is provided for improving the detection capability and the technical level of different types of veterinary detection laboratories nationwide and verifying the reliability of a detection method and a detection reagent.

Drawings

FIG. 1 is the amplification curve of the Newcastle disease virus RT-qPCR method.

FIG. 2 is a standard curve of amplification of Newcastle disease virus RT-qPCR method of the present invention.

FIG. 3 shows the result of annealing temperature optimization by the digital PCR method for Newcastle disease virus.

FIG. 4 is a graph showing a linear relationship between NDV RT-ddPCR according to the present invention.

Detailed Description

The invention establishes a freeze-drying method, which is characterized in that a Newcastle disease virus nucleic acid standard substance mixed with a freeze-drying protective agent is frozen in advance and then is put into a freeze-drying machine for freeze-drying for at least more than 10 hours. And (4) taking out the freeze-drying bottle after freeze-drying is finished, pressing the rubber plug, covering the outer cover tightly, and storing in a refrigerator at the temperature of-20 ℃. The optimum character of the nucleic acid standard substance of the Newcastle disease virus is effectively ensured by groping the freeze-drying process, and the preservation and the transportation of the standard substance are facilitated.

In the quantitative detection of the invention in the process of developing the nucleic acid standard substance of the urban epidemic virus, a micro-drop digital PCR platform is utilized, and compared with the traditional fluorescent quantitative PCR technology, the accuracy and the sensitivity of the digital PCR are better. By using the droplet digital PCR technology, rare mutation can be detected, copy number variation can be accurately determined, and absolute quantification can be carried out on gene expression. According to the invention, through optimization of the newcastle disease virus droplet type digital PCR reaction, including optimization of reverse transcription conditions, selection and performance evaluation of a nucleic acid extraction kit, optimization of reaction conditions (primer probe concentration and annealing temperature), and linearity and minimum detection limit determination of the method, uniformity and stability of a newcastle disease virus nucleic acid standard substance can be evaluated and investigated more accurately and sensitively, and the reliability of an experimental result is greatly improved.

The invention utilizes a one-factor variance analysis method to evaluate the Newcastle disease nucleic acid standard substance. In the process of valuing the nucleic acid standard substance of Newcastle disease, a dixon method and a Grabas method are adopted to test suspicious values of data in a laboratory; adopting a Kokring method to check whether the data between laboratories have equal precision; and calculating the extended uncertainty of the prepared Newcastle disease nucleic acid standard substance according to the uncertainty introduced by uniformity, the uncertainty introduced by stability and the uncertainty brought by a valuing process, wherein the uncertainty brought by the valuing process calculates multiple links such as balance weighing, a liquid transfer device, a digital PCR instrument and the like. The prepared Newcastle disease nucleic acid standard substance is evaluated and valued by adopting a biometrical method, so that the prepared standard substance is more accurate in value.

The following examples are intended to further illustrate the invention but should not be construed as limiting it. Modifications and substitutions may be made thereto without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test materials used, unless otherwise specified, were purchased from conventional biochemicals. The quantitative tests in the following examples, all set up three replicates and the results averaged.