CN110684746A - Preparation method and detection method of freeze-dried Newcastle disease virus nucleic acid standard substance - Google Patents
Preparation method and detection method of freeze-dried Newcastle disease virus nucleic acid standard substance Download PDFInfo
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Abstract
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.
Description
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.
Example 1 preparation of viral fluid of Newcastle disease Virus
(1) SPF chick embryo selection and inoculation location markers
Selecting 9-day-old live embryos (blood vessel branches are obvious and bright red, and chick embryos can normally move), marking the positions of an air outlet chamber, embryos and main blood vessels, and avoiding the positions of blood vessel marking inoculation at the positions of the opposite sides of the embryos, which are about 5mm away from the boundary of the air chamber.
(2) Inoculation with Newcastle disease Virus
The method comprises the steps of taking out a Newcastle disease virus (LaSota strain) separated and stored in a laboratory (in the invention, the LaSota vaccine strain is selected, and the LaSota vaccine strain F gene has nucleotide homology of more than 80% with the current NDV epidemic strain, so that the LaSota vaccine strain is selected to prepare an NDV nucleic acid detection standard substance), melting, diluting by 1000 times with PBS, shaking and mixing uniformly. 20 chick embryos were inoculated per 0.2mL allantoic cavity with a 1mL syringe.
(3) Culture and harvest of newcastle disease virus fluid
Inoculating newcastle disease virus to chick embryos, placing the chick embryos in an incubator at 37 ℃ for incubation, observing death conditions of the chick embryos every 8 hours after 18 hours, and aseptically collecting chick embryo allantoic fluid with the hemagglutination titer larger than 5 after 72 hours. The virus culture was centrifuged at 4500rpm/min at 4 ℃ for 5min and then filtered through a 0.22 μm filter. Storing the virus liquid at-80 deg.C.
Example 2 preparation of nucleic acid Standard substance for Newcastle disease Virus
1. Inactivation of newcastle disease virus
The virus solution of the newcastle disease virus prepared in example 1 was taken and subjected to water bath at 65 ℃ for 2 hours to obtain an inactivated virus solution.
2. Verification of newcastle disease virus inactivation
Inoculating 1mL of the newcastle disease virus solution prepared in the step 1 to SPF chick embryos, culturing, and then quantitatively detecting the inoculated embryo allantoic fluid without hemagglutination characteristics (the detection method can be specifically shown in example 3) through PCR (polymerase chain reaction) to determine that the newcastle disease virus is negative in nucleic acid, so that the newcastle disease virus can be considered to be inactivated.
3. Freeze-drying of newcastle disease virus liquid
1) Preparation before lyophilization
500mL of stock solution of the Newcastle disease virus LaSota strain and 500mL of freeze-drying protective agent are sucked, and the ratio of the virus solution to the protective agent is 1: 1, fully and uniformly mixing, covering a bottle cover, placing the bottle cover into an ultra-low temperature refrigerator for pre-freezing for 24 hours, ensuring that the pre-freezing is in a solid state, and taking out the bottle cover for freeze-drying. Wherein the freeze-drying protective agent is prepared by dissolving 50 g of gelatin and 250 g of sucrose in 1L of purified water, sterilizing at high temperature and high pressure (116 ℃) for 20min, and storing in a refrigerator at 2-8 ℃ for later use.
The inventor discovers that the newcastle disease virus nucleic acid standard substance freeze-dried by the gelatin sucrose freeze-drying protective agent is dry powder, good in physical state, plump in appearance and occasionally provided with cracks through comparison of freeze-drying effects of a large number of experiments on milk sucrose and the gelatin sucrose protective agent. A comparative experiment of the content of the gelatin and the content of the sucrose shows that the content of the gelatin (g/mL) with the mass fraction of 5% and the content of the sucrose (g/mL) with the mass fraction of 25% have no influence on the content of the nucleic acid of the Newcastle disease virus, so that the cryoprotectant containing the gelatin with the mass fraction of 5% and the sucrose with the mass fraction of 25% is selected.
2) Freeze-drying procedure
Turning on an EYELA FDU-1200 freeze dryer main switch, turning off an AUTO indicator light, pressing a refrigeration control button ' Refrigerator RUN/STOP ', starting refrigeration, lighting the Refrigerator RUN/STOP indicator light, quickly putting a pre-frozen sample into a drying chamber and covering the drying chamber with a drying chamber cover when the temperature indicates that ' TRAP TEMP ' is-40 ℃, pressing a ' VAC PUMP ' button, lighting the VAC PUMP ' button indicator light, starting a vacuum PUMP, starting to PUMP vacuum, entering a normal working state to start freeze drying when the vacuum degree indicates that ' VACCUM GUARGE ' is below 20Pa, and freeze-drying for 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 4 ℃.
Example 3 establishment of Newcastle disease Virus nucleic acid Standard substance digital PCR method
1. Design of primer and probe for Newcastle disease virus
And designing a primer probe sequence according to the NDV F gene conserved region.
NDV upstream primer (SEQ ID NO. 1): 5 '-AACAGCTGCACARATAACAGC-3';
NDV downstream primer (SEQ ID NO. 2): 5'-TAACAAACTGCTGCATCTTCCC-3', respectively;
NDV probe (SEQ ID NO. 3): 5 '-FAM-ATCCTCCGRCTTAAGGAGAGCATTGC-BHQ-3'.
The length of the amplification product is 170bp, and the amplification target sequence (SEQ ID NO.4) is as follows:
AACTGCCGCACAAATAACAGCGGCCGCAGCTCTGATACAAGCCAAACAAAATGCTGCCAACATCCTCC GACTTAAAGAGAGCATTGCCGCAACCAATGAGGCTGTGCATGAGGTCACTGACGGATTATCGCAACTAGCAGTGGCGGTTGGGAAGATGCAGCAGTTTGTTAwhere the underlined "-" is the primer position and "═ is the probe position.
2. Verification primer and probe for newcastle disease virus RT-qPCR method
The designed primer and probe are adopted to establish the RT-qPCR method, and the reaction system of the RT-qPCR is 2
Probe qPCR dUTP Master Mix 10. mu.L, GoScript RT Mix for1-step RT-QPCR 0.5. mu.L, 10. mu.M primer 1.2. mu.L, 10. mu.M Probe 0.6. mu.L, water 5.7. mu.L, template 2. mu.L. The amplification condition is 15min at 45 ℃; 2min at 95 ℃; 95 ℃ for 15s, 55 ℃ for 1min, 40 cycles. Newcastle disease virus-extracted nucleic acid (nucleic acid extracted from the standard substance prepared in example 2) was diluted 10-fold in gradient to draw a standard RT-qPCR curve, where y is-3.255 x +37.24 and R is the standard curve2The value is 0.995, the amplification efficiency is 102.9%, and the RT-qPCR method linear relationship established according to the designed primers and probes is good. The amplification curve of the Newcastle disease virus RT-qPCR method is shown in figure 1, and the amplification standard curve is shown in figure 2.
3. Optimization of newcastle disease virus digital PCR method conditions
1) Optimization of primer and probe concentrations
Extracting nucleic acid of the newcastle disease inactivated virus liquid by using an automatic extraction reagent by a paramagnetic particle method, and diluting the nucleic acid in a gradient way 5-4The optimized template is searched for the system, the preparation of the ddPCR experimental system is shown in table 1, wherein the concentrations of the first group of primers and the probe are respectively 600nmol/L and 150 nmol/L; the concentration of the second group of primers and the concentration of the probes are 900nmol/Lol/L and 250nmol/L respectively; the concentration of the third group of primers and probes is 1200nmol/L and 350nmol/L respectively.
TABLE 1 preparation of ddPCR Experimental systems
Reaction procedure: 15min at 45 ℃; 2min at 95 ℃; 15s at 95 ℃, 45s at 55 ℃ and 40 cycles; 10min at 98 ℃; 60min at 12 ℃. The temperature rising and falling speed is 2 ℃/s. The results are shown in Table 2: the RSD values of the three groups are less than 5%, but the copy number of the first group, the third group is obviously lower than that of the second group, therefore, the concentration of the primer is set as 900nmol/L of the second group, and the concentration of the probe is 250 nmol/L.
TABLE 2 amplification copy number analysis of different primer and probe concentrations
2) Optimization of annealing temperature
The annealing temperature is selected from 55 ℃, 57 ℃, 60 ℃ and 62 ℃; reaction system: supermix 5.0. mu.L, Reversetranscriptase 2.0. mu.L, 300mM DTT 1. mu.L, 10. mu.M primer set 1.8. mu.L, 10. mu.M probe 0.5. mu.L, water 7.7. mu.L, template 2.0. mu.L. Reaction procedure: 15min at 45 ℃; 2min at 95 ℃; 95 ℃ for 15s, 55 ℃ for 45s, 40 cycles. The temperature rising and falling speed is 2 ℃/s.
The annealing temperature optimization results are shown in figure 3. At an annealing temperature of 60 ℃, the signal intensity is reduced and the copy number is reduced. Therefore, the annealing temperature was set to 55 ℃.
4. Linear relation and minimum detection limit determination of Newcastle disease virus digital PCR method
Diluting the prepared nucleic acid containing NDV virus by 10 times of dilution ratio in gradient 10-0，10-1，10-2，10-3，10-43 replicates were performed for each concentration.
Reaction system: supermix 5.0. mu.L, Reverse transcriptase 2.0. mu.L, 300mM DTT 1. mu.L, 10. mu.M primer set 1.8. mu.L, 10. mu.M probe 0.5. mu.L, water 7.7. mu.L, template 2.0. mu.L.
Reaction procedure: 15min at 45 ℃; 2min at 95 ℃; 95 ℃ for 15s, 55 ℃ for 45s, 40 cycles. The temperature rising and falling speed is 2 ℃/s.
NDV nucleic acid dilution test results are shown in Table 3, and when the test concentration is lower than 10 copies, the CV value of the test result is 4.97% and is less than 5%, which shows that the method can stably detect a sample with the concentration of 1.8 copies/muL, so the lowest detection limit of the method is 1.8 copies/muL.
TABLE 3 fold dilution (10 fold) NDV nucleic acid assay results
The copy number was determined by NDV nucleic acid gradient dilution and a linear profile was generated, as shown in FIG. 4, R2＝0.9989>0.99, the linear relationship is good.
5 Newcastle disease Virus digital PCR method specificity test
Extracting virus nucleic acids such as H9N2 subtype avian influenza virus (H9N2), avian Infectious Bronchitis Virus (IBV), avian infectious laryngotracheitis virus (AILTV), Egg Drop Syndrome Virus (EDSV), avian adenovirus type 4 (FAdV-4), avian Infectious Bursal Disease Virus (IBDV), Newcastle Disease Virus (NDV), etc., and diluting to approximately 10%3And (4) carrying out RT-ddPCR detection according to the reaction system and the program determined in the steps 3 and 4 by copies/. mu.L, and verifying the specificity of the method. The results are shown in Table 4, and other detections except NDV specific amplification are all negative, which indicates that the method has better specificity.
TABLE 4 specificity test of NDV digital PCR method
6. Newcastle disease virus digital PCR method repeatability verification test
1 part of NDV nucleic acid was subjected to RT-ddPCR assay 10 times in accordance with the reaction system and procedure defined in steps 3 and 4. The results are shown in Table 5, and the coefficient of variation is 2.4%, which shows that the method has good repeatability and stable and reliable detection results.
TABLE 5 NDV repeatability tests
Example 4 Newcastle disease Virus nucleic acid Standard substance packaging
The method is carried out in a level II biological safety cabinet in a ten thousand working area. Bottling into brown 10mL glass bottle, autoclaving, and filling with nitrogen gas at a volume of 500 mg/bottle. And (3) after the freeze-dried powder prepared in the embodiment 2 is subpackaged, adding a rubber soft cover for sealing, and finally adding an aluminum cover for reinforcing and sealing. And finally storing in an environment of (-20 +/-2) DEG C.
Sampling and submitting for inspection. And after the product is qualified, the next procedure can be carried out. In a hundred thousand class plant. And (4) assembling the finished product according to the specification, and storing the finished product in an environment at the temperature of minus 20 +/-2 ℃ to avoid repeated freeze thawing.
Example 5 assay of Newcastle disease Virus nucleic acid Standard substance
1. Physical property test of nucleic acid standard substance of newcastle disease virus
The newcastle disease virus nucleic acid standard substance is white uniform powder.
2. Inactivation test for nucleic acid standard substance of newcastle disease virus
Dissolving a nucleic acid standard substance of the Newcastle disease virus by using nuclease-free water, inoculating 0.2mL of the nucleic acid standard substance into 5 SPF (specific pathogen free) chick embryos of 9 days old, incubating at 37 ℃ for 120h, wherein no dead embryo exists within 48h-120h, the blood coagulation titer of allantoic fluid obtained by inoculating the chick embryos is 0, and the virus inactivation is considered to be complete. The detection result shows that the Newcastle disease virus is completely inactivated.
3. Newcastle disease virus nucleic acid standard substance mycoplasma assay
And detecting whether mycoplasma pollution exists or not by using a mycoplasma culture mode. The results are reported in Table 6, and the Mycoplasma test is negative.
TABLE 6 Mycoplasma detection results recording
4. Other viral assay assays in Newcastle disease Virus nucleic acid standards
Dissolving 100mg of NDV standard substance with 1mL of nuclease-free water, extracting 200 mu L of the NDV standard substance, and performing nucleic acid extraction on the NDV standard substance by adopting a fluorescence quantitative PCR method to obtain 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) nucleic acid; wherein H9N2, IBV and AILTV are detected by adopting a commercial kit and EDSV, IBDV and FAdV-4 fluorescent quantitative PCR detection methods by referring to published documents. The results are shown in Table 7, and the viruses do not generate an S-shaped amplification curve, which indicates that no other viruses exist in the nucleic acid standard substance of the Newcastle disease virus.
TABLE 7 NDV nucleic acid standards other viral assays
Example 6 evaluation of Newcastle disease Virus nucleic acid Standard substance
1. Uniformity evaluation
1) The samples prepared in example 2 were coded sequentially and the number of 15 vials taken was determined using a random number table and 3 replicates were taken.
13-38-48-90-3-61-15-91-69-11-70-43-54-59-36
90-54-15-3-70-59-13-38-91-61-43-69-36-11-48
36-54-13-11-38-3-15-43-59-48-90-70-61-69-91
2) after 100mg of each bottle of sample is dissolved in 1mL of nuclease-free water, the sample nucleic acid is extracted by using a full-automatic nucleic acid extractor.
3) The newcastle disease virus RT-ddPCR method established in example 3 was used for detection.
4) And (3) calculating the average value of results of 3 times of detection of 15 bottles of the Newcastle disease nucleic acid standard substance, and performing homogeneity test on the Newcastle disease nucleic acid standard substance by adopting a one-factor analysis of variance method (F test).
The method using the one-factor analysis of variance in the step 4) includes:
setting:
the sum of the difference between groups:
within-group variance and sum:
degree of freedom between groups: v. of1＝m-1
In-group degree of freedom: v. of2＝N-m
Variance between groups was:
the intra-group variance is:
as statistic F:
it follows that the statistic is a degree of freedom (v)1,v2) F distribution variable of (2).
5) According to degree of freedom (v)1,v2) And given a significance level α, a critical F that can be looked up by the F tableαThe value is obtained. If the F value calculated according to the formula satisfies F<FαNo significant difference between data groups was considered. The sample is homogeneous if F.gtoreq.FαSystematic differences in the components, i.e., differences between samples, are suspected.
In the formula:
Q1The sum of the differences between the groups
Q2A sum of differences in the group
v1-between-groups degree of freedom
v2- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
s1 2-variance between groups
s2 2-within-group variance
Standard deviation s of uniformity between bottlesbbCan be calculated by the formula:
in this case, sbbEquivalent to the uncertainty component u due to inter-bottle inhomogeneitybb
ubb＝sbb
In the formula:
sbb-standard deviation between bottles
n- -number of measurements in the group
ubbComponent of uncertainty due to inter-bottle heterogeneity
If the measurement method for uniformity evaluation has poor repeatability, the method can possibly cause
When, or when, the estimated inter-vial standard, the standard deviation sbbLess than the standard deviation s of repeatabilityrTo sbbCannot adopt a formula
Then (c) is performed. At this time, the repeatability variance is given to sbbCan be formulated
In the formula:
One-way anova (table 10) was performed based on newcastle disease virus nucleic acid standard substance homogeneity evaluation measurement values (table 8) and the mean, variance and number of measurements of the samples tested (table 9).
TABLE 8 measurement of standard material homogeneity evaluation of NDV
TABLE 9 mean, variance and number of measurements of the samples
TABLE 10 analysis of variance
According to the degrees of freedom (v _1, v _2) and the given significance level α, F < F α (F (0.05,14,30) ═ 2.04) can be calculated by looking up the F value of the threshold value in table 8, indicating that there is no statistical difference between the groups, and thus the newcastle disease virus nucleic acid standard substance prepared by the present invention is uniform.
2. Stability evaluation
1) Short term stability assessment (i.e. stability during transport of sample)
3 vials of the Newcastle disease virus nucleic acid standards were randomly withdrawn and stored at 4 deg.C, 25 deg.C or 60 deg.C for1 week, 2 weeks or 4 weeks, respectively, as set forth in Table 11. And extracting the sample nucleic acid by using a fully automatic nucleic acid extractor. The newcastle disease virus RT-ddPCR method established in example 3 was used for detection, and 3 replicates were taken per vial. The short-term stability evaluation of the newcastle disease virus nucleic acid standard substance was performed using T-test according to sampling method and number of tests.
TABLE 11 Experimental Schedule
Note: and 4 ℃ synchronous stability inspection, namely, periodically storing the samples (meeting the stability inspection sampling quantity) in an environment of-80 ℃, and finally, testing all the taken samples under a repeatability condition. The amount of sample placement should be sufficient to detect for the scheduled time of the experiment or longer. The stability test at 25 ℃ and 60 ℃ adopts a timing detection observation result.
1-1) short-term stability test results at 4 ℃
The results of the short-term stability test at 4 ℃ are shown in Table 12.
T(0.95,2)*s(β_1)＝4.3*5.43E+01＝1.54E+03
Results and analysis: the stability results of the NDV standard substance under the storage condition of 4 ℃ are analyzed, and the result beta _1 is less than T (0.95,2) s (beta _1), so that the slope is considered to have no significant difference, no instability is observed, and the requirement of actual measurement can be met. The standard substance was considered to be stable for 4 weeks at 4 ℃.
TABLE 12 copy number of NDV reference substances at 4 ℃
1-2) short-term stability at 25 deg.C
The NDV standard substance is stored for 1-2 weeks at 25 ℃ and the detection results stored at-80 ℃ (Table 13) are subjected to T test statistical analysis (Table 14), wherein the P value is greater than >0.05 when the NDV standard substance is stored for 7 days, so that the detection value of the NDV standard substance stored for1 week at 25 ℃ is not significantly different from the detection value of the standard substance of a control group, and the NDV standard substance is stable for1 week at 25 ℃. Since the P value was less than 0.05 after 14 days of storage, the NDV standard substance was considered to be significantly different from the control standard substance in the test value measured after 2 weeks of storage at 25 ℃, and it was considered that the standard substance was not stable for 2 weeks at 25 ℃.
1-3) short-term stability test results at 60 DEG C
T test statistical analysis (table 16) is carried out on the detection results (table 15) of the newcastle disease virus nucleic acid standard substance stored in the environment of-80 ℃ and the storage time of 7 days at 60 ℃, wherein the P values are all less than 0.05, so that the detection value of the newcastle disease virus nucleic acid standard substance stored in the environment of 60 ℃ for1 week is considered to have significant difference from the detection value of the control group standard substance, and the standard substance can not be placed in the environment of 60 ℃.
TABLE 13 copy number of NDV reference substances at 25 ℃
TABLE 14 NDV Standard substance short-term stability at 25 ℃ paired T test results
TABLE 15 copy number of NDV reference substances at 60 ℃
TABLE 16 NDV Standard substance short-term stability at 60 ℃ paired T test results
2) Evaluation of Long term stability (i.e., stability under specific storage conditions)
Randomly extracting 3 bottles of the Newcastle disease virus nucleic acid standard substance, and respectively storing at-20 ℃ for1, 2, 4 and 6 months. And (3) extracting sample nucleic acid by using a full-automatic nucleic acid extractor, detecting by using the Newcastle disease virus RT-ddPCR method established in the embodiment 3, performing 3 repeated detections on each bottle of sample, and evaluating the long-term stability of the Newcastle disease virus nucleic acid standard substance by using T test according to the sampling method and the detection times. The results are shown in Table 17.
T (0.95,3) is obtained by table lookup: 3.18.
T(0.95,3)*s(β1)＝3.18*4.79E+02＝1.52E+03
calculating to obtain fruit beta1Less than T (0.95,3) s (beta)1) (should be larger than) so that the slope is not considered to have a significant difference, no instability is observed, and the requirement of actual measurement can be met. The standard substance is considered to be stable for 6 months at-20 ℃.
TABLE 17 copy number Table of NDV-20 ℃ Standard substance
Example 7 determination of Newcastle disease Virus nucleic acid standards
The newcastle disease virus nucleic acid standard substance having high uniformity and stability obtained in example 4 was subjected to a fixed value, and the fixed value was expressed as a standard value ± extended uncertainty. The characteristic values of the standard substance are measured by a digital PCR method independently in 8 laboratories which have certain technical authoritativeness, necessary conditions for measuring the characteristic quantity of the standard substance and equivalent technical capability and experience.
1. Standard value of Newcastle disease virus nucleic acid standard substance
1) Measurement of characteristic value of standard substance
Selecting 8 qualified laboratories, and numbering in sequence as follows: A-H, 8 laboratories each independently measured 4 bottles of highly homogeneous and stable Newcastle disease virus nucleic acid standard substance prepared in example 4 using digital PCR method, each bottle of substance was repeated 2 times, providing 8 characteristic values. The measured characteristic values are shown in Table 18.
2) Laboratory data suspect value inspection
The method comprises the steps of respectively checking suspicious values of 8 groups of data of 8 laboratories by a dixon method and a Grabas method, and removing the suspicious values by combining technical judgment.
TABLE 18 NDV nucleic acid Standard substance Cooperation rating test data summary (copies/mg)
TABLE 18.NDV nucleic acid Standard substance Cooperation rating test data summarization (copies/mg)
Wherein the dixon method: the measured data are arranged in the order from small to large, and r is respectively calculated1Value of sum rnValue r1＝(X(2)-X(1))/(X(n)-X(1))，rn＝(X(n)-X(n-1))/(X(n)-X(1)). If r1>rnAnd r is1>f(a,n)Then, X is judged(1)Is an abnormal value; if r1<rnAnd r isn>f(a,n)Then, X is judged(n)Is an abnormal value; if r1And rnAll values are less than f(a,n)All data was retained and the results are shown in table 19.
TABLE 19 data processing and conclusions of the Dixon method for each operator
In the Grabbs method, residual errors
When | vi|>λ(a，n)When s, then xiShould be kicked away. All data of the indoor NDV in this joint quantification are retained, and the results are shown in table 20.
TABLE 20 data processing and conclusions for each operator by the Grabbs method
3) Precision test of data between groups
And performing equal precision test on the standard deviation of each group of data by using a Kokern method, and determining whether to eliminate the data groups with significant differences after technical examination.
And (3) adopting a Koclen method to check whether the average values have equal precision, firstly calculating the variance of each group of n data of the m groups of data, and then calculating the ratio of the maximum variance to the sum of the m variances:
according to the significance level alpha, the data set number m, the repeated measurement times n and the Kekelen test critical value table, the method comprises the following steps: c (5%, 8,8) ═ 0.3043, calculated to give C(NDV)＝0.1940，C<C (5%, 8,8), indicating equal precision between the mean values of the sets of data.
4) Normal distribution test of whole data
The number of the data is 64, and the data are examined by a Dagol stano (D' ago) method appropriately.
The Y value was calculated from the data as-0.48, and when n is 64 and the confidence probability is 95%, the Y value falls within the range of the interval (-2.64 to 1.19), and the data was accepted as a normal distribution.
5) Calculation of standard value of nucleic acid standard substance of Newcastle disease virus
And (4) calculating the average value again according to the 8 average values obtained in the step table 18, wherein the obtained total average value is the standard value of the nucleic acid standard substance of the Newcastle disease virus.
E +05copies/mg2. extended uncertainty of Newcastle disease Virus nucleic acid Standard substance
The uncertainty in the valuing results for the newcastle disease virus nucleic acid standards is due to the uncertainty introduced by the uniformity, the uncertainty introduced by the stability, and the uncertainty introduced by the valuing process.
1) Assessment of uniformity-induced uncertainty
Uniformity-induced uncertainty the uniformity-induced uncertainty u was calculated in step 1 of example 6bb＝sbb1.05E +03copies/mg, relative standard uncertainty urel (bb) 0.0065.
2) Assessment of stability-induced uncertainty
The standard substance stored in an environment of (-20 + -2) ° c was measured once a month. After each detection, the data is processed, and the slope, intercept and uncertainty of the data are calculated. The validity period of the provisional product is 6 months, and the uncertainty contribution of the stability of the validity period t being 6 months is as follows:
us＝s(β1)·X
the value is the mean value of the stability test detection in the previous 6 months, and the calculation result is as follows:
s(β1)＝480copies/mg
us＝2.9E+03copies/mg
relative standard uncertainty urel(s)＝0.018
3) Assessment of uncertainty introduced by a valuating process
The uncertainty introduced by the valuing process includes a class A assessment of uncertainty u (A) and a class B assessment of uncertainty u (B).
The uncertainty u (a) introduced by the repeatability of the fixed-value measurement can be calculated from the relative standard deviation of the measurements.
u(A)＝s(A)＝5.4E+02copies/mg
Relative standard uncertainty urel(A)＝0.0071
The class B assessment comprises two parts: one part is the uncertainty introduced by the balance weighing and the other part is the uncertainty introduced by the detection instrument. Wherein the detection instrument comprises a pipette and a digital PCR.
3-1) evaluation of the uncertainty of the balance
The scale uncertainty refers to the electronic scale used this time, and the model is as follows: ML104/02, manufacturer: METTLER with calibration certificate number: JA18J-AD 000347. Its metering performance requirements, MPE: 0.5 mg; test load: 0.0100 g; its uncertainty U is 0.5 (mg). The weight to be weighed was 100mg, which was calculated
Degree of uncertainty
3-2) assessment of pipette uncertainty
In the combined evaluation process, the total content of 100mg of standard substance used is 1.7X 107copies, first step 200. mu.L of the eluate was taken with a 100-1000. mu.L pipette to elute the nucleic acids into the centrifuge tube (pipette relative uncertainty 2%) and mix the RNA solution homogeneously. The second dilution step is to take 100. mu.L of the RNA solution into a centrifuge tube (2% relative uncertainty of the pipette) by using a 10-100. mu.L pipette, and then to suck 900. mu.L of ddH by using a 100-1000. mu.L pipette2O (pipette relative uncertainty 1%) was mixed well with the RNA solution, and the volume of the solution was set to 1000. mu.L, achieving 10-fold dilution. Calculating the uncertainty introduced by the gradient diluted RNA standard solution, and the synthesis relative to the standard uncertainty u of the diluted standard solutionc(C) Comprises the following steps:
in the formula:
vr(V1) Relative uncertainty of the volume of nucleic acid eluate drawn up to 200. mu.L
vr(V2) Relative uncertainty of the volume of nucleic acid sample solution aspirated, 100. mu.L
vr(V3) ddH of suction2Relative uncertainty of O volume (900. mu.L, 10-fold dilution)
Relative uncertainty of 200. mu.L of solution removed with 100-:
V1relative standard uncertainty of (d):
in the formula: 2% is the relative uncertainty of 200. mu.L of water removed with a pipette; Δ v represents the change in solution volume at different temperatures, and 0.0008 is the correction of solution volume at different temperatures.
Relative uncertainty of 100. mu.L of solution removed with a 10-100. mu.L pipette:
V2relative standard uncertainty of (d):
in the formula: 2% is the relative uncertainty of 100. mu.L of stock solution removed by pipette; Δ v represents the change in solution volume at different temperatures, and 0.0008 is the correction of solution volume at different temperatures.
Relative uncertainty of 900. mu.L of water removed with a 10-1000. mu.L pipette:
in the formula: 1% is the relative uncertainty of 900. mu.L of water removed with a pipette; Δ v represents the change in solution volume at different temperatures, and 0.0008 is the correction of solution volume at different temperatures.
Thus, the relative uncertainty of the synthesis:
the uncertainties introduced by the gradient diluted RNA standard solution were:
3-3) evaluation of uncertainty of digital PCR Instrument
The test uses three types of digital PCR instruments of QX100, QX200 and QuantStaudio 3D, and because the latter two instruments are the latest instruments, no relevant data report on uncertainty exists, so the relative uncertainty of QX100 is uniformly used as the B type relative uncertainty of the test in the test.
The relative uncertainty generated in the measurement process of the digital PCR instrument consists of two parts: the relative standard uncertainty introduced by precision was 1.6% and the relative standard uncertainty introduced by droplet volume was 0.8%. Thus, the class B relative uncertainty caused by the digital PCR instrument is:
synthesis of class B uncertainty:
relative uncertainty introduced during the standard's valuing:
3. magnitude and extended uncertainty of newcastle disease virus nucleic acid standards
The results of the magnitude and extended uncertainty calculations for the newcastle disease virus nucleic acid standards are shown in table 21.
TABLE 21 Newcastle disease Virus nucleic acid Standard Mass values and extended uncertainties
4. Newcastle disease virus nucleic acid standard mass value results
The identification value of the Newcastle disease virus nucleic acid standard substance is 1.7 multiplied by 105copies/mg, extension uncertainty 0.2X 105copies/mg (see Table 22).
TABLE 22 NDV Standard Mass-value results
Sequence listing
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cggattatcg caactagcag tggcggttgg gaagatgcag cagtttgtta 170
Claims (10)
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.
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