Method for measuring virus titer of live attenuated measles, mumps, rubella and varicella combined vaccine

Abstract

Provided are a primer-probe combination for real-time quantitative PCR, a related kit, and a method for using primers to measure the titers of measles, mumps and varicella virus pathogens. The method is applicable to clinical and laboratory detection, is fast and convenient, and has high sensitivity.

Description

A method for detecting the virus titer of live attenuated measles, mumps, rubella and varicella combined vaccine TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly to the use of real-time quantitative PCR technology for detecting the infectious titer of measles, mumps and varicella viruses in live attenuated measles, mumps, rubella and varicella combined vaccine. BACKGROUND

[0002] Measles, mumps, rubella and varicella are common viral infectious diseases in children, which are widely spread and easily outbreak. At present, the live attenuated measles, mumps and rubella combined vaccine has been included in the immunization program in China, and the use of live attenuated varicella vaccine is also being promoted. The development of live attenuated measles, mumps, rubella and varicella combined vaccine (MMRV) not only simplifies the immunization program, but also reduces the vaccination cost and the rate of missed vaccination. Accurate determination of the infectious titer of viruses in the combined vaccine is of great significance for evaluating the quality of the vaccine and ensuring the immunization effect of the vaccine.

[0003] According to the Chinese Pharmacopoeia, the titer detection of the combined vaccine requires the complete neutralization of other viruses except the virus to be tested by using specific antiserum. In actual operation, there are often problems such as the quality, supply and interference with the target virus of the neutralizing serum. Moreover, the neutralization process requires high-quality antiserum and strict verification. If a certain virus is sensitive to the neutralization process, it will lead to inaccurate results or actual titers higher than the measured results, which not only may cause safety risks of over-dosage, but also increases the production cost and social cost. Therefore, it is necessary to develop a detection method without neutralization process for product quality control.

[0004] It is of great significance to develop an accurate and efficient titer detection method for measles, mumps and varicella viruses without neutralization process for the field. SUMMARY

[0005] The purpose of the present application is to provide a method for detecting the virus titer of live attenuated measles, mumps, rubella and varicella combined vaccine and a primer probe combination thereof.

[0006] In the first aspect of the present application, a primer probe combination for detecting the pathogens of measles, mumps and varicella viruses is provided, which specifically binds to the following pathogen genes and is used for amplifying specific amplification products corresponding to the respective genes: measles virus, mumps virus and varicella virus; the primer probe combination comprises a primer pair and a probe selected from the following group:

[0007] a first primer pair and a probe therefor for amplifying a measles virus, the first primer pair and the probe therefor being selected from the group consisting of:

[0008] (A1) an upstream primer as shown in SEQ ID NO: 1, a downstream primer as shown in SEQ ID NO: 2, and a probe as shown in SEQ ID NO: 3,

[0009] (A2) an upstream primer as shown in SEQ ID NO: 4, a downstream primer as shown in SEQ ID NO: 5, and a probe as shown in SEQ ID NO: 6,

[0010] (A3) an upstream primer as shown in SEQ ID NO: 7, a downstream primer as shown in SEQ ID NO: 8, and a probe as shown in SEQ ID NO: 9;

[0011] a second primer pair and a probe therefor for amplifying a mumps virus, the second primer pair and the probe therefor being selected from the group consisting of:

[0012] (B1) an upstream primer as shown in SEQ ID NO: 10, a downstream primer as shown in SEQ ID NO: 11, and a probe as shown in SEQ ID NO: 12,

[0013] (B2) an upstream primer as shown in SEQ ID NO: 13, a downstream primer as shown in SEQ ID NO: 14, and a probe as shown in SEQ ID NO: 15; and

[0014] a third primer pair and a probe therefor for amplifying a varicella virus, the third primer pair and the probe therefor being selected from the group consisting of:

[0015] (C1) an upstream primer as shown in SEQ ID NO: 16, a downstream primer as shown in SEQ ID NO: 17, and a probe as shown in SEQ ID NO: 18,

[0016] (C2) an upstream primer as shown in SEQ ID NO: 19, a downstream primer as shown in SEQ ID NO: 20, and a probe as shown in SEQ ID NO: 21,

[0017] (C3) an upstream primer as shown in SEQ ID NO: 22, a downstream primer as shown in SEQ ID NO: 23, and a probe as shown in SEQ ID NO: 24.

[0018] In another preferred embodiment, the pathogen gene is selected from RNA nucleic acids of the pathogen.

[0019] A second aspect of the present application provides a method of detecting titers of measles, mumps and varicella viruses, the method comprising the steps of:

[0020] (a) providing a detection sample comprising nucleic acid extracts of measles, mumps and rubella viruses;

[0021] (b) performing real-time quantitative PCR amplification reaction on the detection sample using the primer probe combination as described in the first aspect of the present application, with virus reference of measles, mumps and rubella viruses as standard, to calculate the relative titer of measles, mumps and rubella viruses in the detection sample.

[0022] In another preferred embodiment, the detection sample is obtained by inoculating the virus sample to cells and purifying the nucleic acid extracts after cell infection.

[0023] In another preferred embodiment, the cells are selected from the group consisting of diploid cells (MRC-5, 2BS, WI-38), rabbit kidney cells (RK-13), monkey kidney cells (Vero) and chicken embryo fibroblasts (CPE).

[0024] In another preferred embodiment, the inoculation is selected from the group consisting of direct inoculation, adsorption inoculation.

[0025] In another preferred embodiment, the nucleic acid extracts are RNA extracts.

[0026] In another preferred embodiment, the virus reference is selected from the group consisting of single virus reference, multiple virus reference.

[0027] In another preferred embodiment, the multiple virus reference is a mixture of measles, mumps, rubella and varicella viruses in a certain ratio.

[0028] In another preferred embodiment, the measles, mumps and rubella viruses are from measles, mumps and rubella combined attenuated live vaccine.

[0029] In another preferred embodiment, the detection is performed 24h to 60h after the measles, mumps and rubella viruses are inoculated to the cells.

[0030] In another preferred embodiment, the virus sample is inoculated to the cells after dilution; preferably, a dilution gradient of 1.5-5 times, more preferably, a dilution gradient of 2-3 times, more preferably, a dilution gradient of 3 times.

[0031] In another preferred embodiment, 3 to 4 dilution gradients are selected for inoculation in each detection.

[0032] In another preferred embodiment, the titer of measles, mumps and rubella viruses in the detection sample is calculated by using the method of reaction parallel lines.

[0033] In another preferred embodiment, the calculation is determined by using the methods of 3.3, 3.2, 4.3 or 4.2 in the method of reaction parallel lines.

[0034] In another preferred embodiment, the detection is performed in a mode of setting of 4 dilutions of the reference, 4 replicates for each dilution, 3 dilutions of the sample, 4 replicates for each dilution.

[0035] In another preferred embodiment, the detection method has a detection recovery rate substantially consistent with the virus titration method of the Chinese Pharmacopoeia Volume III.

[0036] In another preferred embodiment, the substantial consistency means that under the same detection conditions, the difference between the average detection recovery rate of the detection method and the average detection recovery rate of the virus titration method of the Chinese Pharmacopoeia Volume III is not more than 15%, preferably not more than 10%, more preferably not more than 5%.

[0037] In another preferred embodiment, the volume of the detection sample inoculated is 10-100 μl / well, preferably 10-50 μl / well, more preferably 20-30 μl / well, more preferably 20 μl / well.

[0038] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0039] In another preferred embodiment, the method is in vitro.

[0040] In another preferred embodiment, the PCR amplification system comprises: template RNA, forward primer, reverse primer, probe, and other buffer reagents.

[0041] In another preferred embodiment, the PCR amplification system is: a total volume of 20 μL, wherein the RT-qPCR buffer reagent is 5 μL, each virus' s upstream and downstream primers with a concentration of 10 μmol / ml are 0.8 μL each, each virus' s probe is 0.2 μL, and the RNA template is 8 μL.

[0042] In another preferred embodiment, the PCR amplification program is: 50 ℃ for 5 min; 95 ℃ pre-denaturation for 20 s; 95 ℃ denaturation for 3 s, 60 ℃ annealing for 30 s, 40 cycles.

[0043] The third aspect of the present application provides a kit for detecting the pathogen of measles, mumps and varicella virus, which comprises (i) a container, and (ii) the primer probe combination as described in the first aspect of the present application in the container.

[0044] It should be understood that, within the scope of the present application, each of the technical features described above and in the following (such as the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 shows the proliferation of DNA and RNA after varicella virus infection of cells.

[0046] Figure 2 shows the proliferation of MMRV in human diploid cell substrate.

[0047] Figure 3 shows the linear analysis of the proliferation of MMRV in MRC-5 cell substrate.

[0048] Figure 4 shows the linear analysis of the proliferation of MMRV in 2BS cell substrate.

[0049] Figure 5 shows the linear analysis of the proliferation of MMRV in WI-38 cell substrate.

[0050] Figure 6 shows the detection linear range of the method for detecting single virus.

[0051] Figure 7 shows the accuracy of the detection results using the method and the pharmacopoeia method.

[0052] Figure 8 shows the interference between viruses and the recovery rate.

[0053] Figure 9 shows the consistency of the virus titers detected using the method and the pharmacopoeia method in the presence of other viruses.

[0054] Figure 10 shows the recovery rate of the three virus titers in MMRV calculated using different reference products.

[0055] Figure 11 shows the consistency of the results of the three virus titers calculated using different reference products. DETAILED DESCRIPTION

[0056] The present inventors have established a method for detecting the infectivity titers of measles, mumps and varicella viruses in measles, mumps and rubella combined attenuated live vaccine by RT-qPCR. The method of the present application can be used to simultaneously detect the three viruses in one cell substrate. The initial titer of the sample to be detected is calculated by detecting the content of specific RNA nucleic acid fragments produced by the proliferation of the viruses after infection of cells and comparing it with the known titer reference product.

[0057] Measles virus

[0058] Measles virus is the pathogen of measles, which belongs to the genus of measles virus in the family of paramyxoviridae. Measles is a common acute infectious disease in children, which is highly contagious and characterized by papular rash, fever and respiratory symptoms. Measles virus is spherical or filamentous, with a diameter of about 120nm-250nm, a core of single negative strand RNA, no segment, and a genome of about 16kb in length.

[0059] Mumps virus

[0060] Mumps virus belongs to Paramyxoviridae, Paramyxovirus. The virus is spherical, with a diameter of 100-200 nm, and the genome is single-stranded negative-strand RNA, and the capsid is helical symmetry. There are HA and NA protrusions on the envelope, and the components are glycoproteins. So far, only one serotype of mumps virus has been found. The virus can proliferate in the amniotic cavity of chicken embryos and also in monkey kidney cell culture, and can cause cell fusion to form multinucleated giant cells.

[0061] Varicella virus

[0062] Varicella-zoster virus (VZV) is a round particle with a diameter of 150-200 nm, which includes three layers of nucleocapsid, cortex and envelope from inside to outside. The nucleic acid is DNA, which proliferates in cells. Patients are the only source of infection. VZV exists in respiratory secretions, blisters and blood of patients, and is transmitted by droplets or direct contact with blister fluid. It is known that VZV can be transmitted by medical devices. VZV mainly invades the respiratory tract, grows and reproduces on the mucosa, and then enters the blood and lymph, and reproduces for the second time in reticuloendothelial cells, causing viremia and systemic lesions. The main damage site is the skin, and occasionally involves internal organs.

[0063] Primer pair (primer sequence) of the present application

[0064] As used herein, the term "primer" has the meaning conventionally understood by those skilled in the art. The primer of the present application is specific to the conserved region sequence of the virus.

[0065] In one specific embodiment, the primer sequence comprises:

[0066] a first primer pair for amplifying measles virus, the first primer pair being selected from:

[0067] an upstream primer as set forth in SEQ ID NO: 1, a downstream primer as set forth in SEQ ID NO: 2,

[0068] an upstream primer as set forth in SEQ ID NO: 4, a downstream primer as set forth in SEQ ID NO: 5,

[0069] an upstream primer as set forth in SEQ ID NO: 7, a downstream primer as set forth in SEQ ID NO: 8;

[0070] a second primer pair for amplifying mumps virus, the second primer pair being selected from:

[0071] an upstream primer as set forth in SEQ ID NO: 10, a downstream primer as set forth in SEQ ID NO: 11,

[0072] an upstream primer as set forth in SEQ ID NO: 13, a downstream primer as set forth in SEQ ID NO: 14; and

[0073] a third primer pair for amplifying varicella-zoster virus, the third primer pair being selected from the group consisting of:

[0074] an upstream primer as set forth in SEQ ID NO: 16, a downstream primer as set forth in SEQ ID NO: 17,

[0075] an upstream primer as set forth in SEQ ID NO: 19, a downstream primer as set forth in SEQ ID NO: 20,

[0076] an upstream primer as set forth in SEQ ID NO: 22, a downstream primer as set forth in SEQ ID NO: 23.

[0077] probe

[0078] The term "probe" as used herein has the meaning commonly understood by one of ordinary skill in the art, i.e., a short piece of single-stranded DNA or RNA fragment used to detect a nucleic acid sequence complementary to it.

[0079] In view of the teachings of the present application and the common general knowledge of one of ordinary skill in the art, it should be apparent that, given the primer pair, one of ordinary skill in the art can design a probe according to the template sequence between the forward primer and reverse primer binding sites and detect the technical effect of the probe in combination with the primer pair. In specific embodiments, one of ordinary skill in the art can design a probe according to the need, which can be in a liquid phase or immobilized on a solid phase; which can be combined before amplification or after amplification. Therefore, the probe of the present application is not limited to the probe specifically disclosed in the examples. The primer pair of the present application is also not limited to pairing with the probe specifically disclosed in the examples.

[0080] detection method

[0081] The method of the present application for titer detection of measles, mumps and varicella viruses comprises the following steps:

[0082] (a) providing a detection sample, the detection sample comprising: nucleic acid extract of measles, mumps and varicella viruses;

[0083] (b) performing real-time quantitative PCR amplification reaction on the detection sample using the primer pair of the present application, taking virus reference of measles, mumps and varicella viruses as standard, thereby calculating the relative titer of measles, mumps and varicella viruses in the detection sample.

[0084] In another preferred embodiment, the nucleic acid extract is RNA extract.

[0085] In another preferred embodiment, the virus reference is selected from the group consisting of: single virus reference, multiple virus reference.

[0086] In another preferred embodiment, the detection is performed 24h to 60h after the measles, mumps and varicella virus inoculation to the cell.

[0087] Advantages of the present application:

[0088] 1) The detection method of the present application can realize the detection of the titers of the measles, mumps and varicella viruses in MMRV in one virus inoculation experiment in one cell matrix.

[0089] 2) The detection method of the present application can eliminate the influence of the antiserum in the neutralization process by omitting the neutralization step in the detection of the combined vaccine.

[0090] 3) The detection method of the present application can shorten the time for the detection of the titers of the viruses in MMRV from 8-10 days in the traditional method to 3-5 days, thereby improving the detection throughput.

[0091] 4) The detection method of the present application can realize automation in the operation process, and the detection data can be presented in digital form, and has the advantage of traceability.

[0092] 5) The detection method of the present application has good specificity and strong anti-interference property.

[0093] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples are not specified, and the methods are usually performed according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0094] Example 1. Feasibility analysis of detecting varicella virus at RNA level

[0095] In order to confirm the amplification feasibility of the virus target gene of the present application at the nucleic acid level, considering that the varicella virus is a DNA virus, a comparative experiment of the DNA and RNA proliferation after the varicella virus infects the cell was performed, and the detection results are shown in Figure 1.

[0096] As can be seen from the figure, it is unexpectedly found that during the proliferation process of 0-72h, no obvious proliferation can be detected at the virus DNA level, while the virus RNA level has an obvious proliferation trend, which can verify the feasibility of detecting the virus titer from the varicella virus RNA level.

[0097] Example 2 Primer and probe for detecting nucleotide sequence and virus related to the present application

[0098] The measles virus gene sequence (accession number: FJ416067) was downloaded from the NCBI gene library, and specific primer pairs and TaqMan probes were designed in the conserved region thereof, as shown in Table 1.

[0099] Table 1

[0100] The mumps virus gene sequence (accession number: HQ416906.1, AF338106) was downloaded from the NCBI gene library, and specific primers and TaqMan probes were designed in the conserved region thereof, as shown in Table 2.

[0101] Table 2

[0102] The varicella virus gene sequence (accession number: AB097932) was downloaded from the NCBI gene library, and specific primers and TaqMan probes were designed in the conserved region thereof, as shown in Table 3.

[0103] Table 3

[0104] The regions of the GenBank sequence of the measles virus, mumps virus and varicella virus to which the primers and TaqMan probes of the present application are directed are shown in SEQ ID NOs. 25-32, respectively.

[0105] Measles virus:

[0106] SEQ ID NO. 25:

[0107] SEQ ID NO. 26:

[0108] SEQ ID NO. 27:

[0109] Mumps virus:

[0110] SEQ ID NO. 28:

[0111] SEQ ID NO. 29:

[0112] Varicella virus:

[0113] SEQ ID NO. 30:

[0114] SEQ ID NO. 31:

[0115] SEQ ID NO.32:

[0116] Example 3 primer probe specificity

[0117] Four cell substrates, human diploid cells (MRC-5, 2BS, WI-38), rabbit kidney cells (RK-13), monkey kidney cells (Vero) and chicken embryo fibroblasts (CPE), and four virus stocks, measles virus, mumps virus, varicella virus and rubella virus, were selected. After nucleic acid purification, the nucleic acid of each cell substrate and virus stock was detected by RT-qPCR using the designed and synthesized primer probe. Ct value ≥ 35 was negative, represented as “-”. Ct value < 35 was positive, represented as “+”. The detection results are shown in Table 4. The designed primer probes can meet the requirements of detection specificity, among which SEQ ID NO. 1, NO. 2, NO. 3, NO. 10, NO. 11, NO. 12, NO. 16, NO. 17 and NO. 18 are preferentially selected for subsequent experiments.

[0118] Table 4 primer probe specificity

[0119] Example 4 virus culture and RT-qPCR detection

[0120] Cells growing to a dense monolayer were prepared into a cell suspension with a concentration of 1-1.5×10 5 Cells growing to a dense monolayer were prepared into a cell suspension with a concentration of 1-1.5×10 After the cells were full-grown, the culture solution was discarded, and the virus sample was inoculated into the 96-well plate at 20 μl per well after 3-fold serial dilution. After incubation in a 37℃, 5% CO2 incubator until the due date, harvesting was performed. The culture solution was discarded, and RNA purification was performed using a nucleic acid automatic extractor after adding lysis solution. One-step RT-qPCR detection was performed using Fast Virus 1-Step Master Mix, and the reaction system was as follows: 4× Fast Virus 1-Step Master Mix reagent 5 μl, 10 μmol / ml measles virus upstream primer 0.8 μl, 10 μmol / ml measles virus downstream primer 0.8 μl, 10 μmol / ml measles virus probe 0.2 μl, 10 μmol / ml mumps virus upstream primer 0.8 μl, 10 μmol / ml mumps virus downstream primer 0.8 μl, 10 μmol / ml mumps virus probe 0.2 μl, 10 μmol / ml varicella virus upstream primer 0.8 μl, 10 μmol / ml varicella virus downstream primer 0.8 μl, 10 μmol / ml varicella virus probe 0.2 μl, 8 μl of the RNA template of the sample to be tested, and using RNase-free Water to make the final system 20 μl. The reaction conditions are as follows: 50°C for 5 min, 95°C for 20 s, 95°C for 3 s, 60°C for 30 s, 40 cycles. Taking single virus reference as the standard, the relative titer (lg QPA / ml) of the sample was calculated according to the method 3.3 in the parallel line method of the general rule 1431 in the third part of the Pharmacopoeia.

[0121] Example 5 Confirmation of the optimal cell matrix for the proliferation and detection of MMRV vaccine in cells

[0122] The MMRV vaccine was inoculated into different cells according to Example 4, and after 0, 12, 24, 36, 48 and 60 h of culture, RNA was extracted for RT-qPCR detection. Among them, as shown in Table 5 and FIG. 2, the proliferation of the three viruses in MMRV in MRC-5, 2BS, WI-38 cells. It can be seen that the proliferation trends of measles, mumps and varicella viruses in human diploid cells are consistent, and they show stable growth in the process of 12 to 60 hours. The cells are suitable for the three viruses, and can meet the purpose of detecting the three viruses in one cell matrix.

[0123] Table 5 Proliferation of the three viruses in MMRV in human diploid cells

[0124] Example 6 Confirmation of the optimal detection time of the method

[0125] The MMRV vaccine was inoculated into cells according to Example 4, and according to the linear relationship between the cycle threshold (Ct) and the dilution factor (lg logarithmic value), the optimal detection time was confirmed (FIGS. 3-5). The detection Ct values of the three viruses in different cells cultured for 24 h to 60 h were analyzed by linear regression analysis with the virus dilution factor (lg logarithmic value), and the linear relationship was good (Tables 6-8), R 2 >0.9. It can be confirmed that 24 h to 60 h are suitable detection time windows.

[0126] Table 6 Linear analysis of MMRV proliferation in MRC-5 cells

[0127] Table 7 Linear analysis of MMRV proliferation in 2BS cells

[0128] Table 8 Linear analysis of MMRV proliferation in WI-38 cells

[0129] Example 7 Linear range and accuracy of RT-qPCR method for detecting virus titer

[0130] The measles virus stock, mumps virus stock and varicella virus stock were diluted by 3-fold serial dilution, for a total of 6 dilution levels. The cells were inoculated according to Example 4, with 4 replicate wells for each dilution level. The titer of the serially diluted samples was calculated using the three single viruses as reference, and the titer was also determined using the viral titration method of the third volume of the Pharmacopoeia. The correlation and recovery rate of the titration results of the RT-qPCR method and the Pharmacopoeia method were compared to confirm the linear range and accuracy of the detection method.

[0131] The measles virus stock with a theoretical value of 2.21-4.60 lg CCID 50 / ml, the mumps virus stock with a theoretical value of 5.75-3.36 lg CCID 50 / ml, and the varicella virus stock with a theoretical value of 5.08-2.69 lg PFU / ml were detected for relative titer according to Example 3, and the recovery rate was calculated. The detection recovery rate of the measles virus was 94%-120%, the detection recovery rate of the mumps virus was 86%-115%, and the detection recovery rate of the varicella virus was 93%-113% (Figure 6).

[0132] The linear regression curve was plotted with the theoretical potency value of the sample as the abscissa and the relative titer value measured by the RT-qPCR method or the titer value measured by the Pharmacopoeia method as the ordinate (Figure 7), and the linear relationship of the three viruses was good. Linear regression analysis was performed on the curve, and the correlation coefficient R was not less than 0.9 (measles virus: 0.9700, mumps virus: 0.9174, varicella virus: 0.9589), P < 0.01, and the statistical model was meaningful.

[0133] Example 8 Interference of RT-qPCR method for detecting target viruses

[0134] The virus titration was detected according to the method of Example 4, and each dilution had 4 replicates. The virus dilution was used to replace the interfering virus stock, and the target single virus stock was added in the same proportion, and the titration was detected simultaneously according to the virus titration method of the third part of the pharmacopoeia. By comparing the correlation and recovery rate of RT-qPCR method and pharmacopoeia method, the interference of the detection method was evaluated.

[0135] As shown in Figure 8, the measles virus was in the range of 5.20-2.81 lg CCID 50 / ml titers, and the detection recovery rate was 96-110%. The mumps virus was in the range of 5.52-3.13 lg CCID 50 / ml titers, and the detection recovery rate was 87-105%. The varicella virus was in the range of 5.00-2.61 lg PFU / ml titers, and the detection recovery rate was 87-109%.

[0136] The relative titer value detected by the RT-qPCR method or the titer value detected by the pharmacopoeia method was used as the vertical coordinate, and the theoretical potency value of the sample was used as the horizontal coordinate. The linear regression curve was drawn (Figure 9). The results determined by the RT-qPCR method showed a linear relationship with the theoretical value, and R 2 >0.95. The results of the two detection methods were consistent, and the correlation coefficient R was >0.95. It can be confirmed that when the MMRV virus titer is detected by the method, the infectious titer of the target virus can be accurately determined in the presence of heterologous viruses.

[0137] Example 9: Detection of MMRV accuracy using single virus reference and MMRV reference

[0138] The titration of the serially diluted MMRV sample was detected according to the method described in Example 4, and each dilution had 4 replicates. The titration results of the sample were calculated using the single virus reference and the MMRV reference, respectively. By analyzing the recovery rate of the results calculated by the two references and the consistency of the detection results, the detection accuracy of the MMRV sample was confirmed.

[0139] As shown in Figure 10, the measles virus was in the range of 4.34-2.91 lg CCID 50 / ml titers, and the detection recovery rate was 97-109% when the single virus was used as the reference, and the detection recovery rate was 95-108% when the MMRV was used as the reference. The mumps virus was in the range of 5.65-4.21 lg CCID 50The detection recovery rate of the single virus as the reference was 94-105% in the 4.90-3.46 lg PFU / ml titer range, and the detection recovery rate of the MMRV as the reference was 91-104%. The detection recovery rate of the single virus as the reference was 81-102% in the 4.90-3.46 lg PFU / ml titer range, and the detection recovery rate of the MMRV as the reference was 81-105%.

[0140] The single virus reference and the MMRV reference are prepared in the same way, and the difference is that the single virus reference is prepared by a single virus, and the MMRV reference is prepared by mixing the measles, mumps, rubella and varicella viruses in a certain proportion. The Chinese Pharmacopoeia stipulates that the single virus reference is used for the detection of virus titer of the combined vaccine. The experimental results of the present application show that the two references can be used for the detection of virus titer of the measles, mumps, rubella and varicella combined vaccine.

[0141] The theoretical potency value of the sample is used as the horizontal coordinate, and the sample titer value calculated by using the single virus reference or the MMRV reference is used as the vertical coordinate, and a straight line regression curve is drawn (Figure 11). It can be seen that the calculation results of the two references are consistent, and the correlation coefficient R is greater than 0.98. It can be confirmed that when the MMRV virus titer is detected by using the present method, the single virus reference or the MMRV reference can be selected for the calculation of the sample, and the MMRV vaccine is preferably selected as the reference.

[0142] Discussion

[0143] At present, the detection technology in the field of biological medicine is in the development period. With the nucleic acid detection method included in the pharmacopoeia, the related application of real-time fluorescent quantitative technology in the quality control of biological products is increasingly expanded, and the quality control requirements of virus-related products are gradually strict. The detection of virus potency of each component vaccine of the combined attenuated live vaccine is the key to the quality control of the product, but it is challenging to accurately determine the infectious titer of each virus in the combined vaccine, and often faces the problems of the quality, supply of neutralizing serum and the interference of the target virus.

[0144] The method for detecting the virus titer of the measles, mumps and rubella combined vaccine based on cell infection provided by the present application can avoid the influence of the neutralization process and eliminate the interference of the neutralizing serum without the neutralization step. Moreover, the detection mode of multiplex RT-qPCR has the technical advantage of efficient detection. Whether in the update and development of the detection technology or in the digitization and traceability of the detection data, it has a certain competitiveness.

[0145] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to readers of the disclosure and the appended claims.

Claims

1. A primer probe combination for detecting a pathogen of measles, mumps and varicella virus, characterized by, The primer probe combination specifically binds to the following pathogen genes and is used to amplify specific amplification products corresponding to each of the genes: measles virus, mumps virus, and varicella virus; the primer probe combination includes a primer pair and a probe selected from the group consisting of: a first primer pair and its probe for amplifying measles virus, the first primer pair and its probe being selected from the group consisting of: (A1) an upstream primer as shown in SEQ ID NO: 1, a downstream primer as shown in SEQ ID NO: 2, and a probe as shown in SEQ ID NO: 3, (A2) an upstream primer as shown in SEQ ID NO: 4, a downstream primer as shown in SEQ ID NO: 5, and a probe as shown in SEQ ID NO: 6, (A3) an upstream primer as shown in SEQ ID NO: 7, a downstream primer as shown in SEQ ID NO: 8, and a probe as shown in SEQ ID NO: 9; a second primer pair and its probe for amplifying mumps virus, the second primer pair and its probe being selected from the group consisting of: (B1) an upstream primer as shown in SEQ ID NO: 10, a downstream primer as shown in SEQ ID NO: 11, and a probe as shown in SEQ ID NO: 12, (B2) an upstream primer as shown in SEQ ID NO: 13, a downstream primer as shown in SEQ ID NO: 14, and a probe as shown in SEQ ID NO: 15; and a third primer pair and its probe for amplifying varicella virus, the third primer pair and its probe being selected from the group consisting of: (C1) an upstream primer as shown in SEQ ID NO: 16, a downstream primer as shown in SEQ ID NO: 17, and a probe as shown in SEQ ID NO: 18, (C2) an upstream primer as shown in SEQ ID NO: 19, a downstream primer as shown in SEQ ID NO: 20, and a probe as shown in SEQ ID NO: 21, (C3) an upstream primer as shown in SEQ ID NO: 22, a downstream primer as shown in SEQ ID NO: 23, and a probe as shown in SEQ ID NO:

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2. A method of detecting titers of measles, mumps and rubella viruses, characterized by, The method comprises the steps of: (a) providing a detection sample, the detection sample comprising: nucleic acid extracts of measles, mumps, and varicella viruses; (b) performing real-time quantitative PCR amplification reaction on the detection sample using the primer probe combination as claimed in claim 1, taking virus reference products of measles, mumps, and varicella viruses as standards, to calculate the relative titers of measles, mumps, and varicella viruses in the detection sample.

3. The method of claim 2, wherein, The detection sample is obtained by inoculating a virus sample into cells and purifying nucleic acid extracts after cell infection.

4. The method of claim 3, wherein, The cells are selected from the group consisting of diploid cells (MRC-5, 2BS, WI-38), rabbit kidney cells (RK-13), monkey kidney cells (Vero), and chicken embryo fibroblasts (CPE).

5. The method of claim 2, wherein, The nucleic acid extracts are RNA extracts.

6. The method of claim 2, wherein, The virus reference products are selected from the group consisting of single virus reference products, multiple virus reference products.

7. The method of claim 2, wherein, The measles, mumps and rubella virus is from a measles, mumps and rubella combined attenuated live vaccine.

8. The method of claim 3, wherein, The detection is performed 24h to 60h after inoculation of the measles, mumps and rubella virus to the cells.

9. The method of claim 2, wherein, The titer of the measles, mumps and rubella virus in the test sample is calculated using the method of parallel lines for quantal response.

10. A kit for detecting a measles, mumps and rubella virus pathogen, the kit comprising (i) a container, and (ii) the primer probe combination of claim 1 in the container.

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