Primer pair, probe and kit for rapidly distinguishing mouse hepatitis virus, reovirus type III, mouse poxvirus and mouse parvovirus

Abstract

The invention belongs to the technical field of biological detection, and discloses a primer pair, a probe and a kit for rapidly distinguishing mouse hepatitis virus, reovirus type III, mouse pox virus and mouse parvovirus. Meanwhile, the invention also discloses a multiple fluorescent quantitative PCR method for simultaneously detecting mouse hepatitis virus, reovirus type III, mouse pox virus and mouse parvovirus by adopting the kit. The kit comprises: a) an amplification reaction solution, b) a primer mixed solution, c) an enzyme mixed solution, and d) a positive control. The multiple real-time detection kit provided by the invention can jointly detect mouse hepatitis virus, reovirus type III, mouse pox virus and mouse parvovirus at one time, and has the advantages of rapidness, simplicity, high sensitivity and strong specificity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a multiplex fluorescence quantitative PCR method for rapidly distinguishing mouse hepatitis virus, reovirus type III, ectromelia virus and parvovirus, and a primer pair, a probe and a kit specially used for the method. BACKGROUND

[0002] Mouse hepatitis virus (MHV) belongs to the family of Coronaviridae and the genus of Coronavirus, and is a single-stranded positive RNA virus. The natural host is mouse, and it can also easily infect nude mice and immunodeficient mice. Mouse hepatitis virus generally presents as hidden or subclinical infection, but has high infectivity, and the transmission routes are divided into droplet infection or oral infection according to different types. Mouse hepatitis virus can cause hepatitis, encephalitis and enteritis in mice, and the immune function of infected mice can be decreased, thereby causing acute infection of the mouse population and leading to animal death.

[0003] Mouse reovirus type III (Reo III) belongs to the family of Reoviridae and the genus of Orthoreovirus in classification. Reo III is mainly transmitted through the digestive tract, respiratory tract, air and fecal-oral route, and the main reason for virus pollution is that Reo III can stably survive in the environment. When mice are infected, acute cases are mainly found in newborn and weaned mice, and chronic cases are found in mice over 28 days old, and the clinical manifestations are characterized by oily fur hand effect and fatty diarrhea. The pathological changes are mainly hepatitis, encephalitis and pancreatitis, which can reduce the activities of pancreatic amylase and lipase in infected mice, increase the activity of trypsin, destroy the B cells of pancreatic islets to reduce insulin secretion, and produce metabolic and pathological changes similar to diabetes. Reo III also plays an immune stimulating role in the response of the host to environmental carcinogens, seriously interferes with animal experiments, and is one of the more serious viral diseases of experimental mice.

[0004] Ectromelia virus (ECTV) belongs to the Orthpoxvirus of Chordopoxvirinae of Poxviridae, which is a single-molecule double-stranded DNA virus with brick-shaped virion, 200 nm x 200 nm x 250 nm in size, and resistant to diethyl ether. The infection of mousepox virus is generally divided into acute infection, chronic infection and latent infection. Among them, acute infection and chronic infection are easy to find, while latent infection of mousepox mice is healthy on the surface without any clinical symptoms. The virus gene exists in a certain tissue or cell and does not produce infectious virus, and it is generally not easy to detect by the current routine serological antibody detection method. When the virus is activated under certain conditions, acute onset occurs. Therefore, the latent infection of mouse group is difficult to find in time and is very harmful. Therefore, strengthening detection and improving detection methods should be paid attention to.

[0005] Parvovirus infecting mice belongs to single-strand negative DNA virus, and the infection rate is high in ordinary level mice. Both mouse minute virus (MVM) and mouse parvovirus (MPV) have been isolated from mice. The MVM strain was first discovered in 1966, and subsequent studies found that the virus strain had an inhibitory effect on tumors in vivo. The MPV strain, which was initially considered to be an isolated parvovirus strain, was first isolated from cytotoxic T lymphocytes in 1933. MVM and MPV strains, like other parvoviruses, have strong physical stability and certain tolerance to acid, alkali and temperature. In the production of biological products, many cell lines used in biological products are derived from murine cells such as Chinese hamster ovary cells (CHO) and baby hamster kidney cells, which are easily contaminated by parvovirus strains. Mice naturally infected with parvovirus are usually asymptomatic, but studies have shown that young mice and immunocompromised mice infected with MVM strains develop dwarfism, sudden death and hematopoietic dysfunction. Compared with MVM infection, laboratory and naturally infected mice with MPV strains have no pathological conditions. Due to the weak or asymptomatic symptoms of clinical infection and the strong viability of parvovirus, it has become a very difficult pathogen to eradicate in animal barrier facilities.

[0006] The national standard "Microbiology of Laboratory Animals and Monitoring" (GB14922.2-2011) and "Pharmacopoeia of the People's Republic of China" both stipulate that mouse hepatitis virus, reovirus type III, mousepox virus and mouse parvovirus belong to the mandatory detection items, and the detection results are required to be negative. The mouse virus detection methods specified in the Pharmacopoeia include cell test, animal antibody production test and chicken embryo infection test. These methods detect the potential contamination of mouse viruses from the biological effect, and the detection means is complex, time-consuming, long cycle and has certain requirements for the laboratory, which is not suitable as a routine quality control means for guiding production.

[0007] Real-time fluorescent quantitative PCR technology has the characteristics of high sensitivity and specificity, multiple reactions, high automation, no pollution, real-time and accuracy, which has been widely used in immune analysis, bacterial and viral detection and other fields. So far, real-time fluorescent quantitative PCR technology detection has been used to detect the above four pathogens, but there is no report on the simultaneous detection of four pathogens by real-time fluorescent quantitative PCR technology. SUMMARY

[0008] To solve the above problems, the present application provides a multiplex real-time fluorescent PCR method for simultaneously detecting four viruses that infect mice, i.e., mouse hepatitis virus, reovirus type III, ectromelia virus and mouse parvovirus, as well as a special primer pair, probe and kit thereof.

[0009] In a first aspect, the present application provides a multiplex real-time fluorescent PCR primer set for simultaneously detecting mouse hepatitis virus, reovirus type III, ectromelia virus and mouse parvovirus, wherein the detection primer set comprises primer pair 1 for detecting mouse hepatitis virus, primer pair 2 for detecting reovirus type III, primer set 3 for detecting ectromelia virus and primer pair 4 for detecting mouse parvovirus. The primer pair 1 comprises an upstream primer MHV-F as shown in SEQ ID NO. 1 and a downstream primer MHV-R as shown in SEQ ID NO. 2, The primer pair 2 comprises an upstream primer Roe3-F as shown in SEQ ID NO. 4 and a downstream primer Roe3-R as shown in SEQ ID NO. 5, The primer pair 3 comprises an upstream primer Ectv-F as shown in SEQ ID NO. 7 and a downstream primer Ectv-R as shown in SEQ ID NO. 8, The primer pair 4 comprises an upstream primer MMPV-F as shown in SEQ ID NO. 10 and a downstream primer MMPV-R as shown in SEQ ID NO. 11.

[0010] In a second aspect, the present application further provides a probe set for use in cooperation with the multiplex real-time fluorescent PCR primer set of the first aspect, wherein the probe set comprises probe 1 for detecting mouse hepatitis virus as shown in SEQ ID NO. 3, probe 2 for detecting reovirus type III as shown in SEQ ID NO. 6, probe 3 for detecting ectromelia virus as shown in SEQ ID NO. 9 and probe 4 for detecting mouse parvovirus as shown in SEQ ID NO. 12.

[0011] In some embodiments, the 5' end of the probe is connected with a fluorescent reporter group, and the 3' end is connected with a fluorescent quencher group.

[0012] In some embodiments, the fluorescent reporter group is selected from any one of FAM, JOE, HEX, VIC, and ROX, and the fluorescent quencher group is MGB.

[0013] In some embodiments, probes 1-4 each have different fluorescent reporter groups.

[0014] Thirdly, the present invention also provides a kit for simultaneously detecting mouse hepatitis virus, reovirus type III, mousepox virus, and mouse parvovirus, the kit comprising the multiplex real-time fluorescent PCR primer set described in the first aspect and the probe set described in the second aspect.

[0015] Using the above technical solution, multiplex real-time PCR of four viruses that infect mice can be performed simultaneously: mouse hepatitis virus, reovirus type III, mousepox virus, and mouse parvovirus. This kit has the advantages of being rapid, simple, highly sensitive, and highly specific.

[0016] In some embodiments, the concentrations of the upstream primer MHV-F and the downstream primer MHV-R are 0.15-0.25 μmol / L, the concentrations of the upstream primer Roe3-F and the downstream primer Roe3-R are 0.55-0.65 μmol / L, the concentrations of the upstream primer Ectv-F and the downstream primer Ectv-R are 0.55-0.65 μmol / L, and the concentrations of the upstream primer MMPV-F and the downstream primer MMPV-R are 0.45-0.5 μmol / L.

[0017] In some embodiments, the concentrations of the upstream primer MHV-F and the downstream primer MHV-R are 0.2 μmol / L, the concentrations of the upstream primer Roe3-F and the downstream primer Roe3-R are 0.6 μmol / L, the concentrations of the upstream primer Ectv-F and the downstream primer Ectv-R are 0.6 μmol / L, and the concentrations of the upstream primer MMPV-F and the downstream primer MMPV-R are 0.48 μmol / L.

[0018] In some embodiments, the concentrations of probe 1, probe 3 and probe 4 are 0.06-0.1 μmol / L, preferably 0.08 μmol / L; the concentration of probe 2 is 0.05-0.08 μmol / L, preferably 0.06 μmol / L.

[0019] Using the above technical solution, the present invention has determined the optimal concentration of primers and probes in the kit through experimental optimization, which effectively improves the detection sensitivity of the kit, making the detection sensitivity reach 10 copies / μL.

[0020] In some embodiments, the kit also includes positive controls for mouse hepatitis virus, reovirus type III, mousepox virus, and mouse parvovirus.

[0021] In this invention, the mouse hepatitis virus positive control and the reovirus type III positive control are prepared by adding viral sample DNA / RNA preservation solution to in vitro transcribed RNA; the mousepox virus positive control and the mouse parvovirus positive control are prepared by adding viral sample DNA / RNA preservation solution to positive standard plasmids. The concentration of the corresponding nucleic acid (DNA or RNA) for each viral positive control is 1×10⁻⁶. 7 Copy / mL.

[0022] In some embodiments, the kit further includes a PCR amplification reagent and an enzyme mixture.

[0023] Fourthly, the present invention also provides a multiplex real-time fluorescent PCR detection method for simultaneously detecting mouse hepatitis virus, reovirus type III, mousepox virus, and mouse parvovirus using the kit of the present invention. The annealing temperature of the detection method is 58-62℃, preferably 58℃, 59℃, 60℃, 61℃, or 62℃, and more preferably 60℃.

[0024] In some embodiments, the reaction conditions of the detection method are: 55°C for 5 min; 95°C for 10 min; 95°C for 15 s, 60°C for 1 min, for a total of 40 cycles.

[0025] Compared with the prior art, the present invention has the following technical effects.

[0026] 1) This invention provides a multiplex real-time fluorescent PCR detection reagent and kit that simultaneously targets mouse hepatitis virus, reovirus type III, mousepox virus, and mouse parvovirus. The detection targets include DNA / RNA viruses, enabling the detection of pathogens from different sources.

[0027] 2) The detection kit provided by this invention contains detection reagents, PCR amplification reagents, enzyme mixtures, and positive controls for four pathogens. The positive control for DNA viruses uses a standard plasmid-added sample preservation solution, and the positive control for RNA viruses uses an in vitro transcribed RNA-added sample preservation solution. The performance of the positive control has been verified, thereby effectively ensuring the accuracy of the detection kit of this invention.

[0028] 3) The detection kit provided by this invention can detect mouse hepatitis virus, reovirus type III, mousepox virus and mouse parvovirus in one go, and has the advantages of being rapid, simple, highly sensitive and specific. Attached Figure Description

[0029] Figure 1Standard curves for the kit to detect four pathogens.

[0030] Figure 2 The results show the sensitivity of the kit for detecting four pathogens.

[0031] Figure 3 The results show the specificity evaluation of the kit for detecting four pathogens. Detailed Implementation

[0032] The specific embodiments of the present invention are described below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0033] Example 1: Primer and probe concentration optimization based on DOE (Design of Experiments) 1. Primer and probe design Based on the genome sequences of the four pathogens published on NCBI, primers and probes were designed by selecting conserved specific regions through BLAST comparison analysis. For parvovirus, primers and probes were designed based on the common sequences of MVM and MPV strains. Specific information is shown in Table 1.

[0034] 2. Software settings and skill level JMP10 software was used to optimize the DOE parameters, input data, and analyze results for primer and probe concentration. This study involved a total of eight control factors for primers and probes from four pathogens, with three levels set for each factor, as detailed in Table 2.

[0035] By setting the corresponding parameters on the JMP10 software according to the factors and levels in Table 2, 93 combinations of different primer and probe concentrations can be generated. The corresponding reaction solutions with the corresponding primer and probe concentrations are then prepared for the experiment.

[0036] 3. Group testing The effectiveness of the quadruple real-time PCR amplification system was evaluated using the CT values ​​of four responses: mouse hepatitis, mouse pox, mouse reovirus type III, and mouse parvovirus. Each response measurement was performed three times, and the average value was obtained to reduce error. The reaction system composition is shown in Table 3, where the concentrations of the primer-probe mixture were prepared according to Table 3. The template used was a quadruple plasmid, and the copy number concentration of each plasmid was 1.08 × 10⁻⁶. 3 Copy / μL.

[0037] After inputting the test results into the JMP10 software, select the stepwise regression model. Table 4 is a summary table of the regression model used in this study.

[0038] R, R-squared, and adjusted R-squared represent the goodness of fit, which estimate how well the model fits the observations.

[0039] The R-squared value, ranging from 0 to 1, represents the goodness of fit of the model; generally, a larger R-value is considered a better fit. The adjusted R-squared values ​​of mouse pox and parvovirus showed relatively high fits to the model, indicating a better established regression equation and that the CT values ​​of mouse pox and parvovirus in the amplification response had a higher explanatory power for the independent variables.

[0040] Analysis of variance (ANOVA) is used to investigate whether the parameters of the experimental design have a significant impact on the response. Typically, the F-test is used to check whether the coefficients of the overall equation are significantly different from zero. If the p-value of the F-test is less than 0.05, it indicates that the overall regression is significant, meaning that all variables have a significant impact on the explained variable.

[0041] In Table 5, the F values ​​for hepatitis CT value, mousepox CT value, regurgitation CT value, and parvovirus CT value were 3.5276, 5.3622, 3.2182, and 5.2970, respectively, and the P values ​​were all less than 0.001, indicating that all variables had a significant impact on the explanatory variables of hepatitis CT value, mousepox CT value, regurgitation CT value, and parvovirus CT value.

[0042] Maximizing willingness was predicted using the predictive profiler in JMP10 software. Maximizing willingness refers to the Ct value of mouse hepatitis, mouse pox, mouse reoblast type III, and mouse parvovirus amplification approaching its minimum. The optimal primer-probe concentration combination is shown in Table 6.

[0043] 5. Comparison and verification The reaction system was prepared according to Table 3 in step 3, and a comparative experiment was conducted. In the initial system, all primer concentrations were 0.2 μmol / L, and all probe concentrations were 0.05 μmol / L. In the optimized system, primers and probes were prepared at their optimal concentrations. The comparison results are shown in Table 7. When the template concentration in the sample was 10 copies / μL, the system with the optimized primer and probe concentrations showed a positive result, indicating that the optimized system had higher sensitivity.

[0044] Example 2 Standard Curve The reaction system described in Example 1 was used to detect standard plasmids for four pathogens. The concentration range of the standard plasmids for the four pathogens was 1×10⁻⁶. 6 Up to 10 copies / μL. After detection, plot the corresponding standard curves, as shown below. Figure 1 As shown. The correlation coefficient R of the standard curve for detecting MHV. 2 The value was 0.9925, and the amplification efficiency was 94.39%. The R-value for REO-3 was detected. 2 The value was 0.9978, and the amplification efficiency was 86.30%; the R value for detecting ECTV was... 2 The value was 0.9978, and the amplification efficiency was 88.96%; the R value for detecting MMV was... 2 The value was 0.9925, and the amplification efficiency was 91.69%.

[0045] Example 3 Preparation of positive control As DNA viruses, mousepox virus and mouse parvovirus can be synthesized and prepared into corresponding standard plasmids for positive controls. However, mouse hepatitis virus and reovirus type III, as RNA viruses, require further transcription into RNA fragments using corresponding standard plasmids.

[0046] 1. Construction of recombinant positive plasmids Gene sequences of various pathogens were retrieved and downloaded from the NCBI database, including: mouse hepatitis virus N gene (NCBI sequence NC_048217.1, fragment 29670-31034); mousepox virus crmD gene (NCBI reference sequence NC_004105.1, fragment 5506-6468); reovirus type III M1 gene (NCBI sequence NC_077841.1); and conserved regions shared by mouse parvovirus and mouse microvirus (NCBI sequence NC_001510.1, fragment 2000-2580). These gene fragments were used as vectors in pBluescript II SK+, and plasmids were synthesized and sequenced by Sangon Biotech (Shanghai) Co., Ltd.

[0047] 2. Propagation of positive bacterial suspensions Preparation of ampicillin-resistant LB broth medium: Add 40 mL of deionized water to a clean wide-mouth bottle, followed by 0.8 g of LB broth medium. Autoclave at 120 °C for 20 min, cool, and store at 4 °C for later use. Dissolve 0.5 g of ampicillin sodium powder in 5 mL of deionized water, vortex until completely dissolved, filter through a 0.22 µm filter membrane, dispense, and store at -20 °C. When using, add the ampicillin solution to the sterilized and room-temperature-cooled LB broth medium at a ratio of 1:1000 to achieve a final ampicillin concentration of 100 µg / mL, mix well, and store at 4 °C for later use.

[0048] Bacterial propagation: Positive bacterial culture was inoculated into LB liquid medium containing ampicillin at a volume ratio of 1:100 and cultured with shaking at 37℃ and 200 rpm for 8-10 h for propagation.

[0049] 3. Positive plasmid extraction The obtained positive bacterial suspensions were subjected to plasmid extraction according to the instructions of the plasmid miniprep kit (Tiangen Biotech (Beijing) Co., Ltd.), resulting in high-purity plasmids for subsequent experiments. The extracted plasmid solutions were then labeled after copy number concentration was determined using NanoDrop2000 and stored at -20℃.

[0050] 4. In vitro transcription preparation of mouse hepatitis virus and reovirus type III template RNA 4.1 PCR amplification of the target band The plasmid fragment containing the T7 promoter was obtained by PCR amplification using the automatically generated universal primers M13 and T7 promoter of the pBluescript II SK+ vector. The universal primers used were: upstream primer for M13: 5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO.13); downstream primer for M13: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO.14). The reaction system is shown in Table 8.

[0051] Reaction program: 95℃ pre-denaturation for 5 s; three-step method for 40 cycles: denaturation at 94℃ for 5 s; annealing at 55℃ for 15 s; extension at 72℃ for 10 s; final extension at 72℃ for 30 s.

[0052] The PCR products were subjected to agarose gel electrophoresis. Electrophoresis was performed on a 2% agarose gel at 120 V for 30 min. The target bands were then recovered using the FastPure Gel DNA Extraction Mini Kit (Novizan).

[0053] 4.2 In vitro transcription of RNA RNA in vitro transcription was performed using a high-yield T7 RNA in vitro transcription kit (Shanghai Sangon Biotech) targeting the recovered PCR products. The reaction system was prepared according to the manufacturer's instructions, and transcription was performed followed by product recovery. High-purity RNA was obtained for subsequent experiments. The extracted RNA solution was labeled after copy number concentration determination using a NanoDrop 2000 and stored at -80°C.

[0054] 5. Prepare a positive control. Based on the copy number results of each pathogen-positive plasmid or in vitro transcribed RNA fragment, the in vitro transcribed RNA of mouse hepatitis virus, in vitro transcribed RNA of reovirus type III, positive plasmid of mousepox virus, and positive plasmid of mouse parvovirus were diluted to 1×10⁻⁶ using viral sample DNA / RNA preservation solution (inactivated type, Kangrun Biotechnology). 7 Copy / mL. Four positive controls were obtained: mouse hepatitis virus positive control, reovirus type III positive control, mousepox virus positive control, and mouse parvovirus positive control.

[0055] 6. Performance verification of positive control standard Using a DNA / RNA co-extraction kit (Tiangen Biotech, DP422), positive controls for mouse hepatitis virus, reovirus type III, mousepox virus, and mouse parvovirus were extracted. The performance of the positive controls was evaluated using the primers, probes, and reaction conditions described in Example 1.

[0056] 6.1 Homogeneity verification of reference standards To assess the homogeneity of each positive control, five samples were randomly selected from the prepared positive controls and tested using the corresponding pathogen qPCR detection method. Each sample was tested three times. Analysis of the coefficient of variation showed that the homogeneity of each positive control was good. The experimental results are shown in Table 9 below.

[0057] 6.2. Repeated freeze-thaw test of the reference standard The positive control sample was repeatedly frozen and thawed at -20℃ 1, 5, and 10 times, and detected using the corresponding pathogen qPCR method, with each sample tested three times. Analysis of the coefficient of variation showed no significant change in the positive control sample after 10 freeze-thaw cycles. The experimental results are shown in Table 10.

[0058] 6.3 Stability test of reference standard stored at 4℃ During simulated use, the samples were temporarily stored at 4°C for 0, 1, 2, 4, and 7 days. The results showed that the CV values ​​of all positive controls for each pathogen were <5% within one week, indicating good stability. The test results are shown in Table 11.

[0059] 6.4 Stability test of reference standard stored at -20℃ The stability of each control standard was tested at -20℃ for storage durations of 0 days, 1 week, 1 month, 3 months, and 6 months. Results showed that all positive controls for pathogens were stable at -20℃ for up to six months, with CV values ​​all <5%. The test results are shown in Table 12.

[0060] Example 4: Performance validation of a multiplex qPCR detection kit for mouse hepatitis virus, reovirus type III, mousepox virus, and mouse parvovirus. 1. Multiplex qPCR detection method The primer and probe sequences used in the kits for detecting relevant pathogens are shown in Table 1, and the qPCR reaction systems of the kits are shown in Table 3. Specifically, in the primer-probe mixture, the concentrations of the two primers for mouse hepatitis virus are both 200 nmol / L, and the probe concentration is 80 nmol / L; the concentrations of the two primers for mousepox virus are both 600 nmol / L, and the probe concentration is 60 nmol / L; the concentrations of the two primers for reovirus type III are both 600 nmol / L, and the probe concentration is 80 nmol / L; and the concentrations of the two primers for mouse parvovirus are both 480 nmol / L, and the probe concentration is 80 nmol / L.

[0061] The qPCR reaction procedure for the kit is shown in Table 13.

[0062] 2. Sensitivity Using the reaction system and conditions described in step one, different concentrations of standard plasmids for four pathogens were detected. The minimum detectable value for each target virus was 10 copies / μL. Figure 2 As shown, the corresponding Ct value ranges from Ct=34 to Ct=37.

[0063] 3. Specificity To evaluate the specificity of the test kit, the reaction system and conditions described in step one were used to detect the nucleic acids of mouse hepatitis virus, reovirus type III, mousepox virus, mouse parvovirus, and other common mouse-sensitive viruses (mouse norovirus, Sendai virus, and mouse pneumonia virus). The results are as follows: Figure 3 The results showed that only mouse hepatitis virus, reovirus type III, mousepox virus, and mouse parvovirus were positive, while other nucleic acid samples were negative. Therefore, the kit has good detection specificity.

[0064] 4. Repeatability To evaluate the reproducibility of the test kit, the positive control described in Example 4 was tested using the reaction system and conditions described in step one. The results are shown in Table 14 below. The intra-assay Ct value coefficient of variation was 0.86%–3.77%, and the inter-assay Ct value coefficient of variation was 1.98%–2.60%, both less than 5%, indicating that the kit has good reproducibility.

[0065] 5. Sample testing 5.1 Simulated Sample Detection Samples to be tested: Ten tissue homogenate samples (including heart, liver, spleen, lung, kidney, small intestine, mesenteric lymph nodes, and reproductive system) that tested negative for four pathogens: mouse hepatitis virus, reovirus type III, mousepox virus, and mouse parvovirus. Single or mixed positive controls of the four pathogens were added to the negative samples to prepare simulated samples for different pathogens. The simulated samples were tested using the reaction system and conditions described in step one, and the results were as expected. See Table 15 below.

[0066] 5.2 Actual Sample Testing Using the reaction system and conditions described in step one, 24 clinical samples were tested. The results showed that the detection rate of mouse hepatitis virus was 70.83% (17 / 24); the detection rate of reovirus type III was 45.83% (11 / 24); the detection rate of mousepox virus was 0% (0 / 24); and the detection rate of mouse parvovirus was 19.17% (7 / 24).

[0067] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A multiplex real-time fluorescent PCR primer set for simultaneously detecting mouse hepatitis virus, reovirus type III, ectromelia virus and minute virus of mice, characterized in that, The detection primer set comprises primer pair 1 for detecting mouse hepatitis virus, primer pair 2 for detecting reovirus type III, primer group 3 for detecting ectromelia virus, and primer pair 4 for detecting minute virus of mice; The primer pair 1 comprises an upstream primer MHV-F shown in SEQ ID NO. 1 and a downstream primer MHV-R shown in SEQ ID NO. 2, The primer pair 2 comprises an upstream primer Roe3-F shown in SEQ ID NO. 4 and a downstream primer Roe3-R shown in SEQ ID NO. 5, The primer pair 3 comprises an upstream primer Ectv-F shown in SEQ ID NO. 7 and a downstream primer Ectv-R shown in SEQ ID NO. 8, The primer pair 4 comprises an upstream primer MMPV-F shown in SEQ ID NO. 10 and a downstream primer MMPV-R shown in SEQ ID NO.

11.

2. A probe set for use in conjunction with the multiplex real-time fluorescent PCR primer set of claim 1, characterized in that, The probe set comprises probe 1 for detecting mouse hepatitis virus shown in SEQ ID NO. 3, probe 2 for detecting reovirus type III shown in SEQ ID NO. 6, probe 3 for detecting ectromelia virus shown in SEQ ID NO. 9, and probe 4 for detecting minute virus of mice shown in SEQ ID NO.

12.

3. The probe set of claim 2, wherein, The 5' end of the probe is connected with a fluorescent reporter group, and the 3' end is connected with a fluorescent quencher group.

4. The probe set of claim 3, wherein, The fluorescent reporter group is selected from any one of FAM, JOE, HEX, VIC, and ROX, and the fluorescent quencher group is MGB.

5. The probe set of claim 4, wherein, The probes 1-4 have different fluorescent reporter groups, respectively.

6. A kit for simultaneous detection of mouse hepatitis virus, reovirus type III, ectromelia virus, and minute virus of mice, characterized in that, The kit comprises the multiplex real-time fluorescent PCR primer set of claim 1 and the probe set of any one of claims 2-5.

7. The kit of claim 6, wherein The concentrations of the upstream primer MHV-F and the downstream primer MHV-R are 0.15-0.25 μmol / L, the concentrations of the upstream primer Roe3-F and the downstream primer Roe3-R are 0.55-0.65 μmol / L, the concentrations of the upstream primer Ectv-F and the downstream primer Ectv-R are 0.55-0.65 μmol / L, and the concentrations of the upstream primer MMPV-F and the downstream primer MMPV-R are 0.45-0.5 μmol / L.

8. The kit of claim 6, wherein The concentrations of the upstream primer MHV-F and the downstream primer MHV-R are 0.2 μmol / L, the concentrations of the upstream primer Roe3-F and the downstream primer Roe3-R are 0.6 μmol / L, the concentrations of the upstream primer Ectv-F and the downstream primer Ectv-R are 0.6 μmol / L, and the concentrations of the upstream primer MMPV-F and the downstream primer MMPV-R are 0.48 μmol / L.

9. The kit of claim 6, wherein The concentrations of the probes 1, 3, and 4 are 0.06-0.1 μmol / L, preferably 0.08 μmol / L, and the concentration of the probe 2 is 0.05-0.08 μmol / L, preferably 0.06 μmol / L.

10. The kit of claim 6, wherein The kit also includes positive control for mouse hepatitis virus, reovirus type III, ectromelia virus, and minute virus of mice. The kit also includes positive control for mouse hepatitis virus, reovirus type III, ectromelia virus, and minute virus of mice.

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