CN114934136A - Primer and probe composition for simultaneously detecting mouse parvovirus and mouse thymic adenovirus, detection method, kit and application thereof - Google Patents
Primer and probe composition for simultaneously detecting mouse parvovirus and mouse thymic adenovirus, detection method, kit and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of virus detection, and particularly provides a primer and a probe composition for simultaneously detecting mouse parvovirus and mouse thymic adenovirus. The invention also provides a kit comprising the primer and the probe composition, and a method for simultaneously detecting the mouse parvovirus and the mouse thymic adenovirus. The primer, the probe composition, the kit and the method provided by the invention can specifically amplify the MVM and the MTLV, do not generate cross reaction with other virus nucleic acids, and also do not generate cross reaction with various common cell line genomes possibly existing in a sample, have good specificity, high sensitivity and good repeatability, make up for short plates in the prior art that mouse parvovirus and mouse thymic adenovirus must be detected respectively, and provide a good detection means for preventing and controlling exogenous virus pollution of rodent biological products.
Description
Technical Field
The invention belongs to the technical field of virus detection, and relates to a primer and probe composition for simultaneously detecting mouse parvovirus and mouse thymic adenovirus and application thereof, in particular to a dual-fluorescence quantitative QPCR detection primer and probe composition for simultaneously detecting mouse parvovirus and mouse thymic adenovirus, a detection method, a kit and application thereof.
Background
With the widespread use of recombinant protein drugs in the medical field, the problem of virus safety of biological products is also highlighted, and rodent cell lines such as Chinese hamster ovary cell line (CHO-K1 cell) are commonly used in protein expression production systems to ensure the correct conformation and activity of the product, but no matter the bioactive raw material or the complex production operation process, the risk of virus contamination is inevitable.
Mouse parvovirus (MVM) is a single-stranded DNA virus, has no envelope and extremely strong stability, and is susceptible to infection of rodent cells such as CHO-K1 and BHK21 cells. Mouse Thymic adenovirus Thymic Virus (MTLV; murid hepes Virus 3) belongs to the herpesviridae family, has a Virus particle diameter of about 135nm, is an enveloped DNA Virus, can infect and kill developing T lymphocytes in the thymus of newborn mice, and belongs to a pathogen susceptible to experimental animals. ICH Q5A clearly states that MVM and MTLV, as species-specific viruses in rodent cell lines, require exogenous contamination detection. The detection method commonly used for MVM and MTLV is virus separation, ELISA antigen detection and RT-PCR nucleic acid detection, and compared with the fluorescence quantitative QPCR detection method, the fluorescence quantitative QPCR detection method is more visual and rapid and is suitable for early virus safety risk control.
At present, two fluorescence quantitative QPCR detections must be respectively carried out on mouse parvovirus and mouse thymic adenovirus in a sample, the detection process takes labor and time, and the detection method and the kit which can simultaneously detect MVM and MTLV are lacked in the market.
Disclosure of Invention
The invention aims to solve the problem that mouse parvovirus and mouse thymic adenovirus must be detected respectively in the prior art.
Therefore, the invention provides a primer and a probe composition for simultaneously detecting mouse parvovirus and mouse thymic adenovirus, which comprise an upstream primer P-MVM-F, a downstream primer P-MVM-R and a probe MVM-P for detecting mouse parvovirus, and an upstream primer P-MTLV-F, a downstream primer P-MTLV-R and a probe MTLV-P for detecting mouse thymic adenovirus;
the sequence of the upstream primer P-MVM-F is 5'-AACTTACTTCTTCTGCTGCACA-3';
the sequence of the downstream primer P-MVM-R is 5'-AGACCCAGTAGAAACACCAACC-3';
the sequence of the probe MVM-P is 5 '- (FAM) -CCAACCTGA/iXNA _ C/RGCGRAAACG- (BHQ) -3';
the sequence of the upstream primer P-MTLV-F is 5'-AGGACTTGCTTTACCTGTTG-3';
the sequence of the downstream primer P-MTLV-R is 5'-ATACATACCTCTCCAAGAACCG-3';
the sequence of the probe MTLV-P is 5 '- (FAM) -CCAACCTGA/iXNA _ C/RGCGRAAACG- (BHQ) -3';
wherein/iXNA _ A/represents the locked nucleic acid modification of base A; the/iXNA _ T/represents the locked nucleic acid modification of the base T; the/iXNA _ G/represents the locked nucleic acid modification of the base G; the term,/iXNA _ C/, denotes the locked nucleic acid modification of base C.
The invention also provides a kit for simultaneously detecting the mouse parvovirus and the mouse thymic adenovirus, the kit comprises the primer and a probe composition, the upstream primer P-MVM-F, the downstream primer P-MVM-R, the upstream primer P-MTLV-F and the downstream primer P-MTLV-R are mixed to form a mixed primer, and the probe MVM-P and the probe MTLV-P are mixed to form a mixed probe.
Specifically, the concentration of the upstream primer P-MVM-F in the mixed primer is 0.1-1mM, the concentration of the downstream primer P-MVM-R is 0.1-1mM, the concentration of the upstream primer P-MTLV-F is 0.1-1mM, and the concentration of the downstream primer P-MTLV-R is 0.1-1 mM.
Specifically, the concentration of the probe MVM-P in the mixed probe is 0.05-1mM, and the concentration of the probe MTLV-P is 0.05-1 mM.
Specifically, the kit further comprises a QPCR reaction premix; the QPCR reaction premixed liquid comprises a premixed liquid AceQ Universal U + Probe Master Mix V2 special for Probe method fluorescence quantification and Mg 2+ 。
Specifically, the kit also comprises a quantitative standard substance; the quantitative standard is a mixture of PUC57-MVM and PUC57-MTLV plasmids.
Specifically, the kit further comprises a positive control; the positive control is a mixture of inactivated MVM strain and MTLV pseudovirus strain; the nucleic acid information of the MTLV pseudovirus strain is a UL27 full-length sequence, and an AdMax packaging system is used for assembling.
Specifically, the primer and probe composition or the kit can be used for early monitoring of high-incidence pathogens of murine experimental animals, and can also be used for detecting rodent biological products and raw materials in real time and whether the pollution of mouse parvoviruses and mouse thymic viruses exists in the production process of the rodent biological products and the raw materials.
The invention also provides a double fluorescence quantitative QPCR method for simultaneously detecting the mouse parvovirus and the mouse thymic adenovirus, which comprises the following steps:
(1) extracting a DNA template of a sample to be detected;
(2) performing a quantitative fluorescence QPCR reaction on a DNA template by using the primer and probe composition as claimed in claim 1;
(3) and when the Ct value of the sample to be detected is less than or equal to 35 and an obvious amplification curve exists, the mouse parvovirus and the mouse thymic adenovirus of the sample to be detected are judged to be positive.
Specifically, the QPCR amplification program in the step (2) is 95 ℃ for 10 min; 95 deg.C, 15s, 55 deg.C, 30s, 72 deg.C, 20s, 41 cycles.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the primer, the probe composition and the kit provided by the invention can be used for simultaneously detecting the mouse parvovirus and the mouse thymic adenovirus, can specifically amplify the MVM and the MTLV, do not generate cross reaction with other virus nucleic acid, and also do not generate cross reaction with various common cell line genomes possibly existing in a sample, have good specificity, and make up for short plates which are required to be respectively detected by the mouse parvovirus and the mouse thymic adenovirus in the prior art. The kit can be used for early monitoring of high-incidence pathogens of mouse experimental animals, and can also be used for detecting rodent biological products, raw materials and whether the pollution of mouse parvovirus and mouse thymidyvirus exists in the production process of the rodent biological products and the raw materials in real time so as to take precautionary measures quickly and minimize the influence of the pollution on the biological safety of animal populations, the scientific research safety, the loss of enterprises and the like.
2. The invention establishes a double fluorescence quantitative QPCR detection method for simultaneously detecting the mouse parvovirus and the mouse thymic adenovirus, can specifically detect and distinguish the mouse parvovirus and the mouse thymic adenovirus together, has strong specificity, high sensitivity and good repeatability, can shorten the detection time, reduce the use amount of samples, reduce the detection cost, solve the technical problem of labor and time waste in the existing detection process, provide a good detection means for preventing and controlling the exogenous virus pollution of rodent biological products, and have certain social application prospect.
The present invention will be described in further detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a standard curve of the double fluorescent quantitative PCR detection method in the example of the present invention.
FIG. 2 shows the virus detection results of the double fluorescent quantitative PCR detection method according to the embodiment of the present invention.
FIG. 3 shows MTLV specificity test results in an embodiment of the present invention.
FIG. 4 shows MVM attribute detection results in an embodiment of the present invention.
FIG. 5 is a diagram illustrating the results of MTLV and MVM specialization in an embodiment of the present invention.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art to which the present invention pertains will appreciate that various modifications and changes can be made to the present invention without departing from the scope of the invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.
The effects of the primer and probe composition, kit and detection method of the present invention are examined below with reference to specific examples.
Example 1:
1. design of primers and probes
Comparing with the nucleic acid sequences of MVM and MTLV retrieved from NCBI through MegAlign to find out conserved sequences, and respectively designing a primer pair and a probe pair, wherein the sequences are as follows:
the sequence of the upstream primer P-MVM-F is 5'-AACTTACTTCTTCTGCTGCACA-3';
the sequence of the downstream primer P-MVM-R is 5'-AGACCCAGTAGAAACACCAACC-3';
the sequence of the probe MVM-P is 5 '- (FAM) -CCAACCTGA/iXNA _ C/RGCGRAAACG- (BHQ) -3';
the sequence of the upstream primer P-MTLV-F is 5'-AGGACTTGCTTTACCTGTTG-3';
the sequence of the downstream primer P-MTLV-R is 5'-ATACATACCTCTCCAAGAACCG-3';
the sequence of the probe MTLV-P is 5 '- (FAM) -CCAACCTGA/iXNA _ C/RGCGRAAACG- (BHQ) -3';
wherein/iXNA _ A/represents the locked nucleic acid modification of base A; the/iXNA _ T/represents the locked nucleic acid modification of the base T; the/iXNA _ G/represents the locked nucleic acid modification of the base G; and/iXNA _ C/represents the locked nucleic acid modification of base C.
The primers and the probes are synthesized by Shanghai Biotech company, the upstream primer P-MVM-F, the downstream primer P-MVM-R, the upstream primer P-MTLV-F and the downstream primer P-MTLV-R are mixed to form a mixed primer, and the probe MVM-P and the probe MTLV-P are mixed to form a mixed probe for later use. The concentrations of P-MVM-F, P-MVM-R, P-MTLV-F and P-MTLV-R in the mixed primer are both 1 mM; the concentration of MVM-P and MTLV-P in the mixed probe was 1 mM.
2. Preparation of Positive control
The inactivated MVM was used as a positive control in combination with MTLV pseudovirus containing the desired fragment.
Wherein the MTLV pseudovirus is constructed by the following method: constructing shuttle plasmid GV315-MTLV (UL27), co-transfecting HEK293 cells with the obtained shuttle plasmid and helper plasmid pBHG lox delta E1,3 Cre to obtain recombinant adenovirus Ad/MTLV (UL27), and using Adeno-X TM Virus Purification Kit purified the Virus, and the end point dilution method was used to determine the Virus titer.
3. Extraction of viral DNA
The MVM and MTLV viral DNA was extracted using a commercially available viral nucleic acid purification kit (Hangzhou Xinjing Biochemical development Co., Ltd.).
4. Fluorescent quantitative PCR condition optimization
The fluorescent quantitative PCR reaction system and procedure performed in this example are as follows.
Reaction system: 10 mul of QPCR reaction premix, 1.2 mul of mixed primers, 0.4 mul of mixed probe and 5 mul of sample to be detected are added with RNase-free deionized water to make up to 20 mul.
Wherein the QPCR reaction premixed solution is a Probe method special premixed solution AceQ Universal U + Probe Master Mix V2 for fluorescence quantification (containing 0.5mM Mg with final concentration 2+ )。
And (3) amplification procedure: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 deg.C, 15s, annealing at 55 deg.C, 30s, extension at 72 deg.C, 20s, and 41 cycles.
5. Drawing a standard curve
The pUC57-MVM and pUC57-MTLV plasmids were synthesized and sequenced by Biotechnology engineering (Shanghai) Inc., respectively.
The gene sequence of the conserved region of the PUC57-MTLV standard is as follows:
ATACATACCTCTCCAAGAACCGGTACCGTACATTCCCGCTTCAAACTGTCTAAAGAATTTTAATGGATGAACCAAATAATAAATTTCAACAGGTAAAGCAAGTCCT
the gene sequence of the conserved region of the PUC57-MVM standard is as follows:
AACTTACTTCTTCTGCTGCACAGCAAAGCAGTCAAACCATGAGTGATGGCACCAGCCAACCTGACAGCGGAAACGCTGTCCACTCAGCTGCAAGAGTTGAACGAGCAGCTGACGGCCCTGGAGGCTCTGGGGGTGGGGGCTCTGGCGGGGGTGGGGTTGGTGTTTCTACTGGGTCT
the plasmid is extracted by a commercial kit, and diluted and mixed into a quantitative standard substance 1, a quantitative standard substance 2, a quantitative standard substance 3, a quantitative standard substance 4, a quantitative standard substance 5, a quantitative standard substance 6 and a quantitative standard substance 7 after the concentration is measured. Wherein the quantitative standard 1 is 1.0 × 10 1 The plasmid mixture of pUC57-MVM and pUC57-MTLV was copes/. mu.l, and quantitative standard 2 was 1.0X 10 2 copies/. mu.l of pUC57-MVM and pUC57-MTLV plasmid mixture, quantitative standard 3 is 1.0X 10 3 copies/. mu.l of pUC57-MVM and pUC57-MTLV plasmid mixture, quantitative standard 4 is 1.0X 10 4 copies/. mu.l of a mixture of PUC57-MVM and PUC57-MTLV plasmids, quantitative standard 5 was 1.0X 10 5 The plasmid mixture of pUC57-MVM and pUC57-MTLV was used at a ratio of copies/. mu.l, and the quantitative standard 6 was 1.0X 10 6 copies/. mu.l of pUC57-MVM and pUC57-MTLV plasmid mixture, quantitative standard 7 was 1.0X 10 7 copies/. mu.l of a mixture of PUC57-MVM and PUC57-MTLV plasmids.
The quantitative standards 1 to 7 are used for reaction under the fluorescent quantitative PCR condition, and the results are shown in table 1 and figure 1, wherein Ct mean is the average value of Ct values; ct SD is the standard deviation of Ct values; CV% is the coefficient of variation.
TABLE 1 quantitative standards fluorescent quantitative PCR reaction results
As can be seen from FIG. 1 and Table 1, the quantitation limit and detection limit of the primers and methods provided by the present invention can both reach 10 1 copies/. mu.l at 1.0X 10 1 -1.0×10 7 The amplification efficiency is good in a concentration range, wherein, the MTLV standard amplification curve has a linear correlation coefficient R 2 1, the amplification efficiency is 100.1 percent, and the linear correlation coefficient R of the MVM standard amplification curve 2 The amplification efficiency was 107.8% at 0.998, indicating that the present invention has high sensitivity for detection of MVM and MTLV.
If the Ct value of the test sample is less than or equal to 35 and an obvious amplification curve exists, the exogenous murine virus of the test sample is judged to be positive. Otherwise, the sample is negative.
6. Stability evaluation
Setting a positive control group (a mixed sample of an MVM strain and an MTLV pseudovirus strain) and adopting the fluorescent quantitative PCR condition to carry out detection limit and quantitative limit experiments, wherein each experiment has three groups of parallel samples, carrying out three repeated experiments, calculating standard deviation and variation coefficient between groups, evaluating the stability of the established detection method, and when the MVM virus titer in the solution is 10, the result is shown in figure 2 -1 TCID50/ml, MTLV pseudovirus titer 2X 10 2 PFU/ml, can be detected stably by the primer and the probe provided by the invention.
7. Assessment of specificity
In order to detect the specificity of the kit, the kit is used for detecting the specificity of 3 common cell genomes including Balb/c 3T3, BHK21 and CHO-K1 and 5 murine virus genomes including mouse parvovirus MVM, mouse thymus virus MTLV, mouse polyoma virus MPyV, mouse pneumovirus PVM and reovirus Reo3, an NTC control is arranged, and the detection condition of the primers and the probes provided by the invention on common cell lines is analyzed. The results of the experiment are shown in FIGS. 2 to 4.
As can be seen from FIG. 2, only MTLV can be amplified normally in FAM channel and judged as positive, other susceptible viruses MVM, MPyV, PVM and Reo3, and common cell lines Balb/c 3T3, BHK21, CHO-K1 and a negative control group are all judged as negative without amplification.
As shown in FIG. 3, only MVM can be normally amplified in HEX channel and is positive, other susceptible viruses MTLV, MPyV, PVM and Reo3, and common cell lines Balb/c 3T3, BHK21, CHO-K1 and a negative control group are all in the condition of no amplification and are negative.
As shown in FIG. 4, when both MVM and MTLV viruses exist in the sample, both FAM channel and HEX channel can be amplified normally and are positive, and other susceptible viruses MPyV, PVM and Reo3, and the common cell lines Balb/c 3T3, BHK21, CHO-K1 and the negative control group are negative without amplification.
In conclusion, the kit can specifically amplify the MVM virus and the MTLV virus, does not generate cross reaction with other virus nucleic acids, and also does not generate cross reaction with various common cell line genomes possibly existing in a sample, and the kit and the method have good specificity.
The above examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims and any design similar or equivalent to the scope of the invention.
Claims (10)
1. A primer and probe composition for simultaneously detecting mouse parvovirus and mouse thymic adenovirus comprises an upstream primer P-MVM-F, a downstream primer P-MVM-R and a probe MVM-P for detecting mouse parvovirus, and an upstream primer P-MTLV-F, a downstream primer P-MTLV-R and a probe MTLV-P for detecting mouse thymic adenovirus, and is characterized in that:
the sequence of the upstream primer P-MVM-F is 5'-AACTTACTTCTTCTGCTGCACA-3';
the sequence of the downstream primer P-MVM-R is 5'-AGACCCAGTAGAAACACCAACC-3';
the sequence of the probe MVM-P is 5 '- (FAM) -CCAACCTGA/iXNA _ C/RGCGRAAACG- (BHQ) -3';
the sequence of the upstream primer P-MTLV-F is 5'-AGGACTTGCTTTACCTGTTG-3';
the sequence of the downstream primer P-MTLV-R is 5'-ATACATACCTCTCCAAGAACCG-3';
the sequence of the probe MTLV-P is 5 '- (FAM) -CCAACCTGA/iXNA _ C/RGCGRAAACG- (BHQ) -3';
wherein/iXNA _ A/represents the locked nucleic acid modification of base A; the/iXNA _ T/represents the locked nucleic acid modification of the base T; the/iXNA _ G/represents the locked nucleic acid modification of the base G; the term,/iXNA _ C/, denotes the locked nucleic acid modification of base C.
2. A kit for simultaneously detecting mouse parvovirus and mouse thymic adenovirus is characterized in that: the kit comprises the primer and probe composition of claim 1; the upstream primer P-MVM-F, the downstream primer P-MVM-R, the upstream primer P-MTLV-F and the downstream primer P-MTLV-R are mixed to form a mixed primer, and the probe MVM-P and the probe MTLV-P are mixed to form a mixed probe.
3. The kit for simultaneously detecting mouse parvovirus and mouse thymic adenovirus according to claim 2, wherein: the concentration of the upstream primer P-MVM-F in the mixed primer is 0.1-1mM, the concentration of the downstream primer P-MVM-R is 0.1-1mM, the concentration of the upstream primer P-MTLV-F is 0.1-1mM, and the concentration of the downstream primer P-MTLV-R is 0.1-1 mM.
4. The kit for simultaneously detecting mouse parvovirus and mouse thymic adenovirus according to claim 2, wherein: the concentration of the probe MVM-P in the mixed probe is 0.05-1mM, and the concentration of the probe MTLV-P is 0.05-1 mM.
5. The kit for simultaneously detecting mouse parvovirus and mouse thymic adenovirus according to claim 2, wherein: also included is a QPCR reaction premix.
6. The kit for simultaneously detecting mouse parvovirus and mouse thymic adenovirus according to claim 2, wherein: also comprises a quantitative standard substance; the quantitative standard is a mixture of PUC57-MVM and PUC57-MTLV plasmids.
7. The kit for simultaneously detecting mouse parvovirus and mouse thymic adenovirus according to claim 2, wherein: a positive control is also included; the positive control is a mixture of inactivated MVM strain and MTLV pseudovirus strain.
8. Use of the primer and probe composition of claim 1 or the kit of any one of claims 2-7 for detecting contamination by mouse parvovirus and mouse thymus virus in a rodent biological product.
9. A fluorescence quantitative QPCR method for simultaneously detecting mouse parvovirus and mouse thymic adenovirus is characterized by comprising the following steps:
(1) extracting a DNA template of a sample to be detected;
(2) performing a quantitative fluorescence QPCR reaction on a DNA template by using the primer and probe composition according to claim 1;
(3) and when the Ct value of the sample to be detected is less than or equal to 35 and an obvious amplification curve exists, the mouse parvovirus and the mouse thymic adenovirus of the sample to be detected are judged to be positive.
10. The dual-fluorescence quantitative QPCR method for simultaneous detection of mouse parvovirus and mouse thymic adenovirus according to claim 9, wherein: the QPCR amplification program in the step (2) is 95 ℃ and 10 min; 95 ℃, 15s, 55 ℃, 30s, 72 ℃, 20s, 41 cycles.
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| CN202310046665.5A CN116287447A (en) | 2022-05-24 | 2023-01-31 | Primer and probe composition for simultaneously detecting mouse parvovirus and mouse thymus virus, detection method, kit and application of primer and probe composition |
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| CN202310046665.5A Pending CN116287447A (en) | 2022-05-24 | 2023-01-31 | Primer and probe composition for simultaneously detecting mouse parvovirus and mouse thymus virus, detection method, kit and application of primer and probe composition |
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| CN202310046665.5A Pending CN116287447A (en) | 2022-05-24 | 2023-01-31 | Primer and probe composition for simultaneously detecting mouse parvovirus and mouse thymus virus, detection method, kit and application of primer and probe composition |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506187A (en) * | 2016-02-19 | 2016-04-20 | 苏州药明康德检测检验有限责任公司 | Quantitative PCR detection method of minute virus of mice and primers and probe of quantitative PCR detection method |
| CN106636474A (en) * | 2017-01-10 | 2017-05-10 | 广东省实验动物监测所 | Primer group, kit and method for detecting six mouse viruses by multiple immunity fluorescence |
| CN106893786A (en) * | 2017-03-22 | 2017-06-27 | 中国农业科学院上海兽医研究所 | Differentiate primer pair, probe, kit and the method for detection rat piconavirus and rat parvovirus |
-
2022
- 2022-05-24 CN CN202210571115.0A patent/CN114934136A/en active Pending
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506187A (en) * | 2016-02-19 | 2016-04-20 | 苏州药明康德检测检验有限责任公司 | Quantitative PCR detection method of minute virus of mice and primers and probe of quantitative PCR detection method |
| CN106636474A (en) * | 2017-01-10 | 2017-05-10 | 广东省实验动物监测所 | Primer group, kit and method for detecting six mouse viruses by multiple immunity fluorescence |
| CN106893786A (en) * | 2017-03-22 | 2017-06-27 | 中国农业科学院上海兽医研究所 | Differentiate primer pair, probe, kit and the method for detection rat piconavirus and rat parvovirus |
Non-Patent Citations (3)
| Title |
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| JOACHIM DENNER等: "Comparative Analysis of Roseoloviruses in Humans,Pigs, Mice, and Other Species", 《VIRUSES》 * |
| SWAPNEEL J.PATEL等: "A Murine Herpesvirus Closely Related to Ubiquitous Human Herpesviruses Causes T-Cell Depletion", 《JOURNAL OF VIROLOGY》 * |
| 王淑菁等: "小鼠微小病毒( MVM) 荧光定量 PCR检测方法的建立及初步应用", 《实验动物科学》 * |
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