CN117947220A - Primer and probe combination for detecting rhesus RRV virus and application thereof - Google Patents
Primer and probe combination for detecting rhesus RRV virus and application thereof Download PDFInfo
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Abstract
The invention relates to a primer and probe combination for detecting rhesus monkey RRV virus and application thereof, wherein the primer and probe combination comprises a primer and probe combination for detecting rhesus monkey RRV virus vIL6 gene; the primer and probe combination comprises: forward primer, reverse primer and probe; the nucleotide sequence of the forward primer comprises a sequence shown in SEQ ID NO. 1, and the nucleotide sequence of the reverse primer comprises a sequence shown in SEQ ID NO. 2; the nucleotide sequence of the probe comprises a sequence shown in SEQ ID NO. 3. The invention designs corresponding forward and reverse primers, probes and reaction programs aiming at the rhesus RRV 17577 strain, and establishes a real-time quantitative PCR method with the advantages of good specificity, sensitivity, high variant strain coverage and the like, and the detection method is suitable for complex detection of mass production of biological products.
Description
Technical Field
The invention belongs to the technical field of medical molecular biology, and relates to a primer and probe combination for detecting rhesus RRV virus and application thereof.
Background
By 2022, 6 and 30 days, 162 antibody classes were globally approved. The emerging antibody discovery technology accelerates the development of antibodies, will lead more antibody candidates to clinical development, and the global antibody therapeutic industry and its market will expand greatly in the future.
Most biological agents (such as monoclonal antibodies) are used for disease treatment by injection, and the quality of the biological agents is closely related to the medication safety of patients. Regulatory authorities in various countries have issued a series of regulations to ensure the biosafety of biological drugs, for example, in the regulations, there is a clear need for virus detection of seed bank cells for production, suspensions at the end of cell culture, homogenates of animal tissue raw materials, i.e. for detection of contaminated viruses by various suitable methods.
The advent of biologicals has had very special implications for the improvement of human health and quality of life. A large number of rhesus monkey kidney cells (LLC-MK 2), vero, are used in large quantities in vaccine production, the quality of which directly affects the quality safety of biological products, especially the quality safety of products can be seriously affected by contamination with viral bioactive substances. Meanwhile, LLC-MK2 and Vero are used as one of human and primate virus infection indicator cells, and are widely used in cell line virus infection screening experiments. Many viral vaccine biologicals currently use monkey-derived cells as expression hosts. Therefore, the biosafety detection mechanism needs to develop a method for detecting viruses with convenience, rapidness, sensitivity and strong specificity.
Rhesus monkey RRV virus (Rhesus Macaque Rhadinovirus, RRV) belongs to the Rhadinovirus genus of viruses. At present, rhesus RRV virus is identified in chimpanzees, spider monkeys, african green monkeys, gorillas, tonsils and drilling monkeys, pigtail macaques, squirrel monkeys, and rhesus monkeys. Among them, rhesus monkeys are often used in biomedical experiments. These non-human primates are widely used as an irreplaceable animal model in the research and development of biological products, for example, in basic biological research, quality control of human and veterinary medicine, disease diagnosis, and pharmaceutical toxicology and safety assessment, including scientific experiments such as safety assessment of biopharmaceutical products. Since non-human primates are genetically similar to humans, and only non-human primates possess the same major pharmacodynamic response as humans, they are used in large numbers for biopharmaceutical preclinical evaluation. In addition, both wild monkeys and artificially bred monkeys can be infected with RRV due to the infectious nature of RRV. Wherein, the centralized cage-cultured artificial propagation monkey is extremely easy to cause large-scale RRV infection and transmission, thereby influencing the evaluation result of the biological experiment. Thus, there is a need for a simple, rapid method of detecting RRV virus.
Currently, the virus detection methods recommended in the guidelines for biosafety detection include in vitro methods, in vivo methods, animal antibody production assays, electron microscopy, ELISA, PCR, RT detection, and the like. Among them, in vitro methods employ sensitive cell lines for co-culture detection, and samples are taken at appropriate culture time points (usually 28 days) to detect infectious viruses. When no reliable in vitro test method is available, the inoculation blind test can be carried out by adopting a proper animal, and the in vivo method is adopted to detect whether the infectious virus exists or not. For in vivo and in vitro virus detection methods not suitable, different animals can be used to observe the generation of antibodies against the species-specific virus. The method has the advantages of long detection period, complicated experimental operation, ethical reasons of experimental animals and the like, and limits the application range to a certain extent. The nucleic acid detection technology has the advantages of short detection period, convenient operation, no need of animals, wide application range, high sensitivity and short detection period, and is a good choice for RRV virus detection.
Polymerase Chain Reaction (PCR) is a powerful tool for detecting target nucleic acid fragments, and PCR drives amplification by thermal cycling. Since the invention of this technology in 1981, various improvements have been introduced to meet the scientific needs of the field of molecular biology. Quantitative PCR (qPCR, Q-PCR) is widely used for the quantification of nucleic acids in cells and for detecting the expression level of a target protein. This technique has been developed for use in virus detection and virus clearance studies. The main methodology of qPCR is to detect the content of PCR products by monitoring the fluorescence intensity in real time. In conventional PCR, the amplified products are separated and visualized by electrophoresis. The DNA polymerase used in qPCR has 5 'to 3' endonuclease activity, generating a fluorescent signal by hydrolyzing oligonucleotide probes labeled with fluorescein and a quencher dye. By combining qPCR with fluorescein-labeled probes, it is theoretically possible to detect single viral DNA/RNA, and in practical applications qPCR can detect single digit viral DNA/RNA in a sample, regardless of viral activity. Currently, an example of qPCR used for exogenous factor detection is to perform exogenous viral factor detection, e.g., to detect the presence or absence of a specific virus in a master cell bank (MBC), working cell bank (WBC), of some engineered cell lines used to produce biological products. The Chinese pharmacopoeia prescribes that aiming at human cell lines, human EB virus, human Cytomegalovirus (HCMV), human reverse transcription (HIV-1/2, HTLV1/2, human hepatitis virus (HAV, HBV, HCV), human parvovirus B19, human papillomavirus, human polyoma virus, human adenovirus difficult to culture, human herpesvirus-6/7/8 and the like should be considered to be detected, in addition, the 2020 edition of Chinese pharmacopoeia adds a PCR (qPCR) checking method, and provides a rule basis for the application of fluorescent quantitative technology in virus detection and biological product safety evaluation.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides a primer and probe combination for detecting rhesus RRV virus and application thereof. The invention designs corresponding forward and reverse primers, probes and reaction programs aiming at all different types of rhesus RRV viruses, and establishes a real-time quantitative PCR method with the advantages of good specificity, sensitivity, high variant coverage and the like, and the detection method is suitable for complex detection of mass production of biological products.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a primer and probe combination for detecting rhesus monkey RRV virus, the primer and probe combination comprising a primer and probe combination for detecting rhesus monkey RRV virus vIL6 gene;
the primer and probe combination comprises: forward primer, reverse primer and probe;
The nucleotide sequence of the forward primer comprises a sequence shown in SEQ ID NO. 1, and the nucleotide sequence of the reverse primer comprises a sequence shown in SEQ ID NO. 2;
the nucleotide sequence of the probe comprises a sequence shown in SEQ ID NO. 3.
SEQ ID NO:1：CGGTAACACCTACGTCTCGG。
SEQ ID NO:2：CCTGGGACACGTTGGTTACC。
SEQ ID NO:3：FAM-ACTGCGAGTGGCCACCGAACCGGGAGA-BHQ1。
In the invention, 1 group of forward and reverse primers and probes for real-time quantitative PCR are designed by carrying out homology comparison on all isolated plants (including H26-95 and 17577) of RRV in NCBI database, and the combination of the primers and the probes can efficiently and accurately detect all types of RRV DNA, is not influenced by a large amount of background cell DNA, and is particularly suitable for detecting RRV virus pollution in biopharmaceutical raw materials and products, in particular in cell libraries.
Preferably, the 5 'end of the probe modifies fluorescein and the 3' end of the probe modifies a quencher.
Preferably, the fluorescein is selected from any one of FAM, VIC or HEX.
Preferably, the quencher is selected from any one of MGB, BHQ1 or TAMRA.
Preferably, the nucleotide sequence of the target fragment amplified by the forward primer and the reverse primer comprises the sequence shown in SEQ ID NO. 4.
SEQ ID NO:4：
CCGCGCACCCATTTCTCTTAACCCAGAAGACCAAGCCCTTTACCGAGCGAAAGTTTTGCCGCCTGATCATGGACAACGACCAGCGCAGCGCCGTCAACACCGTCTACCTGGGAAAGCAGCACGTGAGGGTGACCGTGACCCGCCCCCCGGAAACAATCGTCACCGACGGCCCCGTGACGGCGACCCTGTCCCTCACCGGTAATGCGCCAATCGCCTTTCGCCACAACCCATACTTTGAACTCCCGTGGTCGTCCACAACGGCGATATTCACGCCCGTGGTGTACGTGGGCCTGACCGTGTGCATCCCACCCAACTGTAGCAAATTCGTAAGGTACGGTAACACCTACGTCTCGGCATTTAACCGCAAGCTGACGGCGATTATTAGCAATCACGCCCACAACGGCGGGTTCCGGATTCAGGACTGCGAGTGGCCACCGAACCGGGAGATAGAGATTTTGGTAACCAACGTGTCCCAGGCCCCGGTGTACATCAGCACCGGGACGCAGCTGGGGCAAGCCATCTTCGTGTTCGCGCCGCGGTTCGGTGGCCCGGCGAAACTGCGGCAGCTCCTCGGCCACCGATCGCGCGCCCTGGAGCTGCCGGGCGGGGTGACAGTGGACAGCC.
In a second aspect, the invention provides a kit for detecting rhesus monkey RRV virus, the kit comprising the primer and probe combination for detecting rhesus monkey RRV virus according to the first aspect.
Preferably, the kit further comprises a viral nucleic acid standard and a fluorescent quantitative PCR reaction reagent.
Preferably, the viral nucleic acid standard is a DNA fragment of the fragment of interest.
Preferably, the fluorescent quantitative PCR reaction reagent comprises an enzyme solution and a reaction buffer solution, wherein the enzyme solution comprises DNA polymerase.
In a third aspect, the present invention provides a method of using the kit for detecting rhesus monkey RRV virus according to the second aspect for the purpose of non-disease diagnosis and/or treatment, the method of using comprising the steps of:
(1) Collecting and processing a sample to be detected, and preparing a fluorescent quantitative PCR system;
(2) Performing fluorescent quantitative PCR reaction on the sample to be detected and the virus nucleic acid standard to obtain fluorescent signals of the sample to be detected and the virus nucleic acid standard, and performing data processing on the fluorescent signals to obtain a Ct value and an amplification curve;
(3) And drawing a fluorescent quantitative standard curve according to the concentration and Ct value of the virus nucleic acid standard substance, and carrying out qualitative and quantitative detection on the sample to be detected according to the fluorescent quantitative standard curve.
Preferably, the fluorescent quantitative PCR system comprises, in terms of final concentration: taqman Universal PCR MASTER Mix 1X, 800-900nM forward primer (e.g., 800nM, 850nM, 900nM, etc.), 800-900nM reverse primer (e.g., 800nM, 850nM, 900nM, etc.), 220-250nM probe (e.g., 220nM, 240nM, 250nM, etc.).
Other specific point values in the above numerical ranges are selectable, and will not be described in detail herein.
Preferably, the procedure of the fluorescent quantitative PCR reaction comprises:
step 1:94-96 ℃ for 8-12min; step 2:94-96 ℃,13-17sec; step 3:58-62 ℃ for 0.8-1.2min; step 2 and step 3, 40-42 cycles.
Wherein, step 1:94-96 ℃ (e.g., 94 ℃, 95 ℃ or 96 ℃ and the like) for 8-12min (e.g., 8min, 9min, 10min, 11min or 12min and the like);
Step 2:94-96 ℃ (e.g., 94 ℃, 95 ℃ or 96 ℃ and the like), 13-17sec (e.g., 13sec, 14sec, 15sec, 16sec or 17sec and the like);
Step 3:58-62 deg.C (such as 58 deg.C, 59 deg.C, 60 deg.C, 61 deg.C, 62 deg.C, etc.), 0.8-1.2min (such as 0.8min, 0.9min, 1.0min, 1.1min, 1.2min, etc.);
Step 2 and step 3, 40-42 cycles (which may be 40, 41 or 42, for example).
Other specific point values in the above numerical ranges are selectable, and will not be described in detail herein.
In a fourth aspect, the present invention provides a system for detecting rhesus monkey RRV virus, the system comprising:
(1) Sample preparation module: collecting and processing a sample to be detected, and preparing a fluorescent quantitative PCR system, wherein the PCR system comprises the primer and probe combination for detecting the rhesus RRV virus in the first aspect;
(2) And a detection module: carrying out fluorescent quantitative PCR reaction on the fluorescent quantitative PCR system;
(3) And an analysis module: and carrying out qualitative and quantitative detection on the sample to be detected according to the fluorescent quantitative standard curve.
Preferably, the fluorescent quantitative PCR system comprises, in terms of final concentration: taqman Universal PCR MASTER Mix 1X, 800-900nM forward primer, 800-900nM reverse primer, 220-250nM probe.
Preferably, the procedure of the fluorescent quantitative PCR reaction comprises:
step 1:94-96 ℃ for 8-12min; step 2:94-96 ℃,13-17sec; step 3:58-62 ℃ for 0.8-1.2min; step 2 and step 3, 40-42 cycles.
In a fifth aspect, the present invention provides a method for detecting rhesus monkey RRV virus, comprising:
(1) Collecting and processing a sample to be detected, and preparing a fluorescent quantitative PCR system, wherein the PCR system comprises the primer and probe combination for detecting the rhesus RRV virus in the first aspect;
(2) Performing fluorescent quantitative PCR reaction on the sample to be detected and the virus nucleic acid standard to obtain fluorescent signals of the sample to be detected and the virus nucleic acid standard, and performing data processing on the fluorescent signals to obtain a Ct value and an amplification curve;
(3) And drawing a fluorescent quantitative standard curve according to the concentration and Ct value of the virus nucleic acid standard substance, and carrying out qualitative and quantitative detection on the sample to be detected according to the fluorescent quantitative standard curve.
Preferably, the fluorescent quantitative PCR system comprises, in terms of final concentration: taqman Universal PCR MASTER Mix 1X, 800-900nM forward primer, 800-900nM reverse primer, 220-250nM probe.
Preferably, the procedure of the fluorescent quantitative PCR reaction comprises:
step 1:94-96 ℃ for 8-12min; step 2:94-96 ℃,13-17sec; step 3:58-62 ℃ for 0.8-1.2min; step 2 and step 3, 40-42 cycles.
In a sixth aspect, the present invention provides the use of a combination of a primer and a probe for detecting rhesus monkey RRV virus according to the first aspect, a kit for detecting rhesus monkey RRV virus according to the second aspect, or a system for detecting rhesus monkey RRV virus according to the fourth aspect, or a combination of at least two of the primers and the probe for detecting rhesus monkey RRV virus in the preparation of a product for detecting rhesus monkey RRV virus.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention designs 1 group of forward and reverse primers and probes of real-time quantitative PCR by carrying out homology comparison on all RRV isolates (including H26-95 and 17577) in an NCBI database, and the detection method can detect all RRV virus isolates (including H26-95 and 17577) existing in the NCBI database. The amplification detection region selected by the invention is a very conserved region in the RRV genome, and the probability of detecting new variant strains in the future by the method is very high.
(2) The detection method of the invention takes about 2.0 hours, and compared with virus cell culture detection, the time is greatly shortened, and the labor and material cost is reduced.
(3) The detection method of the present invention can detect as low as 100 copies of the viral genome of RRV (including H26-95 and 17577) (FIG. 1). The high sensitivity and the high coverage rate of the method can better ensure the safety of biological products.
(3) Biosafety detection of cell banks requires detection of trace viral contamination in large numbers of cells. When the PCR method is used for detection, the detection sample DNA also contains a large amount of cell DNA and a trace amount of virus DNA (if virus contamination exists). The large amount of cellular DNA often inhibits the PCR reaction. The detection method of the invention is not interfered by the total DNA of the cells, and is particularly suitable for detecting virus pollution in samples containing the cells.
Drawings
FIG. 1 shows the detection results of viral nucleic acid standards.
FIG. 2 shows the amplification results of the primer probe of example 1.
FIG. 3 shows the amplification results of the primer probe of comparative example 1.
FIG. 4 is a graph showing the amplification effect of example 5.
FIG. 5 is a graph showing the amplification effect of example 6.
FIG. 6 is a graph showing the amplification effect of example 7.
FIG. 7 is a graph showing the amplification effect of example 8.
FIG. 8 is a graph showing the amplification effect of example 9.
FIG. 9 is a graph showing the amplification effect of example 10.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
This example provides a primer and probe combination for detecting rhesus RRV virus. In the embodiment, 1 group of forward and reverse primers and probes of real-time quantitative PCR are designed by carrying out homology comparison on all the isolated strains (including H26-95 and 17577) of RRV in NCBI database, and the combination of the primers and the probes can efficiently and accurately detect all types of RRV DNA and is not influenced by a large amount of background cell DNA.
The primers and probes are shown below:
(Forward primer 1) SEQ ID NO. 1: CGGTAACACCTACGTCTCGG.
(Reverse primer 1) SEQ ID NO. 2: CCTGGGACACGTTGGTTACC.
(Probe 1) SEQ ID NO:3:
FAM-ACTGCGAGTGGCCACCGAACCGGGAGA-BHQ1。
The sequence of the target fragment amplified by the forward primer and the reverse primer is shown as SEQ ID NO. 4.
SEQ ID NO:4：
CCGCGCACCCATTTCTCTTAACCCAGAAGACCAAGCCCTTTACCGAGCGAAAGTTTTGCCGCCTGATCATGGACAACGACCAGCGCAGCGCCGTCAACACCGTCTACCTGGGAAAGCAGCACGTGAGGGTGACCGTGACCCGCCCCCCGGAAACAATCGTCACCGACGGCCCCGTGACGGCGACCCTGTCCCTCACCGGTAATGCGCCAATCGCCTTTCGCCACAACCCATACTTTGAACTCCCGTGGTCGTCCACAACGGCGATATTCACGCCCGTGGTGTACGTGGGCCTGACCGTGTGCATCCCACCCAACTGTAGCAAATTCGTAAGGTACGGTAACACCTACGTCTCGGCATTTAACCGCAAGCTGACGGCGATTATTAGCAATCACGCCCACAACGGCGGGTTCCGGATTCAGGACTGCGAGTGGCCACCGAACCGGGAGATAGAGATTTTGGTAACCAACGTGTCCCAGGCCCCGGTGTACATCAGCACCGGGACGCAGCTGGGGCAAGCCATCTTCGTGTTCGCGCCGCGGTTCGGTGGCCCGGCGAAACTGCGGCAGCTCCTCGGCCACCGATCGCGCGCCCTGGAGCTGCCGGGCGGGGTGACAGTGGACAGCC.
Comparative example 1
This comparative example provides a primer and probe combination for detecting rhesus RRV virus. The primers and probes are as follows:
forward primer 2 (SEQ ID NO: 5): GCAATCACGCCCACAACG.
Reverse primer 2 (SEQ ID NO: 6): GTCCCGGTGCTGATGTACAC.
Forward primer 3 (SEQ ID NO: 7): GGATTCAGGACTGCGAGTGG.
Reverse primer 3 (SEQ ID NO: 8): CGAACACGAAGATGGCTTGC.
Forward primer 4 (SEQ ID NO: 9): GCAATCACGCCCACAACG.
Reverse primer 4 (SEQ ID NO: 10): AAGATGGCTTGCCCCAGC.
Probe 2 (SEQ ID NO: 11):
FAM-TTGGTAACCAACGTGTCCCAGGCCCCGGT-BHQ1。
Example 2
The embodiment provides a kit for detecting rhesus RRV virus, which comprises the primer and probe combination for detecting the rhesus RRV virus, a virus nucleic acid standard and a fluorescent quantitative PCR reaction reagent in embodiment 1.
The fluorescent quantitative PCR reaction reagent comprises: taqman Universal PCRMasterMix (2X), ddH2O (DNAse & RNASE FREE).
The virus nucleic acid standard is DNA containing target sequence, and is made into standard sample containing 1×10 5,1×104,1×103,1×102 and 10 target sequence copies per 5 μl after gradient dilution in use.
Example 3
This example uses the kit for detecting rhesus RRV virus described in example 2 to detect rhesus RRV virus nucleic acid. The specific steps of detection are as follows:
(1) Collecting and processing a sample to be detected, and preparing a fluorescent quantitative PCR system; the fluorescent quantitative PCR system is shown in table 1:
TABLE 1
| Composition of the components | Volume (final concentration) |
| Taqman Universal PCR Master Mix(2X) | 1X |
| Forward primer | 900nM |
| Reverse primer | 900nM |
| Probe with a probe tip | 240nM |
| Template | 5μL |
| ddH2O | Supplement to 25 mu L |
Concentration gradient of the viral nucleic acid standard: each 5. Mu.L of standard sample contained 1X 10 5,1×104,1×103,1×102 and 10 copies of the target sequence.
The sample to be tested contains 100 copies of the virus nucleic acid standard.
And synchronously preparing a PCR system for interference experiments. In the interference experiments, group 1 was tested against background, and 0.5. Mu. gHEK293, 0.5. Mu.g CHO-K1 and 0.5. Mu.g Vero cell total DNA was added as background DNA to the reaction system. Group 2 was interference detection, 0.5. Mu.g HEK293, 0.5. Mu.g CHO-K1 and 0.5. Mu.g Vero cell total DNA were added to the reaction system together with 100 copies of viral nucleic acid standard.
(2) And performing fluorescent quantitative PCR reaction on the sample to be detected and the virus nucleic acid standard by adopting an ABI 7500 real-time PCR system to obtain fluorescent signals of the sample to be detected and the virus nucleic acid standard, and performing data processing on the fluorescent signals to obtain a Ct value and an amplification curve. The procedure for fluorescent quantitative PCR reactions is as follows:
Step 1:95 ℃ for 10min; step 2:95 ℃ for 15sec; step 3:60 ℃ for 1min; step 2 and step 3, 40 cycles.
(3) And drawing a fluorescent quantitative standard curve according to the concentration and Ct value of the virus nucleic acid standard substance, and carrying out qualitative and quantitative detection on the sample to be detected according to the fluorescent quantitative standard curve.
The detection result of the virus nucleic acid standard is shown in figure 1, wherein the number (10-10 5) is the copy number of the template, and the result shows that the detection system can detect 10 copies of the template.
Test results: (equation of standard curve: y= -3.551lgx+37.286), (R 2: 0.997).
Analysis of results: the standard curve shows that the detection system can detect 10 copies of the template. In the interference experiment, 0.5. Mu.g HEK293, 0.5. Mu.g CHO-K1 and 0.5. Mu. gVero cell total DNA was added as background RNA to the reaction system, and no fluorescent signal was detected; 100 copies of the viral nucleic acid standard were incorporated into 0.5. Mu. gHEK293 g 293, 0.5. Mu.g CHO-K1 and 0.5. Mu.g Vero cell total DNA and were detected as normal.
Example 4
This example was performed using the primer probes of example 1 and comparative example 1, respectively, and the detection effects of the different primer probes were compared. For specific steps of the assay reference is made to example 3.
Fluorescent quantitative PCR System As shown in reference example 3, the samples to be tested were 0.5. Mu.g HEK293, 0.5. Mu.g CHO-K1 and 0.5. Mu.g Vero cell total DNA spiked with 100 copies of viral nucleic acid standard. Detection was performed with the primer probes in example 1 and comparative example 1, respectively.
The amplification procedure for the fluorescent quantitative PCR reaction is as follows:
Step 1:95 ℃ for 10min; step 2:95 ℃ for 15sec; step 3:60 ℃ for 1min; step 2 and step 3, 40 cycles.
Test results: as shown in FIGS. 2 and 3, the detection using the primer probe of example 1 had an average product fluorescence intensity of about 4.0 units, whereas the detection using the primer probe of comparative example 1 had a product fluorescence intensity of only about 1.5 units, indicating that the amplification of the target sequence by the primer probe of comparative example 1 was inhibited by the background of cellular DNA.
Example 5
This example differs from example 3 only in that the final concentration of the forward and reverse primers was changed to 700nM.
Test results: as shown in FIGS. 2 and 4, the final concentration of the forward and reverse primers in example 3 was used to detect that the product fluorescence intensity was about 4.0 units on average, while the final concentration of the forward and reverse primers in example 5 was used to detect that the product fluorescence intensity was only 2.0 units. It was demonstrated that the final concentration of the forward and reverse primers in example 3 was superior to that in example 5.
Example 6
This example differs from example 3 only in that the final concentration of the forward and reverse primers was changed to 1000nM.
Test results: as shown in FIGS. 2 and 5, the final concentration of the forward and reverse primers in example 3 was used to detect that the product fluorescence intensity was about 4.0 units on average, while the final concentration of the forward and reverse primers in example 6 was used to detect that the product fluorescence intensity was only 1.9 units. It was demonstrated that the final concentration of the forward and reverse primers in example 3 was superior to that in example 6.
Example 7
This example differs from example 3 only in that the final probe concentration is changed to 200nM.
Experimental results: if the final probe concentration in example 3 was used for detection as shown in FIGS. 2 and 6, the average product fluorescence intensity was about 4.0 units, whereas the final probe concentration in example 7 was used for detection, and the average product fluorescence intensity was only 2.0 units. It was demonstrated that the probe concentration in example 3 was better than that in example 7.
Example 8
This example differs from example 3 only in that the final probe concentration is changed to 300nM.
Experimental results: as shown in FIGS. 2 and 7, the final concentration of the probe in example 3 was used to measure the average fluorescence intensity of the product of about 4.0 units, and the final concentration of the probe in example 8 was used to measure the average fluorescence intensity of the product of only 2.1 units. It was demonstrated that the probe concentration in example 3 was better than that in example 8.
Example 9
The only difference between this example and example 3 is that the procedure for the fluorescent quantitative PCR reaction was changed to step 3:55℃for 1min.
Experimental results: as shown in FIGS. 2 and 8, the detection was performed using the fluorescent quantitative PCR reaction procedure in example 3, the product fluorescence intensity was about 4.0 units on average, and the detection was performed using the fluorescent quantitative PCR reaction procedure in example 9, the product fluorescence intensity was only about 0.5 units on average. The procedure for the fluorescent quantitative PCR reaction in example 3 was demonstrated to be superior to that in example 9.
Example 10
The only difference between this example and example 3 is that the procedure for the fluorescent quantitative PCR reaction was changed to step 3:65℃for 1min.
Experimental results: as shown in FIGS. 2 and 9, the detection was performed using the fluorescent quantitative PCR reaction procedure in example 3, the product fluorescence intensity was about 4.0 units on average, and the detection was performed using the fluorescent quantitative PCR reaction procedure in example 10, the product fluorescence intensity was only about 1.9 units on average. The procedure for the fluorescent quantitative PCR reaction in example 3 was demonstrated to be superior to that in example 10.
In summary, the invention provides a primer probe combination for detecting rhesus RRV viruses and application thereof, and the primer probe combination can be used for detecting various rhesus RRV viruses, and has high detection efficiency and high accuracy. The method for detecting RRV virus provided by the invention can detect samples with as low as 10 virus genome copies, and has high sensitivity and high coverage rate. The detection method of the invention is not interfered by the total DNA of the cells, is particularly suitable for detecting virus pollution in samples containing the cells, and has important application prospect in quality control of biological drug development.
The applicant states that the present invention is illustrated by the above examples as a primer and probe combination for detecting rhesus RRV virus and its application, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be practiced depending on the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. A primer and probe combination for detecting rhesus monkey RRV virus, characterized in that the primer and probe combination comprises a primer and probe combination for detecting rhesus monkey RRV virus vIL6 gene;
the primer and probe combination comprises: forward primer, reverse primer and probe;
The nucleotide sequence of the forward primer comprises a sequence shown in SEQ ID NO. 1, and the nucleotide sequence of the reverse primer comprises a sequence shown in SEQ ID NO. 2;
the nucleotide sequence of the probe comprises a sequence shown in SEQ ID NO. 3.
2. The primer and probe combination for detecting rhesus monkey RRV virus according to claim 1, wherein the 5 'end of the probe is modified with fluorescein and the 3' end of the probe is modified with a quencher;
Preferably, the fluorescein is selected from any one of FAM, VIC or HEX;
Preferably, the quencher is selected from any one of MGB, BHQ1 or TAMRA;
preferably, the nucleotide sequence of the target fragment amplified by the forward primer and the reverse primer comprises the sequence shown in SEQ ID NO. 4.
3. A kit for detecting rhesus monkey RRV virus comprising the primer and probe combination of claim 1 or 2.
4. The kit for detecting rhesus monkey RRV virus of claim 3, further comprising a viral nucleic acid standard and a fluorescent quantitative PCR reaction reagent;
Preferably, the viral nucleic acid standard comprises a DNA fragment of the fragment of interest;
Preferably, the fluorescent quantitative PCR reaction reagent comprises an enzyme solution and a reaction buffer solution, wherein the enzyme solution comprises DNA polymerase.
5. Use of the kit for detecting rhesus monkey RRV virus according to claim 3 or 4 for the purpose of non-disease diagnosis and/or treatment, characterized in that it comprises the following steps:
(1) Collecting and processing a sample to be detected, and preparing a fluorescent quantitative PCR system;
(2) Performing fluorescent quantitative PCR reaction on the sample to be detected and the virus nucleic acid standard to obtain fluorescent signals of the sample to be detected and the virus nucleic acid standard, and performing data processing on the fluorescent signals to obtain a Ct value and an amplification curve;
(3) And drawing a fluorescent quantitative standard curve according to the concentration and Ct value of the virus nucleic acid standard substance, and carrying out qualitative and quantitative detection on the sample to be detected according to the fluorescent quantitative standard curve.
6. The method of claim 5, wherein the fluorescent quantitative PCR system comprises, in final concentration: taqman Universal PCRMasterMix 1X, 800-900nM forward primer, 800-900nM reverse primer, 220-250nM probe;
Preferably, the procedure of the fluorescent quantitative PCR reaction comprises:
step 1:94-96 ℃ for 8-12min; step 2:94-96 ℃,13-17sec; step 3:58-62 ℃ for 0.8-1.2min; step 2 and step 3, 40-42 cycles.
7. A system for detecting rhesus monkey RRV virus, the system comprising:
(1) Sample preparation module: collecting and processing a sample to be tested, and preparing a fluorescent quantitative PCR system, wherein the PCR system comprises the primer and probe combination for detecting the rhesus RRV virus according to claim 1 or 2;
(2) And a detection module: carrying out fluorescent quantitative PCR reaction on the fluorescent quantitative PCR system;
(3) And an analysis module: and carrying out qualitative and quantitative detection on the sample to be detected according to the fluorescent quantitative standard curve.
8. A method of detecting rhesus monkey RRV virus, the method comprising:
(1) Collecting and processing a sample to be tested, and preparing a fluorescent quantitative PCR system, wherein the PCR system comprises the primer and probe combination for detecting the rhesus RRV virus according to claim 1 or 2;
(2) Performing fluorescent quantitative PCR reaction on the sample to be detected and the virus nucleic acid standard to obtain fluorescent signals of the sample to be detected and the virus nucleic acid standard, and performing data processing on the fluorescent signals to obtain a Ct value and an amplification curve;
(3) And drawing a fluorescent quantitative standard curve according to the concentration and Ct value of the virus nucleic acid standard substance, and carrying out qualitative and quantitative detection on the sample to be detected according to the fluorescent quantitative standard curve.
9. The method of claim 8, wherein the fluorescent quantitative PCR system comprises, in final concentration: taqman UniversalPCRMasterMix 1X, 800-900nM forward primer, 800-900nM reverse primer, 220-250nM probe;
Preferably, the procedure of the fluorescent quantitative PCR reaction comprises: step 1:94-96 ℃ for 8-12min; step 2:94-96 ℃,13-17sec; step 3:58-62 ℃ for 0.8-1.2min; step 2 and step 3, 40-42 cycles.
10. Use of a primer and probe combination for the detection of rhesus monkey RRV virus according to claim 1 or 2, a kit for the detection of rhesus monkey RRV virus according to claim 3 or 4 or a system for the detection of rhesus monkey RRV virus according to claim 7, or a combination of at least two thereof, for the preparation of a product for the detection of rhesus monkey RRV virus.
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