CN114317821A - Method for rapidly detecting rat leukemia virus - Google Patents
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- CN114317821A CN114317821A CN202111614381.9A CN202111614381A CN114317821A CN 114317821 A CN114317821 A CN 114317821A CN 202111614381 A CN202111614381 A CN 202111614381A CN 114317821 A CN114317821 A CN 114317821A
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- 241001428754 Rat leukemia virus Species 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 24
- 239000000523 sample Substances 0.000 claims abstract description 63
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 125000006853 reporter group Chemical group 0.000 claims abstract description 13
- 238000003753 real-time PCR Methods 0.000 claims abstract description 10
- 241000700605 Viruses Species 0.000 claims description 9
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 6
- 241000714177 Murine leukemia virus Species 0.000 claims description 5
- 238000010791 quenching Methods 0.000 claims description 5
- 230000000171 quenching effect Effects 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 2
- 208000032839 leukemia Diseases 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000011084 recovery Methods 0.000 abstract description 4
- 230000003321 amplification Effects 0.000 description 17
- 238000003199 nucleic acid amplification method Methods 0.000 description 17
- 230000000052 comparative effect Effects 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 238000000137 annealing Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
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Abstract
The invention provides a method for rapidly detecting rat leukemia virus. The method comprises the steps of taking RNA in a biological product to be detected as a template, carrying out fluorescent quantitative PCR reaction by using a primer and a probe, and collecting a fluorescent signal to determine the content of the rat leukemia virus in the biological product to be detected, wherein the primer comprises an upstream primer and a downstream primer, the sequence of the upstream primer is 5'-TCTTGTCCTGTGCGCGTGTT-3', the sequence of the downstream primer is 5'-TCCTTCTCTGACCCGCCCAA-3', the sequence of the probe is 5'-TCAGGACCCTCAGCGCCTCC-3', the 5 'end of the probe is connected with a fluorescent reporter group, and the 3' end of the probe is connected with a fluorescent quencher group. The detection method is simple and rapid, high in sensitivity, good in repeatability, good in accuracy and high in recovery rate.
Description
Technical Field
The invention relates to the technical field of biological kits, in particular to a method for rapidly detecting rat leukemia virus.
Background
The biological products used for clinic use must be put into use by a safety evaluating party, so that in the process of preparing the biological products, viruses must be removed or inactivated by a special method, and meanwhile, the virus removal or inactivation effect needs to be detected in a certain way, so that the safety of the biological products is guaranteed.
The rat leukemia virus is a mark virus of retrovirus and is an important index in virus safety evaluation. Traditional detection modes comprise cell experiments and animal experiments, wherein virus levels are evaluated through antibody production conditions, and omission or false positive is inevitable. In addition, the existing detection method has long time period and high manpower cost, for example, about 8 days is needed before and after cell experiment, and simultaneously, the problems of more pollutants, high virus treatment cost, high danger and the like exist. The fluorescent quantitative PCR technology has the effects of rapidness and high efficiency, is applied more quickly and more widely in virus nucleic acid detection, and has application prospects in biological product safety evaluation, but the accuracy, repeatability, sensitivity, specificity and the like of the detection result of the fluorescent quantitative PCR need to be established on a specially designed primer probe, so that the primer and the probe become important points for providing a method for rapidly detecting the rat leukemia virus.
Disclosure of Invention
The invention aims to provide a method for simply and quickly detecting rat leukemia virus.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for rapidly detecting rat leukemia virus, said method is to regard RNA in the biological products to be detected as the template, use primer, probe to carry on the fluorescent quantitative PCR reaction, gather the fluorescent signal in order to confirm the rat leukemia virus content in the said biological products to be detected, said primer includes upstream primer and downstream primer, the sequence of the said upstream primer is 5'-TCTTGTCCTGTGCGCGTGTT-3', or has at least 80% homologous sequence with it;
the sequence of the downstream primer is 5'-TCCTTCTCTGACCCGCCCAA-3', or a sequence having at least 80% homology thereto;
the sequence of the probe is 5'-TCAGGACCCTCAGCGCCTCC-3', or a sequence with at least 80% homology with the probe, the 5 'end of the probe is connected with a fluorescence reporter group, and the 3' end of the probe is connected with a fluorescence quencher group.
Preferably, the size of the amplified fragment of the primer is 135-140 bp.
Preferably, the length of the upstream primer, the length of the downstream primer and the length of the probe are independently 18-22 bp, and more preferably 19-21 bp.
Preferably, the GC content of the upstream primer is 50-60%, more preferably 52-58%, and even more preferably 54-56%.
Preferably, the GC content of the downstream primer is 55-65%, more preferably 58-64%, and even more preferably 59-61%.
The GC content of the probe is 65-75%, more preferably 68-72%, and still more preferably 69-71%.
Preferably, the 5 'end of the probe is connected with a fluorescence reporter group, and the 3' end of the probe is connected with a fluorescence quencher group.
Preferably, the fluorescent reporter group is FAM; the fluorescence quenching group is TAMRA or BHQ.
According to the embodiment, the reaction system of the fluorescence quantitative PCR is as follows:
the reaction condition is 42 ℃/5 min; 95 ℃/30 sec; 95 ℃/5sec, 60 ℃/30sec for 40 cycles; fluorescence signals were collected during annealing extension at 60 ℃.
A primer and a probe for rapidly detecting rat leukemia virus, wherein the primer comprises an upstream primer and a downstream primer, and the sequence of the upstream primer is 5'-TCTTGTCCTGTGCGCGTGTT-3' or a sequence with at least 80% homology with the upstream primer; the sequence of the downstream primer is 5'-TCCTTCTCTGACCCGCCCAA-3', or a sequence having at least 80% homology thereto; the probe has a sequence of 5'-TCAGGACCCTCAGCGCCTCC-3', or a sequence having at least 80% homology thereto.
Preferably, the 5 'end of the probe is connected with a fluorescence reporter group, and the 3' end of the probe is connected with a fluorescence quencher group; the fluorescent reporter group is FAM; the fluorescence quenching group is TAMRA or BHQ.
A kit for rapidly detecting rat leukemia virus comprises the primer and the probe.
Preferably, the kit further comprises an RNA standard, and the sequence of the RNA standard is TCTTGTCCTGTGCGCGTGTTCAGTTCGTCTCAGTTTTGGACTCAGATCTGGGTTTTGGTCGAAGGAGAAGGCCCAGGGCTTCGGTTTCTCAGGGTTCAGGACCCTCAGCGCCTCCGTTTGGGCGGGTCAGAGAAGGA.
The primer, the probe and/or the kit are/is applied to detecting the rat leukemia virus in biological products.
Compared with the prior art, the invention has the following advantages:
the method for rapidly detecting the rat leukemia virus adopts specific primers and probes to perform fluorescence quantitative PCR reaction, acquires fluorescence signals to determine the content of the rat leukemia virus in the biological product to be detected, and has the advantages of simplicity, rapidness, high sensitivity, high accuracy, good repeatability, strong specificity, high recovery rate and the like.
Drawings
FIG. 1 is a schematic view of the amplification curve obtained in example 1 (template concentration is 2X 10 from left to right in the order of magnitude)7copies/μL、2×105copies/μL、2×103copies/μL)；
FIG. 2 is a graph showing the amplification curve obtained in example 1 (template concentration is 2X 10 from left to right in this order)7copies/μL、2×106copies/μL、2×105copies/μL、2×104copies/μL、2×103copies/μL)；
FIG. 3 is a graph showing the amplification curve obtained in example 1 (template concentration is 2X 10 from left to right in this order)1copies/μL、2×100copies/μL)；
FIG. 4 is a graph showing the amplification curve obtained in comparative example 1 (template concentration: 2X 10)7copies/. mu.L, no apparent amplification curve);
FIG. 5 is a graph showing the amplification curve obtained in comparative example 2 (template concentration: 2X 10)7copies/. mu.L, no apparent amplification curve);
FIG. 6 is a graph showing the amplification curve obtained in example 3 (template concentration: 2X 10)7copies/. mu.L, amplification efficiency is poor);
FIG. 7 is a diagram showing an amplification curve obtained by using the sample DNA of example 2 as a template.
Detailed Description
The present invention will be further described with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are conventional conditions in the industry. The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.
The invention designs and screens a primer probe for fluorescent PCR quantitative detection aiming at an envelope gene sequence (the accession number of a gene bank is M77194.1) of the rat leukemia virus, and the primer probe is verified by a large number of experiments.
According to the invention, the method for rapidly detecting the rat leukemia virus comprises the steps of taking RNA in a biological product to be detected as a template, carrying out fluorescence quantitative PCR reaction by adopting a primer and a probe, and collecting a fluorescence signal to determine the content of the rat leukemia virus in the biological product to be detected, wherein the primer comprises an upstream primer and a downstream primer, and the sequence of the upstream primer is 5'-TCTTGTCCTGTGCGCGTGTT-3' or a sequence with at least 80% homology with the upstream primer;
the sequence of the downstream primer is 5'-TCCTTCTCTGACCCGCCCAA-3', or a sequence having at least 80% homology thereto;
the sequence of the probe is 5'-TCAGGACCCTCAGCGCCTCC-3', or a sequence with at least 80% homology with the probe, the 5 'end of the probe is connected with a fluorescence reporter group, and the 3' end of the probe is connected with a fluorescence quencher group.
According to the invention, the fluorescent reporter group is FAM; the fluorescence quenching group is TAMRA or BHQ.
According to the invention, the kit for rapidly detecting the rat leukemia virus comprises the probe, the primer and the RNA standard substance, wherein the sequence of the RNA standard substance is TCTTGTCCTGTGCGCGTGTTCAGTTCGTCTCAGTTTTGGACTCAGATCTGGGTTTTGGTCGAAGGAGAAGGCCCAGGGCTTCGGTTTCTCAGGGTTCAGGACCCTCAGCGCCTCCGTTTGGGCGGGTCAGAGAAGGA.
Compared with the prior art, the primer probe for fluorescent quantitative PCR detection has the following advantages:
and (3) shortening the detection time: the conventional rat leukemia virus detection method uses cell experiments, requires about 8 days before and after, consumes labor and time, and only takes about 4 hours from the extraction of sample DNA to the acquisition, thereby greatly shortening the operation time.
And (3) reducing the risk: the traditional method has infection risks in the cell experiment process, and the invention can perform proper inactivation treatment on the sample before operation, thereby reducing the risks of pollution and infection.
The sensitivity is high: when the virus content is lower, the existing detection method has certain probability of infecting cells, and the condition that the detection cannot be easily generated is very easy to occur, when the invention detects the rat leukemia virus, the RNA standard substance is 2 multiplied by 103～2×107The linearity is good in the range of copies/. mu.L concentration (R)2Not less than 0.99), the sensitivity reaches 20 copies/mu L.
The repeatability, accuracy and specificity are strong: the repeatability SD of the detection method is less than or equal to 0.5, the RNA standard is used for making a standard curve, the RNA amount of a sample is quantified, the system template is consistent, the accuracy is high, and the specificity is strong because a primer and a probe are designed by the nucleic acid sequence of the rat leukemia virus.
The recovery rate is high: the recovery rate of the fluorescent quantitative PCR method is more than or equal to 80 percent.
The technical scheme and the technical effect of the invention are further illustrated by combining specific examples and comparative examples.
Example 1
(1) The sequence of the envelope gene of the murine leukemia virus was found from NCBI (GenBank accession No. M77194.1).
(2) Primers and probes shown in Table 1 were designed, and the probes were synthesized by Gene Synthesis company, in which the fluorescent reporter group FAM was connected to the 5 'end and the fluorescent quencher group TAMRA was connected to the 3' end.
TABLE 1
(3) Standard of DNA: the sequence was TCTTGTCCTGTGCGCGTGTTCAGTTCGTCTCAGTTTTGGACTCAGATCTGGGTTTTGGTCGAAGGAGAAGGCCCAGGGCTTCGGTTTCTCAGGGTTCAGGACCCTCAGCGCCTCCGTTTGGGCGGGTCAGAGAAGGA (SEQ ID NO.13), the length was 137bp, and was synthesized directly by Gene Synthesis.
(4) Making a standard curve
a) Diluting the synthesized RaLV RNA standard substance to 2, 20 and 2X 102、2×103、2×104、2×105、2×106、2×107copies/. mu.L to make RNA templates of different concentrations;
b) preparing a reaction system:
reaction system (20 μ L) (TAKARA):
c) and (3) PCR reaction conditions: 42 ℃/5 min; 95 ℃/30 sec; 95 ℃/5sec, 60 ℃/30sec for 40 cycles; fluorescence signals were collected during annealing extension at 60 ℃.
d) And C, preparing a standard curve by taking the Cq value obtained by each standard substance concentration as an ordinate and taking the logarithm of the standard substance copy number as an abscissa, wherein the Cq value is the cycle number required when the fluorescence signal in each reaction tube reaches a set threshold value. Linear relationship y-3.589 x +42.173, R2＝1.0000。
The one-step method is adopted and intersected with the traditional two-step method, the pollution probability can be reduced, the detection time is reduced, and when a reaction system is added, other components except the template RNA can be prepared into premixed liquid according to the number of reaction holes and then are subpackaged into eight-connecting pipes or 96-hole plates, so that the error between every two holes is reduced.
The RNA standard is 2X 107SD of 0.12 is repeatedly detected for 10 times when the copies/mu L is detected;
RNA markerThe standard product is 2 × 105SD of 0.05 is repeatedly detected for 10 times when the copies/mu L is detected;
the RNA standard is 2X 103The SD was 0.22 in duplicate at copies/. mu.L.
Comparative example 1
The PCR reaction system and the PCR reaction conditions of this comparative example were substantially the same as those of example 1, except that the sequences of the primers and probes were different, and the primers and probes of this comparative example are shown in Table 2.
TABLE 2
The amplification curve of this comparative example is schematically shown in FIG. 4, with no significant amplification curve.
Comparative example 2
The PCR reaction system and the PCR reaction conditions of this comparative example were substantially the same as those of example 1, except that the sequences of the primers and probes were different, and the primers and probes of this comparative example are shown in Table 3.
TABLE 3
The amplification curve of this comparative example is schematically shown in FIG. 5, with no apparent amplification curve.
Comparative example 3
The PCR reaction system and the PCR reaction conditions of this comparative example were substantially the same as those of example 1, except that the sequences of the primers and probes were different, and the primers and probes of this comparative example are shown in Table 3.
TABLE 3
The amplification curve of the comparative example is schematically shown in FIG. 6, and the detection result is unstable, the amplification efficiency is low, and the linear relationship is poor.
Example 2
(1) Sample (A)Extracting product RNA: using PureLinkTMThe Viral RNA/DNA Mini Kit (Invitrogen) extracted the sample RNA.
(2) The extracted sample RNA was used as a template, and the test was performed by referring to the reaction system and PCR reaction conditions of example 1 using the primer probe of example 1, and the sample amplification curve is shown in FIG. 7.
(3) The nucleic acid content of the rat leukemia virus was accurately quantified according to the standard curve obtained in example 1.
The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.
Claims (10)
1. A method for rapidly detecting rat leukemia virus is characterized in that: the method comprises the steps of taking RNA in a biological product to be detected as a template, carrying out fluorescent quantitative PCR reaction by using primers and probes, and collecting fluorescent signals to determine the content of the rat leukemia virus in the biological product to be detected, wherein the primers comprise an upstream primer and a downstream primer, and the sequence of the upstream primer is 5'-TCTTGTCCTGTGCGCGTGTT-3' or a sequence with at least 80% homology with the upstream primer;
the sequence of the downstream primer is 5'-TCCTTCTCTGACCCGCCCAA-3', or a sequence having at least 80% homology thereto;
the sequence of the probe is 5'-TCAGGACCCTCAGCGCCTCC-3', or a sequence with at least 80% homology with the probe, the 5 'end of the probe is connected with a fluorescence reporter group, and the 3' end of the probe is connected with a fluorescence quencher group.
2. The method for rapidly detecting the murine leukemia virus according to claim 1, wherein the size of the amplified fragment of the primer is 135-140 bp.
3. The method for rapidly detecting murine leukemia virus according to claim 1, wherein the length of the upstream primer, the length of the downstream primer and the length of the probe are independently 18-22 bp.
4. The method for rapidly detecting the rat leukemia virus according to claim 1, wherein the GC content of the upstream primer is 50-60%; the GC content of the downstream primer is 55-65%; the GC content of the probe is 65-75%.
5. The method for rapid detection of leukemia virus of rat according to claim 1, wherein the fluorescent reporter group is FAM; the fluorescence quenching group is TAMRA or BHQ.
6. A primer and a probe for rapidly detecting rat leukemia virus are characterized in that the primer comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is 5'-TCTTGTCCTGTGCGCGTGTT-3' or a sequence with at least 80% homology with the upstream primer;
the sequence of the downstream primer is 5'-TCCTTCTCTGACCCGCCCAA-3', or a sequence having at least 80% homology thereto;
the probe has a sequence of 5'-TCAGGACCCTCAGCGCCTCC-3', or a sequence having at least 80% homology thereto.
7. The primer and probe for rapidly detecting rat leukemia virus of claim 6, wherein the 5 'end of the probe is connected with a fluorescence reporter group, and the 3' end of the probe is connected with a fluorescence quencher group; the fluorescent reporter group is FAM; the fluorescence quenching group is TAMRA or BHQ.
8. A kit for rapidly detecting rat leukemia virus, which comprises the primer and the probe of claim 6 or 7.
9. The kit for rapidly detecting the murine leukemia virus of claim 8, wherein the kit further comprises an RNA standard, wherein the sequence of the RNA standard is TCTTGTCCTGTGCGCGTGTTCAGTT CGTCTCAGTTTTGGACTCAGATCTGGGTTTTGGTCGAAGGAGAAGGCCCAGGGCTTCG GTTTCTCAGGGTTCAGGACCCTCAGCGCCTCCGTTTGGGCGGGTCAGAGAAGGA.
10. Use of a primer, probe according to claim 6 or 7 and/or a kit according to claim 8 or 9 for detecting murine leukemia virus in a biological product.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337359A (en) * | 2011-11-02 | 2012-02-01 | 舒泰神（北京）生物制药股份有限公司 | Primers and probe for detecting mouse leukemia virus and method thereof |
| WO2012051800A1 (en) * | 2010-10-18 | 2012-04-26 | 北京雅康博生物科技有限公司 | Plasmid standard for use in quantitative assays using fluorescent quantitative pcr |
| CN112011648A (en) * | 2020-10-29 | 2020-12-01 | 苏州良辰生物医药科技有限公司 | Method for detecting heterophilic mouse leukemia virus in biological product |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012051800A1 (en) * | 2010-10-18 | 2012-04-26 | 北京雅康博生物科技有限公司 | Plasmid standard for use in quantitative assays using fluorescent quantitative pcr |
| CN102337359A (en) * | 2011-11-02 | 2012-02-01 | 舒泰神（北京）生物制药股份有限公司 | Primers and probe for detecting mouse leukemia virus and method thereof |
| CN112011648A (en) * | 2020-10-29 | 2020-12-01 | 苏州良辰生物医药科技有限公司 | Method for detecting heterophilic mouse leukemia virus in biological product |
Non-Patent Citations (2)
| Title |
|---|
| SHING-YI LEE ET AL.: "Genetic Analysis of the Rat Leukemia Virus: Influence of Viral Sequences in Transduction of the c-ras Proto-Oncogene and Expression of Its Transforming Activity", JOURNAL OF VIROLOGY, vol. 72, no. 12, pages 9906 * |
| 刘艳艳等: "小鼠树突状细胞肉瘤中SRS19-6MuLV病毒的检测", 中华医学会病理学分会2010学术年会论文汇编, pages 176 * |
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