CN108531662B - Specific primer and detection method for heterophilic mouse leukemia virus - Google Patents
Specific primer and detection method for heterophilic mouse leukemia virus Download PDFInfo
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- 241000700605 Viruses Species 0.000 title claims abstract description 24
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- 208000032839 leukemia Diseases 0.000 title claims abstract description 16
- 230000003321 amplification Effects 0.000 claims abstract description 15
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 15
- 238000010839 reverse transcription Methods 0.000 claims abstract description 13
- 238000012408 PCR amplification Methods 0.000 claims abstract description 9
- 238000001502 gel electrophoresis Methods 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims description 14
- 238000000137 annealing Methods 0.000 claims description 5
- 238000004925 denaturation Methods 0.000 claims description 5
- 230000036425 denaturation Effects 0.000 claims description 5
- 238000012257 pre-denaturation Methods 0.000 claims description 5
- 230000004927 fusion Effects 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims 1
- 201000010099 disease Diseases 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 229950010203 nimotuzumab Drugs 0.000 description 2
- 108020004437 Endogenous Retroviruses Proteins 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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Abstract
The invention relates to a specific primer of heterophilic mouse leukemia virus and a detection method, wherein the specific primer comprises a reverse primer 5'-CACTGGAGACCGAGGAATCAT-3'. The detection method comprises the following steps: designing specific primers according to the X-MuLV genome sequence, wherein the specific primers comprise a forward primer 5'-GACAGGACAAACAGCTAACGC-3' and a reverse primer 5'-CACTGGAGACCGAGGAATCAT-3'; extracting total RNA in the biological product; reverse transcription is carried out according to the reverse primer and the total RNA; performing PCR amplification by using the reverse transcription product as a template and using a forward primer and a reverse primer; and taking the amplification product to perform gel electrophoresis to obtain a detection result. The kit can rapidly detect heterophilic mouse leukemia virus in biological products in real time, takes about 3 hours, and obviously improves the detection effect.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a specific primer and a detection method of heterophilic mouse leukemia virus.
Background
At present, the recombinant fusion biological product generally uses Chinese hamster ovary Cells (CHO) as an expression vector, and endogenous retroviruses such as heterophilic mouse leukemia virus (X-MuLV) may exist in the CHO cells. Although this virus is not pathogenic to humans, there are still potential risks. At present, few research reports are reported on X-MuLV detection in China, and the current methods for detecting X-MuLV mainly comprise methods such as virus infection tests, enzyme-linked immunosorbent assays (ELISA), polymerase chain reaction methods (PCR) and the like. The virus infectivity test has the defects of long detection period, complicated steps and the like; ELISA methods are prone to false negatives and are costly; the PCR technology has high sensitivity and specificity, low requirement on workers and low cost, and can be widely popularized.
Disclosure of Invention
An object of the present invention is to provide a specific primer capable of rapidly and sensitively detecting X-MuLV in a biological product.
Another object of the present invention is to provide a detection method capable of rapidly and sensitively detecting X-MuLV in a biological product.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention aims to provide specific primers of heterophilic mouse leukemia virus, which comprise a reverse primer, wherein the reverse primer is 5'-CACTGGAGACCGAGGAATCAT-3'.
Preferably, the specific primer further comprises a forward primer, and the forward primer is 5'-GACAGGACAAACAGCTAACGC-3'.
Another objective of the invention is to provide a method for detecting heterophilic mouse leukemia virus, which comprises the following steps:
(1) designing specific primers according to the X-MuLV genome sequence, wherein the specific primers comprise a forward primer and a reverse primer, the forward primer is 5'-GACAGGACAAACAGCTAACGC-3', and the reverse primer is 5'-CACTGGAGACCGAGGAATCAT-3';
(2) extracting total RNA in the biological product;
(3) designing a reverse primer according to the step (1) and carrying out reverse transcription on the total RNA extracted in the step (2);
(4) performing PCR amplification by using the reverse transcription product obtained in the step (3) as a template and using the forward primer and the reverse primer designed in the step (1);
(5) and (5) carrying out gel electrophoresis on the amplification product obtained in the step (4) to obtain a detection result.
Preferably, the biological product is a recombinant fusion biological product, a monoclonal antibody or a biological tissue extract product.
Preferably, the PCR amplification in step (4) is performed under the following conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; total extension at 72 ℃ for 10 min; storing at 4 ℃.
Preferably, the length of the amplification product is 345 bp.
Preferably, the gel electrophoresis is agarose gel electrophoresis.
The extraction of total RNA, reverse transcription, PCR amplification system, etc. in the present invention are performed by methods conventional in the art.
Due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:
the method can rapidly detect heterophilic mouse leukemia virus in biological products in real time, and takes about 3 hours, while the traditional cell infection test takes 5-7 days, so the detection effect of the method is obviously improved.
Drawings
FIG. 1 is a graph showing the results of the detection.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It is to be understood that these embodiments are provided to illustrate the basic principles, essential features and advantages of the present invention, and the present invention is not limited by the following embodiments. The implementation conditions used in the examples can be further adjusted according to specific requirements, and the implementation conditions not indicated are generally the conditions in routine experiments.
Example 1
The detection method for detecting the X-MuLV in the X-MuLV virus liquid comprises the following steps:
(1) designing specific primers according to the X-MuLV genome sequence, wherein the specific primers comprise a forward primer and a reverse primer, the forward primer is 5'-GACAGGACAAACAGCTAACGC-3' (SEQ ID NO. 1), and the reverse primer is 5'-CACTGGAGACCGAGGAATCAT-3' (SEQ ID NO. 2);
(2) extracting total RNA in X-MuLV virus liquid;
(3) designing a reverse primer according to the step (1) and carrying out reverse transcription on the total RNA extracted in the step (2);
(4) performing PCR amplification by using the reverse transcription product obtained in the step (3) as a template and using the forward primer and the reverse primer designed in the step (1); amplification conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; total extension at 72 ℃ for 10 min; storing at 4 deg.C;
(5) and (3) carrying out 1% gel electrophoresis (120V, 30 min) on the amplification product obtained in the step (4) to obtain a detection result, wherein the result is shown in a figure 1.
Example 2
The detection method for detecting the X-MuLV virus in the nimotuzumab product comprises the following steps:
(1) designing specific primers according to the X-MuLV genome sequence, wherein the specific primers comprise a forward primer and a reverse primer, the forward primer is 5'-GACAGGACAAACAGCTAACGC-3' (SEQ ID NO. 1), and the reverse primer is 5'-CACTGGAGACCGAGGAATCAT-3' (SEQ ID NO. 2);
(2) extracting total RNA in the nimotuzumab product;
(3) designing a reverse primer according to the step (1) and carrying out reverse transcription on the total RNA extracted in the step (2);
(4) performing PCR amplification by using the reverse transcription product obtained in the step (3) as a template and using the forward primer and the reverse primer designed in the step (1); amplification conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; total extension at 72 ℃ for 10 min; storing at 4 deg.C;
(5) and (3) carrying out 1% gel electrophoresis (120V, 30 min) on the amplification product obtained in the step (4) to obtain a detection result, wherein the result is shown in a figure 1.
Comparative example 1
The detection method for detecting the X-MuLV in the MuLV virus liquid comprises the following steps:
(1) designing specific primers according to the X-MuLV genome sequence, wherein the specific primers comprise a forward primer and a reverse primer, the forward primer is 5'-GACAGGACAAACAGCTAACGC-3' (SEQ ID NO. 1), and the reverse primer is 5'-CACTGGAGACCGAGGAATCAT-3' (SEQ ID NO. 2);
(2) extracting total RNA of MuLV virus liquid;
(3) designing a reverse primer according to the step (1) and carrying out reverse transcription on the total RNA extracted in the step (2);
(4) performing PCR amplification by using the reverse transcription product obtained in the step (3) as a template and using the forward primer and the reverse primer designed in the step (1); amplification conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; total extension at 72 ℃ for 10 min; storing at 4 deg.C;
(5) and (3) carrying out 1% gel electrophoresis (120V, 30 min) on the amplification product obtained in the step (4) to obtain a detection result, wherein the result is shown in a figure 1.
In FIG. 1, M represents Marker, 1 is the detection result of the amplification product of example 1, 2 is the detection result of the amplification product of example 2, 3 is the detection result of the amplification product of comparative example 1, and N is a negative control without template. As can be seen from FIG. 1, the obvious band appears at 345bp in example 1 and example 2, but no band appears in comparative example 1, so that the invention can well detect whether X-MuLV virus exists in biological products.
The above embodiments are merely illustrative of the technical concept and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and implement the invention, and not to limit the scope of the invention, and all equivalent changes or modifications made according to the spirit of the present invention should be covered by the scope of the present invention.
Sequence listing
<110> Suzhou Liangchen biomedical science and technology Co., Ltd
<120> specific primer and detection method for heterophilic mouse leukemia virus
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence (rengongxulie)
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gacaggacaa acagctaacg c 21
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<212> DNA
<213> Artificial sequence (rengongxulie)
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cactggagac cgaggaatca t 21
Claims (6)
1. A specific primer of heterophilic mouse leukemia virus, which is characterized in that: the specific primer comprises a reverse primer, and the reverse primer is 5'-CACTGGAGACCGAGGAATCAT-3'; the specific primer also comprises a forward primer, and the forward primer is 5'-GACAGGACAAACAGCTAACGC-3'.
2. A method for detecting heterophilic mouse leukemia virus for non-disease diagnosis, which comprises: the method comprises the following steps:
(1) designing specific primers according to the X-MuLV genome sequence, wherein the specific primers comprise a forward primer and a reverse primer, the forward primer is 5'-GACAGGACAAACAGCTAACGC-3', and the reverse primer is 5'-CACTGGAGACCGAGGAATCAT-3';
(2) extracting total RNA in the biological product;
(3) designing a reverse primer according to the step (1) and carrying out reverse transcription on the total RNA extracted in the step (2);
(4) performing PCR amplification by using the reverse transcription product obtained in the step (3) as a template and using the forward primer and the reverse primer designed in the step (1);
(5) and (5) carrying out gel electrophoresis on the amplification product obtained in the step (4) to obtain a detection result.
3. The method for detecting heterophilic mouse leukemia virus according to claim 2, wherein: the biological product is a recombinant fusion biological product, a monoclonal antibody or a biological tissue extraction product.
4. The method for detecting heterophilic mouse leukemia virus according to claim 2, wherein: the amplification conditions for the PCR amplification in the step (4) are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; total extension at 72 ℃ for 10 min; storing at 4 ℃.
5. The method for detecting heterophilic mouse leukemia virus according to claim 2, wherein: the length of the amplification product is 345 bp.
6. The method for detecting heterophilic mouse leukemia virus according to claim 2, wherein: the gel electrophoresis adopts agarose gel electrophoresis.
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| CN111020060A (en) * | 2019-12-19 | 2020-04-17 | 苏州药明检测检验有限责任公司 | Method for determining heterophilic murine leukemia virus titer by plaque staining |
| CN110923364B (en) * | 2019-12-25 | 2022-04-19 | 苏州药明检测检验有限责任公司 | Method for detecting multiple viruses in sample by utilizing multiple quantitative PCR technology |
| CN112011649B (en) * | 2020-10-29 | 2021-01-15 | 苏州良辰生物医药科技有限公司 | Primer probe and kit for detecting heterophilic mouse leukemia virus and application |
| CN112011648B (en) * | 2020-10-29 | 2021-01-15 | 苏州良辰生物医药科技有限公司 | Method for detecting heterophilic mouse leukemia virus in biological product |
| CN112359139A (en) * | 2020-11-06 | 2021-02-12 | 苏州药明检测检验有限责任公司 | Method for determining heterophilic murine leukemia virus titer by tissue median infection staining |
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| 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 |
| CN106399588A (en) * | 2016-09-19 | 2017-02-15 | 中国农业大学 | Reagent kit for detecting avian leukemia virus J sub-groups |
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| 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 |
| CN106399588A (en) * | 2016-09-19 | 2017-02-15 | 中国农业大学 | Reagent kit for detecting avian leukemia virus J sub-groups |
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