CN112251540A - African swine fever virus (African swine fever) lighting RNA probe detection kit and preparation method and application thereof - Google Patents
African swine fever virus (African swine fever) lighting RNA probe detection kit and preparation method and application thereof Download PDFInfo
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Abstract
An African swine fever virus lighting RNA probe detection kit and a preparation method and application thereof, belonging to the technical field of food biology. Comprises an African swine fever virus lighting RNA probe, and the nucleotide sequence is shown as SEQ ID NO. 1. The probe is specifically combined with a part of specific sequence of African swine fever virus nucleic acid sequence p54, and the nucleotide sequence is shown as SEQ ID NO. 2. An African swine fever virus lighting RNA probe detection kit comprises a detection filter paper sheet, a p54 partial specific sequence SEQ-p54 and a DEPC-aqueous solution. Application of the African swine fever virus lighting RNA probe detection kit in African swine fever virus detection. The lighted RNA probe has strong affinity, and is specifically combined with p54 RNA in a sample to be detected to generate fluorescence. The whole detection process is short in time consumption and low in cost, and can realize rapid and safe detection on the African swine fever in an in-vitro system without living cells and living viruses.
Description
Technical Field
The invention belongs to the technical field of food biology, and particularly relates to an African swine fever virus (African swine fever) lighting RNA probe detection kit, and a preparation method and application thereof.
Background
African Swine Fever (ASF) is a swine virulent infectious disease caused by acute and high-contagious filterable viruses. The ASF has a short course of onset with acute infection mortality rates of up to 100%. The clinical manifestations are fever, fast heartbeat, dyspnea and other symptoms. ASF occurred, spread and epidemic in several countries around the world in succession since 1909 after the first report in kenya. Until now, there has been no specific method for treating ASF. The measures taken by our country to the epidemic situation of African swine fever are mainly prevention and control. Therefore, detection and identification of meat food and clinical suspected virus can provide etiology basis for early discovery and control of African swine fever. With the development of molecular biology techniques, different technical approaches have been developed for the detection of ASF viruses. Common molecular biology techniques such as polymerase chain reaction (PCR, multiplex PCR, real-time fluorescence quantitative PCR), isothermal gene amplification (loop-mediated isothermal amplification (LAMP), nucleic acid sequence dependent amplification (NASBA)), etc. have the advantages of strong specificity, simple operation, large detection amount, intuitive judgment, etc. But also has obvious defects, such as higher cost, limited primer design, insufficient sensitivity and the like. Therefore, a rapid, cheap and high-precision detection method is urgently needed to be developed.
Aptamer technology was primarily used initially for live cell imaging and localization. Aptamers have a stable secondary structure and are synthetic nucleic acid fragments, either DNA or RNA, that bind specifically to target molecules, typically 25 to 60 nucleotides in length. The RNA aptamer probe detection technology based on the luminous effect has the advantages of good specificity, high sensitivity, low cost, simple and convenient detection method operation and the like. However, at present, no RNA probe kit for detecting African swine fever virus exists in China.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provide an African swine fever virus (African swine fever) lighting RNA probe detection kit, and a preparation method and application thereof. The invention designs a lighting RNA probe for detecting African swine fever virus according to the nucleic acid sequence of the p54 protein coded by the African swine fever virus, the RNA probe can be used for quickly, conveniently and efficiently identifying the African swine fever virus, the specificity is good, the sensitivity is high, and the detection method is simple and efficient to operate.
In order to achieve the purpose, the invention adopts the following technical scheme:
an African swine fever virus (African swine fever) lighting RNA probe SEQ-lighting is characterized in that the nucleotide sequence of the probe is shown as SEQ ID NO. 1.
The African swine fever virus lighting RNA probe SEQ-lighting is characterized in that the probe is specifically combined with a partial specific sequence SEQ-p54 of an African swine fever virus nucleic acid sequence p54, and the nucleotide sequence of SEQ-p54 is shown as SEQ ID NO. 2.
The probe SEQ-lighting is applied to preparation of a kit for detecting African swine fever viruses.
An African swine fever virus lighting RNA probe detection kit, which is characterized in thatThe detection filter paper comprises 9 mu L of illuminating RNA probe SEQ-lighting, 64 mu L of cell-free in-vitro transcription system, 1 mu L of 20 mu M DFHBI, 10 mu L of buffer solution 140mM KCl, 10 mu L of 1mM MgCl and a detection filter paper piece, p54 partial specific sequence SEQ-p54 and DEPC-aqueous solution2、10μL 10mM NaH2PO4And then the mixture is dripped and adsorbed on a sterile filter paper sheet with the diameter of 2mm, and the mixture is frozen and dried to obtain the product.
A preparation method of an African swine fever virus (African swine fever) lighting RNA probe detection kit is characterized by comprising the following steps:
(1) preparation of the light RNA probe SEQ-lighting: selecting 22bp base from p54 gene sequence, combining with 54bp base forming stem-loop structure in Spinach to construct RNA probe, totally synthesizing RNA probe and constructing RNA probe plasmid pUC-RNA probe, transforming Escherichia coli DH5a to obtain engineering bacteria, culturing, extracting pUC-RNA probe plasmid, and performing restriction endonuclease treatmentNdeI, performing linearization treatment to obtain cDNA of the RNA probe, performing in-vitro transcription, purifying and concentrating to obtain a lighted RNA probe SEQ-lighting, and storing in a refrigerator at-80 ℃ for later use;
(2) carrying out cell-free in vitro transcription system on the lighted RNA probe SEQ-lighting 9 mu L, 64 mu L, 1 mu L20 mu M MDFHBI, 10 mu L140mM KCl buffer solution and 10 mu L1mM MgCl obtained in the step (1)2、10μL10mM NaH2PO4Dripping the mixture with the pH value of 7.0 to be adsorbed on a sterile filter paper sheet with the diameter of 2mM, freezing and drying the mixture to prepare a detection filter paper sheet, and sealing and storing the detection filter paper sheet, wherein the cell-free in-vitro transcription system comprises a transcriptase system, an NTP buffer Mix and 100mM sucrose;
(3) p54 partial specific sequence SEQ-p 54;
(4) and (3) forming a detection kit by the detection filter paper sheet obtained in the step (2), the p54 partial specific sequence SEQ-p54 and DEPC-aqueous solution.
The preparation method of the African swine fever virus (AFV) lighting RNA probe detection kit is characterized in that the culture of the Escherichia coli engineering bacteria in the step (1) is specifically as follows: (a) preparing an LB liquid culture medium which comprises 10 g/L of NaCl, 10 g/L of peptone, 5 g/L of yeast extract powder and pH 6.8; (b) adding kanamycin antibiotic with the final concentration of 40 mug/mL into a culture medium, and then inoculating escherichia coli engineering bacteria containing pUC-RNA probe plasmid according to the inoculation proportion of 1%; (c) the culture was carried out at 37 ℃ and 200 rpm for 14 h.
The preparation method of the African swine fever virus (AFV) lighting RNA probe detection kit is characterized in that the linearization treatment of the pUC-RNA probe plasmid in the step (1) is specifically as follows: the treatment system consisted of 15. mu.L of the plasmid pUC-RNA probe, 2.5. mu.L of 10 XBuffer, 1. mu.L of the enzyme, 6.5. mu.L of ddH2O, the treatment condition is 37 ℃ for 4 h, and the product after enzyme digestion is purified and recovered by using a San Prep column type PCR product purification kit.
The preparation method of the African swine fever virus lighting RNA probe detection kit is characterized in that the RNA probe in-vitro transcription in the step (1) is specifically as follows: the system is 30 muL and comprises NTP Buffer Mix 10 muL, T7 RNA Polymerase Mix 2 muL, cDNA11 muL or 1 mug of an RNA probe, DEPC-water 7 muL and reaction conditions of 37 ℃ and 16 h.
The use method of the African swine fever virus lighting RNA probe detection kit is characterized by comprising the following steps:
(1) extracting DNA in the sample by using the sample processing agent according to the landmark DB 21/T3256-;
(2) respectively dripping the DNA extracted in the step (1) and part of the specific sequence SEQ-p54 of p54 on a detection filter paper sheet, simultaneously supplementing to 100 mu L by DEPC-water, keeping out of the sun, and preserving the heat for 150min at 37 ℃;
(3) the fluorescence was measured at an excitation wavelength of 460 nm and an emission wavelength of 502 nm.
The African swine fever virus lighting RNA probe detection kit is applied to the detection of the African swine fever virus.
The application of the detection method in detecting and identifying whether the swine fever virus exists in the swine disease-like tissue or not, or the application in detecting and identifying the African swine fever virus is within the protection scope of the invention.
The lighted RNA probe has strong affinity, can be specifically combined with p54 RNA in a sample to be detected to form a stable secondary structure, and the secondary structure can be specifically combined with fluorescent dye to generate fluorescence. Compared with other classical swine fever virus detection methods, the detection kit disclosed by the invention is short in time consumption in the whole detection process, low in cost, convenient for food detection and clinical detection, and has a wide application prospect. And can realize rapid and safe detection of African swine fever in an isolated system without living cells and viruses. The detection method disclosed by the invention has the advantages of good specificity, high sensitivity, convenience, rapidness and high efficiency in identifying the African swine fever virus, simplicity and convenience in operation, high efficiency, convenience in clinical detection and food detection and great application prospect.
Drawings
FIG. 1 shows the secondary structure of the specific sequence of p54 gene after binding with the RNA probe;
FIG. 2 is a graph showing the results of fluorescence detection of specific binding of an RNA probe to a p54 fragment;
FIG. 3 is a diagram showing the result of fluorescence detection in an anti-interference experiment with an RNA probe.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified; all reagents used were commercially available unless otherwise specified. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
Example 1: preparation of a light-Up RNA Probe
Selecting 22bp base from the p54 gene sequence, combining the base with 54bp base which can form a stem-loop structure in Spinach to construct an RNA probe, simulating a secondary structure after the RNA probe is combined with the p54 specific sequence by utilizing NUPACK software, and screening out the RNA probe which can form the stem-loop structure with the p54 specific sequence, wherein the final result is shown in figure 1.
The determined RNA probes were synthesized in their entirety and an RNA probe plasmid pUC-RNA probe was constructed. Transforming Escherichia coli DH5a to obtain engineering bacteria. The culture steps of the escherichia coli engineering bacteria are as follows: (1) preparing LB liquid culture medium (NaCl 10 g/L, peptone 10 g/L, yeast extract powder 5 g/L, pH 6.8); (2) adding kanamycin antibiotic with the final concentration of 40 mug/mL into a culture medium, and then inoculating escherichia coli engineering bacteria containing pUC-RNA probe plasmid according to the inoculation proportion of 1%; (3) the culture was carried out at 37 ℃ and 200 rpm for 14 h.
The pUC-RNA probe plasmid extraction was carried out according to the method provided by the SanPrep column type plasmid DNA miniprep kit (Shanghai Biotech Co., Ltd.).
The extracted pUC-RNA probe plasmid was digested with restriction endonucleaseNdeI is subjected to linearization treatment, the treatment system comprises 15 mu L of plasmid, 2.5 mu L of 10 Xbuffer, 1 mu L of enzyme and 6.5 mu L of ddH2And (C) O. The treatment conditions were 37 ℃ for 4 h. And (3) purifying and recovering the product after enzyme digestion by using a San Prep column type PCR product purification kit to obtain cDNA of the RNA probe.
RNA probe in vitro transcription. The in vitro transcription system of the RNA probe is 30 muL and comprises NTP Buffer Mix 10 muL, T7 RNA Polymerase Mix 2 muL, cDNA11 muL (1 mug) of the RNA probe and DEPC-water 7 muL. The reaction conditions were 37 ℃ 16 h. The required in vitro transcription kit was purchased from NEB. The obtained RNA was purified and concentrated using a column type RNA rapid concentration and purification kit (Shanghai Biotech Co., Ltd.), and stored in a refrigerator at-80 ℃.
Example 2: preparation of rapid detection kit
The reagents used in the preparation process of the rapid detection kit are prepared as follows: (1) reaction buffer was prepared with DEPC-water: 140mM KCl, 1mM MgCl2And 10 mM NaH2PO4. (2) Preparing 20 mu M DFHBI by using DMSO, and storing in a dark place after preparation; (3) cell-free in vitro transcription systems (including but not limited to the transcriptase system, NTP buffer Mix); (4) 100mM sucrose was formulated using DEPC-water.
9 μ L of the RNA probe prepared in example 1 was dropped onto a 2mm sterile filter paper sheet; mu.L of 140mM KCl and 10. mu.L of 1mM MgCl were added dropwise2And 10. mu.L of 10 mM NaH2PO4(ii) a 4 mu L of transcriptase system and 50 mu L of LNTP buffer Mix are dripped; 10 mu L of 100mM sucrose is added dropwise; 1 μ L of 20 μ M DFHBI was added dropwise. And (4) freeze-drying the sterile filter paper sheet to obtain a detection filter paper sheet, and sealing and storing in a dark place.
The detection filter paper obtained in example 1, p54 partial specific sequence SEQ-p54 and DEPC-water solution constitute a detection kit.
Example 3: detection of specific sequence of African swine fever virus p54 gene
Coli containing plasmid pUC-p54 was obtained by total synthesis of the p 54-specific sequence identified in example 1. Coli culture and plasmid extraction procedures reference example 1. The extracted pUC-p54 plasmid was subjected to restriction endonucleasePstI is subjected to linearization treatment, and the treatment system, the treatment conditions and the purification process refer to example 1. The resulting linear pUC-p54 plasmid was used for concentration determination. 7 μ LpUC-p54 (100 nM) was added to the test filter paper slide, and 93 μ LDEPC-water was added. Protected from light and incubated at 37 ℃ for 150 min. Exciting at 37 deg.C with a microplate reader to reach excitation wavelength of 460 nm; the emission wavelength was 502 nm, the measurement was carried out for 45min, and the fluorescence value was measured every minute. The results show that the addition of p54 significantly improved the fluorescence of the solution compared to the sample containing only the test filter paper sheet, as shown in FIG. 2.
Example 4: anti-interference capability of lighting RNA probe
Coli and listeria monocytogenes DNA were used as negative controls. The cultivation of Escherichia coli and Listeria monocytogenes adopts LB culture medium, and the cells in logarithmic phase are collected for DNA extraction. The DNA extraction process was carried out by referring to the method provided by the bacterial genome rapid extraction kit (Baozi physician Biotech Co., Ltd.). The concentration was determined after DNA extraction and stored at-20 ℃ until use. The detection procedure was performed with reference to example 3 above, wherein 8. mu.L of E.coli DNA and 6.5. mu.L of Listeria monocytogenes DNA were added, and 85. mu.L and 86.5. mu.L of DEPC-water were added, respectively. Protected from light and incubated at 37 ℃ for 150 min.
The assay procedure was as in example 3. The assay results show that the RNA probes are less likely to bind to E.coli RNA or Listeria monocytogenes RNA, as shown in FIG. 3. The detection of the p54 gene cannot be obviously interfered by the independent existence or simultaneous existence of the Escherichia coli RNA and the Listeria monocytogenes RNA in the reaction system. Therefore, the RNA probe structure designed by the invention aiming at the African swine fever virus p54 gene has good anti-interference performance.
Sequence listing
<110> Zhejiang university of industry and commerce
ZHEJIANG WUWEIHE FOOD Co.,Ltd.
<120> African swine fever virus lighting RNA probe detection kit, and preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 76
<212> DNA
<213> Lighting RNA Probe (SEQ-lighting)
<400> 1
gatgccgcgg atggcgaagg acgggtccag tgcgttcgcg cactgttgag tagagtgtga 60
gcgccaaacc ggttga 76
<210> 2
<211> 22
<212> DNA
<213> partial specific sequence of p54 (SEQ-p54)
<400> 2
tctgaatttt ttcaaccggt tt 22
Claims (10)
1. An African swine fever virus (African swine fever) lighting RNA probe SEQ-lighting is characterized in that the nucleotide sequence of the probe is shown as SEQ ID NO. 1.
2. The African swine fever virus (African swine fever) lighting RNA probe SEQ-lighting according to claim 1, wherein the probe specifically binds to a part of specific sequence SEQ-p54 of African swine fever virus nucleic acid sequence p54, and the nucleotide sequence of SEQ-p54 is shown as SEQ ID NO. 2.
3. Use of the probe SEQ-lighting according to claim 1 for the preparation of a kit for detecting african swine fever virus.
4. An African swine fever virus lighting RNA probe detection kit is characterized by comprising a detection filterA paper sheet, p54 partial specific sequences SEQ-p54 and DEPC-water solution, wherein the detection filter paper sheet consists of 9 mu L of lighting RNA probe SEQ-lighting, 64 mu L of cell-free in vitro transcription system, 1 mu L of 20 mu M DFHBI, 10 mu L of buffer solution 140mM KCl, and 10 mu L of 1mM MgCl2、10μL 10mM NaH2PO4And then the mixture is dripped and adsorbed on a sterile filter paper sheet with the diameter of 2mm, and the mixture is frozen and dried to obtain the product.
5. A preparation method of an African swine fever virus (African swine fever) lighting RNA probe detection kit is characterized by comprising the following steps:
(1) preparation of the light RNA probe SEQ-lighting: selecting 22bp base from p54 gene sequence, combining with 54bp base forming stem-loop structure in Spinach to construct RNA probe, totally synthesizing RNA probe and constructing RNA probe plasmid pUC-RNA probe, transforming Escherichia coli DH5a to obtain engineering bacteria, culturing, extracting pUC-RNA probe plasmid, and performing restriction endonuclease treatmentNdeI, performing linearization treatment to obtain cDNA of the RNA probe, performing in-vitro transcription, purifying and concentrating to obtain a lighted RNA probe SEQ-lighting, and storing in a refrigerator at-80 ℃ for later use;
(2) carrying out cell-free in vitro transcription system on the lighted RNA probe SEQ-lighting 9 mu L, 64 mu L, 1 mu L20 mu M MDFHBI, 10 mu L140mM KCl buffer solution and 10 mu L1mM MgCl obtained in the step (1)2、10μL10mM NaH2PO4Dripping the mixture with the pH value of 7.0 to be adsorbed on a sterile filter paper sheet with the diameter of 2mM, freezing and drying the mixture to prepare a detection filter paper sheet, and sealing and storing the detection filter paper sheet, wherein the cell-free in-vitro transcription system comprises a transcriptase system, an NTP buffer Mix and 100mM sucrose;
(3) p54 partial specific sequence SEQ-p 54;
(4) and (3) forming a detection kit by the detection filter paper sheet obtained in the step (2), the p54 partial specific sequence SEQ-p54 and DEPC-aqueous solution.
6. The method for preparing the African swine fever virus (African swine fever) lighted RNA probe detection kit according to claim 5, wherein the cultivation of the Escherichia coli engineering bacteria in the step (1) is specifically as follows: (a) preparing an LB liquid culture medium which comprises 10 g/L of NaCl, 10 g/L of peptone, 5 g/L of yeast extract powder and pH 6.8; (b) adding kanamycin antibiotic with the final concentration of 40 mug/mL into a culture medium, and then inoculating escherichia coli engineering bacteria containing pUC-RNA probe plasmid according to the inoculation proportion of 1%; (c) the culture was carried out at 37 ℃ and 200 rpm for 14 h.
7. The method for preparing the African swine fever virus (AFV) lighting RNA probe detection kit according to claim 5, wherein the linearization treatment of the pUC-RNA probe plasmid in the step (1) is specifically as follows: the treatment system consisted of 15. mu.L of the plasmid pUC-RNA probe, 2.5. mu.L of 10 XBuffer, 1. mu.L of the enzyme, 6.5. mu.L of ddH2O, the treatment condition is 37 ℃ for 4 h, and the product after enzyme digestion is purified and recovered by using a San Prep column type PCR product purification kit.
8. The method for preparing the African swine fever virus (African swine fever) lighting RNA probe detection kit according to claim 5, wherein the RNA probe in-vitro transcription in the step (1) is specifically as follows: the system is 30 muL and comprises NTP Buffer Mix 10 muL, T7 RNA Polymerase Mix 2 muL, cDNA11 muL or 1 mug of an RNA probe, DEPC-water 7 muL and reaction conditions of 37 ℃ and 16 h.
9. The use method of the African swine fever virus lighting RNA probe detection kit is characterized by comprising the following steps:
(1) extracting DNA in the sample by using the sample processing agent according to the landmark DB 21/T3256-;
(2) respectively dripping the DNA extracted in the step (1) and part of the specific sequence SEQ-p54 of p54 on a detection filter paper sheet, simultaneously supplementing to 100 mu L by DEPC-water, keeping out of the sun, and preserving the heat for 150min at 37 ℃;
(3) the fluorescence was measured at an excitation wavelength of 460 nm and an emission wavelength of 502 nm.
10. The African swine fever virus (African swine fever virus) lighted RNA probe detection kit according to claim 4, for use in African swine fever virus detection.
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| CN110760620A (en) * | 2019-12-12 | 2020-02-07 | 黑龙江八一农垦大学 | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method |
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| CN110791590A (en) * | 2019-11-12 | 2020-02-14 | 南宁众册生物科技有限公司 | Dual real-time fluorescence detection primer probe set, kit and method for genes VP72 and CD2V of African swine fever virus |
| CN110760620A (en) * | 2019-12-12 | 2020-02-07 | 黑龙江八一农垦大学 | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method |
| CN111218528A (en) * | 2020-03-11 | 2020-06-02 | 山东省滨州畜牧兽医研究院 | PCR primer group and kit for detecting African swine fever virus based on double genes and application |
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| Title |
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| 宋红芹;姜翠翠;张鑫宇;夏晓莉;孙怀昌;: "非洲猪瘟病毒NP419L基因shRNA表达质粒的构建与筛选", 中国兽医科学 * |
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