Nucleic acid probe and kit for simultaneously detecting multiple tomato viruses and application of nucleic acid probe and kit
阅读说明：本技术 一种同时检测多种番茄类病毒的核酸探针、试剂盒及其应用 (Nucleic acid probe and kit for simultaneously detecting multiple tomato viruses and application of nucleic acid probe and kit ) 是由 张志想 张煜泓 李世访 于 2021-08-19 设计创作，主要内容包括：本发明提供一种同时检测多种番茄类病毒的核酸探针、试剂盒及其应用,属于生物工程和分子检测技术领域。本发明通过深入分析马铃薯纺锤块茎形类病毒科中的各个类病毒的序列,以发现的一段约为60bp的、在6种类病毒基因组中均存在的共有序列,以PSTVd质粒为模板,设计特异性引物,通过PCR扩增获得该序列,插入pGEM-T载体,用限制性酶消化重组质粒,再以体外转录的方法获得一种通用核酸探针。使用上述核酸探针,一次可同时检测6种不同的番茄类病毒。这不仅降低了制备核酸探针的成本,而且大大提高了检测效率。从而可用于田间番茄样品的大规模检测,能快速、准确地进行病害诊断,因此具有良好的实际应用之价值。(The invention provides a nucleic acid probe for simultaneously detecting various tomato viruses, a kit and application thereof, belonging to the technical field of biological engineering and molecular detection. The invention discloses a universal nucleic acid probe which is obtained by deeply analyzing the sequences of various viroids in potato spindle tuber viroid, using a segment of a found 60bp consensus sequence existing in 6 viroid genomes, using a PSTVd plasmid as a template, designing a specific primer, obtaining the sequence through PCR amplification, inserting a pGEM-T vector, digesting a recombinant plasmid by using a restriction enzyme, and then carrying out in vitro transcription. The nucleic acid probe can be used for simultaneously detecting 6 different tomato viruses at one time. This not only reduces the cost of preparing nucleic acid probes, but also greatly improves the detection efficiency. Therefore, the method can be used for large-scale detection of field tomato samples, and can quickly and accurately diagnose diseases, thereby having good practical application value.)
1. A primer pair, which is characterized by consisting of a primer F1 and a primer R1;
the primer F1 is a1) or a2) as follows:
a1) a nucleotide sequence shown as a sequence 1 in a sequence table;
a2) a nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1;
the primer R1 is a3) or a4) as follows:
a3) a nucleotide sequence shown in a sequence 2 of a sequence table;
a4) and (b) the nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2.
2. Use of the primer pair of claim 1 for preparing a nucleic acid probe; the application of the nucleic acid probe is b1) or b 2):
b1) identifying or assisting in identifying the tomato viruses;
b2) identifying or assisting in identifying whether the sample to be detected contains the tomato viruses;
preferably, the tomato viroid includes, but is not limited to, any one or more of PSTVd, PCFVd, TASVd, TCDVd, CLVd and TPMVd.
3. A nucleic acid probe, wherein the nucleic acid probe is c1) or c 2):
c1) a nucleotide sequence shown as a sequence 3 in a sequence table;
c2) a nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3;
preferably, the nucleic acid probe is a cRNA probe, and the use thereof is b1) or b 2):
b1) identifying or assisting in identifying the tomato viruses;
b2) and identifying or assisting in identifying whether the sample to be detected contains the tomato viruses.
4. The method for preparing a nucleic acid probe according to claim 3, which comprises: the viroid genome sequence is amplified by the primers of claim 1, cloned into an expression vector, digested with restriction enzymes and purified, and synthesized by in vitro transcription.
5. Use of a primer pair according to claim 1 and/or a nucleic acid probe according to claim 3 for the preparation of a kit; the use of the kit is b1) or b 2):
b1) identifying or assisting in identifying the tomato viruses;
b2) and identifying or assisting in identifying whether the sample to be detected contains the tomato viruses.
6. A kit comprising the nucleic acid probe of claim 3.
7. The nucleic acid probe of claim 6, wherein the kit further comprises other nucleic acid probes for detecting tomato-like viruses, the nucleic acid probes comprising d1) or d2) as follows:
d1) a nucleotide sequence shown in any one of sequences 4 to 9 of a sequence table;
d2) and (b) a nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more nucleotides in any one of the sequences 4-9 and has the same function as the sequences 4-7.
8. Use of the nucleic acid probe of claim 3 and/or the kit of claim 6 or 7 in b1) or b2) as follows:
b1) identifying or assisting in identifying the tomato viruses;
b2) identifying or assisting in identifying whether the sample to be detected contains the tomato viruses;
preferably, the tomato viroid includes, but is not limited to, any one or more of PSTVd, PCFVd, TASVd, TCDVd, CLVd and TPMVd.
9. A method for identifying or aiding in the identification of a tomato virus, comprising: specifically hybridizing a nucleic acid of a virus-like to be tested using the nucleic acid probe of claim 3 and/or the kit of claim 6 or 7.
10. A method for identifying or assisting in identifying whether a sample to be tested contains a tomato-like virus or not, which is characterized by comprising the following steps: extracting and purifying nucleic acids in a sample, specifically hybridizing using the nucleic acid probe of claim 3 and/or the kit of claim 6 or 7;
preferably, the tomato viroid includes, but is not limited to, any one or more of PSTVd, PCFVd, TASVd, TCDVd, CLVd and TPMVd;
preferably, the sample is derived from a plant sample, including a plant sample susceptible to the aforementioned viroids, further including potato, tomato, pepper, avocado, chrysanthemum, citrus, goldfish, bloodamaranth, and solanum cordifolia;
preferably, when the nucleic acid probe shown in the sequence 3 is used, the hybridization temperature is 50-65 ℃, including 50 ℃, 55 ℃, 60 ℃ or 65 ℃, and preferably 55 ℃;
preferably, when one or more of the nucleic acid probes shown in any one of the sequences 4 to 9 are used, the hybridization temperature is 60 to 70 ℃, including 60 ℃, 65 ℃, 68 ℃ or 70 ℃, preferably 68 ℃.
Technical Field
The invention belongs to the technical field of biological engineering and molecular detection, and particularly relates to a nucleic acid probe and a kit for simultaneously detecting various tomato viruses and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Viroids (viroids) are a class of circular, non-coding, single-stranded, low-molecular-weight RNAs which are the smallest plant pathogens and which cause serious economic losses after infestation of plants (Flores R, Hern a dez C, Alba AEMD, Dar oa JA, Di series f. Various potatosubulaviridae viroids, such as: potato spindle tuber viroid (PSTVd), tomato chlorotic dwarf viroid (TCDVd), tomato topping viroid (TASVd), Capsicum fructide (PCFVd), tomato male Plant viroid (TPMVd) and Kinsellosa latent viroid (CLVd) can infect tomato (Lycopersicon esculentum) (Matsushita Y, Tsuda S.Seed Transmission of liquid to spindle viroid, tomato chloroplastic to vision, and Columbia latifolia later in biological plants [ J ]. European Journal of Plant Pathology,2016,145: 1007. 1011). And some viroids spread with the tomato seed (Fiona C, Grant C, Lindsay P, Andrew D, Joanne M, Kevin D, Bredan R, Mark G. Viroid-induced tomato and capsule seed shifts to Autoclavia [ J ]. Virus, 2019,11(2): 98). Therefore, early detection and diagnosis of tomato-like viruses are important measures for disease control.
The nucleic acid molecule hybridization has high reliability, is suitable for detecting a large number of samples, and is an indispensable or deficient technology for the conventional detection of viroid. Typically, detection is performed using nucleic acid molecule hybridization, which can only detect one type of virus at a time. However, in actual production, plants are usually infected with multiple viroids simultaneously, which requires that multiple different viroids can be detected at one time. Improved, nucleic acid molecules are currently usedHybridization allows simultaneous detection of a number of different viroids (James, D, Varga, A, P a llas, V, and Candrese, T.Strategies for multiple plant detection [ J. ]]Canadian Journal of Plant Pathology.2006,28: 16-29). Initially, different probes were mixed to simultaneously detect multiple viroids (Saldarelli, P, Barbarossa, L, Grieco, F, and Gallitelli, D.Digoxigenin-labelled riboprobes applied to virology center of tomato in Italy [ J].Plant Disease.1996,80:1343–1346. Sánchez-Navarro,J.A,M.C,Cano,E.A,and Pallás,V.Simultaneous detection of five carnation viruses by non-isotopic molecular hybridization[J]Journal of viral methods 1999,82: 167-175.), but the hybridization background tends to be too deep due to the mixing of multiple probes, affecting the outcome determination (Pall S V, S-nchez N, Jesus A, James D.Recent advances on the multiplex molecular detection of plants and viruses [ J.]Frontiers in Microbiology 2018,9: 2087-. The above disadvantages can be overcome by connecting probes of different virus types in series to form a multimeric probe. The use of multimeric probes to detect a variety of different viroids allows for multiple, rapid, time-saving objectives (Zhang, Z X. Peng, S. Jiang, D M.Pan, S.Wang, H Q.Li, S F. Development of a multiprobe for the detection of four viral genes in viral plants [ J.]European Journal of Plant Pathology 2012,132: 9-16). The inventors have found that the preparation of multimeric probes requires successive ligation of sequences of different virus types by multiple subcloning. This not only requires careful design and selection of appropriate cleavage sites, but also requires a cumbersome and time-consuming preparation process. Taking the detection of 6 types of viruses (PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd) infecting tomato as an example, 5 subclones are times are required for preparing a multimeric probe capable of simultaneously detecting the 6 types of viruses, which is very complicated.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a nucleic acid probe for simultaneously detecting a plurality of tomato viruses, a kit and application thereof. According to the invention, through research, a common sequence existing among 6 tomato viruses (PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd) is discovered unexpectedly, a nucleic acid probe capable of detecting 6 different viruses infecting tomatoes at the same time with high sensitivity is prepared, the preparation process is simpler and more convenient, time-saving and low in cost, and the invention is completed based on the research results.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided a primer pair, which consists of a primer F1 and a primer R1;
the primer F1 is a1) or a2) as follows:
a1) a nucleotide sequence shown as a sequence 1 in a sequence table;
a2) a nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1;
the primer R1 is a3) or a4) as follows:
a3) a nucleotide sequence shown in a sequence 2 of a sequence table;
a4) and (b) the nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2.
In a second aspect of the invention, there is provided the use of said primer pair in the preparation of a nucleic acid probe; the application of the nucleic acid probe is b1) or b 2):
b1) identifying or assisting in identifying the tomato viruses;
b2) and identifying or assisting in identifying whether the sample to be detected contains the tomato viruses.
Wherein, the tomato viruses include any one or more of PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd.
In a third aspect of the present invention, there is provided a nucleic acid probe, wherein the nucleic acid probe is c1) or c 2):
c1) a nucleotide sequence shown as a sequence 3 in a sequence table;
c2) and (b) the nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3.
The nucleic acid probe is a cRNA probe, and the application of the nucleic acid probe is b1) or b 2):
b1) identifying or assisting in identifying the tomato viruses;
b2) and identifying or assisting in identifying whether the sample to be detected contains the tomato viruses.
Wherein, the tomato viruses include any one or more of PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd.
In yet another embodiment of the present invention, the nucleic acid probe may be labeled to facilitate subsequent in situ hybridization detection. Currently, commonly used labeling methods include radioisotope labeling and non-radioisotope labeling; wherein the non-radioactive isotope labeling comprises using biotin, digoxigenin or fluorescent labels. In one embodiment of the present invention, digoxin is selected for labeling the nucleic acid probe based on a combination of cost and safety considerations.
In a fourth aspect of the present invention, there is provided a method for producing the nucleic acid probe of the third aspect, the method comprising: the viroid genome sequence is amplified by the primer of the first aspect, cloned into an expression vector, digested with restriction enzymes and purified, and synthesized by an in vitro transcription method.
The virus used in the viral genome is a tomato virus to be detected, and includes but is not limited to any one or more of PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd; PSTVd (GenBank: U23058.1) is preferred.
The expression vector can be any one or more of a viral vector, a plasmid, a phage, a phagemid, a cosmid, an F cosmid, a phage or an artificial chromosome; the viral vector may comprise an adenoviral vector, a retroviral vector, or an adeno-associated viral vector, the artificial chromosomes comprising a Bacterial Artificial Chromosome (BAC), a bacteriophage P1 derived vector (PAC), a Yeast Artificial Chromosome (YAC), or a Mammalian Artificial Chromosome (MAC); further preferably a plasmid; in one embodiment of the invention, the expression vector selected is a pGEM-T plasmid.
The restriction enzyme is not particularly limited, and in one embodiment of the present invention, the restriction enzyme Spe I is selected.
In a fifth aspect of the present invention, there is provided a use of the primer set of the first aspect and/or the nucleic acid probe of the third aspect for preparing a kit. The use of the kit is b1) or b 2):
b1) identifying or assisting in identifying the tomato viruses;
b2) and identifying or assisting in identifying whether the sample to be detected contains the tomato viruses.
In a sixth aspect of the present invention, there is provided a kit comprising the nucleic acid probe of the third aspect.
In another embodiment of the present invention, the kit may further comprise other nucleic acid probes for detecting the above-mentioned tomato viruses, wherein the nucleic acid probes comprise the following d1) or d 2):
d1) a nucleotide sequence shown in any one of sequences 4 to 9 of a sequence table;
d2) and (b) a nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more nucleotides in any one of the sequences 4-9 and has the same function as the sequences 4-9.
In a seventh aspect of the present invention, there is provided a use of the nucleic acid probe and/or the kit of the sixth aspect as described in the following b1) or b 2):
b1) identifying or assisting in identifying the tomato viruses;
b2) and identifying or assisting in identifying whether the sample to be detected contains the tomato viruses.
Wherein, the tomato viruses include any one or more of PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd.
In an eighth aspect of the present invention, there is provided a method for identifying or aiding in identifying a tomato virus, comprising: specifically hybridizing a nucleic acid of a test viroid using the nucleic acid probe of the third aspect and/or the kit of the sixth aspect.
In a ninth aspect of the present invention, there is provided a method for identifying or assisting in identifying whether a sample to be tested contains a tomato virus, comprising: nucleic acids in the sample are extracted and purified, and specific hybridization is performed using a probe.
Wherein, the tomato viruses include any one or more of PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd.
The sample may be derived from a plant sample, particularly a plant sample susceptible to the aforementioned viroids, such as potato, tomato, pepper, avocado, chrysanthemum, citrus, goldfish, bloodamaranth, and solanum cordifolia, among others.
In any of the above methods, in order to further improve the detection sensitivity, the hybridization temperature is optimized, in one embodiment of the present invention, when the universal nucleic acid probe (sequence 3) is used, the hybridization temperature is 50 to 65 ℃, such as 50 ℃, 55 ℃, 60 ℃ or 65 ℃, and the hybridization result indicates that the hybridization signal for detecting the above six types of viruses is stronger than the rest of the hybridization temperatures at 55 ℃, so that the optimal hybridization temperature for the detection sensitivity of the universal probe is 55 ℃, and the detection sensitivity of the universal nucleic acid probe is the highest. The universal nucleic acid probe can detect 6 kinds of viruses simultaneously and has strong hybridization signal.
In one embodiment of the present invention, when specific nucleic acid probes (any one or more of sequences 4 to 9) are used, the hybridization temperature is 60 to 70 ℃, such as 60 ℃, 65 ℃, 68 ℃ or 70 ℃, preferably 68 ℃, and each specific probe can detect one or more other viroids except itself, but the hybridization signal for detecting other viroids is weak.
The beneficial technical effects of one or more technical schemes are as follows:
according to the technical scheme, through deep analysis of sequences of various viroids in the potato spindle tuber viroid family, a discovered consensus sequence of about 60bp and existing in 6 viroid genomes is used, a PSTVd plasmid is used as a template, a specific primer is designed, the sequence is obtained through PCR amplification, a pGEM-T vector is inserted, a recombinant plasmid is digested by restriction enzyme, and then a universal nucleic acid probe is obtained through an in vitro transcription method.
As the exchange of tomato propagation materials or germplasm resources is more frequent in the world and the region, the quarantine of tomato viruses puts higher requirements. This requires a simpler, faster detection technique as a support. The technical scheme meets the requirement to a certain extent. The nucleic acid probe provided by the technical scheme can be used for simultaneously detecting 6 different tomato viruses at one time. This not only reduces the cost of preparing nucleic acid probes, but also greatly improves the detection efficiency.
In conclusion, the nucleic acid probe and the corresponding detection technology provided by the technical scheme can be used for large-scale detection of field tomato samples, and can be used for quickly and accurately diagnosing diseases, so that the nucleic acid probe and the corresponding detection technology have good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a flow chart showing the procedures for cloning cDNA and preparing probes in example 1 of the present invention.
FIG. 2 shows the alignment results of PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd sequences and the positions of the universal probe sequences in the PSTVd sequence in example 1 of the present invention.
FIG. 3 shows the result of the electrophoresis detection of the PCR product in example 1 of the present invention.
FIG. 4 shows the detection results of the restriction enzyme products of the plasmid vector with the universal nucleic acid probe in example 1. Wherein, 1: a non-linearized plasmid; 2: the plasmid was linearized.
FIG. 5 shows the results of the enzyme digestion products PSTVd, PCFVd, TASVd, TCDVd, TPMVd and CLVd in example 1 of the present invention. Wherein, in diagram A, 1: PSTVd non-linearized plasmid; 2: PSTVd linearized plasmid for in vitro transcription to prepare specific probe; 3: a TASVd non-linearized plasmid; 4: TASVd linearized plasmid for in vitro transcription to prepare its positive strand RNA; 5: TPMVd non-linearized plasmid; 6: TPMVd linearized plasmid is used for preparing the positive strand RNA thereof by in vitro transcription; 7: PCFVd non-linearized plasmid; 8: the PCFVd linearized plasmid is used for preparing the positive strand RNA thereof by in vitro transcription; 9: CLVd non-linearized plasmid; 10: CLVd linearized plasmid for in vitro transcription preparation of its positive strand RNA; in panel B, 1: TCDVd non-linearized plasmid; 2: TCDVd linearized plasmid for in vitro transcription to prepare its positive strand RNA; 3: TCDVd linearized plasmid for in vitro transcription to prepare specific probe; 4: a TASVd non-linearized plasmid; 5: TASVd linearized plasmid used for extracorporeally transcribing and preparing a specific probe; 6: PCFVd non-linearized plasmid; 7: PCFVd linearized plasmid for use in the preparation of specific probes for in vitro transcription. In panel C, 1: TPMVd non-linearized plasmid; 2: TPMVd linearized plasmid is used for preparing a specific probe through in vitro transcription; 3: CLVd non-linearized plasmid; 4: CLVd linearized plasmid for in vitro transcription preparation of specific probe; 5: PSTVd non-linearized plasmid; 6: the PSTVd linearized plasmid is used for preparing the positive strand RNA thereof by in vitro transcription.
FIG. 6 is a result of comparison of hybridization sensitivity and specificity of universal nucleic acid probes at different hybridization temperatures in example 1 of the present invention.
FIG. 7 shows the results of example 1 of the present invention under certain concentration conditions (RNA dilution gradient of 5)-3) And comparing the detection sensitivity and specificity of the different virus-like specific probes with that of the universal nucleic acid probe. The hybridization temperature of the universal nucleic acid probe was 55 ℃ and the hybridization temperature of the specific nucleic acid probe was 68 ℃. A: PSTVd; b: PCFVd; c: TCDVd; d: TPMVd; e: TASVd; f: CLVd. .
FIG. 8 shows the spot hybridization results of the universal nucleic acid probe and the single viroid-specific probe on tomato samples in example 1 of the present invention. The hybridization temperature of the universal nucleic acid probe was 55 ℃ and the hybridization temperature of the specific nucleic acid probe was 68 ℃. A: PSTVd + RNA of healthy tomato; b: PCFVd + RNA of healthy tomato; c: TCDVd + RNA of healthy tomatoes; d: TPMVd + RNA of healthy tomatoes; e: TASVd + RNA of healthy tomatoes; f: CLVd + RNA of healthy tomato.
FIG. 9 shows the restriction enzymes and RNA polymerases used for in vitro transcription of plus and minus-strand RNA by 6 kinds of viruses.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments of the invention. As used herein, the singular forms "a", "an", and/or "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the features, steps, operations, devices, components, and/or combinations thereof. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
As mentioned above, the preparation of the multimeric probes is complicated and time-consuming, and the experimental cost is relatively high. Taking the detection of 6 types of viruses (PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd) infecting tomato as an example, 5 times of subcloning are required for preparing a multimeric probe capable of simultaneously detecting the 6 types of viruses, and the process is very complicated.
In view of the above, the inventors have found out the common sequence among the above 6 tomato viruses by sequence comparison, and further designed a universal nucleic acid probe, the length of which is 63 nucleotides (sequence 3 in the sequence table), which is easy to prepare, and which can simultaneously detect 6 different viruses (PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd) infecting tomatoes, thereby solving the technical problem of complicated preparation of the multimeric probe.
In another embodiment of the present invention, there is provided a method for preparing the above-mentioned universal nucleic acid probe, comprising the steps of:
(1) finding out a common sequence among the 6 tomato viruses through sequence comparison, designing specific primers Pospi6-probe-F1 and Pospi6-probe-R1 as shown in sequence 1 and sequence 2 of a sequence table, taking a recombinant plasmid containing a PSTVd genome sequence (GenBank: U23058.1) as a template, carrying out PCR amplification, purifying and recovering, and cloning to a vector pGEM-T to obtain a plasmid for preparing a probe;
(2) digesting the plasmid obtained in the step (1) by using a restriction enzyme Spe I, and purifying a digestion product by using a PCR product purification kit;
(3) synthesizing a universal nucleic acid probe by an in vitro transcription method for the purified linearized plasmid obtained in the step (2).
In yet another embodiment of the present invention, a kit is provided, which comprises the above-described universal nucleic acid probe.
In another embodiment of the present invention, there is provided a spot hybridization method for simultaneously detecting the above 6 tomato viruses, comprising the steps of:
(1) transcribing and obtaining each viroid positive strand RNA; extracting total RNA of a sample to be detected to obtain an RNA extract;
(2) applying said RNA to a hybridization membrane;
(3) hybridization assays are performed using the universal nucleic acid probes and/or kits described above.
The method for obtaining each viroid positive-strand RNA through transcription in the step (1) is an in-vitro transcription method, and the method for extracting the total RNA of the sample to be detected is a Trizol method;
the hybridization temperature in the step (2) is 50 ℃ to 65 ℃ (such as 50 ℃, 55 ℃, 60 ℃ and 65 ℃, and more preferably 55 ℃), and the hybridization time is 16 h.
In another embodiment of the present invention, there is provided a cRNA viroid-specific probe of PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd, wherein the sequence of the cRNA viroid-specific probe is represented by sequences 4 to 9 in the sequence table.
In another embodiment of the present invention, there is provided a method for preparing the cRNA viroid-specific probe, comprising the steps of:
and (3) carrying out enzyme digestion by using a plasmid preserved in a laboratory, and preparing a PSTVd-cRNA, a PCFVd-cRNA, a TCDVd-cRNA, a TPMVd-cRNA, a TASVd-cRNA and a CLVd-cRNA probe by an in vitro transcription method according to the specification of a digoxin labeling kit.
In another embodiment of the present invention, there is provided a dot hybridization method for detecting the above 6 tomato viruses (PSTVd, PCFVd, TCDVd, TPMVd, TASVd and CLVd), comprising the steps of:
(1) transcribing and obtaining positive strand RNA of various viruses; extracting total RNA of a sample to be detected to obtain an RNA extract;
(2) applying said RNA to a hybridization membrane;
(3) performing hybridization detection by using the cRNA viroid-specific probe (such as any one or more of PSTVd-cRNA, PCFVd-cRNA, TCDVd-cRNA, TPMVd-cRNA, TASVd-cRNA and CLVd-cRNA probes);
the method for obtaining each viroid positive-strand RNA through transcription in the step (1) is an in-vitro transcription method, and the method for extracting the total RNA of the sample to be detected is a Trizol method;
in the step (2), the hybridization temperature is 60-70 ℃, such as 60 ℃, 65 ℃, 68 ℃ or 70 ℃, preferably 68 ℃, and the hybridization time is 16 h.
Primer and probe
The primer of the invention comprises the primer pair. The probe of the present invention includes the above-mentioned universal nucleic acid probe.
The nucleotide sequences of the primers and probes of the present invention also include modified forms thereof, as long as the amplification effect of the primers or the hybridization effect of the probes are not significantly affected. The modification may be, for example, the addition of one or more nucleotide residues in or at both ends of the nucleotide sequence, the deletion of one or more nucleotide residues in the nucleotide sequence, or the substitution of one or more nucleotide residues in the sequence with another nucleotide residue, for example, the substitution of a for T, the substitution of C for G, etc. It is clear to a person skilled in the art that primers or probes formed by such modifications are also encompassed by the present invention, especially within the scope of the claims.
One end (e.g., the 5 'end or the 3' end) of the nucleic acid probe can be labeled, e.g., with a radioisotope or non-radioisotope label; wherein the non-radioactive isotope label comprises biotin, digoxigenin or fluorescence. In one embodiment of the present invention, digoxin is selected for labeling the nucleic acid probe, based on a combination of cost and safety considerations.
Each nucleotide in the primer set and the probe of the present invention can be chemically synthesized using, for example, a general-purpose DNA synthesizer (e.g., model 394, manufactured by Applied Biosystems). Any other method well known in the art may also be used to synthesize oligonucleotides, such as primers and probes.
Preferably, the viroid gene is amplified using PCR. The PCR method per se is well known in the art. The term "PCR" includes derivative forms of the reaction, including, but not limited to, reverse transcription PCR, real-time PCR, nested PCR, multiplex PCR, and fluorescent quantitative PCR, among others.
PCR is performed by repeating a cycle of annealing, extension and denaturation steps approximately 30 to 50 times (e.g., 45 times) in the presence of a primer, a template DNA (i.e., a viroid gene) and a thermostable DNA polymerase using a primer hybridizing to a sense strand (reverse primer) and a primer hybridizing to an antisense strand (forward primer).
The nucleic acid probe of the present invention capable of detecting a viroid can be subjected to a specific hybridization reaction with a nucleic acid in a sample, for example, a viroid nucleic acid, thereby achieving quantitative or qualitative detection of an amplification product. The probe of the present invention may be labeled with a label including, for example, any one of a fluorescent substance, biotin and digoxigenin, to facilitate detection by an instrument. Among them, examples of the fluorescent label include FITC, Cy3, Cy5, Cy5.5, Cy7, TAMRA, Dabcyl, ROX, TET, rhodamine, Texas Red, HEX, Cyber Green, FAM, MGB, BHQ1 and the like. Preferred fluorescent labels may include, but are not limited to, FAM, MGB, and BHQ1, among others. By observing the fluorescence intensity derived from such a fluorescent substance by optical detection means, various fluorescent substances used for fluorescent labeling can be detected with high sensitivity. The label may also include a reporter fluorophore and a quencher fluorophore. The reporter fluorophore can be, for example, FAM, HEX, or TET; the quenching fluorescent group can be, for example, TAMRA, MGB, or BHQ 1.
Sample (I)
The sample used in the present invention may be a quantity of material of biological, environmental origin. In one aspect, it may comprise a specimen or culture. On the other hand, it may also include, but is not limited to, biological samples and non-biological samples (e.g., environmental samples, industrial samples, etc.). Biological samples may include samples taken from plant material, particularly plants susceptible to the aforementioned viroids, such as potato, tomato, pepper, avocado, chrysanthemum, citrus, goldfish, bloodamaranth, and solanum cordifolia, among others. Of course, these examples are not to be construed as limiting the type of sample that is suitable for use in the present invention.
Methods for extracting nucleic acids from a sample are well known in the art and can be performed, for example, by RNA extraction using phenol and chloroform, or by using commercially available RNA extraction reagents. For example, extraction can be performed using a column kit.
Nucleic acids can be purified by purification methods conventional in the art. Viral nucleic acid purification can be achieved, for example, by further purification using phenol chloroform extraction as described above. The purified viral RNA is free of proteins, nucleases and other impurities and has high recovery rate.
Of course, other known methods such as an automated instrument platform can be used for nucleic acid extraction and purification.
Reagent kit
The invention relates to a kit for detecting tomato-like viruses, comprising a combination of probes according to the invention. The invention also relates to the use of a primer pair or a combination of a primer pair and a probe in the preparation of a kit for detecting a viroid in a sample.
The kit may comprise materials or reagents (including primer sets and/or probes) for performing the methods of the invention. The kit may include storage reagents (e.g., primers, probes, enzymes, etc. in suitable containers) and/or support materials (e.g., buffers, instructions for performing the assay, etc.). For example, a kit may comprise one or more containers (e.g., cassettes) containing the corresponding reaction reagents and/or support materials. Such contents may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for the assay, a second container contains a primer set, and a third container contains a probe. The kit may also contain a compartment suitable for holding the reagent or container. As an example, the kit may contain probes, reaction buffers, instructions for use. The kit can also contain internal standards for quality control, positive and negative controls and the like. The kit may further comprise reagents for preparing the above-described tomato-like virus from a sample.
It is to be noted that the kit of the present invention may further comprise any other primer and/or probe other than the primer and probe of the present invention, for example, a probe capable of effectively detecting the above-mentioned viroid: PSTVd-cRNA, PCFVd-cRNA, TCDVd-cRNA, TPMVd-cRNA, TASVd-cRNA and CLVd-cRNA. The sequences of the cRNA nucleic acid probes are shown as sequences 4, 5, 6, 7, 8 and 9 in a sequence table. The above examples are not to be construed as limiting the kits and their contents suitable for use in the present invention.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Six tomato-like viruses appear in the examples: potato spindle tuber viroid (PSTVd), pepper fruit viroid (PCFVd), Tomato top dwarf viroid (TASVd), Tomato chlorotic dwarfism (TCDVd), Tomato male viroid (TPMVd) and goldfish latent viroid (Columnea latentivirid, CLVd).
Example 1
1 Material
1.1 test plasmid:
recombinant plasmids containing the genomic sequences of PSTVd, PCFVd, TASVd, TCDVd, TPMVd and CLVd.
1.2 main reagents and instruments:
the main reagents are as follows: restriction endonucleases and T7/T3/SP6 RNA polymerase were purchased from TaKaRa, reagents required for PCR amplification and PCR product purification kits were purchased from Beijing Quanjin Biotechnology Co., Ltd (TransGen Biotech), Biotechnology engineering, Inc., Shanghai, DNA gel recovery kits and plasmid DNA Mini kit (Corning Life sciences Co., Ltd.), pGEM-T vectors were purchased from Promega, Beijing Promega, and Probe preparation related Reagents (ROCHE) were purchased from Synergic Biotech, Inc., Beijing.
The main apparatus is as follows: Bio-Rad PCR instrument, KCBIO-2800 gel imaging system, DYY-6B type voltage-stabilizing electrophoresis apparatus, constant temperature water bath, constant temperature incubator, AIRTECH SW-CJ-1FD super clean bench, ultraviolet spectrophotometer, and micro ultraviolet spectrophotometerND-1000UV-Vis Spectrophotometer (NanoDrop, America), nucleic acid molecule hybridization oven, UV cross-linking instrument.
Primer synthesis and DNA sequencing were performed by Biotechnology engineering (Shanghai) Inc.
2 method
2.1 preparation of Universal nucleic acid probes
The preparation process is shown in figure 1.
2.1.1 PCR primer design for Probe Synthesis (shown in sequence 1, 2 of the sequence listing)
Pospi6-probe-F1：GACAGGAGTAATCCCMGCCG
Pospi6-probe-R1：GAGGAAGGAAACCMGAAGA
2.1.2 PCR
PCR amplification was performed using the PSTVd plasmid stored in the laboratory (fig. 3).
PCR reaction system
PCR cycling parameters: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, Tm annealing for 30s, extension at 72 ℃ for 15s, and circulation for 25 times; further extension at 72 deg.C for 5min, and storage at 4 deg.C. Taking 2 mu L of PCR product, analyzing by 1.5% agarose gel electrophoresis, carrying out electrophoresis for about 30min at 150V in an DYY-6B type voltage-stabilizing electrophoresis apparatus, taking pictures of electrophoresis gel in a KCBIO-2800 gel imaging analyzer, and purifying and recovering the amplification product by using a chloroform extraction method when an amplification target band is displayed.
1) DEPC water was added to the PCR product (. about.50. mu.L) to 200. mu.L.
2) Then 200. mu.L of chloroform was added thereto, and the mixture was thoroughly mixed, centrifuged at 12000rpm for 15 min.
3) The supernatant was aspirated, transferred to a fresh centrifuge tube, added with an equal volume of chloroform (-200. mu.L), mixed well, centrifuged at 12000rpm for 15 min.
4) After centrifugation, the supernatant was placed in a fresh centrifuge tube and 20. mu.L NaAC (3mol/L) was added.
5) Adding 2 times volume of absolute ethyl alcohol (400 mu L), standing at 20 ℃ and storing for 30-60 min.
6) Centrifuging at 12000rpm for 10min, washing the precipitate with 70% anhydrous ethanol, 7500rpm, and centrifuging for 5 min.
7) After the precipitate is dried, DEPC water is added for dissolving.
2.1.3 cloning and sequencing
The PCR purified product was ligated into pGEM-T vector in the following reaction system:
ligation reaction System
After mixing, ligation was performed overnight at 4 ℃. Transferring the ligation product into 50 mu L of Escherichia coli DH5 a competent cells, adding 700 mu L of LB liquid culture medium (without ampicillin) into a centrifuge tube, mixing the mixture gently by a pipette, carrying out shaking culture at 37 ℃ and 180rpm for 60min, centrifuging the cultured bacteria solution at 5000rpm for 2min, carrying out colony aggregation, removing part of upper layer bacteria solution in a super clean workbench, mixing the rest bacteria solution uniformly, coating 100 mu L of the bacteria solution on LB solid culture medium (with ampicillin), sealing the culture dish, and carrying out inversion culture at 37 ℃ overnight. Single colonies were picked and cultured in 600. mu.L of LB (ampicillin-containing) medium. Taking 1 mu L of cultured bacterial colony PCR, detecting the PCR product of the bacterial liquid by using 1.5% agar gel electrophoresis, selecting 3 positive clones according to the electrophoresis result, sending the bacterial liquid to the company Limited in Biotechnology engineering (Shanghai) for sequence determination, and verifying the correctness and directionality of the connecting sequence. Then, the verified bacterial liquid is selected for propagation culture, plasmids are extracted by using a plasmid DNA miniextract kit, and the plasmids are stored at the temperature of minus 20 ℃ for later use.
2.1.4 preparation of negative sense Universal nucleic acid probes
The process comprises the steps of enzyme digestion, enzyme digestion product purification, transcription product purification and the like. Digesting the recombinant plasmid with a restriction enzyme Spe I (TaKaRa), cutting the circular plasmid into a linear shape and purifying (FIG. 4); digoxin-labeled negative strand RNA was transcribed in vitro using T7 RNA polymerase using the purified linearized plasmid as a template. The reaction system is as follows:
enzyme digestion reaction system
Flicking, mixing, incubating at 37 deg.C for 3h, subjecting 2 μ L of enzyme digestion product to 1% agarose gel electrophoresis, electrophoresing under 150V for 30min in DYY-6B type voltage-stabilized electrophoresis apparatus, collecting picture with electrophoresis gel in KCBIO-2800 gel imaging analyzer, and detecting whether digestion is sufficient. After confirming that the cleavage was sufficient (FIG. 4), the cleavage product was purified using a PCR product purification kit. Taking the purified linear plasmid as a template, and carrying out in-vitro transcription according to the following system:
probe preparation system
After mixing, incubation was carried out at 37 ℃ for 2h, 2. mu.L of RNase-free DNase I (ROCHE) was added, and incubation was carried out at 37 ℃ for 15 min. Storing at-80 deg.C for use.
2.2 preparation of Virus-like specificity probes
The recombinant plasmid containing the whole genome of the viroid was prepared at 4 to 5. mu.g (FIG. 5). The specific method and steps are the same as above. The PSTVd and TCDVd recombinant plasmids were digested with the restriction enzyme Spe I (Takara), and the TASVd, PCFVd, TPMVd and CLVd recombinant plasmids were digested with the restriction enzyme EcoR I (Takara). The TASVd, PCFVd, TPMVd and CLVd recombinant plasmids transcribe digoxin-labeled negative strand RNA in vitro using T3 RNA polymerase, and the PSTVd and TCDVd recombinant plasmids transcribe digoxin-labeled negative strand RNA in vitro using T7 RNA polymerase.
The restriction enzyme digestion system of the TASVd, TPMVd, PCFVd, CLVd, PSTVd and TCDVd recombinant plasmids is as follows:
enzyme digestion reaction system
Flicking, mixing, incubating at 37 deg.C for 3h, subjecting 2 μ L of enzyme digestion product to 1% agarose gel electrophoresis, electrophoresing under 150V for 30min in DYY-6B type voltage-stabilized electrophoresis apparatus, collecting picture with electrophoresis gel in KCBIO-2800 gel imaging analyzer, and detecting whether digestion is sufficient. After confirming that the cleavage was sufficient (FIG. 5), the cleavage product was purified using a PCR product purification kit. Taking the purified linear plasmid as a template, and carrying out in-vitro transcription according to the following system:
probe preparation system
After mixing, incubation was carried out at 37 ℃ for 2h, 2. mu.L of RNase-free DNase I (ROCHE) was added, and incubation was carried out at 37 ℃ for 15 min. Storing at-80 deg.C for use.
2.3 preparation of Virus-like Linear Positive-stranded RNA
Recombinant plasmids containing the whole genome of the viroid (4. mu.g to 5. mu.g) were used for the preparation (FIG. 5). The steps of the enzyme digestion and the purification method are the same as above. The PSTVd, PCFVd, TPMVd, TASVd and CLVd recombinant plasmids were digested with restriction enzyme Spe I (Takara), and the TCDVd recombinant plasmids were digested with restriction enzyme Nco I (Takara). The PSTVd recombinant plasmid uses T3 RNA polymerase to transcribe the positive-strand RNA in vitro, the TCDVd recombinant plasmid uses SP6 RNA polymerase to transcribe the positive-strand RNA in vitro, and the PCFVd, TPMVd, TASVd and CLVd recombinant plasmids use T7 RNA polymerase to transcribe the positive-strand RNA in vitro.
The restriction enzyme digestion system of the TASVd, TPMVd, PCFVd, CLVd, PSTVd and TCDVd recombinant plasmids is as follows:
enzyme digestion reaction system
The linear positive strand RNA in vitro transcription system is as follows:
in vitro transcription reaction system
After mixing, incubation was carried out at 37 ℃ for 2h, 2. mu.L of DNase I without RNase was added, and incubation was carried out at 37 ℃ for 15 min. Storing at-80 deg.C for use.
The in vitro transcribed viroid positive-strand RNA was purified by chloroform extraction.
1) DEPC water was added to the in vitro transcripts (. about.100. mu.L) to 200. mu.L.
2) Then 200. mu.L of chloroform was added thereto, and the mixture was thoroughly mixed, centrifuged at 12000rpm for 15 min.
3) The supernatant was aspirated, transferred to a fresh centrifuge tube, added with an equal volume of chloroform (-200. mu.L), mixed well, centrifuged at 12000rpm for 15 min.
4) After centrifugation, the supernatant was placed in a fresh centrifuge tube and 20. mu.L NaAC (3mol/L) was added.
5) Adding 2.5 times volume of absolute ethyl alcohol (500 mu L), standing at 20 ℃ and storing for 30-60 min.
6) Centrifuging at 12000rpm for 10min, washing the precipitate with 70% anhydrous ethanol, 7500rpm, and centrifuging for 5 min.
7) After the precipitate is dried, it is dissolved in water without RNase.
The purified in vitro transcripts were examined on denaturing agar gels. Convenient for quantifying the RNA of each different virus. 2.4 detection sensitivity determination and detection specificity verification of nucleic acid probes
The solution of the synthesized viroid linear positive-strand RNA was subjected to a 5-fold concentration gradient (concentration gradient of 5 in this order)0，5-1，5-2，5-3，5-4，5-5，5-6，5-7) Prepared into a series of solutions, and added to the hybridization membrane. The ideal hybridization temperature is explored, and the detection sensitivity of the universal nucleic acid probe and the specific probe is compared.
2.4.1 sample preparation and spotting: sequentially diluting the positive-strand RNA transcribed from each viroid in vitro by 5-fold concentration gradient, wherein the initial concentration is 75 ng/mu L, and the end point of the dilution is 5-70.96 pg/. mu.L; total RNA of the plant sample (about 0.1g of leaf sample, volume of 50. mu.L after RNA extraction) was extracted by the Tirzol method, and the utility of the universal nucleic acid probe was determined.
2.4.2 dot blot hybridization: after the nylon membrane is dried, ultraviolet crosslinking (1200 multiplied by 100 uJ/cm)2). Placing the UV-crosslinked nylon membrane into a hybridization bottle, and adding hybridization solution (per 100 cm)2Membrane required 12.5mL), prehybridization>And (4) 1 h. Then 1. mu.L of probe was added and hybridization was carried out overnight. Discarding the hybridization solution, adding sufficient 2 XSSC and 0.1% SDS solution, washing the membrane for 2 times at normal temperature, each time for 5 min. Adding 0.1 XSSC, 0.1% SDS solution (1-5 mL/cm)2) Washing the membrane for 2 times, each time for 15min under the hybridization temperature condition. The washing solution was discarded, and 15mL of a blocking agent (1% blocking agent, 0.1mol/L maleic acid solution) was added and blocked at room temperature for 30 min. The blocking agent was discarded, 15mL of antibody solution (diluted 1000 times with blocking solution) was added, and the reaction was carried out at room temperature for 30 min. Abandoning antibody liquid, adding 25mL of washing solution (0.3% Tween 20, 1mol/L maleic acid solution), washing the membrane twice at normal temperature, 15min each time. Add 25mL of detection buffer to equilibrate for 5 min. Taking out the nylon membrane, putting the nylon membrane into a hybridization bag with the sample application face up. Adding an appropriate amount of diluted CSPD (the CSPD is diluted by a detection solution according to the proportion of 1: 1-3 and then used), sealing the nylon membrane, and repeatedly and lightly pushing for several times to uniformly diffuse the CSPD on the membrane. Excess liquid is squeezed out and the hybridization membrane is sealed. Finally, Chemidoc chemiluminescence imaging was performed and pictures were taken.
3 results
3.1 identification of sensitivity and specificity of Universal nucleic acid probes at different hybridization temperatures
A universal nucleic acid probe is prepared from the common sequences of 6 tomato viruses for dot hybridization, and the 6 tomato viruses can be theoretically detected under the condition of proper hybridization temperature.
For finding the optimum hybridization temperature, hybridization was carried out at 4 temperatures, 50 ℃, 55 ℃, 60 ℃ and 65 ℃. The results show (FIG. 6) that at 55 ℃, the hybridization signal for detecting 6 types of viruses is stronger than the rest of the hybridization temperature. Therefore, the hybridization temperature optimum for the detection sensitivity of the universal probe is 55 ℃.
Experiments show that the detection sensitivity of the universal nucleic acid probe is high. The universal nucleic acid probe detects that the final concentration of TCDVd and CLVd is 5-4The final concentrations of PSTVd, PCFVd and TASVd were 5-5And detecting the final concentration of TPMVd to be 5-6. The result shows that the concentration of the common RNA for detecting the stronger hybridization signal of the 6 types of viruses is 5 at the optimal hybridization temperature of 55 DEG C-3。
3.2 detection sensitivity and specificity comparison of Universal nucleic acid probes with Virus-like specific probes
Based on the above results, the concentration of 5 was used under the conditions of the optimum hybridization temperature between the universal probe and each of the viroid-specific probes-3(0.6 ng/. mu.L) of RNA, the detection sensitivity and specificity of the universal probe and the specific probe were compared.
The results of the universal probe for 6 types of viruses at 55 ℃ show that the 6 types of viruses can be detected under the condition of the same RNA concentration (FIG. 7). At 68 ℃ the 6 types of viruses were detected by hybridization with the 6 types of virus-specific probes (PSTVd-probe, PCFVd-probe, TCDVd-probe, TPMVd-probe, TASVd-probe and CLVd-probe), respectively, and the results showed (FIG. 7) that each type of virus-specific probe was not able to detect the 6 types of viruses. Although each virus-specific probe can detect other virus-like species than itself, the sensitivity for detecting other virus-like species is significantly lower than that of the universal probe. Specific probes for PSTVd can only detect PSTVd, TCDVd and TPMVd simultaneously. Specific probes for PCFVd can only detect PCFVd and TPMVd simultaneously. The specific probe for TCDVd can only detect TCDVd and TPMVd simultaneously. The TPMVd specific probe can only detect PSTVd, TCDVd, TPMVd and TASVd simultaneously. Probes specific for TASVd and CLVd can only detect TASVd, TPMVd and CLVd simultaneously.
3.3 detection of tomato samples Using Universal probes
The above-described test for verifying the detection sensitivity and specificity of the probe uses in vitro transcribed RNA. The RNA of tomato samples was used for the actual assay. It is contemplated that tomato RNA is more complex than in vitro transcribed RNA, since tomato RNA contains various proteins and metabolites. Therefore, it is necessary to verify the reliability of universal nucleic acid probes for detecting tomato samples.
Extracting RNA of healthy tomato, mixing with positive-strand RNA transcribed in vitro from various viruses, measuring the concentration, unifying the concentration of each RNA (100 ng/. mu.L), taking 4. mu.L, sequentially spotting on a nylon membrane (figure 8), and sequentially hybridizing by using a universal nucleic acid probe and various virus specific probes at 55 ℃ and 68 ℃ respectively. The results show (FIG. 8) that the universal nucleic acid probe can detect 6 kinds of viruses simultaneously and has strong hybridization signals. Although each specific probe can detect one or more other viroids besides itself, the hybridization signal for detecting other viroids is weaker. In summary, the universal nucleic acid probe can be used for detection of field samples.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.