CN114438226B - Rapid detection method and application of potato golden nematode RPA-LFD - Google Patents
Rapid detection method and application of potato golden nematode RPA-LFD Download PDFInfo
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Abstract
The application discloses a rapid detection method and application of a potato golden nematode RPA-LFD, and belongs to the technical field of biology. The application discloses a rapid detection primer and a probe for a potato golden nematode RPA-LFD, which are characterized in that the primer sequence is an upstream primer sequence shown as SEQ ID NO. 1 and a downstream primer sequence shown as SEQ ID NO. 2, and the probe sequence is shown as SEQ ID NO. 3. The primer and the probe are utilized to construct an RPA-LFD detection method, the golden nematode DNA of the golden nematode is extracted, the golden nematode RPA system of the golden nematode is amplified isothermally, and the golden nematode of the potato can be detected specifically and rapidly by adopting an LFD test strip. The detection method has the advantages of strong specificity, high sensitivity, simple and convenient operation and high application value in the aspect of rapid quarantine detection of the golden nematode of the potato.
Description
Technical Field
The application relates to the technical field of biology, in particular to a rapid detection method and application of a potato golden nematode RPA-LFD.
Background
The potato gold nematodes (Globodera rostochiensis) and potato Bai Xianchong (Globodera pallida), collectively referred to as potato cyst nematodes (Potato cyst nematode, PCN for short), are a class of pests with high specialization and economic importance to potato. PCN, a single pathogen infestation, harms potatoes, which can result in a 30% reduction in yield. The nematodes are important quarantine pests in potato production. PCN is highly productive and can survive for long periods of time under adverse environmental conditions, thus being difficult to eradicate thoroughly once the population is established. At present, the potato golden nematode occurs in local areas of China and is an important quarantine pest for internal and external detection of China. PCN morphological characteristics are very similar and difficult to distinguish, and the traditional potato cyst nematode identification method must be carried out by means of a certain number of characteristic cysts or females and combining the characteristics of 2-year-old larvae, so that the potato cyst nematode detection has extremely strong specificity, and a sufficient number of specimens are needed. In order to protect the safety of agricultural production in China and prevent the potato white nematodes from being transmitted into China and the potato golden nematodes from spreading in China, the development of specific and rapid molecular detection technology research of potato cyst nematodes has important practical significance.
The research of molecular identification of the potato cyst nematodes starts in the 80 th century, and researchers develop and design a plurality of specific probes based on protein differences of the potato white nematodes and the potato golden nematodes by using a protein electrophoresis technology, so that the specific probes can be used for detecting and distinguishing two PCNs. Monoclonal antibodies specific to two species of PCN are obtained by screening by using serology technology, schots and the like, and can be quantitatively identified. Nucleic acid detection techniques aimed at classification began in 1985, but the current nucleic acid detection methods were time-consuming, required a large amount of nucleic acid, and were not sensitive. Along with the continuous optimization of the PCR technology, the reaction sensitivity and the efficiency are continuously improved, and the detection can be realized by using a small amount of materials. The PCR method is more suitable for the study of nematode DNA. By designing PCR primers, two PCN species are amplified, and two potato cyst nematodes can be well distinguished by detecting PCR products. Based on PCR technology, various molecular marker technologies developed based on DNA molecular polymorphism are involved in PCN detection and identification application, such as RAPD, AFLP, RFLP, real-time fluorescence PCR and some isothermal amplification technologies LAMP are also applied in PCN detection.
The recombinase polymerase amplification technique (RPA) was first proposed by Olaf piebenburg group in england in 2006, and has been used to detect a variety of pathogens, such as ambrotin (2018) using RPA to amplify the sequence of the IGS rRNA fragment of the root-knot nematode of e.g. otobean, and using real-time fluorescence to detect the amplified product, a sensitivity of 1/10 of that of a two-instar larva. Ju Yuliang (2019) establishes a rapid detection method for the RPA of meloidogyne incognita, meloidogyne javanica, meloidogyne arachidis and meloidogyne auriculata. The method for detecting the RPA-LFD of the root-knot nematode of Java is established by the Kaiyan (2020), and the visual detection result of the positive sample can be realized within 20min under the constant temperature condition of 37-42 ℃. However, there is no report on the golden nematode of potato.
Disclosure of Invention
The application aims to provide a rapid detection method of a potato gold nematode RPA-LFD and application thereof, which are used for solving the problems in the prior art, and the rapid detection method of the potato gold nematode RPA-LFD is established by designing an RPA specific primer and an LFD specific probe, so that the potato gold nematode can be rapidly detected with high sensitivity and specificity.
In order to achieve the above object, the present application provides the following solutions:
the application provides a rapid detection primer and a probe for a potato golden nematode RPA-LFD, wherein the primer sequence is an upstream primer sequence shown as SEQ ID NO. 1 and a downstream primer sequence shown as SEQ ID NO. 2, and the probe sequence is shown as SEQ ID NO. 3.
Preferably, biotin is added to the 5' end of the downstream primer.
Preferably, fam is added to the 5 'end of the probe, one tetrahydrofuran is added in the middle of the 5' end and the 3 'end, and a Spacer-C3 phosphoramidite is added to the 3' end.
The application also provides a rapid detection kit for the potato golden nematode RPA-LFD, which comprises the primer and the probe.
Preferably, the kit further comprises a test strip, wherein the test strip is a streptavidin and Fam antibody modified gold nano-labeled nucleic acid detection test strip.
Preferably, the method further comprises a DNA extraction reagent, wherein the DNA extraction reagent comprises a lysate A and a lysate B.
The application also provides a rapid detection method of the potato golden nematode RPA-LFD, which utilizes the primer and the probe to carry out the RPA reaction, points the final product of the RPA reaction on an LF test strip, stands the observation result, and judges whether the potato golden nematode is the potato golden nematode according to the observation result.
Preferably, the RPA reaction system comprises:
1) Reaction mixture: 25. Mu.L of rehydration solution, 2.1. Mu.L of each of the upstream primer and the downstream primer, 0.6. Mu.L of probe, 2.5. Mu.L of magnesium acetate, all the above solutions were dissolved in dry enzyme powder, and sterilized double distilled water was added to supplement 48. Mu.L;
2) 2. Mu.L of DNA template.
Preferably, the RPA reaction conditions are: after the reaction mixture and the DNA template are uniformly mixed, the mixture is incubated for 30min at 37-40 ℃.
The application also provides the primer and the probe, or the kit, or the application of the method, which are applied to any one of the following (a) - (c):
(a) Detecting the condition of carrying the golden nematode in import and export plant products;
(b) Detecting occurrence of potato anoectochilus formosanus in soil;
(c) And (5) identifying the golden nematode of the potato.
The application discloses the following technical effects:
according to the application, 1 RPA forward primer, 1 biotin modified reverse primer and one Fam modified probe are designed, and amplification products with biotin and Fam are generated under the action of polymerase and recombinase, so that the amplification products are detected by a streptavidin and Fam antibody modified gold nano-labeled nucleic acid detection test strip. The detection of the potato golden nematode by using the designed specific primer pair and the probe has the following advantages:
the detection specificity is strong: the primer and the probe used by the RPA are only amplification reaction of potato golden nematode template DNA, so that the dual specificity is ensured, and the method has higher fidelity than other detection methods.
And (II) the detection time is short: the time from the extraction of the nematode nucleic acid to the detection of the RPA-LFD is only 45min, the one-step extraction and the one-step detection are carried out, and no other procedure is needed in the early stage and the later stage.
(III) the detection condition is low in requirement: the RPA can reach the reaction temperature at 37-40 ℃ without any heating instrument such as a PCR instrument, and the detection can be completed by a test strip without a gel electrophoresis and imaging system.
And fourthly, the detection steps are few, the operation is simple, the result is obvious, the whole detection process does not involve complex instruments and equipment, the detection can be completed by general personnel, the result is easy to distinguish, the result can be observed by naked eyes, and complicated electrophoresis and ultraviolet observation are not needed.
And fifthly, the method is friendly to human and environment, no toxic reagents such as EB and the like are needed in the detection process, and the method is very safe to human and environment.
In conclusion, compared with the existing method for detecting the golden nematode, the method has higher specificity, sensitivity and portability, and can be applied to detection in field in actual production. The technology can be applied to rapid molecular detection on soil samples and plants carrying potato anoectochilus formosanus import and export articles, and has important practical application value.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the design of RPA primers and probes for golden nematode potato;
FIG. 2 is a graph showing the result of the specific detection of the RPA of the golden nematode of potato; a: amplifying an electrophoresis chart by using a PCR specific primer of the golden nematode of the potato; b: detecting the RPA-LFD specificity of the golden nematode of the potato;
FIG. 3 is a graph of the detection result of the sensitivity of the RPA of the golden nematode of potato; a: PCR sensitivity detection of the potato golden nematode; b: detecting the sensitivity of the RPA-LFD of the golden nematode of the potato;
FIG. 4 is a graph showing the results of the detection of the RPA of the golden nematode in soil.
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The kits used in the examples below are all commercially available, the nematodes used are quarantine nematodes, and the laboratory of the national academy of agricultural sciences of plant protection is kept in small amounts for research only.
Experimental materials: cyst nematode samples are shown in table 1 below.
Table 1 cyst nematode sample codes and population sources
The main reagent comprises: the nucleic acid extraction kit was purchased from zhenhai baichuan biotechnology limited of Ningbo city (cat No. BC 002); the TwistAmp nfo KIT is available from TwistDx corporation of England (cat. No. TANFO02 KIT); milenia genline hybridetect test strip detection kit was purchased from Milenia biotec company (product No. millenia 01) germany; DNA markers were purchased from TaKaRa; primers and probes were synthesized by Shanghai Biotechnology Co.
Example 1 RPA detection method of potato cyst nematode
1. Extraction of cyst nematode DNA
Absorbing 10 mu L to 200 mu L of the lysis solution A, adding 2 mu L of the lysis solution B for mixing, picking up the potato gold nematode monospora or single nematode, putting into the mixed lysis solution, crushing the cyst, putting the EP pipe at 95 ℃ for 15min, then carrying out instantaneous centrifugation, adding 20 mu L of sterilized water for dilution, and directly using the supernatant as a nematode DNA template for RPA detection or freezing and preserving at-20 ℃ for standby.
2. RPA primer design
According to the ITS sequencing result of the golden nematode of the potato as a template, a pair of specific primers and 1 detection probe (see figure 1) are designed, and the primers and the probes are synthesized by Shanghai bioengineering technology service limited company. The primer sequences were as follows:
(1) the upstream primer GrF (SEQ ID NO: 1): 5 '-CTGTGTATTGGCTGGCACATTGACCAACA-3';
(2) downstream primer GrR (SEQ ID NO: 2): 5 '-Biotin-TACGGCACGTACAACATGGAGTAGCAGCTAC-3';
(3) probe GrP (SEQ ID NO: 3): 5 '-Fam-CGGAGGAAGCACGCCCACAGGGCACCCTAACG (THF) CTGTGCTGGCGTCTGT-C3 spacer-3'.
Preparing an RPA reaction system: 25. Mu.L of rehydration solution, 2.1. Mu.L of GrF and GrR (10. Mu. Mol/L) each, 0.6. Mu.L of GrProbe (10. Mu. Mol/L) and 2.5. Mu.L of magnesium acetate (280 mM) are added into the enzyme dry powder packaged in the kit, and the mixture is fully and uniformly mixed for dissolution and sterilization of ddH 2 O is added to 48 mu L, and a reaction mixed solution is obtained; 2. Mu.L of DNA to be detected is added and incubated at 37-40℃for 30min.
3. The final product of the RPA reaction is taken to the tail end of the test strip, and the reaction product is stood and observed.
EXAMPLE 2 detection of specific for potato nematode RPA
13 different groups of potato Bai Xianchong, ai Qiubao cyst nematodes, beet cyst nematodes, soybean cyst nematodes and the like (see table 1) are collected, the DNA of the 13 different groups is respectively extracted as a template, and RPA detection is carried out together with a potato golden nematode DNA template, so that the specificity of the potato golden nematode RPA detection method is detected.
After the reaction mixture was prepared and uniformly mixed according to the above example 1, 2. Mu.L of template DNA was added, and the amplification reaction was carried out according to the reaction conditions of example 1, and after the reaction was completed, the midpoint of the final product of the RPA reaction was taken to the end of the test strip, and the reaction was allowed to stand for observation.
As shown in FIG. 2, the golden-thread nematodes can amplify specific bands (the gene sequence of the specific fragment is shown as SEQ ID NO: 4), have NO cross with other nematodes and have specificity (see FIG. 2A).
GrJ, grN1, grN2, grN3, grN5, grF were potato golden nematode DNA, and it was observed that the detection lines were distinct, and that other samples and negative controls had only distinct bands on the control lines, and that the detection lines were not striped (see FIG. 2B). The result shows that the RPA primer, the probe and the reaction system have certain specificity in detecting the golden nematode.
SEQ ID NO. 4 is as follows:
CTGTGTATGGGCTGGCACATTGACCAACAATGTACGGACAGCGCCCTGTGGGCACATGAGTGTTGGGGTGTAACCGATGTTGGTGGCCCTATGGGTGAGCCGACGATTGCTGCTGTCGTCGGGTCGCTGCGCCAACGGAGGAAGCACGCCCACAGGGCACCCTAACGGCTGTGCTGGCGTCTGTGCGTCGTTGAGCGGTTGTTGCGCCTTGCGCAGATATGCTAACATGGAGTGTAGCTGCTACTCCATGTTGTACGTGCCGTA。
example 3 detection of sensitivity of RPA from golden nematode
Diluting the DNA template extracted from the two-instar larvae and the monospore of the single-headed potato golden nematode into 1.0X10 by 10 times -1 ～1.0×10 -3 And taking 2 mu L of DNA as a template at each concentration, preparing and uniformly mixing and dissolving the DNA according to the reaction mixture of the example 1, carrying out the reaction according to the reaction conditions of the example 1, taking the midpoint of the final product of the RPA reaction to the tail end of a test strip after the reaction is finished, and standing for observation.
As shown in FIG. 3, the result of the PCR gel electrophoresis shows that 1/10 2-year-old larvae show specific bands, positive amplification is shown, other concentrations are subjected to the PCR, the gel electrophoresis has no specific bands, and the system detection of the nucleic acid with the content below 1/10 2-year-old larvae has no effective amplification. The RPA-LF detection result shows that 1/100 2-year-old larvae have fuzzy detection lines, and the detection of nucleic acid with the content below 1/100 2-year-old larvae is ineffective. The detection of 1/1000 single cysts through PCR and RPA-LF has specific bands and detection lines, which shows that the detection of the two detection methods is effective under the concentration.
Example 4 detection of Solanum tuberosum in soil
And respectively filling 3 parts of collected soil samples into barrels, adding water, fully stirring, standing for about 2 minutes, pouring the suspension into a 35-mesh and 60-mesh screen, washing the filtrate of the 60-mesh screen onto filter paper by using clear water, and picking out cysts by using tweezers under a split microscope. According to the method for extracting cyst nucleic acid of example 1, 3 cysts were picked up from each soil sample and nucleic acid extraction of single cysts was performed.
After the reaction mixture was prepared and uniformly mixed in accordance with the above-mentioned example 1, 2. Mu.L of template DNA was added, and the amplification reaction was carried out in accordance with the reaction conditions of example 1, and after the reaction was completed, the midpoint of the final product of the RPA reaction was taken to the end of the test strip, and the reaction was allowed to stand for observation.
As shown in FIG. 4, the total 9 cyst samples in 3 soil can observe obvious detection line strips, the potato gold-wire worm positive control sample can observe control lines and detection lines, the negative control sample only has obvious strips at the positions of the control lines, and the detection result shows that the 3 soil samples contain potato gold-wire nematodes.
The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.
Sequence listing
<110> Chinese national institute of inspection and quarantine science, national institute of plant protection, national academy of agricultural sciences
<120> potato golden nematode RPA-LFD rapid detection method and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ctgtgtattg gctggcacat tgaccaaca 29
<210> 2
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tacggcacgt acaacatgga gtagcagcta c 31
<210> 3
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
cggaggaagc acgcccacag ggcaccctaa cgctgtgctg gcgtctgt 48
<210> 4
<211> 264
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ctgtgtatgg gctggcacat tgaccaacaa tgtacggaca gcgccctgtg ggcacatgag 60
tgttggggtg taaccgatgt tggtggccct atgggtgagc cgacgattgc tgctgtcgtc 120
gggtcgctgc gccaacggag gaagcacgcc cacagggcac cctaacggct gtgctggcgt 180
ctgtgcgtcg ttgagcggtt gttgcgcctt gcgcagatat gctaacatgg agtgtagctg 240
ctactccatg ttgtacgtgc cgta 264
Claims (4)
1. The application of the rapid detection primer and the probe of the golden nematode RPA-LFD is characterized by being applied to any one of the following (a) - (c):
(a) Detecting the condition of carrying the golden nematode in import and export plant products;
(b) Detecting occurrence of potato anoectochilus formosanus in soil;
(c) Identifying a golden nematode of potato;
the primer sequence is an upstream primer sequence shown as SEQ ID NO. 1 and a downstream primer sequence shown as SEQ ID NO. 2, and the probe sequence is shown as SEQ ID NO. 3;
adding Biotin to the 5' -end of the downstream primer;
adding Fam to the 5 'end of the probe, adding tetrahydrofuran between the 5' end and the 3 'end, and adding Spacer-C3 phosphoramidite to the 3' end;
the rapid detection method of the potato golden nematode RPA-LFD comprises the steps of carrying out an RPA reaction by using a primer and a probe, spotting a final product of the RPA reaction on an LF test strip, standing an observation result, and judging whether the potato golden nematode is the potato golden nematode according to the observation result;
the RPA reaction system comprises:
1) Reaction mixture: 25. Mu.L of rehydration solution, 2.1. Mu.L of each of the upstream primer and the downstream primer, 0.6. Mu.L of probe, 2.5. Mu.L of magnesium acetate, all the above solutions were dissolved in dry enzyme powder, and sterilized double distilled water was added to supplement 48. Mu.L;
2) 2. Mu.L of DNA template;
the RPA reaction conditions are as follows: after the reaction mixture and the DNA template are uniformly mixed, the mixture is incubated for 30min at 37-40 ℃.
2. A rapid detection kit for a potato gold nematode RPA-LFD is characterized by comprising the primer and the probe according to claim 1.
3. The kit of claim 2, further comprising a test strip of streptavidin and Fam antibody-modified gold nano-labeled nucleic acid detection test strips.
4. The use of a kit according to claim 2, for any one of the following (a) - (c):
(a) Detecting the condition of carrying the golden nematode in import and export plant products;
(b) Detecting occurrence of potato anoectochilus formosanus in soil;
(c) And (5) identifying the golden nematode of the potato.
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| CN202210145503.2A CN114438226B (en) | 2022-02-17 | 2022-02-17 | Rapid detection method and application of potato golden nematode RPA-LFD |
| NL2032194A NL2032194B1 (en) | 2022-02-17 | 2022-06-16 | RPA-LFD Rapid Detecting Method of Globodera rostochiensis and its Application |
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