CN114438226A - Rapid detection method and application of potato gold thread insect RPA-LFD - Google Patents
Rapid detection method and application of potato gold thread insect RPA-LFD Download PDFInfo
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Abstract
The invention discloses a rapid detection method and application of potato golden thread insect RPA-LFD, belonging to the technical field of biology. The invention discloses a potato gold thread insect RPA-LFD rapid detection primer and a probe, 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 primers and the probes are utilized to construct an RPA-LFD detection method, DNA of the potato golden thread insects is extracted, an RPA system of the potato golden thread insects is subjected to isothermal amplification, and the LFD test strip is adopted to specifically and rapidly detect the potato golden thread insects. 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 potato golden thread insects.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a rapid detection method and application of potato anoectochilus roxburghii RPA-LFD.
Background
The nematodes of Anoectochilus solani (Globodera rostochiensis) and Leuconostoc pallida (Globodera pallida), collectively known as Potato Cyst Nematodes (PCNs), are a class of pests with high specificity and economic importance for potatoes. PCN infects potatoes as a single pathogen, which can cause a 30% reduction in yield. The nematode is an important quarantine harmful organism in potato production. PCN is highly productive and can survive long periods of time in adverse environmental conditions, and is therefore difficult to eradicate once a population is established. At present, the potato golden thread worms occur in local areas of China, and are important quarantine pests for internal and external inspection in China. PCN morphological characteristics are very similar and difficult to distinguish, and the traditional potato cyst nematode identification method must be used for identifying by means of a certain quantity of characteristic cysts or female worms and combining the characteristics of 2-instar larvae, so that the specificity of potato cyst nematode detection is particularly strong, and meanwhile, a sufficient number of samples are required. In order to protect the safety of agricultural production in China, prevent the introduction of the potato white nematodes into China and the propagation and diffusion of the potato golden thread nematodes in China, the development of the specific and rapid molecular detection technical research of the potato cyst nematodes has important practical significance.
The molecular identification of potato cyst nematode starts from the 80 th century, researchers developed and designed multiple specific probes based on the protein difference of potato white nematode and potato gold thread nematode by using protein electrophoresis technology, and can be used for detecting and distinguishing two PCNs. Monoclonal antibodies specific to two species of PCN are obtained by screening with serology technology, Schots and the like, and can be quantitatively identified. Nucleic acid detection techniques aimed at classification began in 1985, but the current methods of nucleic acid detection were time-consuming, required large amounts of nucleic acid, and were not sensitive. With the continuous optimization of PCR technology, the reaction sensitivity and efficiency are continuously improved, and the detection can be realized only by using a small amount of materials. The PCR method is more suitable for researching nematode DNA. By designing PCR primers and amplifying two PCN species, the detection of PCR products can well distinguish two potato cyst nematodes. Based on PCR technology, various molecular marker technologies developed based on DNA molecular polymorphism are all involved in the detection and identification of PCN, such as RAPD, AFLP, RFLP, real-time fluorescence PCR and some isothermal amplification technologies LAMP, are also applied in the detection of PCN.
Recombinase polymerase amplification technology (RPA) was first proposed in 2006 by Olaf pineburg group, uk, and has been used to detect a variety of pathogens, such as, for example, cobotin (2018) amplified the sequence of the enterolobium hiemale IGS rRNA fragment using RPA and its amplification product detected with real-time fluorescence, a second instar larva with a sensitivity of 1/10. Ju Yu Liang (2019) establishes the RPA rapid detection method of Meloidogyne incognita, Meloidogyne javanicus, Meloidogyne arachidis and Meloidogyne enterolobii. A root knot nematode javanica RPA-LFD detection method is established in the Zymuan Kai (2020), and a visual detection result of a positive sample can be realized within 20min at the constant temperature of 37-42 ℃. However, no related report is available on the potato golden thread.
Disclosure of Invention
The invention aims to provide a potato golden thread insect RPA-LFD rapid detection method and application, which aim to solve the problems in the prior art, establishes the potato golden thread insect RPA-LFD rapid detection method by designing an RPA specific primer and an LFD specific probe, and can rapidly detect the potato golden thread insects with high sensitivity and specificity.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a potato gold thread insect RPA-LFD rapid detection primer and a probe, 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, tetrahydrofuran is added between the 5' end and the 3 'end, and Spacer-C3 phosphoramidite is added to the 3' end.
The invention also provides a kit for rapidly detecting the potato gold thread insect RPA-LFD, which comprises the primer and the probe.
Preferably, the kit further comprises a test strip, wherein the test strip is a nucleic acid detection test strip with streptavidin and Fam antibody modified gold nano-markers.
Preferably, the kit further comprises a DNA extraction reagent, wherein the DNA extraction reagent comprises lysate A and lysate B.
The invention also provides a rapid detection method of the potato anoectochilus roxburghii RPA-LFD, which comprises the steps of carrying out RPA reaction by using the primer and the probe, spotting a final product of the RPA reaction on an LF test strip, standing and observing a result, and judging whether the potato anoectochilus roxburghii is detected according to the observed result.
Preferably, the RPA reaction system comprises:
1) reaction mixture liquid: 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 and 2.5 mu L of magnesium acetate, dissolving all the solutions in the enzyme dry powder, and adding sterilized double distilled water to supplement to 48 mu L;
2) mu.L of DNA template.
Preferably, the RPA reaction conditions are: and uniformly mixing the reaction mixed solution and the DNA template, and incubating for 30min at 37-40 ℃.
The invention 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) to (c):
(a) detecting the condition of carrying the potato golden thread insects in the imported and exported plant products;
(b) detecting the occurrence condition of the potato golden thread insects in the soil;
(c) and (5) identifying the potato golden thread insects.
The invention discloses the following technical effects:
the invention designs 1 RPA forward primer, 1 biotin-modified reverse primer and a Fam-modified probe, and generates an amplification product with biotin and Fam under the action of polymerase and recombinase, so that the amplification product is detected by the nucleic acid detection test strip with streptavidin and Fam antibody-modified gold nano markers. The method for detecting the potato golden thread insects by using the designed specific primer pair and the probe has the following advantages:
the detection specificity is strong: the primers and the probes used by the RPA are only the DNA of the potato gold thread worm template to perform amplification reaction, so that the dual specificity is ensured, and the detection method has higher fidelity than other detection methods.
(II) short detection time: the extraction of nematode nucleic acid to RPA-LFD detection only needs 45min, the one-step extraction detection does not need any other procedure in the early stage and the later stage.
And (III) the detection condition requirement is low: the RPA can reach the reaction temperature at 37-40 ℃ without any temperature rising instrument such as a PCR instrument, and the detection can be completed by the test strip without a gel electrophoresis and imaging system.
And (IV) the detection steps are few, the operation is simple, the result is obvious, the whole detection process does not relate to complex instruments and equipment, the detection can be finished by common 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 (V) the method is friendly to human and environment, toxic reagents such as EB (Epstein-Barr) and the like are not 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 potato golden thread insects, the method has higher specificity, sensitivity and portability, and can be applied to field detection in actual production. The technology can be applied to rapid molecular detection of articles carrying the potato golden thread insects to import and export, including soil samples and plants, and has important practical application value.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings 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 invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic diagram of the RPA primer and probe design of potato Anoectochilus roxburghii;
FIG. 2 is a diagram showing the results of specific detection of potato Anoectochilus roxburghii RPA; a: a PCR specific primer amplification electrophoretogram of the potato gold thread worm; b: detecting the specificity of the potato gold thread RPA-LFD;
FIG. 3 is a graph showing the results of the detection of the sensitivity of the RPA of the potato Anoectochilus roxburghii; a: detecting PCR sensitivity of the potato gold thread insects; b: detecting the sensitivity of the potato golden thread RPA-LFD;
FIG. 4 is a graph showing the results of detection of the RPA of the potato Anoectochilus roxburghii in soil.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
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 invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
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. 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 invention. All documents mentioned in this specification are incorporated by reference herein 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 present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The kits used in the following examples are all commercially available, the nematodes used are quarantine nematodes, and the laboratory of the plant protection research institute of the academy of agricultural sciences of China stores a small amount of them for research only.
Experimental materials: cyst nematode samples are shown in table 1 below.
TABLE 1 cyst nematode sample code and population sources
The main reagents are as follows: the nucleic acid extraction kit is purchased from Ningbo city Zhenhai Baichuan Biotech limited (cargo number BC 002); the twist Amp nfo KIT was purchased from twist Dx corporation, England (cat number TANFO02 KIT); the Milenia genine hybrid test strip detection kit is purchased from Milenia biotec, Germany (Cat. MILENIA 01); DNA marker was purchased from TaKaRa; primers and probes were synthesized by Shanghai Biotechnology Ltd.
Example 1 method for detecting RPA of Potato cyst nematode
1. Extraction of DNA from cyst nematodes
Absorbing 10 mu L of lysate A to 200 mu L of EP tube, adding 2 mu L of lysate B for mixing, picking potato Anoectochilus formosanus monospora or single nematode into the mixed lysate, crushing the cyst, placing the EP tube at 95 ℃ for 15min, then performing 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 storing at-20 ℃ for standby.
2. RPA primer design
According to the method, a pair of specific primers and 1 detection probe (shown in figure 1) are designed according to the ITS sequencing result of the potato anoectochilus roxburghii as a template, and the primers and the probes are synthesized by Shanghai bioengineering technology service company Limited. The primer sequences are as follows:
(ii) 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: adding 25 μ L of rehydration solution, 2.1 μ L of GrF and GrR (10 μmol/L), 0.6 μ L of GrProbe (10 μmol/L), and 2.5 μ L of magnesium acetate (280mM) into enzyme dry powder packaged in kit, mixing well and dissolving, sterilizing ddH2Supplementing O to 48 mu L to obtain reaction mixed liquor; adding 2 mu L of DNA to be detected, and incubating for 30min at 37-40 ℃.
3. And taking the final product of the RPA reaction to the tail end of the test strip, and standing for observation.
Example 2 detection of specificity of Potato Anoectochilus roxburghii RPA
Collecting 13 different populations (shown in table 1) of potato white nematode, eimeria cyst nematode, beet cyst nematode, soybean cyst nematode and the like, respectively extracting DNA of the populations as templates, and carrying out RPA detection together with the potato gold thread nematode DNA template so as to detect the specificity of the potato gold thread nematode RPA detection method.
After the reaction mixture was prepared and mixed uniformly as in example 1 above, 2. mu.L of template DNA was added, amplification reaction was carried out under 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 test strip was allowed to stand for observation.
As shown in FIG. 2, the DNA sequence of the specific fragment of the Helichrysum solani can amplify a specific band (the gene sequence of the specific fragment is shown in SEQ ID NO: 4), and the DNA sequence has NO cross with other nematodes and has specificity (see FIG. 2A).
GrJ, GrN1, GrN2, GrN3, GrN5 and GrF are DNA of potato golden thread insects, and the obvious detection line band can be observed, other samples and negative controls only have obvious control line bands, and the detection line has no band (see figure 2B). The result shows that the RPA primer, the probe and the reaction system have certain specificity when detecting the potato golden thread insects.
SEQ ID NO 4 as follows:
CTGTGTATGGGCTGGCACATTGACCAACAATGTACGGACAGCGCCCTGTGGGCACATGAGTGTTGGGGTGTAACCGATGTTGGTGGCCCTATGGGTGAGCCGACGATTGCTGCTGTCGTCGGGTCGCTGCGCCAACGGAGGAAGCACGCCCACAGGGCACCCTAACGGCTGTGCTGGCGTCTGTGCGTCGTTGAGCGGTTGTTGCGCCTTGCGCAGATATGCTAACATGGAGTGTAGCTGCTACTCCATGTTGTACGTGCCGTA。
example 3 Potato Anoectochilus roxburghii RPA sensitivity detection
Diluting DNA template extracted from single head potato gold thread insect second instar larva and single spore capsule by 10 times to 1.0 × 10-1～1.0×10-3At each concentration of 4, 2. mu.L of DNA was used as a template, the reaction mixture was prepared according to example 1, mixed and dissolved uniformly, and then the reaction was carried out according to the reaction conditions of example 1, 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 test strip was allowed to stand for observation.
The results are shown in FIG. 3, the PCR gel electrophoresis result shows that 1/10 2 nd larvae show specific bands and show positive amplification, and other concentrations have no specific bands through PCR and gel electrophoresis, which indicates that 1/10 nucleic acids with the content of less than 2 nd larvae are not effectively amplified by the system. The RPA-LF detection result shows that 1/100 2-instar larvae have fuzzy detection lines, which indicates that nucleic acid detection of less than 1/100 2-instar larvae is ineffective. The PCR and RPA-LF detection of 1/1000 monosporal cysts shows specific bands and detection lines, which indicates that the detection of the two detection methods is effective under the concentration.
Example 4 detection of Potato Anoectochilus Roxburghii in collected soil
And respectively putting the collected 3 parts of soil samples into barrels, adding water, fully stirring, standing for about 2 minutes, pouring the upper suspension into a 35-mesh and 60-mesh sleeve sieve, washing the filtrate of the 60-mesh sieve into filter paper by using clear water, and picking the cysts out by using tweezers under a body type microscope. The extraction of nucleic acids from the cysts was carried out according to the method of example 1 above, with 3 cysts per soil sample being picked up and the single cysts being extracted.
After the reaction mixture was prepared and mixed uniformly as in example 1 above, 2. mu.L of template DNA was added, amplification reaction was carried out under 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 test strip was allowed to stand for observation.
The results are shown in fig. 4, obvious detection line strips can be observed for 9 cyst samples in 3 parts of soil, control lines and detection lines can be observed for positive control samples of the potato golden thread worms, only obvious strips are arranged on the positions of the control lines for negative control samples, and the detection results show that the 3 parts of soil samples contain the potato golden thread worms.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> plant protection research institute of Chinese academy of agricultural sciences, China institute of inspection and quarantine science
<120> method for rapidly detecting potato gold thread insects RPA-LFD and application thereof
<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 (10)
1. A primer and a probe for fast detecting potato gold thread RPA-LFD 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.
2. The primers and probes as claimed in claim 1, wherein Biotin is added to the 5' end of the downstream primer.
3. The primer and probe of claim 1, wherein Fam is added to the 5 'end of the probe, tetrahydrofuran is added between the 5' end and the 3 'end, and Spacer-C3 phosphoramidite is added to the 3' end.
4. A kit for rapidly detecting the RPA-LFD of the potato golden thread insect, which is characterized by comprising the primer and the probe of any one of claims 1 to 3.
5. The kit of claim 4, further comprising a test strip, wherein the test strip is a streptavidin and Fam antibody modified gold nano-labeled nucleic acid detection test strip.
6. The kit of claim 4, further comprising a DNA extraction reagent comprising lysate A and lysate B.
7. A rapid detection method of potato golden thread insect RPA-LFD is characterized in that the primer and the probe in claim 1 are used for carrying out RPA reaction, the final product of the RPA reaction is spotted on an LF test strip, the test strip is kept still for observation, and whether the test strip is the potato golden thread insect or not is judged according to the observation result.
8. The method of claim 7, wherein the RPA reaction system comprises:
1) reaction mixture liquid: 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 and 2.5 mu L of magnesium acetate, dissolving all the solutions in the enzyme dry powder, and adding sterilized double distilled water to supplement to 48 mu L;
2) mu.L of DNA template.
9. The method of claim 8, wherein the RPA reaction conditions are: and uniformly mixing the reaction mixed solution and the DNA template, and then incubating for 30min at 37-40 ℃.
10. The primer and probe according to claim 1, or the kit according to claim 2, or the use of the method according to claim 7, wherein the primer and probe is used in any one of the following (a) to (c):
(a) detecting the condition of carrying the potato golden thread insects in the imported and exported plant products;
(b) detecting the occurrence condition of the potato golden thread insects in the soil;
(c) and (5) identifying the potato golden thread insects.
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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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| CN111676296A (en) * | 2020-06-17 | 2020-09-18 | 河北省农林科学院植物保护研究所 | Test strip RPA primer for detecting potato rot stem nematode and detection kit thereof |
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