CN110878373A - Recombinase polymerase amplification detection kit for phytophthora infestans and application thereof - Google Patents
Recombinase polymerase amplification detection kit for phytophthora infestans and application thereof Download PDFInfo
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- CN110878373A CN110878373A CN201911114857.5A CN201911114857A CN110878373A CN 110878373 A CN110878373 A CN 110878373A CN 201911114857 A CN201911114857 A CN 201911114857A CN 110878373 A CN110878373 A CN 110878373A
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a recombinase polymerase amplification kit for detecting phytophthora infestans and application thereof, wherein a primer probe composition consists of a forward primer PiRPA-F, a reverse primer PiRPA-R and a probe PiProbe, the forward primer PiRPA-F has a nucleotide sequence shown as SEQ ID No.1, the reverse primer PiRPA-R has a nucleotide sequence shown as SEQ ID No.2, and the probe PiProbe has a nucleotide sequence shown as SEQ ID No. 3. Compared with the traditional detection technology for identifying the phytophthora infestans according to morphological characteristics, the method has higher accuracy, sensitivity and practicability, is convenient to operate and good in practicability, provides a new technical platform for detecting the phytophthora infestans, and can be used for high-sensitivity rapid detection of the potato late blight caused by the phytophthora infestans.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a recombinase polymerase amplification kit for detecting phytophthora infestans and application thereof.
Background
Epidemic diseases caused by Phytophthora infestans infecting crops such as potatoes and tomatoes are one of destructive diseases in global agricultural production, and once the disease directly causes that 1845-1850-year potatoes in Ireland and partial European regions are dead and dead by millions of people, and the disease is known as' Ireland hunger 39313. Late blight causes crop losses of nearly billions of dollars worldwide on average each year, and is one of the major potential hazards threatening human food safety. The potato late blight occurs in main potato planting areas in China, and in areas with serious diseases, tens of thousands of hectares of potatoes suffer from disasters every year. Phytophthora infestans can also infect other solanaceous crops, causing hundreds of millions of economic losses, and diseases caused by phytophthora infestans have become the biggest obstacle restricting the production and realization of industrialization of solanaceous crops. Therefore, the rapid and sensitive detection method is used for strengthening early warning and forecasting of the late blight caused by the phytophthora infestans, and has important significance for guiding the field control work of the late blight in time.
The traditional method for detecting phytophthora infestans is to separate and culture pathogenic bacteria from diseased tissues and observe the forms of the pathogenic bacteria. The method plays an important role in phytophthora infestans detection, but the method is time-consuming and labor-consuming, and requires operators to have professional phytophthora separation, morphological identification knowledge and rich practical experience. With the development of nucleotide identification methods, Polymerase Chain Reaction (PCR) and real-time fluorescence quantitative PCR (real time PCR) have been successfully applied to the detection of Phytophthora infestans. Although PCR and real-time PCR have greatly improved specificity and sensitivity, they rely on sophisticated and expensive temperature cycling equipment, require specialized technicians and complex reagents, and still require relatively long detection times. Therefore, it is crucial to establish a simple, fast, sensitive and accurate method for monitoring the occurrence of late blight in the field.
Recombinase Polymerase Amplification (RPA) is a nucleic acid detection technique invented by TwistDx corporation, uk, which can replace PCR. Because the kit can detect the monomolecular nucleic acid within 15min at normal temperature and has low requirements on hardware equipment, the kit is particularly suitable for the fields of in-vitro diagnosis, biosafety, agriculture and the like. The method is to design a pair of specific primers capable of amplifying fragments of no more than 500bp on a target gene, utilize a micro-filament formed by recombinase and the primers, search a sequence on a template DNA for complete complementary pairing with the template DNA, melt the template DNA with the help of single-stranded DNA binding protein, initiate the pairing of the primers and the template DNA, and perform replication and extension under the action of DNA polymerase. The reaction temperature is controlled between 37 ℃ and 42 ℃, and the amplification product with a detectable level can be obtained within 10min generally. Because the RPA amplification process depends on the specific primer of the long chain, the reaction specificity is strong, and the nucleic acid amplification process is carried out under the condition of normal temperature, the reaction requirements can be met by a common water bath kettle or heat preservation equipment, the detection cost is greatly reduced, and the portable rapid nucleic acid detection can be really realized.
The method is portable and simple to operate, is a necessary condition for detecting pathogenic bacteria in fields or other fields, does not need precise and expensive temperature control equipment for detecting the phytophthora infestans based on the recombinase polymerase amplification technology, needs short time and is simple to operate, so that the recombinase polymerase amplification detection method becomes a hot spot for detecting the pathogenic bacteria in actual production. However, the screening of the primer composition with strong specificity and high sensitivity is the key for detecting the phytophthora infestans based on a recombinase polymerase amplification method.
Disclosure of Invention
Aiming at the problems of long required period, poor specificity and low sensitivity of the detection method in the prior art, the invention aims to provide a primer, a probe and a kit for detecting phytophthora infestans based on a recombinase polymerase amplification method.
In order to realize the purpose, the invention is realized by the following technical scheme:
a primer probe composition for detecting phytophthora infestans based on a recombinase polymerase amplification method comprises a forward primer PiRPA-F, a reverse primer PiRPA-R and a probe PiProbe, wherein the forward primer PiRPA-F has a nucleotide sequence shown as SEQ ID No.1, the reverse primer PiRPA-R has a nucleotide sequence shown as SEQ ID No.2, and the probe PiProbe has a nucleotide sequence shown as SEQ ID No. 3. The sequences of the primers and probes are shown below:
forward primer PiRPA-F: TTAACTAACGGTTCTCCTATTTCAACAGTGGGACA
Reverse primer PiRPA-R: Biotin-CCATCACTTAGCTCTTTCTTTTCTGTAGGTCCATT
Probe PiProbe:
FAM-GAGCGTTTCCGCACGATCACTAGCAGTTAC-THF-ACCGCGGTGCCCACG-C3 space
the primer probe composition is applied to preparation of a kit for detecting phytophthora infestans.
A kit for detecting phytophthora infestans based on a recombinase polymerase amplification method comprises the primer probe composition. As a preferred technical scheme, the concentration and the dosage of each reagent in the kit are as follows: mu.L of 10. mu.M forward primer PiRPA-F, 2.1. mu.L of 10. mu.M reverse primer PiRPA-R, 0.6. mu.L of 10. mu.M probe PiProbe, 29.5. mu.L regeneration Buffer, 12.2. mu.L DEPC treated water, 2.5. mu.L of 280mM MgAC and enzyme dry powder were prepared into a 49. mu.L reaction system.
The kit is applied to the detection of phytophthora infestans.
The primer probe composition is applied to detection of phytophthora infestans.
A method for detecting phytophthora infestans based on a recombinase polymerase amplification method comprises the steps of extracting genome DNA of a sample to be detected as a reaction template, carrying out recombinase polymerase amplification by adopting the primer probe composition, detecting an amplification product by using a lateral flow chromatography test strip, and judging a detection result: when the detection line and the control line have indicator bands, the pathogenic bacteria to be detected are phytophthora infestans. As a preferred technical scheme, the method comprises the following specific process parameters: extracting genome DNA of a sample to be detected, taking 1 mu L of DNA of a microorganism to be detected as a reaction template, adding 49 mu L of detection solution in the kit of claim 3 or 4 for recombinase polymerase amplification, and carrying out reaction procedures as follows: amplifying at 39 ℃ for 20 min.
The method comprises the following specific steps: extracting the genome DNA of a sample to be detected, taking 1 mu L of sample DNA as a reaction template, and carrying out recombinase polymerase amplification reaction by using the detection kit system; because the probe comprises a carboxyfluorescein (FAM) label and a 3' end blocker, the downstream primer comprises biotin B, exonuclease in an RPA reaction system can specifically recognize and cut THF molecules in a probe sequence, and the cut probe and the downstream primer form a double-labeled amplicon which is provided with both the FAM label and the biotin B. Mu.l of the amplification product was mixed with 95. mu.l of HybriDetect assay buffer, a lateral flow strip was placed vertically, the sample end was immersed in the mixture, and the results were observed after 3min at room temperature.
The sample end of the lateral flow chromatography test strip carries the gold nanoparticles, and the detection line contains a biotin antibody. After target genes are amplified by using a biotin-labeled primer and a FAM-labeled probe, the sample end of the test strip is immersed into an amplification solution, and a biotin antibody-nucleic acid-nanogold particle complex is formed on a detection line to form a dark red strip. Therefore, after the reaction is finished, the existence of the phytophthora infestans is judged through the color change of the detection line on the lateral flow chromatography test strip; the detection line and the control line both have indicator strips to indicate that the detection is positive and phytophthora infestans exists; only the control line has an indicator band, and the detection line has no band, which indicates that the detection is negative and has no phytophthora infestans.
Compared with the prior art, the invention has the advantages and positive effects that:
(1) the practicability is good: the common PCR reaction needs precise and expensive temperature control equipment, and the product is easy to diffuse when being subjected to gel electrophoresis, which is one of the main sources of laboratory pollution; ethidium Bromide (EB) is extremely toxic and can accumulate carcinogenesis; the long-term observation of the ultraviolet lamp can also cause certain damage to the experimenters. The recombinase polymerase amplification reaction is only carried out in a constant-temperature water bath or heat preservation equipment, and the result can be directly judged through the color change of the indicator strip of the test strip detection line after the reaction is finished, so that the application value of the recombinase polymerase amplification reaction in the field is increased.
(2) The normal temperature amplification is realized, the PCR method and the loop-mediated isothermal amplification (LAMP) technology are not required to be subjected to thermal cycle or high temperature incubation, so that the dependence on a thermal cycle instrument and a stable heat source is eliminated, the recombinase polymerase amplification reaction can be carried out at normal temperature, and the application range of the PCR technology is greatly expanded. The recombinase polymerase amplification reaction can be mainly dependent on three enzymes at normal temperature: a recombinase capable of binding single-stranded nucleic acids, a single-stranded DNA binding protein (SSB), and a strand-displacing DNA polymerase. The mixture of these three enzymes is also active at ambient temperature, with an optimum reaction temperature around 37 ℃.
(3) The detection speed is high: the traditional phytophthora infestans detection technology is used for identification according to morphological characteristics, and is tedious in operation and time-consuming. The detection time of 1-2 hours is also required for common PCR reaction and LAMP detection. The whole process of recombinase polymerase amplification reaction is very fast, the amplification product with detectable level can be obtained within 10-20min generally, and the detection of the amplification product by a lateral flow chromatography test strip only needs 5 min. The use of the recombinase polymerase amplification technology is not limited by a detection field, and is particularly suitable for the rapid detection of pathogenic bacteria on a base layer.
The recombinase polymerase amplification detection method for phytophthora infestans provided by the invention overcomes the problems of tedious biological detection method, time and labor waste, poor specificity and long required period of the traditional phytophthora infestans, and the problems that PCR and LAMP detection technologies require a thermal cycler and high-temperature incubation equipment and can not rapidly detect the phytophthora infestans. The detection method disclosed by the invention can be used for quickly, conveniently, efficiently, specifically and sensitively detecting the phytophthora infestans within 20min under the constant temperature condition of 25-45 ℃, does not need a complex instrument, can well meet the requirement on the field detection of the phytophthora infestans, provides a new technical platform for detecting the epidemic pathogenic phytophthora infestans, can be used for quickly detecting the pathogenic phytophthora infestans carried by the potatoes in and out of the customs and other solanaceae crops with high sensitivity, and can also be used for early diagnosis and disease monitoring of the field pathogenic phytophthora infestans.
Drawings
FIG. 1 shows the specificity verification result of the primer probe composition for the recombinase polymerase detection of Phytophthora infestans:
the specific primer probe combination of the phytophthora infestans is used for carrying out recombinase polymerase amplification reaction on different pathogenic bacteria, and then lateral flow chromatography test paper strips are used for carrying out result judgment on reaction products. The results show that: only the detection line and the control line of the phytophthora infestans have indicator bands to show a positive detection result, and the detection lines of other phytophthora infestans do not have indicator bands to show a negative detection result.
FIG. 2 shows the sensitivity of the detection of the polymerase enzyme of the Phytophthora infestans recombinase:
amplifying genomic DNA of different concentrations of Phytophthora infestans: the reaction system containing 50. mu.L from left to right contained 1ng, 100pg, 10pg, 1pg, 100fg, 10fg DNA and the amplification results without genomic DNA, respectively. And (4) judging the detection sensitivity of the phytophthora infestans recombinase polymerase by detecting the line strip. The results show that: the detection lines respectively containing 1ng, 100pg, 10pg, 1pg and 100fg of phytophthora infestans DNA in a 50-microliter reaction system can observe strips, and positive reaction is realized; A50-mu-L reaction system contains 10fg phytophthora infestans DNA and a detection line without the DNA has no strip, and the reaction is negative. The strip color development result of the test strip detection line shows that the detection sensitivity of the phytophthora infestans recombinase polymerase is 100 fg.
FIG. 3 shows the detection results of recombinase polymerase in the indoor inoculated diseased leaves:
after the potato leaves are inoculated with phytophthora infestans for 60 hours, the inoculated leaves are divided into four regions, DNA is respectively extracted from plant tissues of the four regions, and then recombinase polymerase detection is respectively carried out. The results show that: indicator bands appear on the detection lines and the control lines of the first, second and third regions to indicate positive detection results, while the detection line of the fourth region does not have indicator bands to indicate negative detection results.
Detailed Description
In order to make the purpose and technical solution of the present invention more clear, the present invention is further described with specific examples, but is not limited to these examples.
Example 1: design, screening and verification of specific primer and probe composition of phytophthora infestans
In order to obtain a primer and probe composition specific to phytophthora infestans, a bioinformatics analysis means is firstly used to find that the sequence of a Ypt1 gene contains enough variable regions to distinguish different phytophthora infestans; then, the Ypt1 sequences of Phytophthora infestans and other 57 kinds of Phytophthora infestans were downloaded from the NCBI database, specific primer compositions were designed in the regions where the sequences of Phytophthora infestans and other Phytophthora infestans are different by multiple sequence alignment, 3 sets of primer compositions were preliminarily selected from the designed primer compositions, primer composition 1: f1(TTAACTAACGGTTCTCCTATTTCAACAGTGGGACA), R1 (CCATCACTTAGCTCTTTCTTTTCTGTAGGTCCATT); primer composition 2: f2(ACATTTGTCCCCGCGTGATTTCCTATTTAACTAAC), R2 (TTAGCTCTTTCTTTTCTGTAGGTCCATTCCGTAAAC); primer composition 3: f3(CGCGTGATTTCCTATTTAACTAACGGTTCTCCTAT), R3 (TCTTTCTTTTCTGTAGGTCCATTCCGTAAACAAGAC). Then, a primer composition with strong specificity and high sensitivity is screened in a common PCR amplification mode, and the result shows that only the primer composition 1 can realize specific amplification on the phytophthora infestans, and other primer compositions either cannot realize the specific amplification on the phytophthora infestans or have extremely low amplification efficiency. Thus, the primer composition 1 is adopted for subsequent recombinase polymerase detection of phytophthora infestans, F1 is the forward primer PiRPA-F, R1 is the reverse primer PiRPA-R, and a specific probe PiProbe is designed on the basis.
Example 2: specificity test of primer and probe composition for Phytophthora infestans
Primer and probe combinations for detecting phytophthora infestans: the forward primer PiRPA-F has a nucleotide sequence shown as SEQ ID No.1, the reverse primer PiRPA-R has a nucleotide sequence shown as SEQ ID No.2, and the probe PiProbe has a nucleotide sequence shown as SEQ ID No. 3.
Forward primer PiRPA-F: TTAACTAACGGTTCTCCTATTTCAACAGTGGGACA
Reverse primer PiRPA-R: Biotin-CCATCACTTAGCTCTTTCTTTTCTGTAGGTCCATT
Probe PiProbe:
FAM-GAGCGTTTCCGCACGATCACTAGCAGTTAC-THF-ACCGCGGTGCCCACG-C3 space
the primer probe composition is adopted to prepare a kit, and the concentration and the dosage of each reagent in the kit are as follows: mu.L of 10. mu.M forward primer PiRPA-F, 2.1. mu.L of 10. mu.M reverse primer PiRPA-R, 0.6. mu.L of 10. mu.M probe PiProbe, 29.5. mu.L regeneration Buffer, 12.2. mu.L DEPC treated water, 2.5. mu.L of 280mM MgAC and enzyme dry powder were prepared into a 49. mu.L reaction system.
Extracting the genome DNA of a sample to be detected, taking 1 mu L of sample DNA as a reaction template, adding 49 mu L of detection solution in the kit for recombinase polymerase amplification, wherein the reaction procedure is as follows: performing reaction amplification at 39 ℃ for 20min, detecting the amplification product by using a lateral flow chromatography test strip, and judging the detection result.
To verify the specificity of the primers and probes for Phytophthora infestans, the specificity was evaluated using Phytophthora infestans and five other closely related Phytophthora infestans (Phytophthora parasitica, P.ipomoea, P.sojae, P.mirabilis, P.capsicii) as test strains. And (3) displaying a detection result: only the detection line and the control line of phytophthora infestans showed an indicator band indicating a positive detection result, while the detection lines of other phytophthora infestans showed no indicator band indicating a negative detection result (fig. 1).
Example 3: sensitivity test for detecting phytophthora infestans recombinase polymerase
In order to determine the sensitivity of the recombinase polymerase detection method, the genomic DNA of the phytophthora infestans is subjected to 10-fold gradient dilution after the concentration is measured by a spectrophotometer, the concentration range of the DNA is set to be 1ng-10fg, 1 muL of diluted DNA diluent with each concentration is taken as a template, DEPC treated water is taken as a negative control, 49 muL of kit solution is added for recombinase polymerase amplification reaction, and the reaction program is as follows: incubate at 39 ℃ for 20 min. And (3) detecting the amplified product by using a lateral flow chromatography test strip after the reaction is finished, and judging the detection result according to the color change of the detection line indicator strip. The results show that: the test strip line was observed to have a band at a DNA concentration in the range of 1ng-100fg, a DNA concentration of 10fg and no band at the negative control test strip line, indicating that a DNA concentration of 100fg is sufficient for detection by the recombinase polymerase method (FIG. 2).
Example 4: recombinase polymerase detection test of diseased leaves after indoor inoculation of phytophthora infestans
To evaluate the utility of the established recombinase polymerase detection method, diseased plants were detected. Inoculating phytophthora infestans on potato leaves for 60 hours, dividing the inoculated leaves into four regions, wherein the inoculation disease point is in the first region, extracting DNA from plant tissues of the four regions respectively, then carrying out recombinase polymerase amplification reaction respectively, and then carrying out result judgment on reaction products by using a lateral flow chromatography test strip. The results show that: indicator bands appeared on the first, second and third detection lines and the control line, indicating a positive detection result, while the detection line of the fourth area showed no indicator band, indicating a negative detection result (FIG. 3). The above description shows that the established recombinase polymerase detection method can detect phytophthora infestans at the early stage of pathogenesis.
Sequence listing
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Claims (8)
1. A primer probe composition for detecting phytophthora infestans based on a recombinase polymerase amplification method is characterized by comprising a forward primer PiRPA-F, a reverse primer PiRPA-R and a probe PiProbe, wherein the forward primer PiRPA-F has a nucleotide sequence shown as SEQ ID No.1, the reverse primer PiRPA-R has a nucleotide sequence shown as SEQID No.2, and the probe PiProbe has a nucleotide sequence shown as SEQ ID No. 3.
2. Use of the primer probe composition of claim 1 for the preparation of a kit for the detection of phytophthora infestans.
3. A kit for detecting Phytophthora infestans based on a recombinase polymerase amplification method, comprising the primer-probe composition of claim 1.
4. The kit of claim 3, wherein the concentration and amount of each reagent in the kit are: mu.L of 10. mu.M forward primer PiRPA-F, 2.1. mu.L of 10. mu.M reverse primer PiRPA-R, 0.6. mu.L of 10. mu.M probe PiProbe, 29.5. mu.L regeneration Buffer, 12.2. mu.L DEPC treated water, 2.5. mu.L of 280mM MgAC and enzyme dry powder were prepared into a 49. mu.L reaction system.
5. Use of the kit according to claim 3 or 4 for the detection of phytophthora infestans.
6. The primer probe composition of claim 1, wherein the primer probe composition is used for detecting Phytophthora infestans.
7. A method for detecting phytophthora infestans based on a recombinase polymerase amplification method is characterized by comprising the following steps: extracting genome DNA of a sample to be detected as a reaction template, carrying out recombinase polymerase amplification by using the primer probe composition of claim 1, detecting an amplification product by using a lateral flow chromatography test strip, and judging a detection result: when the detection line and the control line have indicator bands, the pathogenic bacteria to be detected are phytophthora infestans.
8. The method of claim 7, wherein: the method comprises the following specific process parameters: extracting genome DNA of a sample to be detected, taking 1 mu L of DNA of a microorganism to be detected as a reaction template, adding 49 mu L of detection solution in the kit of claim 3 or 4 for recombinase polymerase amplification, and carrying out reaction procedures as follows: amplifying at 39 ℃ for 20 min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112575113A (en) * | 2020-12-28 | 2021-03-30 | 福建省农业科学院植物保护研究所 | Phytophthora infestans LFD-RPA detection primer and probe and application thereof |
| CN114045358A (en) * | 2021-10-15 | 2022-02-15 | 南京农业大学 | Primer composition for detecting twelve potato disease pathogenic bacteria based on loop-mediated isothermal amplification technology and detection method |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020000043A (en) * | 2000-06-20 | 2002-01-04 | 하서현 | A DNA marker for Phytophthora infestans in Potato and Tomato |
| CN101643788A (en) * | 2009-05-12 | 2010-02-10 | 福建省农业科学院植物保护研究所 | Detection primer of potato late blight bacterium molecules and use method thereof |
| CN101921832A (en) * | 2010-04-13 | 2010-12-22 | 南京农业大学 | Primer, kit and detection method for detecting phytophthora infestans |
| CN104313175A (en) * | 2014-11-13 | 2015-01-28 | 南京林业大学 | LAMP detection primer composition for phytophthora infestans and LAMP detection kit and LAMP detection method of LAMP detection primer composition |
| CN106636378A (en) * | 2016-12-06 | 2017-05-10 | 福建省农业科学院植物保护研究所 | LAMP (Loop-mediated Isothermal Amplification) primer combination for detecting tomato phytophthora infestans and application |
| CN106987653A (en) * | 2017-06-09 | 2017-07-28 | 福建省农业科学院植物保护研究所 | A kind of phytophthora infestans LAMP detection primer and its visible detection method |
| CN108624655A (en) * | 2017-03-17 | 2018-10-09 | 河北农业大学 | Phytophthora infestans real-time fluorescence LAMP detection method and kit |
| CN108676851A (en) * | 2018-07-10 | 2018-10-19 | 南京农业大学 | It is a kind of detection phytophthora infestans loop-mediated isothermal amplification (LAMP) primer composition and its application |
| CN109943624A (en) * | 2019-03-14 | 2019-06-28 | 南京林业大学 | A kind of method and its primer special and probe combinations based on RPA- Sidestream chromatography technology detection soybean phytophthora |
| JP6562586B1 (en) * | 2019-01-30 | 2019-08-21 | 国立大学法人宇都宮大学 | Primer set for amplifying a nucleic acid derived from a disease-causing fungus and a method for detecting the disease |
-
2019
- 2019-11-14 CN CN201911114857.5A patent/CN110878373A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020000043A (en) * | 2000-06-20 | 2002-01-04 | 하서현 | A DNA marker for Phytophthora infestans in Potato and Tomato |
| CN101643788A (en) * | 2009-05-12 | 2010-02-10 | 福建省农业科学院植物保护研究所 | Detection primer of potato late blight bacterium molecules and use method thereof |
| CN101921832A (en) * | 2010-04-13 | 2010-12-22 | 南京农业大学 | Primer, kit and detection method for detecting phytophthora infestans |
| CN104313175A (en) * | 2014-11-13 | 2015-01-28 | 南京林业大学 | LAMP detection primer composition for phytophthora infestans and LAMP detection kit and LAMP detection method of LAMP detection primer composition |
| CN106636378A (en) * | 2016-12-06 | 2017-05-10 | 福建省农业科学院植物保护研究所 | LAMP (Loop-mediated Isothermal Amplification) primer combination for detecting tomato phytophthora infestans and application |
| CN108624655A (en) * | 2017-03-17 | 2018-10-09 | 河北农业大学 | Phytophthora infestans real-time fluorescence LAMP detection method and kit |
| CN106987653A (en) * | 2017-06-09 | 2017-07-28 | 福建省农业科学院植物保护研究所 | A kind of phytophthora infestans LAMP detection primer and its visible detection method |
| CN108676851A (en) * | 2018-07-10 | 2018-10-19 | 南京农业大学 | It is a kind of detection phytophthora infestans loop-mediated isothermal amplification (LAMP) primer composition and its application |
| JP6562586B1 (en) * | 2019-01-30 | 2019-08-21 | 国立大学法人宇都宮大学 | Primer set for amplifying a nucleic acid derived from a disease-causing fungus and a method for detecting the disease |
| CN109943624A (en) * | 2019-03-14 | 2019-06-28 | 南京林业大学 | A kind of method and its primer special and probe combinations based on RPA- Sidestream chromatography technology detection soybean phytophthora |
Non-Patent Citations (4)
| Title |
|---|
| MEHRAN KHAN ET AL: "Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene", 《FRONT MICROBIOL》 * |
| SI AMMOUR M ET AL: "Development of Real-Time Isothermal Amplification Assays for On-Site Detection of Phytophthora infestans in Potato Leaves", 《PLANT DISEASE》 * |
| 孙祖越 周莉主编: "《药物生殖与发育毒理学》", 31 January 2015, 上海科学技术出版社 * |
| 孟军: "以Ypt1基因为靶标的致病疫霉菌Phytophthora infestans的分子检测", 《江苏农业学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112575113A (en) * | 2020-12-28 | 2021-03-30 | 福建省农业科学院植物保护研究所 | Phytophthora infestans LFD-RPA detection primer and probe and application thereof |
| CN114045358A (en) * | 2021-10-15 | 2022-02-15 | 南京农业大学 | Primer composition for detecting twelve potato disease pathogenic bacteria based on loop-mediated isothermal amplification technology and detection method |
| CN114045358B (en) * | 2021-10-15 | 2023-08-18 | 南京农业大学 | Primer composition for detecting twelve potato disease pathogenic bacteria based on loop-mediated isothermal amplification technology and detection method |
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