CN114277166A - RPA detection primer, probe and detection method for melon bacterial fruit blotch - Google Patents
RPA detection primer, probe and detection method for melon bacterial fruit blotch Download PDFInfo
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Abstract
An RPA detection primer, a probe and a detection method of melon bacterial fruit blotch germs comprise an RPA primer pair Ac-F1, Ac-R1 and a detection probe Ac-nfo of the melon bacterial fruit blotch germs, wherein the amplification products of the RPA primer pair Ac-F1 and Ac-R1 are 342bp in size, the 5 'tail end of the primer Ac-R1 is marked with biotin, the 5' tail end of the detection probe Ac-nfo is marked with FAM, the 3 'tail end of the detection probe Ac-nfo is marked with C3Spacer, and the 32bp position at the 5' tail end in the probe is modified by dSpacer; the RPA primer group has high detection sensitivity, and the detection limit reaches 2.5 multiplied by 104cfu/mL; provides a detection method for detecting melon bacterial fruit blotch germs based on RPA combined with lateral flow test paper, establishes a rapid isothermal visual detection method for melon bacterial fruit blotch germs, can complete the detection of samples within 15-20min, realizes the detection under the constant temperature environment,the rapid diagnosis, identification and detection of the bacterial fruit blotch of melons have the advantages of high sensitivity, good specificity and the like, and provide technical support for quarantine, detection, prevention and control of the diseases.
Description
Technical Field
The invention belongs to the technical field of RPA detection, and particularly relates to an RPA detection primer, a probe and a detection method for melon bacterial fruit blotch.
Background
The pathogeny of Bacterial Fruit Blotch (BFB) of melons is acidophilic watermelon (Acidovorax citrulli, A.citrulli for short), seriously harms cucurbitaceous crops such as watermelon, melon and the like, and is an important Bacterial quarantine disease. The disease is a disease which can be transmitted through seeds, leaves, fruits and seeds in the growth period can be diseased, the yield and the quality of the fruits are affected, and destructive damage can be caused in serious cases. In 90 years of the last century, the reports are first reported in China, and then occasionally reported in Xinjiang, Hainan, Shanghai, Zhejiang and other places, and in the List of agricultural plant quarantine pests in China (2016) occurring in various regions, 12 provinces and cities occur in China.
The disease has already started to seriously threaten the industrial development of important cucurbitaceae crops such as watermelons, melons and the like in China, and the disease symptoms caused by the disease are very similar to the symptoms of another bacterial leaf spot and are difficult to distinguish by naked eyes. The common detection methods for the bacterial diseases comprise methods such as symptom identification, PCR detection, nested PCR detection, enzyme-linked immunosorbent assay, quantitative PCR (Qpcr) detection and the like, and the methods have low sensitivity, long time consumption or expensive reagent instruments. Therefore, a rapid and simple method for detecting and diagnosing the bacterial disease is urgently needed to be found.
Disclosure of Invention
The invention aims to provide an RPA detection primer, a probe and a detection method of melon bacterial fruit blotch, wherein the specific primer and the probe are designed according to the specific gene sequence of pathogenic bacteria, the rapid isothermal visual detection method of melon bacterial fruit blotch is established, rapid diagnosis, identification and detection of melon bacterial fruit blotch are realized under the constant temperature environment, and the RPA detection primer, the probe and the detection method have the advantages of high sensitivity, good specificity and the like, and provide technical support for quarantine, detection and prevention and control of the disease.
In order to achieve the purpose, the invention provides the following technical scheme:
an RPA primer group for detecting melon bacterial fruit blotch, which comprises an RPA primer pair Ac-F1, Ac-R1 and a detection probe Ac-nfo of melon bacterial fruit blotch, wherein the amplification product size of the RPA primer pair Ac-F1 and Ac-R1 of the melon bacterial fruit blotch is 342bp, the 5 'end of the primer Ac-R1 is labeled with biotin, the 5' end of the detection probe Ac-nfo is labeled with FAM, the 3 'end is labeled with C3Spacer, the 32bp position at the 5' end in the probe is modified by dSpacer, and the specific nucleotide sequence is as follows:
Ac-F1:5’-ACAACGATGTTGTCATTAAATTGGCGAGGATGG-3’；
Ac-R1:5’-Biotin-TGAAGTCCAAGCCAGCTGAAAGTACTACCTTTG-3’；
Ac-nfo:5’-[6FAM]-GCCGCATTGAAAACCGTCGTCGTGATGGAGA[THF]GACGCCGCCAGAAGC-[C3Spacer]-3’。
an RPA kit for detecting melon bacterial fruit blotch, which comprises an RPA primer pair Ac-F1, Ac-R1 and a probe Ac-nfo of the melon bacterial fruit blotch.
Furthermore, the RPA kit for detecting the bacterial fruit blotch of cucurbit also comprises a corresponding nucleic acid detection test strip.
A detection method for detecting melon bacterial fruit blotch based on RPA combined with lateral flow test paper comprises the following steps:
a. adding template DNA of melon bacterial fruit blotch germ into an RPA reaction system, wherein the RP primer pair is Ac-F1, Ac-R1 and a detection probe Ac-nfo;
incubating the reaction tube at a constant temperature of 36-38 ℃ for 15-20min, sucking the reaction liquid in the reaction tube, and uniformly mixing the reaction liquid with sterile deionized water to a new reaction tube;
c. and (c) taking sterile water as a negative control, inserting the testing end of the nucleic acid detection test strip into the reaction tube mixed in the step (b), reacting for 2-5min, and judging a detection result according to a test strip color development result after the reaction is finished.
Further, in the RPA reaction system in step a): the final concentrations of Ac-F1 and Ac-R1 were 0.4 pmol/. mu. L, Ac-nfo 0.12 pmol/. mu.L, and magnesium acetate 14 mmol/. mu.L, respectively.
Preferably, in the RPA reaction system: the final concentrations of Ac-F1 and Ac-R1 were 0.4 pmol/. mu. L, Ac-nfo 0.12 pmol/. mu.L, and magnesium acetate 14 mmol/. mu.L, respectively.
And the template DNA of the melon bacterial fruit blotch is melon bacterial liquid of the melon bacterial fruit blotch.
Further, in the step a), plant leaves are collected, sterile water is added, ground juice is obtained through full grinding, and PRA detection is directly carried out by taking the ground juice as template DNA.
In the invention, the target sequence for detecting the melon bacterial fruit blotch germ is a specific sequence CP029373.1(896040-896693) selected from the whole genome sequence of the melon bacterial fruit blotch germ, the length is 651bp, the specific nucleotide sequence is shown as SEQ ID NO.1, the specific nucleotide sequence is an Open Reading Frame (ORF) of a hypothetical gene, and the existing detection technology does not detect the sequence. The invention designs a primer and an nfo probe with good specificity and high sensitivity for the specific sequence of the whole genome of the pathogen, and applies the RPA technology to carry out constant-temperature amplification and nucleic acid test paper detection on the specific sequence, thereby being capable of being efficiently and visually used for detecting the pathogen.
Compared with the prior art, the invention has the following beneficial effects:
the RPA technology has the advantages of high sensitivity, good specificity, no need of complex or precise instruments and the like, the primer pair Ac-F1 and Ac-R1 have high sensitivity to the pathogenic bacteria suspension, and the detection limit reaches 2.5 multiplied by 104cfu/mL。
The detection method for detecting the bacterial fruit blotch of melons based on Recombinase Polymerase Amplification (RPA) combined with lateral flow test paper can be used for rapid detection of field samples, is particularly suitable for rapid screening of large-batch samples of enterprises, basic levels, farmers and the like without complex instruments and equipment, and has the advantages of simplicity, rapidness, visual results and the like.
Compared with the existing detection technology, the detection method can finish the detection of the sample within 15-20min, so that enterprises, grass roots, farmers and the like can realize the identification, diagnosis and detection of diseases through the method, the planting risk is reduced, and the economic benefit of the enterprises, grass roots, farmers and the like is guaranteed. Meanwhile, the method provides technical support for government supervision departments such as quarantine enforcement and the like, and has important significance for controlling quarantine diseases.
Drawings
FIG. 1 is a screening of different RPA primers in example 1 of the present invention, wherein: m is 2k plus II Marker, A-O is the result of 1% gel electrophoresis of amplified A.citrulli DNA by different primer pairs.
FIG. 2 is a diagram of the specific sequence primer fragment of the present invention.
FIG. 3 is a diagram showing the specific detection of the RPA primers in example 2 of the present invention, wherein: m is 2k plus II Marker, A is the result of 1% gel electrophoresis of the primer pair in the D band in FIG. 1, A.citrulli DNA, B-I is the result of 1% gel electrophoresis of the primer pair in the D band in FIG. 1, the reference bacterial strains are Pseudomonas syringae (melon bacterial leaf spot), Erwinia carotovora (Chinese cabbage soft rot), E.chrysogenum (rice basic rot), Xanthomonas oryzae (rice bacterial leaf spot), Microbacterium arborescens (Microbacterium arborescens), Pectobacterium carotovorum subsp.
Fig. 4 is a sensitivity detection chart of RPA in embodiment 3 of the present invention, in which: m is 2k plus II Marker; A-I is at a concentration of 2.5X 109-2.5×101cfu/mL of A.citrulli bacterial liquid; negative control.
FIG. 5 is a sensitivity test chart of the lateral flow test strip in example 3 of the present invention, wherein: m is 2k plus II Marker; A-I is at a concentration of 2.5X 109-2.5×101cfu/mL of A.citrulli bacterial liquid; NC is negative control.
FIG. 6 is a LF-RPA detection diagram of a specimen of melon bacterial fruit blotch artificially inoculated in a simulated field with lateral flow test paper in example 3 of the present invention, wherein: A. b is a positive control, C, D is laboratory inoculation melon sample detection, E is a healthy plant, and F is a negative control.
Detailed Description
The invention is further illustrated by the following examples and figures.
Example 1 design and screening of specific primer pairs
Designing a primer:
for the ORF specific sequence CP029373.1(896040-896693) with the length of 651bp of the melon bacterial fruit blotch germ, the concrete nucleotide sequence is shown as SEQ ID NO.1, 12 pairs of different RPA primers are designed, wherein the TM value of the primer pair is set between 65-75 ℃, the amplification length is between 150-294bp, and the alternative RPA primer pairs are respectively configured into solutions with the concentration of 10 mu M.
The specific sequences of 13 different RPA primer pairs were designed, see table 1, from 5 'to 3'.
TABLE 1
The screening process comprises the following steps:
separating and purifying melon bacterial fruit blotch, extracting DNA for later use according to a conventional method, and carrying out PRA detection by taking the extracted DNA as a template, wherein an RPA reaction system is as follows: 29.5 mu L of re-solution, 13.0 mu L of sterile deionized water, 2.0 mu L of each forward/reverse primer of 10 mu mol/L, 1.0 mu L of template of 2.5 mu L, DNA of magnesium acetate solution of 280mmol/L and 50.0 mu L of total volume; the RPA reaction conditions were: keeping the temperature at 37 ℃ or bathing for 15 min.
50.0. mu.L of phenol chloroform isoamyl alcohol (25: 24: 1) was added to the RPA reaction tube, and after mixing, centrifugation was carried out at 12000rpm for 1min, and 3.0. mu.L of the mixture was subjected to 1% agarose gel electrophoresis, and the results of the RPA analysis are shown in FIG. 1.
As can be seen from FIG. 1, there are many 13 pairs of primer pairs designed by the present invention, which can specifically amplify target pathogens, the concentration of the amplification product is high, the primer pair D, E, F, etc., has a good amplification effect on the DNA of the pathogens, high brightness, no miscellaneous bands, good specificity, high efficiency and high sensitivity, and the present invention selects the D primer pair, and is named as the RPA primer pair Ac-F1 and Ac-R1, see FIG. 2.
Example 2 specificity test of primers for detecting bacterial fruit blotch of melon
DNA templates were from reference bacteria: pseudomonas syringae (bacterial leaf spot of melon), Erwinia carotovora (soft rot of chinese cabbage), e.chrysogenanthemi (basic rot of rice), Xanthomonas oryzae (bacterial leaf spot of rice), Microbacterium arborescens (micro-trelliferae), petobacterium carotovorum subsp.
Purifying melon bacterial fruit blotch and each reference bacterium, respectively preparing primer pairs Ac-F1 and Ac-R1 into solutions with the concentration of 10.0 mu mol/L, extracting DNA for later use according to a conventional method, and carrying out RPA detection by taking the extracted DNA as a template, wherein the RPA reaction system is as follows: 29.5 mu L of re-solution, 13.0 mu L of sterile deionized water, 2.0 mu L of each forward/reverse primer of 10 mu mol/L, 1.0 mu L of template of 2.5 mu L, DNA of magnesium acetate solution of 280mmol/L and 50.0 mu L of total volume; the RPA reaction conditions were: keeping the temperature at 37 ℃ or bathing for 15 min.
50.0. mu.L of phenol chloroform isoamyl alcohol (25: 24: 1) was added to each RPA reaction tube, and after mixing, centrifugation was carried out at 12000rpm for 1min, and 3. mu.L of the mixture was subjected to 1% agarose gel electrophoresis, and the results of RPA were shown in FIG. 3.
As can be seen from FIG. 3, the RPA primer pairs Ac-F1 and Ac-R1 have good amplification effect on the DNA of the melon bacterial fruit blotch pathogen, have good specificity, and have no amplification band on the reference strain.
The test shows that: the RPA primer pair designed by the invention has good specificity for Ac-F1 and Ac-R1, has no amplified band for a reference strain, and the selected primer pair can be used for RPA detection of melon bacterial fruit blotch.
EXAMPLE 3 sensitivity test for detecting primer and test paper for detecting pathogenic bacteria RPA of melon bacterial fruit blotch
1) Purifying melon bacterial fruit blotch, culturing, and preparing into 2.5 × 10 with sterile water9-2.5×101cfu/mL of bacterial liquid suspension;
2) according to the primer specificity results of examples 1 and 2, probes are designed and detected in the amplified fragments of the primer pairs Ac-F1 and Ac-R1, are modified and named as Ac-nfo, and the primer pairs Ac-F1, Ac-R1 and the probes Ac-nfo are respectively prepared into solutions with the concentration of 10 mu M;
3) PRA detection is carried out by taking the bacterial liquid suspension prepared in the step 1) as a template, and an RPA reaction system is as follows: 29.5 mu L of re-solution, 12.4 mu L of sterile deionized water, 0.6 mu L of Ac-nfo with 10 mu mol/L, 2.0 mu L of Ac-F1/Ac-R1 with 10 mu mol/L, 1.0 mu L of magnesium acetate solution with 280mmol/L of 2.5 mu L, DNA template and 50.0 mu L of total volume. The RPA reaction conditions were: keeping the temperature at 36-38 deg.C or in water bath for 10-15 min.
4) To the RPA reaction tube was added 50.0 μ L phenol chloroform isoamyl alcohol (25: 24: 1) after mixing, the mixture was centrifuged at 12000rpm for 1min, and 3. mu.L of the mixture was subjected to 1% agarose gel electrophoresis, and the results of RPA were shown in FIG. 4.
As is clear from FIG. 4, the primer pair Ac-F1/Ac-R1 has high sensitivity for RPA amplification of a bacterial suspension of the pathogenic bacteria.
5) Sucking 10 mu L of reaction liquid from the reaction tube in the step 3), and uniformly mixing the reaction liquid with 90 mu L of sterile deionized water into a new 200 mu L tube;
6) inserting the test end of the nucleic acid test strip into the tube in the step 5, judging the test result according to the color development result of the test strip, and judging the result within 5min, wherein the test result of the test strip is shown in figure 5.
As can be seen from FIG. 5, the primer pair Ac-F1/Ac-R1 has high detection sensitivity on the bacterial suspension test paper of the pathogenic bacteria.
Therefore, the primer pair Ac-F1 and Ac-R1 designed by the invention has high sensitivity to the pathogenic bacteria suspension, and the detection limit reaches 2.5 multiplied by 104cfu/mL, the test paper is consistent with the RPA detection limit, which shows that the primers and the detection probes of the method can be used for PRA detection and test paper detection of bacterial liquid without extracting DNA.
EXAMPLE 4 detection of bacterial fruit blotch disease in melon plants
1) Inoculating a disease-susceptible melon plant with purified melon bacterial fruit blotch germs, collecting symptomatic leaves after the plant is diseased, and mixing the leaves according to the mass-volume ratio of 1: 10, adding sterile water into the diseased leaf tissue, and fully grinding to obtain ground juice as an object to be detected; selecting the primer of the embodiment 1 and the probe of the embodiment 3, and respectively preparing a primer pair Ac-F1/Ac-R1 and the probe Ac-nfo into solutions with the concentration of 10 mu M;
2) carrying out PRA detection by taking the ground juice prepared in the step 1) as a template, wherein an RPA reaction system is as follows: 29.5 mu L of redissolution, 12.2 mu L of sterile deionized water, 2.1 mu L of each of 10 mu mol/L Ac-nfo, 0.6 mu L of 10 mu mol/L Ac-F1/Ac-R1, 1.0 mu L of 280mmol/L magnesium acetate solution 2.5 mu L, DNA template, and 50 mu L of total volume, wherein the RPA reaction conditions are as follows: keeping the temperature at 37 ℃ or bathing for 15 min.
3) Sucking 8-15 mu L of reaction liquid from the reaction tube in the step 2), diluting the reaction liquid by 10 times with sterile deionized water, and uniformly mixing the diluted reaction liquid and the sterile deionized water into a new 200 mu L tube;
4) inserting the test end of the nucleic acid test strip into the tube in the step 3), judging the test result according to the color development result of the test strip, wherein the result needs to be judged within 5min, and the test result of the test strip is shown in figure 6.
FIG. 6 is a LF-RPA test chart of artificially inoculated bacterial fruit blotch of melon, and from FIG. 6, it can be seen that both the A, B (positive control) and C, D (laboratory inoculated melon samples) test strips appear as two dark bands, and the E (healthy sample) and F (negative control) only show the dark control band.
Therefore, the primer, the probe and the detection method for detecting the bacterial fruit blotch of melons based on Recombinase Polymerase Amplification (RPA) combined with lateral flow test paper can accurately detect pathogenic bacteria of the inoculated melon bacterial fruit blotch and can be conveniently used for directly detecting disease samples.
The primers Ac-F1 and Ac-R1 and the probe Ac-nfo for detecting the bacterial fruit blotch of cucurbit based on Recombinase Polymerase Amplification (RPA) combined with lateral flow test paper and the detection method can be used for directly detecting the leaf grinding juice without extracting DNA, have good specificity, are not influenced by plant tissues, and are a rapid and convenient detection method.
Sequence listing
<110> Shanghai city academy of agricultural sciences
<120> RPA detection primer, probe and detection method of melon bacterial fruit blotch
<130> 2111207
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 651
<212> DNA
<213> Acidovorax citrulli bacterium
<400> 1
atgactgttt ctgtgctgct tgacaacgat gttgtcatta aattggcgag gatggacgcc 60
taccttgatg cccttttgag catggggctg acacctcgac aggtcggctc ccttaagccg 120
atgctgcggt acatggggct atatgacaat agtcgtctcg tccgatttac ggacaacgca 180
gcggaggcgg ctcggctcct tgccgcattg aaaaccgtcg tcgtgatgga gatgacgccg 240
ccagaagccg cgctcgcgac cagggtcgca aaggtagtac tttcagctgg cttggacttc 300
aatgaaggtg agctgatgat gtgcgtggtc gcggtcatca gaggcaacct agacttctgt 360
actgcagaca agcgagcact gaacagcttg ccgggtgttg aagcttgctg ggcggatgta 420
gggaacatgc gcggcagatg catttgcttc gagcaggtgt tctacgctct atgccaaaaa 480
ttcggtttgg cgcgcgtcac ccaggctgtg cgaacttgtc ccactacgga gaaatccatg 540
acgcgacttc acaacgtctc ggttgcacgc gggcaggacg atttcctacg cgcacttgat 600
ctgtacgtcc aagaccgaat cagccggccc gcacgaggct ggctgaaatc g 651
Claims (8)
1. An RPA primer group for detecting melon bacterial fruit blotch, which comprises an RPA primer pair Ac-F1, Ac-R1 and a detection probe Ac-nfo of melon bacterial fruit blotch, wherein the amplification product size of the RPA primer pair Ac-F1 and Ac-R1 of the melon bacterial fruit blotch is 342bp, the 5 'end of the primer Ac-R1 is labeled with biotin, the 5' end of the detection probe Ac-nfo is labeled with FAM, the 3 'end is labeled with C3Spacer, the 32bp position at the 5' end in the probe is modified by dSpacer, and the specific nucleotide sequence is as follows:
Ac-F1:5’-ACAACGATGTTGTCATTAAATTGGCGAGGATGG-3’；
Ac-R1:5’-Biotin-TGAAGTCCAAGCCAGCTGAAAGTACTACCTTTG-3’；
Ac-nfo:5’-[6FAM]-GCCGCATTGAAAACCGTCGTCGTGATGGAGA[THF]GACGCCGCCAGAAGC-[C3Spacer]-3’。
2. an RPA kit for detecting melon bacterial fruit blotch, comprising the RPA primer pair Ac-F1, Ac-R1 and probe Ac-nfo of melon bacterial fruit blotch as claim 1.
3. The RPA kit for detecting bacterial fruit blotch of cucurbit of claim 2, wherein said kit further comprises a corresponding nucleic acid detection test strip.
4. A detection method for detecting melon bacterial fruit blotch based on RPA combined with lateral flow test paper comprises the following steps:
a. adding template DNA of melon bacterial fruit blotch pathogen, the RPA primer pair Ac-F1, Ac-R1 and the detection probe Ac-nfo as the claim 1 into the RPA reaction system;
incubating the reaction tube at a constant temperature of 36-38 ℃ for 15-20min, sucking the reaction liquid in the reaction tube, and uniformly mixing the reaction liquid with sterile deionized water to a new reaction tube;
c. and (c) taking sterile water as a negative control, inserting the testing end of the nucleic acid detection test strip into the reaction tube mixed in the step (b), reacting for 2-5min, and judging the control of the detection result according to the test strip color development result after the reaction is finished.
5. The method according to claim 4, wherein the RPA reaction system in step a) comprises: the final concentrations of Ac-F1 and Ac-R1 were 0.4 pmol/. mu.L each, and 14 mmol/. mu.L magnesium acetate.
6. The method for detecting bacterial fruit blotch citrullinia according to claim 4, wherein in step a) in the RPA reaction system: the final concentrations of Ac-F1 and Ac-R1 were 0.4 pmol/. mu. L, Ac-nfo 0.12 pmol/. mu.L, and magnesium acetate 14 mmol/. mu.L, respectively.
7. The method for detecting melon bacterial fruit blotch disease according to any one of claims 4-6 wherein the template DNA of melon bacterial fruit blotch disease is melon bacterial fruit blotch liquid.
8. The method for detecting melon bacterial fruit blotch disease according to any one of claims 4-6 wherein in step a), plant leaves are collected, sterile water is added, the mixture is ground sufficiently to obtain ground juice, and the ground juice is used as template DNA for PRA detection directly.
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| CN109517911A (en) * | 2017-09-18 | 2019-03-26 | 上海市农业科学院 | The double PCR detection primer and detection method of melon bacterial Acidovorax avenae subsp and leaf spot fungi |
| CN113151522A (en) * | 2021-04-27 | 2021-07-23 | 中国检验检疫科学研究院 | LFD-RPA technology-based rice bacterial leaf streak germ detection kit, primer probe composition and application thereof |
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| CN109517911A (en) * | 2017-09-18 | 2019-03-26 | 上海市农业科学院 | The double PCR detection primer and detection method of melon bacterial Acidovorax avenae subsp and leaf spot fungi |
| CN113151522A (en) * | 2021-04-27 | 2021-07-23 | 中国检验检疫科学研究院 | LFD-RPA technology-based rice bacterial leaf streak germ detection kit, primer probe composition and application thereof |
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| CN115125319A (en) * | 2022-05-17 | 2022-09-30 | 上海师范大学 | Molecular marker of rice amylose content gene ACF1 allele and primer and method for detecting same |
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