CN113430296B - Dual PCR primer and detection method for synchronously and rapidly detecting peronophythora litchi and colletotrichum - Google Patents
Dual PCR primer and detection method for synchronously and rapidly detecting peronophythora litchi and colletotrichum Download PDFInfo
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- CN113430296B CN113430296B CN202110805657.5A CN202110805657A CN113430296B CN 113430296 B CN113430296 B CN 113430296B CN 202110805657 A CN202110805657 A CN 202110805657A CN 113430296 B CN113430296 B CN 113430296B
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Abstract
The invention discloses a dual PCR primer and a detection method for synchronously and rapidly detecting peronophythora litchi and colletotrichum. The primers include PlF1/PlR1 and CgF1/CgR1 primer pairs. The invention takes sample DNA containing peronophythora litchi and colletotrichum litchi as a template, adds the double PCR primers into the same PCR reaction system, and obtains specific amplification products of 694bp and 286bp respectively through double PCR amplification and agarose gel electrophoresis. The dual PCR detection primer and the detection method for peronophythora litchi and colletotrichum litchi established by the primers have the advantages of strong specificity, high sensitivity, and rapid and accurate synchronous detection and identification of two pathogenic bacteria in litchi in a system, can be used for early diagnosis of field diseases and monitoring and identification of pathogenic bacteria, and effectively guide prevention and control of peronophythora litchi and colletotrichum.
Description
Technical Field
The invention belongs to the technical field of molecular detection of plant pathogens, and particularly relates to a dual PCR primer and a detection method for synchronously and rapidly detecting peronophythora litchi and colletotrichum.
Background
Litchi (lichi chinensis) has good taste, beautiful color and rich nutrition, is known as the king of fruits, is a fruit in tropical and subtropical regions, is distributed in provinces such as Hainan, Fujian, Guangxi, Guangdong, Taiwan and the like, and has important economic value. Peronophythora litchii and litchi anthracnose caused by peronotrichum peronospora litchii and litchi anthracnose are the most important diseases in litchi production at present, and the litchi yield, the litchi quality, the fresh fruit storage and transportation and the external selling are seriously influenced. The two litchi diseases are all serious in Fujian, Guangdong, Guangxi, Hainan, Yunnan, Taiwan and the like at present, and the direct consequence of the damage is that the litchi yield and the fruit quality are reduced, so that the development of the litchi industry is restricted. The two diseases are serious damages to nearly mature fruits, tender tips, leaves, spica, fruiting branches, fruit stalks and young fruits, cause a great amount of fruit drop and rotten fruits, the yield loss is up to more than 80 percent, and even the fruits are harvested absolutely, so that huge economic loss is caused. In recent years, the prevalence frequency and prevalence degree of the two litchi diseases gradually increase, and the two litchi diseases become an important obstacle in litchi production. Although the pathogenic bacteria of the two litchi diseases are different, the pathogenic conditions of the two litchi diseases are similar, the litchi parts (such as fruits close to maturity, young shoots, leaves, spica, fruiting twigs, fruit stalks and young fruits) are basically harmed, and the litchi diseases have the characteristics of large fruit drop and rotten fruits caused by early symptoms and latent infection. Therefore, in view of the huge destructiveness of the peronophythora litchi and the anthracnose of litchi, the method has important significance for establishing early rapid detection, identification and distinguishing pathogenic bacteria causing the two diseases and effectively guiding disease control in order to prevent the disease from spreading.
The detection method of plant pathogenic bacteria generally has biological, serological and molecular biological means. However, as for peronophythora litchi and colletotrichum litchi, because the two germs cause symptoms on litchi leaves and fruits, the early symptoms are very similar, and the 2 diseases are very difficult to be identified only from the symptoms; serology has the advantages of rapidness, simplicity, convenience, high flux and the like, but has higher requirements on the specificity of the prepared antibody; the molecular detection technology is widely applied to the rapid detection and identification of plant pathogenic bacteria and the early detection and diagnosis of diseases due to the advantages of rapidness, sensitivity, specificity, accuracy and the like. With the development of biotechnology, nucleic acid-based multiplex detection technologies play an increasingly important role in the field of nucleic acid diagnosis, mainly including multiplex PCR, nucleic acid isothermal amplification and gene chip-based multiplex nucleic acid detection technologies, which can simultaneously detect multiple targets, and have the characteristics of high speed, high throughput, less sample consumption and the like. The multiplex PCR is a PCR derivation method, its basic principle, reaction reagent and operation are identical to those of conventional PCR, and its difference lies in that the multiplex PCR system contains 2 pairs or several pairs of primers, and respectively amplifies different templates, and can simultaneously detect several targets. The double PCR (duplex polymerase chain reaction) used by the invention is a PCR technology for amplifying a plurality of target fragments in different regions of 2 DNA templates or the same template in the same reaction system, can realize the detection of 2 pathogenic bacteria at one time, and is characterized by short time consumption and improved accuracy and sensitivity. The double PCR technology has the advantages of saving time, reducing cost and improving efficiency, and has wide application prospect in identification and detection of plant pathogenic bacteria, especially detection of symptom similarity or pathogenic bacteria complex infection.
At present, molecular detection technologies for 2 types of pathogenic bacteria, namely peronophythora litchi and colletotrichum litchi, are reported at home and abroad, but the researches only detect one type of pathogenic bacteria independently, and reports for synchronously distinguishing and detecting peronophythora litchi and colletotrichum litchi are not seen at present, and the dual PCR technology can simultaneously detect 2 types of pathogenic bacteria in one reaction, so that the detection time can be greatly saved, the detection efficiency can be improved, and the detection cost can be reduced. Therefore, the establishment of a simple, rapid, specific and sensitive dual PCR technology for distinguishing and detecting peronophythora litchi and colletotrichum litchi is urgently needed in the field.
Disclosure of Invention
In view of the defects of the prior art, the invention provides a dual PCR primer and a detection method for synchronously and rapidly detecting peronophythora litchi and colletotrichum.
The technical scheme of the invention mainly comprises the following contents:
according to the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, double PCR primers for synchronously and rapidly detecting phytophthora litchi (Peronothora litchii) and Colletotrichum gloeosporioides (Colletotrichum gloeosporioides) are designed, the double PCR primers consist of primers PlF1/PlR1 and CgF1/CgR1, and the nucleotide sequences of the primers are as follows:
PlF1：5’-TTCGGTCTGAACTAGTAGCT-3’(SEQ ID NO.3)
PlR1：5’-TTCAGCGGGTAATCTTGCCT-3’(SEQ ID NO.4)
CgF1：5’-TCTTCTGAGTGGTACAAGC-3’(SEQ ID NO.5)
CgR1：5’-TAAAGTTACTACGCAAAGGA-3’(SEQ ID NO.6)。
the invention also relates to a kit, which comprises the dual PCR special primers and other necessary PCR reaction reagents. The kit and the primers can be applied to detecting and distinguishing peronophythora litchi and colletotrichum litchi.
On the other hand, the invention also provides a method for synchronously detecting peronophythora litchi and colletotrichum litchi, which comprises the following steps:
1) extracting DNA of a sample to be detected;
2) taking the DNA of a sample to be detected as a template, and carrying out double PCR amplification by using the double PCR primer;
3) and (4) judging a result: and (4) carrying out agarose gel electrophoresis on the double PCR amplification product, and judging the result according to the size of the amplification product. If the 694bp single-purpose strip is obtained by amplification, the sample to be detected contains peronophythora litchi; the 286bp single-purpose strip obtained by amplification indicates that the sample to be detected contains the lichee anthracnose pathogen; the 2 bands with 694bp and 286bp respectively obtained by amplification show that the sample to be detected contains peronophythora litchi and colletotrichum litchi.
Preferably, the reaction system for the double PCR amplification is:
2 XTaq PCR Master Mix II 20. mu.L, 10. mu.M primer PlF1/PlR1 total 0.5. mu.L, 10. mu.M primer CgF1/CgR1 total 2. mu.L, 50. mu.g/mL-1mu.L of DNA template and 40. mu.L of sterile water.
Preferably, the reaction procedure for the double PCR amplification is: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles, and extension at 72 ℃ for 5 min.
The invention has the following effects:
the method is suitable for rapid and reliable detection and identification for synchronously distinguishing peronophythora litchi and colletotrichum litchi in plants, and has important practical value for disease control caused by distinguishing peronophythora litchi and colletotrichum litchi in agricultural production.
Compared with the prior art, the invention has the following technical advantages and positive effects:
1. the specificity is strong: the detection method of the invention designs a pair of specific primer combinations for detection according to the characteristics of interspecies variation and mutual interval of conservation regions and evolution regions of ITS sequences conserved in seeds. Peronophythora litchi, colletotrichum litchi and other different kindred germs from different areas are verified, and the result has strong specificity;
2. the practicability is good: the designed specific primer combination can be used for high-sensitivity rapid molecular detection of peronophythora litchi and colletotrichum litchi, and compared with the traditional symptom identification, pathogenic bacterium form identification and common single primer PCR or LAMP, the established dual PCR detection method has strong practicability and can meet the requirements of rapid and reliable detection and identification of peronophythora litchi and colletotrichum litchi in a strain plant;
3. the operation is simple, convenient and quick: by applying the method, the result can be judged after DNA extraction, PCR amplification and conventional agarose gel electrophoresis of the peronophythora litchi and/or the anthracnose of litchi, and restriction enzyme digestion of the amplified product is not needed. Generally, the whole detection process can be completed within hours;
4. labour saving and time saving, it is with low costs to detect: peronophythora litchi and colletotrichum litchi are the most important and common pathogenic bacteria in litchi production, the attack parts of the peronophythora litchi and colletotrichum litchi are similar, early symptoms are not easy to distinguish, and the conventional method needs pathogen separation and identification. The invention can detect two pathogenic bacteria at one time, complete the reaction within 2 hours, and save time and labor. Meanwhile, the invention does not need expensive fluorescent PCR instrument, and does not need to synthesize expensive probe and corresponding reagent, the detection cost is low, the operation is simple, and the invention is beneficial to popularization and application.
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FIG. 1: example 1 specific detection result of peronophythora litchi. The sequence of lanes in the figure is M: DL2000 DNA marker; 1: phytophthora litchi SCILZY 1; 2: phytophthora melonis; 3: phytophthora capsici leonian; 4: phytophthora strawberry; 5: phytophthora taro; 6: phytophthora parasitica; 7: phytophthora capsici; 8: phytophthora cinnamomi; 9: phytophthora cowpea; 10: phytopythium (citrus); 11: phytophthora infestans; 12: phytophthora sojae; 13: phytophthora crypticola; 14: phytophthora drechsler; 15: pythium aphanidermatum; 16: lichee anthrax; 17: lichee anthrax; 18: watermelon anthrax; 19: pepper anthrax; 20: phytophthora infestans;
FIG. 2: example 2 Peronophythora litchi nested PCR sensitivity test result graph. M: DL2000 DNA marker; 1-8, 20 μ L of the system respectively contains 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg and 1fg DNA;
FIG. 3: example 3 result graph of detecting peronophythora litchi and colletotrichum litchi by double PCR. M: DL2000 DNAmarker; 1: phytophthora litchi SCILZY 1; 2: peronophythora litchii XP-19; 3: litchi anthrax FZX-14; 4: litchi colletotrichum LZW-9; 5: mixing DNA of peronophythora litchi SCILZY1 and litchi colletotrichum FZX-14; 6: mixing DNA of Peronophythora litchi XP-19 and Laurel anthracis LZW-9; 7: negative control (dd.h)2O)。
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
Example 1: specific amplification of detection primer for peronophythora litchi
(1) Specific detection PCR reaction system: 25.0 μ L of each 0.5 μ L of Peronophythora litchi primer (PlF1/PlR1) containing 2 XTaq PCR Master mix12.5 μ L and 10 μmol/L of 50ng DNA template, the balance being dd H2And (4) complementing O. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles, and extension at 72 ℃ for 5 min.
PlF1：5’-TTCGGTCTGAACTAGTAGCT-3’
PlR1：5’-TTCAGCGGGT AATCTTGCCT-3’
(2) And (3) detection results: specificity of detection: apart from 694bp products which can be specifically amplified by phytophthora litchi DNAs from different regions, the DNA of 14 different oomycetes and 5 fungi are detected to be incapable of amplifying any product, and the specificity is very strong.
Example 2: sensitivity detection of primer on peronophythora litchi
DNA concentration dilution: the extracted peronophythora litchi genome DNA is diluted by adopting a series of concentrations after the concentration is determined by a spectrophotometer.
2. Sensitivity detection of peronophythora litchi
The extracted peronophythora litchi DNA is sequentially diluted into 8 different concentration gradients of 10 ng/mu L, 1 ng/mu L, 100 pg/mu L, 10 pg/mu L, 1 pg/mu L, 100 fg/mu L, 10 fg/mu L and 1 fg/mu L by a 10-fold concentration serial dilution method, and the sensitivity of the primers is measured by adopting nested PCR.
Respectively taking 1 mu L of phytophthora litchi genome DNA with different concentrations as templates, and carrying out first round of PCR amplification by using primers PlF1/PlR1, wherein the reaction system is 25.0 mu L, the reaction system comprises 2 XTaq PCR Master Mix12.5 mu L, 10 mu mol/L primers (PlF1/PlR1) which are respectively 1.0 mu L and 50ng of DNA template, and the deficient part is dd H2And (4) complementing O. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles, and extension at 72 ℃ for 5 min. After the reaction, the PCR product was diluted 100 times, and 1. mu.L of each was used as a DNA template for the second PCR amplification with primers (PlF2/PlR2), and 25. mu.L of the reaction system included: 2 XTaq PCR Master mix 12.5. mu.L, 10. mu. mol/L primers (PlF2/PlR2) 0.5. mu.L each, 25ng DNA template, the remainder being dd H2And (4) complementing O. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 59 ℃ for 30s, and extension at 72 ℃ for 30s for 28 cycles; extension at 72 ℃ for 5 min.
Pl F2：5’-CAGACGTGAAGTGTCTTGCA-3’(SEQ ID NO.7)
Pl R2：5’-TACATAGCTACGGTTCACCAA-3’(SEQ ID NO.8)
3. And (3) detection results: in a 25 mul reaction system, the phytophthora litchi genome DNA can obtain an obvious amplification strip, and the detection sensitivity can reach 10fg/25 mul reaction system.
Example 3: double PCR amplification for synchronously detecting peronophythora litchi and colletotrichum litchi
(1) Extracting the DNA of the sample to be detected, and carrying out double PCR amplification by using the DNA of the sample to be detected as a template. The dual PCR reaction system is as follows: 20 μ L of 2 XTaq PCR Master Mix II, 0.5 μ L of 10 μ M primers PlF1/PlR1 (0.25 μ L for 2 strips), 2 μ L of 10 μ M primers CgF1/CgR1 (1 μ L for 2 strips), 50 μ g/mL-1DNA template 1. mu.L, sterile water make up 40. mu.L. The double PCR reaction procedure was: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles, and extension at 72 ℃ for 5 min.
(2) And (3) detection results: and (3) performing agarose gel electrophoresis on the dual PCR amplification product, wherein if a 694bp single-mesh strip is obtained, the sample to be detected contains peronophythora litchi, if a 286bp single-mesh strip is obtained, the sample to be detected contains peronophythora litchi anthracnose, and if 2 strips of 694bp and 286bp respectively obtained by amplification, the sample to be detected contains peronophythora litchi and peronophythora litchi.
The above description is only exemplary of the present invention and should not be taken as limiting the invention, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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Claims (3)
1. A kit for synchronously detecting peronophythora litchi and colletotrichum litchi is characterized by comprising double PCR primers; the double PCR primers comprise primers PlF1/PlR1 and CgF1/CgR1, and the sequences of the primers are as follows:
PlF1：5’-TTCGGTCTGAACTAGTAGCT-3’
PlR1：5’-TTCAGCGGGTAATCTTGCCT-3’
CgF1：5’-TCTTCTGAGTGGTACAAGC-3’
CgR1：5’-TAAAGTTACTACGCAAAGGA-3’。
2. the kit of claim 1, wherein the kit further comprises PCR reagents.
3. Use of the kit of claim 1 or claim 2 for the detection of peronophythora litchi and colletotrichum litchi.
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| CN101974651B (en) * | 2010-12-02 | 2012-06-27 | 中国农业科学院植物保护研究所 | Fluorescence quantitative polymerase chain reaction (PCR) detection method and detection kit for phytophthora capsici leonian |
| MX2017003228A (en) * | 2014-09-11 | 2017-06-19 | Agrofresh Inc | Methods for pathogen detection and disease management on meats, plants, or plant parts. |
| CN111534621A (en) * | 2020-05-12 | 2020-08-14 | 海南大学 | Primer and detection method for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of colletotrichum gloeosporioides |
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| CN113430296A (en) | 2021-09-24 |
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