CN112280900A - Molecular marker primer combination and method for rapidly and synchronously identifying citrus huanglongbing disease, canker disease, recession disease, leaf shattering disease and split skin disease - Google Patents
Molecular marker primer combination and method for rapidly and synchronously identifying citrus huanglongbing disease, canker disease, recession disease, leaf shattering disease and split skin disease Download PDFInfo
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Abstract
The invention discloses a group of molecular marker primer combinations for rapidly and synchronously identifying citrus huanglongbing, canker, declining disease, leaf shattering disease and splitting disease and an identification method. The invention establishes a synchronous rapid extraction and one-step RT-PCR reaction system of citrus leaf tissue RNA and DNA, develops a group of multiple specific molecular marker primer combinations capable of synchronously identifying citrus yellow shoot, canker, decline, leaf shattering and split skin, is suitable for high-throughput typing detection platforms such as fluorescence capillary electrophoresis, has high sensitivity, and can be used for rapid identification of citrus diseases. The developed molecular marker primer combination is repeatedly tested and optimized, the 5 citrus common diseases can be synchronously detected, and the method has important technical guidance significance for detecting large-scale citrus sample diseases, and has the advantages of simplicity in operation, high efficiency, high speed and the like.
Description
Technical Field
The invention relates to a group of molecular marker primer combinations for rapidly and synchronously identifying citrus huanglongbing, canker, declining disease, leaf shattering disease and splitting disease and a method thereof, belonging to the field of molecular biology.
Background
Citrus yellow shoot (HLB), Citrus Canker Disease (CBCD), Citrus Tristeza Virus (CTV), Citrus Leaf-crushing Virus (CTLV) and Citrus split-skin Virus (CEVd) are five important diseases affecting Citrus production, seriously restrict the development of the Citrus industry in China, and establish a quick and accurate detection method which is one of the essential conditions for preventing and controlling the spread of the diseases. At present, single rapid detection methods are established for the pathogenic pathogens, including serology, RT-PCR, nested PCR, detection methods based on loop-mediated isothermal nucleic acid amplification technology and the like, and the method is widely used for detection and identification of various pathogens such as citrus bacteria and viruses; meanwhile, some pathogenic pathogen detection methods based on the multiplex PCR technology are also constructed, but the synchronous detection of various large-span pathogenic pathogens (such as viruses, bacteria, viroids and the like) cannot be realized; the two types of pathogens are generally divided into two types and are respectively identified and analyzed independently, the operation steps are relatively complex, toxic reagents such as chloroform are generally required to be used, the consumed time is long, the workload and the detection cost are also high, and the method is not beneficial to large-scale popularization and application.
Generally, DNA and RNA of plant pathogenic viruses or bacteria are mainly obtained from infected plant tissues, which are also the raw materials for conventional pathogen detection, and the extraction techniques are very mature, but most of them are extracted separately, and the research reports on simultaneous extraction of DNA and RNA from infected plant tissues are very few, and related extraction kit products are expensive, and are not suitable for large-scale popularization and application, and most of them adopt a two-step precipitation method, usually, after RNA is precipitated, DNA is reprecipitated, and toxic organic solvents such as chloroform and the like are often used in the test process, and the steps are tedious and time-consuming. In view of the above, for the extraction of nucleic acids of pathogens of different types from citrus, if the operation steps can be simplified, i.e., the synchronous extraction can be realized, while ensuring the quality and yield of RNA and DNA extraction, the work efficiency of identification analysis and related detection can be greatly improved. In addition, the RT-PCR technology has the characteristics of simple and rapid operation and high sensitivity, and is widely applied to the detection and identification of the plant pathogenic microorganisms. The multiplex RT-PCR technology can improve the detection efficiency by adding various primers, optimizing a reaction system and the like, realizes the simultaneous detection of various pathogens, is suitable for the detection of large sample amount, and has important significance in the detection and identification of the pathogens in actual production. However, in the multiplex PCR reaction system, the complexity is multiplied as the number and types of primers are increased, and it is necessary to comprehensively consider and consider a plurality of factors such as the concentration and Tm value of the primers, the size of amplified fragments, the buffer components, the template, and the amount of the amplification enzyme to be used, and the composition of the reaction system and the reaction conditions need to be repeatedly adjusted according to the experimental results to meet the need for amplifying a plurality of fragments simultaneously. Therefore, a rapid synchronous detection technology system for citrus virus and bacterial diseases, which is simple and convenient to operate and stable and reliable in result, is established, and has great significance for reducing economic loss of fruit growers.
Disclosure of Invention
The invention aims to provide a group of molecular marker primer combinations for rapidly and synchronously identifying citrus huanglongbing, canker, recession disease, leaf shattering disease and split skin disease.
The primer sequences are shown in Table 1.
TABLE 1 Citrus huanglongbing, canker, tristeza, leaf shattering and splitting pathogens detection related primer sequences
The joint sequence aims to improve the specificity of an amplification product and form a fluorescent product by matching and combining with a primer, so that visual detection is realized. The M13 linker is used more frequently in the field, but other sequences can be used without affecting the achievement of the purpose of the invention, so that other base structures adopted by the linker sequence also belong to the protection scope of the invention;
the invention also discloses a method for rapidly and synchronously identifying the citrus huanglongbing disease, the canker disease, the decline disease, the leaf shattering disease and the dehiscence disease, which comprises the following steps:
s1 synchronous extraction of citrus leaf DNA and RNA
S11, taking a fresh citrus leaf sample with veins, fully grinding the fresh citrus leaf sample in liquid nitrogen, and then adding a total RNA extraction reagent for placing for a period of time;
s12, filtering the liquid mixture in the tube through gauze, transferring the filtrate to a clean centrifugal tube, adding isopropanol with the same volume, uniformly mixing in a vortex mode, and standing at room temperature for a period of time;
s13, centrifuging, removing the supernatant, taking the precipitate, and finally adding RNase-free ddH2Fully dissolving O to obtain a mixture of the DNA and RNA of the citrus leaves;
s2, one-step RT-PCR amplification and result analysis
Performing quintuple RT-PCR amplification on the mixture of the citrus leaf DNA and the RNA obtained in the step S1 by using the primer combination, analyzing whether fragments with the sizes corresponding to five diseases exist according to the fragments of the amplified product, and if so, determining the fragments to be positive types and being infected plants; if no PCR amplification product with the size appears, the plant can be regarded as a negative type, and is a plant without the disease, wherein the length of the Huanglongbing amplification product is 174bp, the length of the ulcer amplification product is 437bp, the length of the recession disease amplification product is 318bp, the length of the broken leaf disease amplification product is 394bp, and the length of the dehiscence disease amplification product is 218 bp.
The whole process of RNA extraction and RT-PCR must be put on a disposable mask and a sanitary cap, and the environment is ensured to be clean; meanwhile, any items such as centrifuge tubes, suction heads, gauzes and the like used in the test process need to be subjected to RNase removal treatment.
Preferably, the PCR amplification reaction system in step S2 is: DNA and RNA mixed template 5.0. mu.L, Abstart Taq 2.0. mu.L, 5 Xone-step RT-PCR buffer 5.0. mu.L, Solution I (10X) 2.5. mu.L, forward primer (10. mu. mol/L) 0.4. mu.L 5, reverse primer (10. mu. mol/L) 0.4. mu.L 5, adaptor primer (10. mu. mol/L) 2.0. mu.L, ddH2O is complemented to 25 mu L; the RT-PCR amplification program is as follows: carrying out 36 cycles of 30min at 42 ℃, 5min at 95 ℃, 30s at 94 ℃, 30s at 52 ℃ and 30s at 72 ℃; and (3) carrying out typing detection by directly using a fluorescence capillary electrophoresis platform after the PCR reaction is finished at 72 ℃ for 10 min.
Preferably, 5 mixed extracts of DNA and RNA of the determined affected tissues are added as internal references in step S2 to reduce typing detection errors.
Preferably, in step S11, the total RNA extraction reagent is added in a ratio of 200mg of leaf sample to 1ml of total RNA extraction reagent.
Preferably, in step S12, the multiple layers of gauze are pre-cut into the diameter of the centrifuge tube, and placed on the upper layer of the liquid mixture in the tube, and then pushed to the bottom of the tube, and the filtrate is taken from above the gauze.
Preferably, the centrifugation is performed at 12,000rpm and 4 ℃ for 5min in step S13.
According to the invention, the citrus leaf tissue is improved based on the traditional guanidinium isothiocyanate extraction method according to the characteristic that the citrus leaf tissue is rich in substances such as polysaccharide, polyphenol and the like, so that the operation steps are simplified.
Compared with the prior art, the invention has the beneficial effects that:
(1) the synchronous extraction method of the citrus leaf tissue RNA and DNA is simple to operate, toxic organic solvents such as chloroform and the like are not needed, the safety risk of experiment technicians is reduced, the whole experiment process only needs to be centrifuged once (the conventional method usually needs to be centrifuged for more than three times), the extraction steps are simplified to a great extent, the extraction time is effectively shortened, and the detection cost is further reduced;
(2) the multiple RT-PCR detection system established by the invention can simultaneously detect huanglongbing, canker, decline, leaf shattering and splitting, the amplified strips are clear and specific, easy to distinguish, and free from background signal interference such as non-specific amplified miscellaneous bands, the lengths of the amplified product fragments are all less than 500bp, the required PCR amplification time is short, and the 5 diseases can be synchronously detected; in addition, the molecular marker primer combination of the invention is added with a joint sequence in advance, and a single or multiple joint primers added with fluorescent groups can be combined into a monochromatic multiple detection system for fluorescent capillary electrophoresis typing analysis only by synthesizing and adding single or multiple joint primers added with fluorescent groups, so that the detection working efficiency and sensitivity are improved.
The invention is described in more detail below with reference to the following examples and the accompanying drawings. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
Drawings
FIG. 1 shows the effect of DNA and RNA synchronous extraction products of citrus leaf tissue detected by 1.2% agarose electrophoresis.
FIG. 2 shows the effect of the specific multiplex PCR primer combination for citrus greening disease, canker, declining disease, leaf shattering disease and dehiscence on the synchronous typing detection on the fluorescence capillary electrophoresis platform.
Detailed Description
Example 1
A method for rapidly and synchronously identifying citrus huanglongbing, canker, decline, leaf shattering and splitting diseases comprises the following steps:
(1) primer synthesis: the relevant biotechnology companies were entrusted with the synthesis of the following primers;
TABLE 1 Citrus Huanglongbing, canker, tristeza, leaf shattering and splitting pathogens detection 5-fold PCR primer combination sequence information
(2) Synchronously and quickly extracting DNA and RNA of a citrus leaf sample to be detected (table 2):
1) taking a fresh citrus leaf sample (with veins) 200mg, fully grinding in liquid nitrogen, adding 1ml of total RNA extraction reagent (Trizol) (Shanghai biological engineering Co., Ltd.), and standing for 5 min;
2) adding four layers of gauze (which is folded in advance and cut into the diameter of a centrifugal tube), placing the gauze on the upper layer of the liquid mixture in the tube, and slowly pushing the gauze to the bottom of the tube by using a pipette tip; absorbing the liquid filtered from the upper part of the gauze, transferring the liquid into a clean centrifugal tube, adding isopropanol with the same volume, uniformly mixing by vortex, and standing at room temperature for 10 min;
3) centrifuging at 12,000rpm at 4 deg.C for 5min, removing supernatant, sucking residual liquid, leaving precipitate, and adding 50 μ l RNase-free ddH2Dissolving O completely, detecting the extraction effect of 5 μ l on 1.2% agarose gel, and placing in-80 deg.C refrigerator;
(3) one-step RT-PCR amplification and result analysis:
performing 5-fold RT-PCR amplification based on the citrus leaf DNA and RNA mixture by using all primers in the table 1; the reaction system is as follows: can be constructed by directly using an Abstart one-step RT-PCR premix solution kit (Shanghai biological engineering Co., Ltd.) after improvement; wherein, the DNA and RNA mixed template is 4.0 muL, Abstart Taq 2.0 muL, 5 xOne-step RT-PCR buffer is 5.0 muL, Solution I (10X) is 2.5 muL, the forward primer (10 mumol/L) is 0.4 muL 5, the reverse primer (10 mumol/L) is 0.4 muL 5, the adaptor primer (10 mumol/L) is 2.0 muL, ddH2O is complemented to 25 mu L; the whole process of RNA extraction and RT-PCR must be put on a disposable mask and a sanitary cap, and the environment is ensured to be clean; meanwhile, any items such as centrifuge tubes, suction heads, gauzes and the like used in the test process need to be subjected to RNase removal treatment.
The RT-PCR amplification procedure was as follows: 30min at 42 ℃, 5min at 95 ℃, 35 cycles (94 ℃ for 30s, 52 ℃ for 30s, 72 ℃ for 30s), 10min at 72 ℃; after the PCR reaction is finished, directly using a fluorescent capillary electrophoresis platform ABI 3730XL to carry out STR typing detection; wherein, if the length of the amplified product is consistent with the size of the internal reference sample fragment (5 determined infected tissue DNA and RNA mixed extracts are added in advance as the internal reference), the amplified product can be regarded as a positive type and is an infected plant; if no PCR amplification product with the size appears, the plant is regarded as a negative type and is a plant without the disease.
Table 2 information and identification results of 16 citrus leaf samples used in the examples of the present invention
Note: specific amplification products of the nucleic acid samples of the internal reference of Huanglongbing, the internal reference of ulcerative diseases, the internal reference of degenerative diseases, the internal reference of leaf shattering diseases and the internal reference of dehiscent skin diseases are respectively numbered A, B, C, D and E, and if the specific amplification products with the same size as the fragments in the internal reference sample appear in the sample to be detected, the infection is determined.
Claims (9)
1. A group of molecular marker primer combinations for rapidly and synchronously identifying citrus huanglongbing, canker, recession, leaf shattering and splitting diseases comprise the following primers:
huanglongbing-F: 5 '-linker sequence-CTAGAGTTTAGGAGAGG-3'
huanglongbing-R: 5'-GGACTACCAGGGTATCTAATC-3', respectively;
ulcer disease-F: 5 '-linker sequence-TATTGGGAGATTG-3'
Ulcer disease-R: 5'-GCTCGTAGAACTGACTTCCA-3', respectively;
degenerative disease-F: 5 '-linker sequence-AACCGAACCGTGAAGGAA-3'
Degenerative disease-R: 5'-CTACATCATCAGTCACAACAG-3', respectively;
leaf shattering disease-F: 5 '-linker sequence-AGTTCTGAGGATGAGATTGC-3'
Leaf shattering disease-R: 5'-CCTATGTTCTGTGGATGGTAT-3', respectively;
split skin disease-F: 5 '-linker sequence-GTCCAGCGGAGAAACAGGA-3'
dehiscence-R: 5'-CTAGGGTTCCGAGGGCTTT-3' are provided.
2. The molecular marker primer combination for rapidly and synchronously identifying the citrus huanglongbing, the canker, the declining disease, the leaf crushing disease and the splitting disease according to claim 1 is characterized in that the linker sequence is a < FAM >/< HEX >/< NED/< PET > -M13 linker sequence.
3. The molecular marker primer combination for rapidly and synchronously identifying citrus huanglongbing, canker, declining disease, leaf shattering and splitting disease according to claim 2 is characterized in that the linker sequence is < FAM >/< HEX >/< NED/< PET > TGTAAAACGACGGCCAGT.
4. A method for rapidly and synchronously identifying citrus huanglongbing, canker, decline, leaf shattering and splitting diseases comprises the following steps:
s1 synchronous extraction of citrus leaf DNA and RNA
S11, taking a fresh citrus leaf sample with veins, fully grinding the fresh citrus leaf sample in liquid nitrogen, and then adding a total RNA extraction reagent for placing for a period of time;
s12, filtering the liquid mixture in the tube through gauze, transferring the filtrate to a clean centrifugal tube, adding isopropanol with the same volume, uniformly mixing in a vortex mode, and standing at room temperature for a period of time;
s13, centrifuging, removing the supernatant, taking the precipitate, and finally adding RNase-free ddH2Fully dissolving O to obtain a mixture of the DNA and RNA of the citrus leaves;
s2, one-step RT-PCR amplification and result analysis
Carrying out quintuple RT-PCR amplification on the mixture of the citrus leaf DNA and the RNA obtained in the step S1 by using the primer combination of claim 1, analyzing whether fragments with the sizes corresponding to five diseases exist according to the fragments of the amplification products, and if the fragments exist, determining the fragments to be positive types and being infected plants; if no PCR amplification product with the size appears, the plant can be regarded as a negative type, and is a plant without the disease, wherein the length of the Huanglongbing amplification product is 174bp, the length of the ulcer amplification product is 437bp, the length of the recession disease amplification product is 318bp, the length of the broken leaf disease amplification product is 394bp, and the length of the dehiscence disease amplification product is 218 bp.
5. The method of claim 4, wherein: the PCR amplification reaction system in the step S2 is as follows: DNA and RNA mixed template 5.0. mu.L, Abstart Taq 2.0. mu.L, 5 Xone-step RT-PCR buffer 5.0. mu.L, Solution I (10X) 2.5. mu.L, forward primer (10. mu. mol/L) 0.4. mu.L 5, reverse primer (10. mu. mol/L) 0.4. mu.L 5, adaptor primer (10. mu. mol/L) 2.0. mu.L, ddH2O is complemented to 25 mu L; the RT-PCR amplification program is as follows: 42 deg.C30min, 95 ℃ for 5min, 94 ℃ for 30s, 52 ℃ for 30s, and 72 ℃ for 30s for 36 cycles; and (3) carrying out typing detection by directly using a fluorescence capillary electrophoresis platform after the PCR reaction is finished at 72 ℃ for 10 min.
6. The method of claim 4, wherein: in step S2, 5 mixed extracts of DNA and RNA of the determined infected tissues are added as internal references to reduce typing detection errors.
7. The method according to any one of claims 4-6, wherein: in step S11, the total RNA extraction reagent was added in a ratio of 200mg of leaf sample to 1ml of total RNA extraction reagent.
8. The method of claim 7, wherein: in step S12, the multiple layers of gauze are pre-cut into the size of the diameter of the centrifuge tube, placed in the upper layer of the liquid mixture in the tube, pushed to the bottom of the tube, and the filtrate above the gauze is taken.
9. The method of claim 8, wherein: in step S13, the mixture was centrifuged at 12,000rpm at 4 ℃ for 5 min.
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