CN106929572B - Specific primer for identifying mulberry sclerotinia sclerotiorum and method for identifying mulberry sclerotinia sclerotiorum by using specific primer - Google Patents
Specific primer for identifying mulberry sclerotinia sclerotiorum and method for identifying mulberry sclerotinia sclerotiorum by using specific primer Download PDFInfo
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Abstract
The invention discloses a specific primer for identifying mulberry sclerotinia sclerotiorum caused by carumforrestii, which is characterized by comprising the following components in percentage by weight: the primer consists of an upstream primer CC-18S-F and a downstream primer CC-18S-R, wherein the nucleotide sequence of the upstream primer CC-18S-F is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer CC-18S-R is shown as SEQ ID NO: 2, respectively. The invention also discloses a method for identifying mulberry sclerotinia sclerotiorum caused by the carumforbesii by applying the specific primer, and the mulberry sclerotinia sclerotiorum can be quickly identified in the early disease stage of the mulberry sclerotinia sclerotiorum.
Description
Technical Field
The invention belongs to the technical field of sclerotinia sclerotiorum, and particularly relates to a specific primer for identifying mulberry sclerotinia sclerotiorum caused by carumforrestii and application thereof.
Background
Morous sclerotinia, also known as white fruit disease, is the main disease of morous alba, which is caused by fungi, and the germs start to invade when morous alba blossoms. Healthy mulberries are purplish red when mature, and mulberries infected by cupule of mulberry become white or off-white, and flowers are significantly enlarged. After the mulberry falls to the ground, the flower quilt is partially separated and becomes black sclerotia. With the continuous expansion of mulberry planting area, mulberry sclerotinia continuously erupts in large area and is in spreading trend, serious orchard even particle is not harvested, the economic benefit of growers is greatly influenced, and the development of mulberry industry is threatened. The main pathogenic bacteria of mulberry sclerotinia sclerotiorum are 4 kinds, which are sclerotinia sclerotiorum, sorosis, caruncle-like sclerotium and sclerotium rolfsii respectively. Through research, the mulberry sclerotinia sclerotiorum pathogenic bacteria in Guangdong area are mainly carumforbesii.
Currently, the main method for identifying morula sclerotiorum is phenotypic identification. Phenotypic identification is to determine whether morbid sclerotinia sclerotiorum occurs by observing morbid phenotypic characteristics of morous alba. The phenotype of the plant diseases can be observed only in the middle and later period of the disease, and pathogenic bacteria invade the body when the mulberry blossoms, so the phenotype identification method has little significance for preventing and controlling the mulberry sclerotinia rot.
Disclosure of Invention
The first purpose of the invention is to provide a specific primer for identifying mulberry sclerotinia sclerotiorum, and the primer can be used for detecting the mulberry sclerotinia sclerotiorum caused by the carumforrestii.
The second purpose of the invention is to provide a method for identifying mulberry sclerotinia sclerotiorum caused by caruncle-like cupule bacteria by applying the specific primer, and the method can rapidly identify the mulberry sclerotinia sclerotiorum at the early stage of the morbid stage of the mulberry sclerotinia sclerotiorum.
The third purpose of the invention is to provide the application of the specific primer for identifying the mulberry sclerotinia sclerotiorum in the preparation of the medicine with the function of identifying the mulberry sclerotinia sclerotiorum.
The first purpose of the invention is realized by the following technical scheme:
a specific primer for identifying mulberry sclerotinia sclerotiorum caused by carumforrestii is characterized by comprising the following components in percentage by weight: the primer consists of an upstream primer CC-18S-F and a downstream primer CC-18S-R, wherein the nucleotide sequence of the upstream primer CC-18S-F is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer CC-18S-R is shown as SEQ ID NO: 2, respectively.
The fluorescent PCR specific primers (CC-18S-F: GAGTGAGCATAAGCTCACCCCGA as shown in SEQ ID NO: 1; CC-18S-R: CTCCACCCCCCGAGAGCGGTC as shown in SEQ ID NO: 2) of the above-mentioned P.carneus were designed and screened using Primer Premier 5.0 and Oligo6.0 based on the 18SrRNA (GenBank ID: KY449059) sequence characteristics of P.multocida in NCBI database. The theoretical amplified fragment size of the primer pair is 344bp, and the primer can be synthesized by Huada gene and other companies.
The specific primer of the invention can be used for conventional PCR and fluorescent quantitative qPCR.
The second purpose of the invention is realized by the following technical scheme:
the method for identifying the mulberry sclerotinia sclerotiorum by applying the specific primer is characterized by comprising the following steps:
(1) taking 10 diseased mulberries and healthy mulberries respectively, and extracting DNA of the mulberries;
(2) performing fluorescent quantitative qPCR by using the extracted DNA of the diseased mulberry and the healthy mulberry as a template and using the specific primer of the carumforrestii; recording Ct values of qPCR reaction systems of diseased mulberry and healthy mulberry DNA; taking the minimum value of the Ct values of the qPCR reaction system of all the DNA of the healthy mulberry as a critical value for judging whether the mulberry has the mulberry sclerotinia rot or not;
(3) taking mulberry to be detected, and extracting DNA of the mulberry;
(4) taking the DNA of the mulberry to be detected extracted in the step (3) as a template, performing fluorescence quantitative qPCR by using the specific primer of the carumforrestii, and recording the Ct value of a qPCR reaction system of the mulberry DNA to be detected;
(5) when the Ct value of the qPCR reaction system of the mulberry DNA to be detected is smaller than the critical value, indicating that the mulberry to be detected is infected with the mulberry sclerotinia sclerotiorum; and when the Ct value of the qPCR reaction system of the mulberry DNA to be detected is greater than or equal to the critical value, indicating that the mulberry to be detected is healthy mulberry.
In the above method, the extraction of mulberry DNA is preferably performed using a DNA extraction kit.
In a preferred embodiment of the present invention, the reaction system of the fluorescence quantitative qPCR comprises: SYBRPremix Ex Taq II (2X) 10.0. mu.L, 10. mu.M upstream primer CC-18S-F0.8. mu.L, 10. mu.M downstream primer CC-18S-R0.8. mu.L, template 2.0. mu.L, and double distilled water 6.4. mu.L.
The fluorescent quantitative qPCR amplification procedure in step (2) is preferably as follows: the first step is pre-denaturation at 95 ℃ for 30s, the second step is denaturation at 95 ℃ for 5s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 15s, for 45 cycles.
In a preferred embodiment of the present invention, the critical value is 35.
The third purpose of the invention is realized by the following technical scheme:
the application of the specific primer for identifying mulberry sclerotinia sclerotiorum in preparing the medicine with the function of identifying the mulberry sclerotinia sclerotiorum is provided.
The medicine comprises a kit and the like.
Has the advantages that:
the specific primer for identifying the mulberry sclerotinia sclerotiorum caused by the carumforbesii is sensitive, efficient and good in specificity. The mulberry sclerotinia sclerotiorum identification method obtained by combining the specific primer with the fluorescent quantitative PCR can quickly identify the mulberry sclerotinia sclerotiorum at the early stage of pathogenesis and provide reliable basis for the early prevention and treatment of the mulberry sclerotiorum.
Drawings
FIG. 1 shows the specific detection of the fluorescent PCR primers of Ralstonia solanacearum in example 1. Lane 1 template is mulberry caruncle-like cupula DNA; lane 2 template trichoderma DNA; lane 3 template sclerotinia sclerotiorum DNA; lane 4 template Botrytis DNA; lane 5 template Aspergillus niger DNA; lane 6 template aspergillus flavus DNA; lane 7 template yeast DNA; m is 2000bp DNA Ladder;
FIG. 2 is Ct values of morous and healthy morous samples of example 3.
Detailed Description
In the examples below, the conventional PCR apparatus used was manufactured by ABIThe Taq PCR Mix (2 ×), DNAiso reagent kit and T-easy vector used were all produced by Takara Shuzo.Ltd.qPCR was performed using LightCycler 480 real-time fluorescent PCR machine produced by Roche and SYBR Premix Ex Taq produced by Takara ShuzoTMAnd (4) carrying out kit.
Example 1
Design and verification of mulberry fleshy goby fungus specific primer
1. According to the sequence characteristics of 18SrRNA (GenBank ID: KY449059) of the mulberry caruncle californica in NCBI database, a PCR specific Primer (CC-18S-F: GAGTGAGCATAAGCTCACCCCGA/CC-18S-R: CTCCACCCCCCGAGAGCGGTC) of the caruncle californica is designed and screened by utilizing Primer Premier 5.0 and Oligo6.0, the size of a theoretically amplified fragment of the Primer pair is 344bp, the Primer is synthesized by Huada gene company, 1 × 10 is adopted before Primer dilution under the condition of 4 DEG C4Centrifuging at rpm for 2min, adding deionized water to obtain 10 μ M stock solution, and storing at-20 deg.C;
2. taking 6 fungi such as trichoderma, sclerotinia sclerotiorum, grey mould, aspergillus niger, aspergillus flavus, yeast and the like as a control, inoculating all experimental strains into a PDA culture medium, and culturing for 24 hours in a constant-temperature gas bath shaker at the rotating speed of 200rpm under the condition of 28 ℃. Extracting DNA of each strain by adopting DNAiso reagent kit, and storing at-20 ℃ for later use;
3. taking DNA of the caruncle-like california and each control strain as templates, and carrying out conventional PCR amplification by using fluorescence PCR specific primers CC-18S-F and CC-18S-R of the caruncle-like california, wherein a PCR reaction system is as follows: a25. mu.L reaction system contained 12.5. mu.L of Taq PCR Mix (2X), 8.5. mu.L of double distilled water, 2. mu.L of template, 1. mu.L of forward primer CC-18S-F, and 1. mu.L of reverse primer CC-18S-R. The amplification reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 20s for 30 cycles; extending for 5min at 72 ℃, and storing at 4 ℃. After the PCR is finished, detecting the product by 1.2% agarose gel electrophoresis;
4. and meanwhile, cutting and recovering the electrophoresis band, connecting the electrophoresis band to a T-easy vector, converting the electrophoresis band into E.coli, obtaining a positive colony and sequencing the colony. The sequencing results were aligned at NCBI to determine the genus type.
Results
Taking 6 fungi such as trichoderma, sclerotinia sclerotiorum, grey mould, aspergillus niger, aspergillus flavus and yeast as reference, and utilizing the fluorescence PCR specific primers CC-18S-F and CC-18S-R of the carumforrestii to carry out conventional PCR amplification, wherein the electrophoresis analysis result is shown in figure 1: only the caruncle-like california can amplify the strip, but 6 fungi such as the control strains of trichoderma, sclerotinia, gray mold, aspergillus niger, aspergillus flavus, yeast and the like do not amplify the strip.
After the electrophoresis band is recovered, transformed and sequenced, the known sequence of the carumforrestii 18SrRNA (KY449059) on NCBI is compared with the sequencing result, and the comparison result shows that the sequence is the characteristic sequence of the carumforrestii 18 SrRNA.
In conclusion, the primer for the carumforrestii has good specificity and can be used for a subsequent fluorescent quantitative analysis experiment of the carumforrestii.
Example 2
Method for quickly identifying mulberry sclerotinia rot in early stage
Determination of Ct value
1. Taking 10 diseased mulberries and 10 healthy mulberries respectively, and extracting DNA of the mulberries by adopting a DNAiso reagent kit;
2. and (3) performing a fluorescent quantitative qPCR experiment by using the DNA extracted in the step (1) as a template and using the specific primer CC-18S-F/CC-18S-R of the carumforrestii. The 20 μ L qPCR reaction system included: SYBR Premix Ex Taq II (2X) 10.0uL, upstream primer CC-18S-F (10. mu.M) 0.8. mu.L, downstream primer CC-18S-R (10. mu.M) 0.8. mu.L, template 2.0. mu.L, double distilled water 6.4. mu.L. The amplification procedure for fluorescent quantitative qPCR was: the first step is pre-denaturation at 95 ℃ for 30s, the second step is denaturation at 95 ℃ for 5s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 15s, for 45 cycles. And immediately performing dissolution curve analysis after the PCR is finished, and verifying the specificity of amplification. Each qPCR reaction was repeated 3 times. Ct values of the qPCR reaction system were recorded and are recorded in table 1;
3. as shown in the data in Table 1, Ct values of 10 healthy morous alba ranged from 35 to 38, while those of 10 morous alba ranged from 11 to 29. Taking Ct values of qPCR reaction systems of all healthy mulberry DNA, namely 35, as critical values for detecting whether the mulberry has mulberry sclerotinia sclerotiorum, namely when the Ct value detected by qPCR of a mulberry sample is less than 35, showing that carumforrestii exists, wherein the sample is diseased mulberry; when the Ct value detected by qPCR of a mulberry sample is more than 35, the mulberry caruncle-like california does not exist, and the sample is healthy mulberry.
Identification of morula sclerotinia
1. Taking mulberry samples and healthy mulberry samples with the disease degree of mulberry sclerotinia sclerotiorum being at the 1 stage (only mulberry small fruit is not enlarged and the inside is slightly infected), the 2 stage (mulberry small fruit is enlarged and seriously infected), the 3 stage (mulberry small fruit is infected to blackish and is partially infected with external flower quilt) and the 4 stage (mulberry small fruit and flower quilt are integrally infected and blackened), washing the mulberry samples and healthy mulberry samples with tap water, sterilizing the surfaces of the mulberry samples and healthy mulberry samples with 70% alcohol for 1min, washing the mulberry samples and healthy mulberry samples with sterile water for 1 time, quickly freezing the mulberry samples and healthy mulberry samples with liquid nitrogen, and storing the mulberry samples and healthy mulberry samples at the temperature of-80 ℃ for later use;
2. taking 1g of mulberry sample as a material, adopting DNAiso reagent kit to extract DNA of all mulberry samples, carrying out agarose gel electrophoresis detection, and storing at-20 ℃ for later use;
3. for each mulberry sample, 1. mu.L of mulberry sample DNA was used as a template, and a fluorescent quantitative qPCR experiment was performed using the primers CC-18S-F/CC-18S-R in example 1. qPCR was performed using LightCycler 480 real-time fluorescent PCR instrument and SYBRPremix Ex TaqTMThe kit is used for carrying out 20 mu L qPCR reaction system, wherein SYBR Premix Ex Taq II (2 ×) is 10.0uL, an upstream primer CC-18S-F (10 mu M) is 0.8 mu L, a downstream primer CC-18S-R (10 mu M) is 0.8 mu L, a template is 1.0 mu L, and double distilled water is 6.4 mu L, the amplification program comprises the following steps of pre-denaturation at 95 ℃ for 30S in the first step, denaturation at 95 ℃ for 5S in the second step, annealing at 55 ℃ for 30S, extension at 72 ℃ for 15S, 45 cycles, and dissolution curve analysis is carried out immediately after PCR is finished, the amplification specificity is verified, and each qPCR reaction is repeated for 3 times;
4. ct values were recorded for each sample qPCR reaction system and the experimental results are summarized in figure 2.
As can be seen from fig. 2:
(1) the Ct value of the healthy mulberry sample is 36.5 which is larger than the critical value of 35.
(2) The Ct values of the mulberry samples at the disease onset stages 1, 2, 3 and 4 are respectively 25.4, 18.3, 14.2 and 12.2, which are all less than the critical value 35.
Therefore, the method can be used for identifying the mulberry sclerotiniose.
TABLE 1
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Bombycis of Guangdong province academy of agricultural sciences and institute of agricultural product processing
<120> a specific primer for identifying morula sclerotiorum and a method for identifying morula sclerotiorum by using the same
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ctccaccccc cgagagcggt c
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Sequence listing
<110> Bombycis of Guangdong province academy of agricultural sciences and institute of agricultural product processing
<120> a specific primer for identifying morula sclerotiorum and a method for identifying morula sclerotiorum by using the same
<160>2
<170>BiSSAP 1.3.6
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<213> Artificial sequence
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ctccaccccc cgagagcggt c 21
Claims (7)
1. A specific primer for identifying mulberry sclerotinia sclerotiorum caused by carumforrestii is characterized by comprising the following components in percentage by weight: the primer consists of an upstream primer CC-18S-F and a downstream primer CC-18S-R, wherein the nucleotide sequence of the upstream primer CC-18S-F is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer CC-18S-R is shown as SEQ ID NO: 2, respectively.
2. A method for identifying morula sclerotiorum by using the specific primer of claim 1, which is characterized by comprising the following steps:
(1) taking 10 diseased mulberries and healthy mulberries respectively, and extracting DNA of the mulberries;
(2) performing fluorescent quantitative qPCR by using the extracted DNA of the diseased mulberry and the healthy mulberry as a template and using the specific primer of the carumforrestii; recording Ct values of qPCR reaction systems of diseased mulberry and healthy mulberry DNA; taking the minimum value of the Ct values of the qPCR reaction system of all the DNA of the healthy mulberry as a critical value for judging whether the mulberry has the mulberry sclerotinia rot or not;
(3) taking mulberry to be detected, and extracting DNA of the mulberry;
(4) taking the DNA of the mulberry to be detected extracted in the step (3) as a template, performing fluorescence quantitative qPCR by using the specific primer of the carumforrestii, and recording the Ct value of a qPCR reaction system of the mulberry DNA to be detected;
(5) when the Ct value of the qPCR reaction system of the mulberry DNA to be detected is smaller than the critical value, indicating that the mulberry to be detected is infected with the mulberry sclerotinia sclerotiorum; and when the Ct value of the qPCR reaction system of the mulberry DNA to be detected is greater than or equal to the critical value, indicating that the mulberry to be detected is healthy mulberry.
3. The method for identification of morula sclerotiorum disease using specific primers according to claim 2, wherein: and extracting mulberry DNA by adopting a DNA extraction kit.
4. The method for identification of morula sclerotiorum disease using specific primers according to claim 2, wherein: the reaction system of the fluorescence quantitative qPCR comprises: SYBR Premix Ex Taq II (2X) 10.0. mu.L, 10. mu.M upstream primer CC-18S-F0.8. mu.L, 10. mu.M downstream primer CC-18S-R0.8. mu.L, template 2.0. mu.L, and double distilled water 6.4. mu.L.
5. The method for identification of morula sclerotiorum disease using specific primers according to claim 2, wherein: the fluorescent quantitative qPCR amplification program in the step (2) comprises the following steps: the first step is pre-denaturation at 95 ℃ for 30s, the second step is denaturation at 95 ℃ for 5s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 15s, for 45 cycles.
6. The method for identification of morula sclerotiorum disease using specific primers according to claim 2, wherein: the critical value is 35.
7. Use of the specific primers for identifying morula sclerotiorum caused by cupule caruncle as set forth in claim 1 for the preparation of a medicament having a function of identifying morula sclerotiorum.
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