CN114395617B - LAMP (loop-mediated isothermal amplification) visual rapid detection method and application of powdery mildew of strawberry - Google Patents
LAMP (loop-mediated isothermal amplification) visual rapid detection method and application of powdery mildew of strawberry Download PDFInfo
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Abstract
The application discloses a LAMP visual rapid detection method and application of powdery mildew of strawberries, which are characterized in that DNA of a sample to be detected is used as a template, primers are used for carrying out LAMP reaction amplification, and then detection is carried out through a real-time fluorescent amplification curve and a fluorescent dye visual observation method. The primers include 1 pair of outer primers F3 and B3,1 pair of inner primers FIP and BIP, and 1 pair of loop primers LF and LB. The LAMP primer for the powdery mildew of the strawberry has the advantages of strong specificity, good amplification effect, quick, simple and convenient construction, strong specificity and high sensitivity, can be used for the detection of the powdery mildew of the strawberry in the early stage and the early stage of the disease, has the advantages of short detection time, low requirements on operators, direct visual observation of results, low detection cost and the like, and has important significance for early diagnosis, monitoring, early prevention and determination of optimal prevention and control time of the powdery mildew of the strawberry.
Description
Technical Field
The application relates to the technical field of strawberry powdery mildew, in particular to a LAMP visual rapid detection method and application of strawberry powdery mildew.
Background
The strawberry powdery mildew is one of strawberry diseases with larger disease area and higher disease frequency of the protected area, the pathogenic bacteria of the strawberry powdery mildew is ascomycota fungus, namely the pinus pinnatifida oocyst fungus (Sphaerotheca aphanis), belongs to air-borne diseases, mainly damages the leaves and fruits of the strawberries, sometimes damages the pedicel and petioles of the strawberries, seldom occurs on the stolons, the occurrence of the strawberry powdery mildew penetrates through each stage of strawberry growth, and is frequently generated in the high-yield season of strawberry production. The powdery mildew of strawberry belongs to the obligate parasitic bacteria, is distributed all over the world, mainly occurs in areas with relatively cold climatic conditions, and has serious morbidity in the cold North China plain. Strawberry powdery mildew is a big bottleneck which restricts the strawberry industry in China, in recent years, especially the strawberry powdery mildew in the protected area has great influence on strawberry fruits and plants, the strawberry is easy to attack even in a greenhouse with good cultivation conditions, once the strawberry is in attack, the strawberry in the whole greenhouse can be infected, the attack symptoms are not obvious during picking, the powdery mildew quantity on the surface of the fruits is too small, and although the strawberry is not found to be infected during picking, the strawberry can also be subjected to powdery mildew in two to three days during the shelf life, and the strawberry is not suitable for being sold at the moment. The fruit rate exceeds 50%, the leaf rate exceeds 45%, the quality of strawberries is deteriorated, the surfaces of the fruits lose luster, the yield of strawberries is reduced, and great economic loss is brought to planting farmers. Varieties suitable for picking such as red color, zhang Ji and the like are sensitive to powdery mildew of strawberries; the prevention and control technology is not in place, and a plurality of medicaments are frequently used by farmers, so that the pathogen resistance is higher. Powdery mildew pathogenic bacteria on fruits are more difficult to control than the leaves, so that 'safeners' are frequently used in production, and the safe production of strawberries is threatened.
At present, the most main control means of strawberry powdery mildew is still to spray chemical pesticides, but a series of problems such as fruit quality reduction, environmental pollution, pathogen resistance generation and the like occur along with the increase of the application dosage. The occurrence period and the epidemic intensity of powdery mildew can be predicted timely and accurately, the medicament use period, times and dosage can be determined, the medicament effect can be improved, the medicament application times can be reduced, the production cost can be reduced, the negative effects caused by pesticide use, such as environmental pollution and the like, can be reduced, and a basis is provided for the comprehensive prevention and treatment of powdery mildew. Therefore, the development of the prediction and forecast research of the powdery mildew of the strawberries has important significance for scientifically preventing and treating the powdery mildew of the strawberries. At present, no report on a rapid detection technology of the powdery mildew of the strawberry exists, so that a high-efficiency and rapid early detection technology is established to provide a new thought for scientific prevention and control of the powdery mildew of the strawberry, and promote healthy development of the strawberry industry.
The traditional powdery mildew early detection method is often identified according to the symptoms of the diseased tissue, the characteristics of pathogenic bacteria and the experience of researchers, but the early-stage green fading caused by powdery mildew is similar to the symptoms caused by other pathogens, and is difficult to accurately judge. On the other hand, powdery mildew is an obligate parasitic bacterium, and cannot be artificially cultured, so that the detection difficulty is high by the traditional methods of separation culture, serum identification, ELISA and the like.
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology published by Japanese school Notomi in 2000, which can identify 4 forward and reverse outward primers and forward and reverse inward primers of 6 specific regions on a target sequence, and specifically and efficiently amplify a target gene by using a high-activity strand displacement DNA polymerase at a constant temperature of 60-65 ℃. The sensitivity of the technology is 100 times of that of the common PCR, and the target gene can be efficiently amplified within 15-60 min. The LAMP reaction product can be detected by a turbidity meter, a real-time PCR (polymerase chain reaction) instrument and a gel electrophoresis instrument, and can be identified by naked eyes after being dyed by SYBR Green I, calcein and hydroxynaphthol blue. Because the LAMP reaction is simple, quick, efficient and economical, no special equipment is needed, and the detection result can be judged by naked eyes, the LAMP reaction is very suitable for popularization and application in basic production departments, and has very wide application prospects. The LAMP detection is mainly applied to detection of human and animal pathogens, food safety and environmental sanitation, few reports are generated in detection of plant pathogenic bacteria, and no report is generated at home and abroad on LAMP detection of powdery mildew of strawberry, so that the development of a visual LAMP detection technology for powdery mildew of strawberry has important application value for early diagnosis and pathogenic bacteria identification of powdery mildew of strawberry, and timely and efficient control of occurrence and spread of powdery mildew of strawberry.
Disclosure of Invention
The application aims to provide a LAMP visual rapid detection method and application of powdery mildew of strawberry, which overcome the defects in the prior art.
The technical problems to be solved by the application are realized by the following technical scheme:
the LAMP visual rapid detection method for powdery mildew of strawberries comprises 1 pair of outer primers F3 and B3,1 pair of inner primers FIP and BIP and 1 pair of loop primers LF and LB, wherein the sequences are as follows:
F3:5’-CGGTATTCCGAGGGGCAT-3’，
B3:5’-CCTGATCCGAGGTCATCCAA-3’，
FIP:5’-CTGTTTAGGGGACGCCGAGCTGTTCGAGCGTCAGAACATC-3’，
BIP:5’-GGCGGTACCGTTGTGCTCTCTAGGTGGGTTTTGGCAGGT-3’，LF:5’-ACCAAGCCAGGCTTGAGAG-3’，
LB:5’-GTAGTCACGTATCTCGCGACAGAG-3’。
preferably, in the above technical solution, the method includes the following steps: and (3) taking DNA of a sample to be detected as a template, carrying out LAMP reaction amplification by using the primer, and detecting by a real-time fluorescent amplification curve and a fluorescent dye visual observation method.
Preferably, in the above technical scheme, the LAMP reaction system is: 10X Isothermal Amplification Buffer 2.5. Mu.L, 8mmol/L MgSO 4 1.5. Mu.L, 1.6mmol/LdNTPs 4.0. Mu.L, 1.2mmol/L betaine 0.6. Mu.L, 320u/mLBSTDNA polymerase 1. Mu.L, DNA template, calcein, 1.6. Mu.M FIP and BIP each 1. Mu.L, 0.2. Mu.M each 1. Mu.L of each of MF3 and B3, 0.4. Mu.M each 1. Mu.L of LoopF and LoopB, sterile deionized water make up 25. Mu.L.
Preferably, in the above technical solution, the real-time fluorescent amplification curve is: the fluorescence signal is collected, the amplification curve is positive in the S shape, and the linear type of the amplification fluorescence signal is not collected and is negative.
Preferably, in the above technical solution, the visual observation method of fluorescent dye: after the reaction is finished, the reaction solution is observed to be positive in green fluorescence and negative in orange color by naked eyes.
Preferably, in the above technical scheme, the LAMP reaction conditions are as follows: incubation was carried out at 65℃for 60min, fluorescence was read at 1min intervals, followed by inactivation at 80℃for 5min, and the reaction was terminated.
The LAMP visual rapid detection method for the powdery mildew of the strawberry is applied to diagnosis, monitoring and identification of the powdery mildew of the strawberry.
The LAMP visual rapid detection method of the powdery mildew of the strawberries is applied to the detection of the bacterial carrying tissue of the powdery mildew plant of the strawberries and the early stage and early stage of the disease.
The LAMP visual rapid detection method of powdery mildew of strawberry is applied to rapid detection of transgenic strawberries and processed products thereof.
The technical scheme of the application has the following beneficial effects:
the LAMP primer for the powdery mildew of the strawberry provided by the application has the advantages of strong specificity, good amplification effect, quick, simple and convenient construction, strong specificity and high sensitivity, can be used for carrying out the detection on the bacterial carrying tissue of the powdery mildew of the strawberry and carrying out the early stage and the initial stage of the disease, has short detection time, does not depend on a noble instrument, has low requirements on operators, can directly observe results by naked eyes, has low detection cost and the like, and has important guiding significance for early diagnosis and monitoring of the powdery mildew of the strawberry, early prevention and determination of the optimal prevention and treatment time.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description, serve to explain the principles of the application.
FIG. 1 is a diagram showing the results of LAMP primer specific detection according to the present application. Wherein Sample1 is powdery mildew of strawberry (Sphaerotheca aphanis), sample2 is powdery mildew of capsicum (Oidiopsis taurica), sample3 is powdery mildew of cucumber (Sphaerotheca cucurbitae), sample4 is Botrytis cinerea (Botrytis cinerea), sample5 is root rot of strawberry (Fusarium sp.), sample6 is anthracnose of strawberry (Colletotrichum gloeosporioides), sample7 is a negative control of clear water.
FIG. 2 is a graph showing the result of optimizing the final concentration of dNTPs in the LAMP detection system of the present application.
FIG. 3 is a graph showing the result of optimizing the final concentration of dNTPs in the LAMP detection system of the present application.
FIG. 4 shows the Mg in the LAMP detection system of the present application 2+ Optimized results for final concentrationGraph diagram.
FIG. 5 shows the Mg in the LAMP detection system of the present application 2+ And (5) optimizing a final concentration result diagram.
FIG. 6 is a graph showing the results of optimizing the final betaine concentration in the LAMP detection system of the present application.
FIG. 7 is a graph showing the results of optimizing the final betaine concentration in the LAMP detection system of the present application.
FIG. 8 is a graph showing the results of optimizing the final concentration of BstDNA polymerase in the LAMP detection system of the present application.
FIG. 9 is a graph showing the results of optimizing the final concentration of BstDNA polymerase in the LAMP detection system of the present application.
FIG. 10 is a graph showing the result of optimizing the temperature in the LAMP detection system of the present application.
FIG. 11 is a graph showing the result of optimizing the temperature in the LAMP detection system of the present application.
FIG. 12 is a graph showing the detection results of LAMP amplification sensitivity of the present application.
FIG. 13 is a graph showing the results of the reproducibility of the LAMP optimization system of the present application.
FIG. 14 is a diagram showing the results of LAMP field test of the present application.
Detailed Description
Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that: the relative arrangement of the components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present application unless it is specifically stated otherwise.
EXAMPLE 1 Rapid extraction of genomic DNA from test strains
A small amount of strawberry powdery mildew hypha (preserved in a laboratory of a plant protection institute of Tianjin national academy of agricultural sciences) is picked up by using a sterilized toothpick, placed in a sterilized PCR tube, 50 mu L of Lysis Buffer A solution is added, after incubation for 10min at 95 ℃, centrifugation is carried out at 10000rpm for 20s, 25-30 mu L of supernatant is taken, an equal volume of Dilution Buffer B solution is added, and the mixture is uniformly mixed by using an oscillator to obtain a DNA solution, and the DNA concentration is measured by using an ultraviolet spectrophotometer and placed at-20 ℃ for LAMP detection of a test DNA template. The whole genome DNA extraction process takes about 15 min.
Example 2 design of LAMP primer for powdery mildew of strawberry
According to published ITS sequences of powdery mildew (Podosphaera aphanis) in NCBI, DNAStar6.0 software is adopted for sequence comparison, and primer design is carried out according to the ITS sequences of the highly conserved regions of powdery mildew. According to the principle of LAMP primer design, primePremier4.0 software is utilized to design a set of specific LAMP primer groups of the powdery mildew of strawberry for 2 specific areas, wherein the specific LAMP primer groups consist of 1 pair of outer primers F3/B3, 1 pair of inner primers FIP/BIP and 1 pair of loop primers LF/LB, and the sequences of the F3/B3, FIP/BIP and LF/LB primers are as follows:
F3:5’-CGGTATTCCGAGGGGCAT-3’(SEQ ID NO.1)，
B3:5’-CCTGATCCGAGGTCATCCAA-3’(SEQ ID NO.2)，
FIP:5’-CTGTTTAGGGGACGCCGAGCTGTTCGAGCGTCAGAACATC-3’(SEQ ID NO.3)，
BIP:5’-GGCGGTACCGTTGTGCTCTCTAGGTGGGTTTTGGCAGGT-3’(SEQ ID NO.4)，
LF:5’-ACCAAGCCAGGCTTGAGAG-3’(SEQ ID NO.5)，
LB:5’-GTAGTCACGTATCTCGCGACAGAG-3’(SEQ ID NO.6)。
example 3 LAMP primer specificity verification of strawberry powdery mildew
The method is characterized in that the DNA of test strains such as powdery mildew (Sphaerotheca aphanis) of strawberry, powdery mildew (Sphaerotheca cucurbitae) of cucumber, powdery mildew (Oidiopsis taurica) of pepper, powdery mildew (Leveillula taurica) of tomato, downy mildew (Plasmopara viticola) of grape, downy mildew (Pseudoperonospora cubensis) of cucumber, powdery mildew (Cladosporium cladosporioides) of melon, root rot of strawberry (Fusarium sp.), gray mold (Botrytis cinerea) of strawberry, colletotrichum fragrans (Colletotrichum gloeosporioides) of strawberry, corynespora cucumeris (Corynespora cassiicola), gray leaf spot bacteria (Stemphylium solani) of tomato, black spot bacteria (Alternaria raphani) of radish and the like are taken as templates, the LAMP amplification is carried out by using F3, B3, FIP, BIP, LF and LB, and the reaction result is judged by using a method combining a real-time amplification curve and visual observation of fluorescent dye.
LAMP detection reaction initial System (25. Mu.L): 10 times Isothermal Amplification Buffer 2.5. Mu.L MgSO with a final concentration of 8mmol/L 4 1.5. Mu.L, 1.4mmol/LdNTPs 3.5. Mu.L, 0.8mmol/L betaine 0.4. Mu.L, 80U/. Mu.L BstDNA polymerase 0.25. Mu. L, DNA template 2.0. Mu.L, calcein 1. Mu.L, FIP and BIP 1.6. Mu.M each 1. Mu.L, 0.2. Mu. M F3 and B3 each 1. Mu.L, loopF and LoopB 1. Mu.L each, sterile deionized water make up 25. Mu.L. LAMP reaction conditions: incubation was carried out at 63℃for 60min, followed by inactivation at 80℃for 5min. In the reaction process of the LAMP reaction system, fluorescence is read every 1min, and fluorescence signals are collected.
Judging the reaction result, and evaluating the detection result according to the real-time amplification curve and visual observation of fluorescent dye: (1) Detecting a real-time fluorescent amplification curve, wherein the fluorescent signal is collected, the amplification curve is positive in an S shape, and the linear type of the amplification curve, which is not collected, is negative; (2) dye color reaction detection: after the completion of the reaction, 1. Mu.L of calcein fluorescent dye (Loopamp FD) was added to the LAMP reaction system, and the reaction mixture was observed to be positive in green fluorescence and negative in orange color.
Primer specificity verification results: the real-time amplification result shows (as shown in figure 1), the primer group only shows an S-shaped curve of real-time amplification for the powdery mildew of the strawberry in the period of nearly 30min, and no curve is amplified by other pathogenic bacteria and negative control; and combining fluorescent color development observation, only the EP tube with the strawberry powdery mildew DNA as a template presents green fluorescence, namely the strawberry powdery mildew is detected in the sample, and other pathogenic bacteria and negative control are orange, namely the sample does not contain the strawberry powdery mildew.
The experiment shows that the strawberry powdery mildew F3, B3, FIP, BIP, loopF and LoopB can distinguish the strawberry powdery mildew from other pathogenic bacteria, have the species specificity, and can be used for detecting and identifying the strawberry powdery mildew.
EXAMPLE 4 establishment and optimization of the LAMP reaction System of powdery mildew of strawberry
LAMP detection System and Condition optimization based on the reaction initiation System of example 3
(1) Optimization of dNTPs final concentration
According to the initial system and procedure of LAMP reaction, the other components are kept unchanged in dosage and condition, the LAMP reaction is carried out with 6 dNTPs final concentration gradients (0.8 mmol/L, 1.0mmol/L, 1.2mmol/L, 1.4mmol/L, 1.6mmol/L, 1.8 mmol/L), and a negative control (ddH is used 2 O is the template).
The real-time amplification curve results show (as shown in FIG. 2), that as the final concentration of dNTPs increases, the amplification start time is earlier (which means that the concentration of template DNA after amplification is higher), the peak value is higher (which means that the fluorescence signal intensity is higher), the amplification efficiency is optimal when the final concentration of dNTPs is 1.6mmol/L, and neither the final concentration is 1.8mmol/L nor the negative control is amplified for 60 min.
The fluorescent dye color development result shows (as shown in figure 3), the final concentration of 0.8-1.6mmol/L is green, the fluorescent green of 1.6mmol/L is the deepest and the most obvious, the fluorescent dye color development result shows that 1.8mmol/L and the negative control are orange, and the optimal final concentration of dNTPs is determined to be 1.6mmol/L by combining the double determination result.
(2)Mg 2+ Optimization of final concentration
On the basis of the optimized result, the dosage and the conditions of other components are unchanged, 4mmol/L, 6mmol/L, 8mmol/L, 10mmol/L and 12mmol/L of 5 Mg are respectively arranged 2+ And (3) final concentration gradient, and setting negative control at the same time, and performing LAMP visual detection. Screening the optimal Mg through a real-time amplification curve and a fluorescent color development result after the reaction is finished 2+ Final concentration.
The results are shown in FIGS. 4 and 5, mg 2+ A typical S curve can be amplified at a final concentration of 6-12 mmol/L, wherein 8mmol/L amplification has the shortest detection time, highest efficiency and Mg 2+ S curve does not appear in the final concentration of 4mmol/L, and the detection result is positive; the fluorescent color development result shows that Mg 2+ The final concentration of 6mmol/L-12mmol/L is fluorescent green, and the treated 4mmol/L and negative control are orange, and comprehensively judges the optimal Mg 2+ The final concentration was 8mmol/L.
(3) Betaine concentration optimization
On the basis of the optimized result, other dosage and conditions are kept unchanged, and final concentration gradients of 0mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L and 1.2mmol/L of 7 betaines are respectively set, and negative control is set at the same time for LAMP visual detection. And screening the optimal final concentration of the betaine through a real-time amplification curve and a fluorescent chromogenic dual result.
The real-time amplification result shows (shown in figure 6), the treatment of betaine with different final concentrations has little influence on the amplification starting time, the final concentration of 0-1.2mmol/L can be amplified to obtain an obvious S-shaped curve, and the ordinate of the final concentration of 1.2mmol/L shows a stronger fluorescence signal; the visual results of the fluorescent dye show that the treatment of 7 final concentrations is fluorescent green, the negative control is orange, and the optimal final concentration of betaine is finally determined to be 1.2mmol/L according to the amplification curve results.
(4) BstDNA polymerase concentration optimization
Based on the optimized result, the dosage and conditions of other components are kept unchanged, 5 BstDNA polymerase final concentration gradients of 40u/mL, 80u/mL, 160u/mL, 320u/mL and 340u/mL are respectively set, and meanwhile, negative control is set for LAMP visual detection. And after the reaction is finished, comprehensively judging and screening the optimal final concentration of the BstDNA polymerase by using a real-time amplification curve and observing a fluorescent color development result by naked eyes.
The real-time amplification result shows (shown in figure 8), the final concentration of BstDNA polymerase is 40-340u/mL, the obvious S-shaped curve can be amplified, and the earlier the amplification starting time is along with the increase of the final concentration, wherein the time of the curves appearing at the final concentration of 320u/mL and 340u/mL is close, and the amplification efficiency is equivalent;
the visual results of the fluorescent dye show that the 5 final concentration treatments are all fluorescent green, the negative control is orange, and the optimal final concentration of the BstDNA polymerase is 320u/mL according to the amplification curve results.
(5) Optimization of reaction temperature
On the basis of the optimized result, the dosage and the conditions of other components are kept unchanged, the reaction is carried out according to the optimized reaction system, the reaction temperature is set to 63 ℃,65 ℃ and 67 ℃ respectively, the LAMP reaction is carried out, and meanwhile, the negative control is set. After the reaction is finished, the optimal reaction temperature is screened through a real-time amplification curve and a fluorescent color development result.
The real-time amplification result shows (as shown in figure 10), the amplification curve can appear at the temperature of 63 ℃,65 ℃ and 67 ℃, wherein the time of the 65 ℃ amplification curve appears earliest and the amplification efficiency is highest; the visual results of the fluorescent dye show that the fluorescent dye is green fluorescent after 3 temperature treatments (as shown in figure 11), the color of the fluorescent dye is darker and brighter at 65 ℃, the negative control is orange, and the optimal reaction temperature at 65 ℃ is finally determined.
LAMP detection optimizes the reaction system (25. Mu.L) according to the above results: 10X Isothermal Amplification Buffer 2.5.5. Mu.L MgSO at a final concentration of 8mmol/L 4 1.5. Mu.L, 1.6mmol/LdNTPs 4.0. Mu.L, 1.2mmol/L betaine 0.6. Mu.L, 320u/mLBSTDNA polymerase 1. Mu.L, DNA template 2.0. Mu.L, calcein 1. Mu.L, FIP and BIP 1.6. Mu.M each, 0.2. Mu.MF 3 and B3 each, loopF and LoopB 1. Mu.L each, sterile deionized water make up 25. Mu.L.
LAMP reaction conditions: incubation was carried out at 65℃for 60min, followed by inactivation at 80℃for 5min. In the reaction process of the LAMP reaction system, fluorescence is read every 1min, and fluorescence signals are collected.
Example 5 LAMP detection sensitivity determination of powdery mildew of strawberry
(1) Preparation of genomic DNA at different concentrations
Diluting the genomic DNA of powdery mildew with sterile ultrapure water to obtain 1, 1×10 -1 、1×10 -2 、1×10 -3 、1×10 -4 、1×10 -5 ng/μL、1×10 -6 The ng/. Mu.L series of concentrations were ready for use.
(2) LAMP detection method sensitivity measurement
And (3) performing an LAMP sensitivity test by taking 2 mu L of strawberry powdery mildew DNA with different mass concentrations as a template, using deionized water to replace the DNA template as a blank control, performing amplification by using an LAMP optimal reaction system and optimal reaction conditions established in the embodiment 2, and comprehensively determining the LAMP detection sensitivity by adopting a real-time fluorescence amplification curve and a double result of a fluorescent dye visual observation method after the LAMP reaction is finished. The real-time fluorescent amplification detection result is positive in the S shape, and the linear result is negative; the visual inspection result of the fluorescent dye shows that green fluorescence is positive, and orange is negative.
(3) LAMP amplification sensitivity detection results
LAMP amplification sensitivity test results showed that 1, 1×10 -1 、1×10 -2 、1×10 -3 、1×10 -4 The genomic DNA chromogenic result of the powdery mildew at the concentration of ng/mu L can observe green fluorescence, a real-time fluorescent amplification curve is typical S, the chromogenic result of other concentrations and negative control is orange, and the real-time amplification is linear, which shows that the detection sensitivity of the designed primer composition F3, B3, FIP, BIP, LF and LB for the powdery mildew can reach 1 multiplied by 10 through the optimized LAMP amplification -4 ng/. Mu.L (as shown in FIG. 12).
Example 6 LAMP detection method for powdery mildew of strawberry repeatability verification
The method comprises the steps of collecting 5 parts of strawberry leaves, fruits and fruit stalks infected with powdery mildew as positive samples in a strawberry greenhouse (the greenhouse is a natural disease nursery for powdery mildew of strawberry, the disease of the powdery mildew of strawberry is serious, the disease rate is more than 70 percent, and the medicament prevention and control are not carried out in the whole growth period) in Wuqing district of Tianjin city; collecting 5 healthy and undeveloped leaves, fruits and fruit stalks as negative samples in a Hangu strawberry greenhouse in Tianjin, wherein fertilizer and water management of the greenhouse is good, early-stage medicament prevention is good, and powdery mildew of the strawberries is hardly generated; 3 parts of deionized water are used as negative control. Diseased tissue was subjected to rapid whole genome DNA extraction in example 1, and its reproducibility and reliability were verified by the LAMP detection method established in example 2.
Visual inspection showed that all positive samples infected with powdery mildew could be stained with fluorescent dye, exhibited significant green fluorescence, while all healthy negative samples and negative control samples were all shown to be orange, indicating that they were not infected with powdery mildew of strawberry (as shown in fig. 13, wherein 1-leaf (light onset), 2-fruit (light onset), 3-fruit stem (light onset), 4-leaf (severe onset), 5-fruit (severe onset), 6-fruit stem (severe onset), 7-healthy fruit), which was consistent with the previously known onset and health condition at the time of collection, so that the reliability and good reproducibility of the above-established LAMP detection method for powdery mildew of strawberry could be demonstrated.
Example 7 detection of field strawberry samples using established LAMP visual detection methods
And (3) performing field detection on the strawberry powdery mildew in the strawberry test greenhouse of the Wuqing base in Tianjin city by using the established LAMP detection method for the strawberry powdery mildew.
The sample collection method comprises the following steps: samples of different parts of the plant and different disease degrees are collected.
The leaf is respectively collected into five leaves with three disease degrees of light disease (only the back of the leaf has a chlorosis spot, no white mould layer is seen on the disease spot), light disease (a small amount of sparse white mould layer is arranged at the back disease spot) and serious disease (a large amount of thick white mould layer is generated at the front and back disease spots of the leaf), and the five healthy leaves are taken as positive control.
Fruits with three degrees of incidence of light disease (slightly deformed fruits but without any disease spots and white mold layers), light disease (a small amount of sparse mold layers on the surfaces of the fruits) and serious disease (the whole fruits are soft and the fruits are wrapped by the white mold layers) are respectively collected, and five healthy and fresh fruits are used as positive controls.
The fruit stalks are respectively used as positive controls, wherein five samples of three disease degrees of lighter disease degree (white mould layer is not seen at the fruit stalks, but reddish brown color is seen), heavier disease degree (a small amount of sparse mould layer is arranged on the surfaces of the fruit stalks) and serious disease degree (the whole fruit stalks are covered by the white mould layer) and five healthy and non-disease fruit stalks are respectively collected.
The collected powdery mildew tissue samples are subjected to DNA rapid extraction and are detected by using an optimized and established LAMP detection method, and the result shows that (as shown in figure 14, the number 1-7 is a strawberry leaf sample, 1 is a very light leaf of the disease, 2 and 3 are heavier, 4 and 5 are serious, 6 and 7 are healthy leaves, the number 8-14 is a strawberry fruit sample, 8 and 9 is a relatively light leaf, 10 is a heavier leaf of the disease, 11 and 12 are serious, 13 and 14 are healthy fruits, the number 15-21 is a strawberry fruit handle sample, 15 is a relatively light leaf, 16 and 17 are serious, 18 and 19 are serious, 20 and 21 are healthy fruit handles), the samples of the strawberry leaf, fruit and fruit handle are all of different degrees of the disease and are all of green fluorescence in the LAMP system detection period, the collected leaf, fruit and fruit handle are of different degrees of the disease, the leaf of the disease is accurately detected when the disease is not obvious, the typical symptoms of the powdery mildew layer are not shown, if the leaf of the powdery mildew is light leaf, the strawberry leaf is only has the light leaf of the powdery mildew, the strawberry is only has the quality of the disease, the surface of the powdery mildew is only has been clearly judged by the rapid detection of the quality of the powdery mildew, and the surface quality of the powdery mildew is only has been clearly judged by the surface characteristics of the powdery mildew; and the healthy leaves and the negative control are orange after fluorescent staining, which indicates that the healthy leaves and the negative control are not infected with the strawberry powdery mildew and are consistent with the actual sample condition.
The results prove that the screened LAMP primer group and the established LAMP reaction system are used for detecting the strawberry powdery mildew sample, a more reliable scientific basis is provided for purposefully developing daily spot inspection monitoring work and strawberry powdery mildew risk assessment in the future, and the method has good production practice feasibility and wide application prospect in the aspect of strawberry powdery mildew detection.
Compared with the prior art, the application has the following technical advantages and positive effects:
1. high efficiency, high speed and short time consumption:
the whole amplification reaction is carried out at a constant temperature of 65 ℃, the process of heating and annealing for several times is not needed, the time of the amplification reaction is obviously shortened, and the detection is completed within 60 minutes.
2. The specificity is strong:
the LAMP primer design is complex, 4 primers respectively specifically target 6 regions of the target gene, and the 6 regions have strict sequence requirements, so that the LAMP method has high specificity.
3. The operation is convenient and the requirement on instrument equipment is not high:
the LAMP reaction is completed in the whole process at a fixed temperature of 65 ℃, so that the LAMP reaction can meet the requirements only by ensuring a constant-temperature environment and having low requirements on instruments, and particularly, a cheap and common water bath kettle for a basic laboratory.
4. The result judgment is simple:
a large amount of white byproduct magnesium pyrophosphate precipitate is generated during the LAMP reaction or a color change is observed by adding a color-developing agent. The amplification reaction can be observed by naked eyes, a real-time fluorescence quantitative PCR instrument can be used for carrying out whole-process monitoring when the method is established, a DNA amplification fluorescent signal is collected through a VIC channel, the data quantization is carried out on the time and the quantity generated by the DNA amplification fluorescent signal, the amplification efficiency and effect are judged, the condition optimization efficiency is improved, and the sensitivity of the method is accurately mastered; in particular, the amplification products of the LAMP do not need to be analyzed through complicated gel electrophoresis and ultraviolet detection processes, so that uncapping detection is avoided, and a large amount of amplification products of the LAMP are exposed to the air to cause serious aerosol pollution to a laboratory.
Although the present application has been described with reference to the above embodiments, it should be understood that the present application is not limited thereto, and that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present application, and the scope of the present application is defined by the appended claims and their equivalents.
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Claims (5)
1. The LAMP visual rapid detection method for the powdery mildew of strawberries is characterized in that the primers in the detection method comprise 1 pair of outer primers F3 and B3,1 pair of inner primers FIP and BIP and 1 pair of loop primers LF and LB, and the sequences are as follows:
F3: 5’-CGGTATTCCGAGGGGCAT-3’，
B3: 5’-CCTGATCCGAGGTCATCCAA-3’，
FIP: 5’-CTGTTTAGGGGACGCCGAGCTGTTCGAGCGTCAGAACATC-3’，BIP: 5’- GGCGGTACCGTTGTGCTCTCTAGGTGGGTTTTGGCAGGT-3’，
LF: 5’-ACCAAGCCAGGCTTGAGAG-3’，
LB: 5’-GTAGTCACGTATCTCGCGACAGAG-3’；
the LAMP visual rapid detection method for the powdery mildew of the strawberry comprises the following steps: taking DNA of a sample to be detected as a template, performing LAMP reaction amplification by using the primer, and detecting by a real-time fluorescent amplification curve and/or a fluorescent dye visual observation method;
the real-time fluorescent amplification curve is as follows: in the reaction process of the LAMP reaction system, fluorescence is read every 1min, fluorescence signals are collected, an amplification curve is positive in an S shape, and a linear type without collecting the amplified fluorescence signals is negative; visual observation of fluorescent dyes: adding 1 mu L of calcein fluorescent dye into the LAMP reaction system, and after the reaction is finished, visually observing that the reaction solution is positive in green fluorescence and negative in orange color;
the LAMP reaction system comprises the following steps: 10X Isothermal Amplification Buffer 2.5. Mu.L, 8mmol/L MgSO 4 1.5 mu.L, 1.6mmol/LdNTPs 4.0. Mu.L, 1.2mmol/L betaine 0.6. Mu.L, 320. 320u/mLBSTDNA polymerase 1. Mu.L, DNA template 2.0. Mu.L, calcein 1. Mu.L, FIP and BIP 1.6. Mu.M each 1. Mu.L, 0.2. Mu.MF 3 and B3 each 1. Mu.L, 0.4. Mu.M LoopF and LoopB each 1. Mu.L, sterile deionized water make up 25. Mu.L.
2. The rapid LAMP visualization detection method for powdery mildew of strawberry of claim 1, wherein the LAMP reaction conditions are as follows: after incubation at 65℃for 60min, inactivation was performed at 80℃for 5min.
3. The application of the LAMP visual rapid detection method for powdery mildew of strawberry according to any one of claims 1-2 in diagnosis, monitoring and identification of powdery mildew of strawberry.
4. The application of the LAMP visual rapid detection method for powdery mildew of strawberry according to any one of claims 1-2 in the detection of the powdery mildew plant bacterial infection tissue and early stage of disease occurrence in field bacterial infection.
5. The application of the LAMP visual rapid detection method for powdery mildew of strawberry according to any one of claims 1-2 in rapid detection of transgenic strawberries and processed products thereof.
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