Method for detecting content of gibberella zeae in soil based on real-time fluorescent quantitative PCR
阅读说明：本技术 一种基于实时荧光定量pcr检测土壤小麦赤霉病菌含量的方法 (Method for detecting content of gibberella zeae in soil based on real-time fluorescent quantitative PCR ) 是由 赵炳梓 周云鹏 于 2021-09-15 设计创作，主要内容包括：本发明公开了一种基于实时荧光定量PCR检测土壤小麦赤霉病菌含量的方法,属于生物技术领域。本发明将纯培养的小麦赤霉病菌的菌丝DNA倍比稀释制成系列浓度梯度的标准曲线模板DNA溶液,对待测土壤样品模板DNA溶液和标准曲线模板DNA溶液进行实时荧光定量PCR扩增,绘制出标准曲线,同时将检测的样品循环阈值与标准曲线对照,进一步得到待测土壤样品模板DNA中的小麦赤霉病菌DNA浓度及土壤样品每克干土的小麦赤霉病菌含量。本发明方法可以快速、准确地检测出土壤小麦赤霉病菌含量,便于对土壤残留目标菌源量进行实时动态检测,为小麦赤霉病害的早期防控提供了技术支持。(The invention discloses a method for detecting the content of gibberella zeae in soil based on real-time fluorescent quantitative PCR (polymerase chain reaction), and belongs to the technical field of biology. The method comprises the steps of diluting hypha DNA of pure-cultured wheat scab germs by a multiple ratio to prepare a standard curve template DNA solution with a series of concentration gradients, carrying out real-time fluorescent quantitative PCR amplification on the soil sample template DNA solution to be detected and the standard curve template DNA solution, drawing a standard curve, and comparing a detected sample circulation threshold value with the standard curve to further obtain the wheat scab germ DNA concentration in the soil sample template DNA to be detected and the wheat scab germ content in each gram of dry soil of the soil sample. The method can quickly and accurately detect the content of the gibberella zeae in the soil, is convenient for carrying out real-time dynamic detection on the residual target bacteria source quantity of the soil, and provides technical support for early prevention and control of the gibberella zeae disease of the wheat.)
1. A method for detecting the content of soil wheat scab germs based on real-time fluorescent quantitative PCR is characterized by comprising the following steps:
1) extracting total DNA of a fresh soil sample, and diluting to prepare a template DNA solution of the soil sample to be detected;
2) extracting hypha DNA of the pure cultured wheat scab germ, measuring the concentration of the hypha DNA, and diluting the hypha DNA in multiple proportions to prepare a standard curve template DNA solution with a series of concentration gradients;
3) performing real-time fluorescent quantitative PCR amplification on a soil sample template DNA solution to be detected and a standard curve template DNA solution, and drawing a standard curve according to a logarithmic value of the standard curve template DNA concentration and a cycle number Ct value to be determined, wherein y is ax + b; wherein y is the cycle number Ct, and x is the logarithm of the DNA concentration of the wheat scab germ;
4) calculating the concentration of the wheat scab germ DNA in the soil sample template DNA to be detected to be 10(Ct-b)/aIn ng/. mu.L;
5) calculating the content of the wheat scab germs of each gram of dry soil of the soil sample as C multiplied by d multiplied by V/M, and the unit is ng/g; in the formula, C is the DNA concentration of the wheat scab in the DNA solution of the soil sample template to be detected, and the unit is ng/mu L; d is the dilution multiple of the total DNA of the soil sample; v is the total DNA volume of the extracted soil sample, and the unit is mu L; m is the soil sample dry weight in g.
2. The method for detecting the content of the fusarium graminearum in the soil based on the real-time fluorescent quantitative PCR as claimed in claim 1, wherein the reaction system of the real-time fluorescent quantitative PCR amplification is as follows:Premix Ex TaqTM12.5. mu.L of each of the upstream and downstream primers, 2. mu.L of the DNA template, and sterile double distilled water to 25. mu.L.
3. The method for detecting the content of the fusarium graminearum in the soil based on the real-time fluorescent quantitative PCR as claimed in claim 1, wherein the reaction conditions of the real-time fluorescent quantitative PCR are as follows: 95 ℃ for 2min, 95 ℃ for 15s, 60 ℃ for 30s, 72 ℃ for 45s, 40 cycles.
4. The method for detecting the content of the fusarium graminearum in the soil based on the real-time fluorescent quantitative PCR as claimed in claim 1, wherein the melting curve program of the real-time fluorescent quantitative PCR is as follows: the temperature was gradually increased from 65 ℃ to 95 ℃ in increments of 0.5 ℃/5 s.
5. The specific primer pair for detecting or assisting in detecting the soil fusarium graminearum is characterized by comprising an upstream primer and a downstream primer, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively as follows:
the sequence of the upstream primer Fg16F is 5'-CTCCGGATATGTTGCGTCAA-3',
the sequence of the downstream primer Fg16R is 5'-GGTAGGTATCCGACATGGCAA-3'.
6. PCR reagent for detecting or assisting in detecting soil gibberella zeae, characterized in that the reagent comprises the specific primer pair of claim 5.
7. A PCR kit for detecting or assisting in detecting soil gibberella zeae, characterized in that the kit contains the specific primer pair of claim 5.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting the content of wheat scab germs in soil based on real-time fluorescent quantitative PCR.
Background
Wheat scab caused by Fusarium graminearum (Fusarium graminearum) is a very devastating and difficult-to-control fungal disease in the global scope, frequently occurs in warm and humid areas, has the characteristic of periodic circulation, and is one of the important reasons for great reduction of the yield and the quality of wheat. Wheat scab mainly infects ear of wheat, when climatic conditions such as temperature and humidity are appropriate, pathogenic bacteria can form a large amount of pink mildew layer on the ear of wheat, and various mycotoxins generated by metabolism of the pathogenic bacteria can cause great harm to the health of people and livestock. The incidence area of wheat scab in China is the largest globally, the wheat scab is the most serious in wheat areas in the south of Huaihe river and the middle and lower reaches of Yangtze river, and the wheat areas in the south China and the northeast China are also frequently in a disaster. At present, with the popularization of straw returning, the enlargement of irrigation area and global warming, the scab of wheat has gradually expanded to the northwest of Huang-Huai-Hai winter wheat area.
Wheat scab belongs to a multicycle fungal disease, and pathogenic bacteria usually take the form of an ascocarp and live through winter and summer on crop diseased and damaged tissues or soil surface straw residues. When the wheat is in the heading and flowering stage in the spring of the next year, a large amount of ascospores on the diseased residues are released into the air from an orifice at the top of the ascocarp shell after the ascocarp shell grows mature, and the wheat ears are infected by the aid of medium such as wind, rainwater or insects by scattering and serving as a primary infection source. And a large number of pathogenic conidia carried on the susceptible wheat ears can be further spread and spread under the action of wind and rain, and become a secondary infection source of the gibberellic disease. Therefore, ascospores and conidia carried on the diseased residues often infect the wheat head at the same time, and the cycle of gibberellic disease is accelerated.
In recent years, in order to promote the sustainable development of agriculture, returning straws to fields is vigorously advocated in China, but the direct returning of the straws to fields often aggravates the occurrence of crop diseases and insect pests. Wheat scab germs taking field straws as a main growth substrate usually remain in soil along with disease residues such as straws and the like for a long time, and when the residual accumulation amount of the pathogenic bacteria in the field soil is increased, the incidence rate and the harm degree of the wheat scab germs are correspondingly increased. Because wheat scab has the characteristics of prevention, controllability and no cure, early prevention is particularly important in practical production. Therefore, a technology for quickly and accurately detecting the soil wheat scab is established, the real-time dynamic detection of the residual bacteria source amount of the soil is realized, and the method has important significance for the comprehensive prevention and control of the wheat scab.
The current quantitative detection method for soil wheat scab usually adopts a selective culture medium to carry out germ isolation culture and colony counting. The method has the advantages of low accuracy, time and labor consumption, high possibility of being influenced by mixed bacteria and high experience. The real-time fluorescence quantitative PCR technology is a method for the quantitative research of microorganisms newly developed in recent years, has the characteristics of high specificity, sensitivity, rapidness and the like, can carry out absolute quantitative research on target pathogenic bacteria, has more accurate results compared with the traditional counting method, and avoids the complicated separation and culture process. Application No. 2020103703729 discloses a primer, a probe, a kit and a method for detecting the RT-QPCR of fusarium graminearum, the method has high specificity and good sensitivity, can accurately detect the content of wheat scab bacteria on plant tissues, but cannot detect the content of the wheat scab bacteria in the soil environment; application No. 201110419678X discloses a method for establishing fusarium copy number in rhizosphere soil in a growth period of transgenic wheat through fluorescent real-time quantitative PCR, wherein the method can quickly and accurately detect the content of fusarium in the soil, but cannot represent the content of fusarium graminearum in the soil on a seed level.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for detecting the content of the gibberella zeae in soil based on real-time fluorescent quantitative PCR.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for detecting the content of soil wheat scab germs based on real-time fluorescent quantitative PCR comprises the following steps:
1) extracting total DNA of a fresh soil sample, and diluting to prepare a template DNA solution of the soil sample to be detected;
2) extracting hypha DNA of the pure cultured wheat scab germ, measuring the concentration of the hypha DNA, and diluting the hypha DNA in multiple proportions to prepare a standard curve template DNA solution with a series of concentration gradients;
3) performing real-time fluorescent quantitative PCR amplification on a soil sample template DNA solution to be detected and a standard curve template DNA solution, and drawing a standard curve according to a logarithmic value of the standard curve template DNA concentration and a cycle number Ct value to be determined, wherein y is ax + b; wherein y is the cycle number Ct, and x is the logarithm of the DNA concentration of the wheat scab germ;
4) calculating the concentration of the wheat scab germ DNA in the soil sample template DNA to be detected to be 10(Ct-b)/aIn ng/. mu.L;
5) calculating the content of the wheat scab germs of each gram of dry soil of the soil sample as C multiplied by d multiplied by V/M, and the unit is ng/g; in the formula, C is the DNA concentration of the wheat scab in the DNA solution of the soil sample template to be detected, and the unit is ng/mu L; d is the dilution multiple of the total DNA of the soil sample; v is the total DNA volume of the extracted soil sample, and the unit is mu L; m is the soil sample dry weight in g.
When a standard curve is drawn, the Fusarium graminearum strain to be tested is Fusarium graminearum FG0609, and after the Fusarium graminearum strain is cultured on a potato glucose agar solid culture medium at a constant temperature in a dark place for 7 days at a temperature of 28 ℃, a sterile inoculation shovel is used for gently scraping fresh hyphae on the surface layer and placing the hyphae in liquid nitrogen to be ground into powder; by usingExtracting the total DNA of the powder by a Fungal DNA Kit, determining the concentration of the total DNA, and finally diluting the total DNA of the wheat scab hyphae by using sterilized double-distilled water according to a ratio of 10 times to prepare the DNA multiplied by 101ng/μL、×100ng/μL、×10-1ng/μL、×10-2ng/μL、×10-3ng/μL、×10-4ng/. mu.L and X10-5ng/. mu.L, etc. of 7 series concentration gradient standard curve template DNA solutions.
In the present invention, the standard curve equation is-3.5627 x +18.961, R2＝0.9985。
Further, the reaction system of the real-time fluorescence quantitative PCR amplification is as follows:Premix Ex TaqTM12.5. mu.L of each of the upstream and downstream primers and 2. mu.L of the DNA template, and sterilizingMake up to 25 μ L with double distilled water.
Further, the reaction conditions of the real-time fluorescence quantitative PCR are as follows: 95 ℃ for 2min, 95 ℃ for 15s, 60 ℃ for 30s, 72 ℃ for 45s, 40 cycles.
Further, the melting curve program of real-time fluorescence quantitative PCR is as follows: the temperature was gradually increased from 65 ℃ to 95 ℃ in increments of 0.5 ℃/5 s.
The melting curve of the real-time fluorescent quantitative PCR is a single peak, and the melting temperature is 84.5 ℃; the amplification curve is in a better S shape, and the amplification efficiency is 90.8%.
The specific primer pair for detecting or assisting in detecting the soil fusarium graminearum comprises an upstream primer and a downstream primer, and the nucleotide sequences of the specific primer pair are respectively as follows:
the sequence of the upstream primer Fg16F is 5'-CTCCGGATATGTTGCGTCAA-3',
the sequence of the downstream primer Fg16R is 5'-GGTAGGTATCCGACATGGCAA-3'.
The PCR reagent for detecting or assisting in detecting the soil wheat scab germ comprises the specific primer pair.
The PCR kit for detecting or assisting in detecting the soil wheat scab germ contains the specific primer pair.
Has the advantages that: compared with the prior art, the invention has the advantages that:
the method can quickly and accurately detect the content of the gibberella zeae in the soil, is convenient for carrying out real-time dynamic detection on the residual target bacteria source quantity of the soil, and provides technical support for early prevention and control of the gibberella zeae disease of the wheat. The method overcomes the defects of low accuracy, time and labor consumption, strong experience and the like of the traditional flat plate bacterial colony counting method, can be used for carrying out absolute quantitative research on target pathogenic bacteria, has more accurate results compared with the traditional counting method, and greatly improves the detection efficiency of the soil wheat scab bacteria.
Drawings
FIG. 1 is a standard curve diagram established with the logarithmic value of the standard curve template DNA concentration as the abscissa and the cycle number Ct value as the ordinate;
FIG. 2 is a melting curve diagram of real-time fluorescence quantitative PCR of a standard curve and a soil template DNA to be detected, wherein the melting temperature is 84.5 ℃;
FIG. 3 is a graph showing the standard curve and the amplification curve of real-time fluorescent quantitative PCR of soil template DNA to be detected.
Detailed Description
The invention is further described with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. In the following examples, unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The following examples refer to strains and media:
the test Fusarium graminearum strain Fusarium graminearum FG0609 originated from the agricultural academy of Jiangsu province;
potato Dextrose Agar (PDA) medium: 20g of glucose, 200g of potatoes (the potatoes are washed, peeled, chopped and boiled for 30min, filtered by four layers of gauze, and the filtrate is taken), 20g of agar and 1L of distilled water, wherein the pH value is 7.0.
Reagents and apparatus to which the following examples refer:
the fungal genome DNA extraction kit comprisesFungal DNA Kit (Omega Bio-tek, USA) with a format of 50 times per cassette, cat # D3390-01;
the kit for extracting the total DNA of the soil comprisesSpin Kit for Soil (MP Bio, USA) with specification of 50 times/box, cat # 116560-;
Premix Ex TaqTMthe kit is purchased from Dalibao bioengineering GmbH, Cathaki No. RR 420A;
the fluorescent quantitative PCR plate and the sealing film are purchased from Stannless State department of bioengineering, Inc., the standard of the PCR plate is 96-hole, 100 mu L, PP, white, half skirt edge, sterilization, nuclease-free, and the product number is CP 2001.
The examples relate to a fluorescent quantitative PCR instrument model CFX96, available from Burley, USA, and the data analysis is performed by the software of CFX96 quantitative PCR instrument.
Example 1
The soil used in this example was abandoned 5 years old (32 ° 0 'N, 119 ° 4' E) from a abandoned land around a mountain of south beijing, south beige, jiangsu province, and the soil type was yellow brown soil. And (3) after an original soil sample is screened by a 4mm sieve, uniformly mixing the prepared diseased wheat grains and the soil in proportion, potting, and carrying out a wheat pot experiment. The soil sample to be detected is non-rhizosphere soil of wheat in the jointing stage; the treatment 1 is soil for planting wheat of the A variety, the treatment 2 is soil for planting wheat of the B variety, and the treatment 3 is blank control.
The specific operation steps are as follows:
1) sieving soil sample to be tested with 2mm sieve to remove impurities such as cobblestone, root system, etc., weighing 500mg fresh soil, and adoptingExtracting total DNA of Soil by using a Spin Kit for Soil DNA extraction Kit, measuring the concentration of the total DNA, and freezing the total DNA at the temperature of-20 ℃ for later use.
2) Inoculating the purified fusarium graminearum strain FG0609 to a potato glucose agar (PDA) solid culture medium, and culturing for 7 days at a constant temperature and in a dark place at the temperature of 28 ℃. The fresh hyphae overgrowing the PDA medium were scraped off with a sterile inoculating spatula and ground into powder in liquid nitrogen. Weighing 100mg fusarium graminearum mycelium powder for useThe total DNA of hyphae was extracted with the Fungal DNA Kit and used as a standard curve template DNA stock solution, which was frozen in a freezer at-20 ℃ for future use.
The concentration of the standard curve template DNA mother liquor is measured to be 56.0 ng/mu L, and the standard curve template DNA mother liquor is sequentially diluted by sterilized double distilled water according to the ratio of 10 timesIs prepared into 5.6X 101ng/μL、5.6×100ng/μL、5.6×10-1ng/μL、5.6×10- 2ng/μL、5.6×10-3ng/μL、5.6×10-4ng/. mu.L and 5.6X 10-5ng/. mu.L, etc. of 7 series concentration gradient standard curve template DNA solutions.
3) A soil total DNA sample with a known concentration is diluted to 20 ng/. mu.L by using sterilized double distilled water and is used as a soil template DNA solution to be detected. On a 96-well fluorescence quantitative plate, 9.9 muL of sterilized double distilled water, 0.3 muL of 10 mumol/L upstream primer Fg16F solution and 0.3 muL of 10 mumol/L downstream primer Fg16R solution are sequentially added in sequence,Premix Ex TaqTM12.5 mul, 2 mul of standard curve or soil template DNA solution to be detected; each sample was run in 3 replicates. Sealing the 96-hole fluorescent quantitative plate by using a transparent sealing film, and centrifuging and uniformly mixing by using a microporous plate centrifuge;
the sequence of the upstream primer Fg16F is 5'-CTCCGGATATGTTGCGTCAA-3', and the sequence of the downstream primer Fg16R is 5'-GGTAGGTATCCGACATGGCAA-3'. The Fg16F/Fg16R primer set was synthesized by Biotechnology engineering (Shanghai) Ltd.
4) And (3) performing real-time fluorescent quantitative PCR amplification by using a CFX96 real-time fluorescent quantitative PCR instrument. The amplification conditions were: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 45s for 40 cycles. And (3) carrying out melting curve analysis after the real-time fluorescent quantitative PCR reaction is finished, wherein the program is as follows: the temperature was raised from 65 ℃ to 95 ℃ in increments of 0.5 ℃ per 5 s.
5) A standard curve is drawn by taking the logarithmic value of the template DNA concentration of the standard curve and the Ct value of the determined cycle number. The standard curve equation of fig. 1 is-3.5627 x +18.961, and R2 is 0.9985; wherein y is the measured cycle number Ct, and x is the logarithm value of the DNA concentration of the wheat scab germ;
in the embodiment, the melting curve of the real-time fluorescent quantitative PCR is shown in FIG. 2, the melting curve is a single peak, and the melting temperature is 84.5 ℃; the amplification curve is shown in FIG. 3, which shows a better "S" shape, and the amplification efficiency is 90.8%.
6) Calculating in the template DNA of the soil sample to be testedThe DNA concentration (ng/. mu.L) of wheat scab germ is 10: (18.961 -Ct)/3.5627。
7) Calculating the content (ng/g) of the wheat scab germs in each gram of dry soil of the soil sample as C multiplied by d multiplied by V/M, wherein C is the DNA concentration of the wheat scab germs in the template DNA solution of the soil sample to be detected, and the unit is ng/mu L; d is the dilution multiple of the total DNA of the soil sample; v is the extraction volume of the total DNA of the soil sample, and the unit is mu L; m is the soil sample dry weight in g. As shown in Table 1, the real-time fluorescent quantitative PCR detection method disclosed by the invention has high sensitivity and specificity, and can accurately detect the content of the soil wheat scab.
TABLE 1 content of Fusarium graminearum (ng/g) in soils treated differently
It is to be noted that the above-mentioned list is only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
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