CN111534626A

CN111534626A - LAMP (loop-mediated isothermal amplification) detection primer composition for pythium bellatus, detection kit and visual detection method of LAMP detection primer composition

Info

Publication number: CN111534626A
Application number: CN202010330834.4A
Authority: CN
Inventors: 高春生; 余永廷; 纪灏君; 王吐虹; 李智敏; 陈佳; 程毅; 郭利桃; 聂纯武; 文传如
Original assignee: Institute of Bast Fiber Crops of CAAS
Current assignee: Institute of Bast Fiber Crops of CAAS
Priority date: 2020-04-24
Filing date: 2020-04-24
Publication date: 2020-08-14

Abstract

The invention relates to the technical field of biology, in particular to an LAMP (loop-mediated isothermal amplification) detection primer composition, a detection kit and a visual detection method for pythium bellatus, wherein genomic DNA of a microorganism to be detected is extracted, the genomic DNA solution is taken as a reaction template, and the detection solution in the LAMP kit is added for LAMP reaction, and the LAMP reaction is carried out by the following procedures: and (3) carrying out reaction amplification at 64 ℃ for 60min, and observing the color change of an amplification product, wherein if the color is changed from purple to blue, the pythium clotocidum exists in the object to be detected, and if the color is not changed and is still purple, the pythium clotocidum does not exist in the object to be detected. Compared with the traditional detection technology depending on morphology and molecular PCR, the method has higher accuracy, sensitivity and effectiveness, and is simple to operate. The method can be used for quickly detecting the pythium closterium and provides technical support for monitoring and preventing diseases caused by the pythium closterium.

Description

LAMP (loop-mediated isothermal amplification) detection primer composition for pythium bellatus, detection kit and visual detection method of LAMP detection primer composition

Technical Field

The invention relates to the technical field of biology, in particular to an LAMP (loop-mediated isothermal amplification) detection primer composition for pythium bellatus, a detection kit and a visual detection method thereof.

Background

Pythium closterium (Phytopythium vexans) as an important pathogenic bacterium of oomycetes can infect a plurality of important economic crops such as tobacco, citrus, ramie and the like. The ramie is a perennial herb of the genus ramie of the family urticaceae, is native to east asia, is one of the oldest fiber crops in the world, and has high economic value. The root brown rot caused by pythium closterium is a new destructive disease of ramie root discovered in recent years, the disease occurs in a plurality of main ramie production areas, the harm is serious, and a method capable of rapidly and accurately identifying the pathogenic bacteria in the early stage of ramie development is needed in production. At present, the identification method of pythium bellatus is mainly characterized by culturing pathogenic bacteria and observing and identifying, but the identification of the pythium bellatus is very difficult because the separation and culture of the pythium bellatus are difficult, the shapes of hypha, sporangium and oospore among related species are very similar, and the caused disease symptoms are similar to other plant diseases and are difficult to distinguish. Therefore, the traditional identification method needs abundant pathogenic bacteria form identification knowledge and experience, is long in time consumption, difficult to judge accuracy, not beneficial to analysis and identification of large-capacity samples, and cannot meet the requirements on field production. With the development of molecular biology, various detection technologies mainly based on Polymerase Chain Reaction (PCR) and real-time fluorescence quantitative PCR have been developed and widely applied, and have become the main means for rapidly, accurately and specifically identifying pathogenic bacteria, thereby avoiding the defect of uncertainty in morphological identification. However, these detection techniques are often carried out in laboratories with specialized instruments, reagents and harsh environmental conditions, which are expensive and require high operational requirements, complicated procedures and still long detection times, which make them unsuitable for use in basic environments.

Loop-mediated isothermal amplification (LAMP) is a newly developed molecular detection technique, which uses 4 specific primers and Bst DNA polymerase with strand displacement activity to realize a large amount of amplification of target DNA in a short period under a constant temperature condition. The detection result of the LAMP technology has various detection modes, and under the condition of high-efficiency amplification, pyrophosphate ions separated from deoxyribonucleic acid triphosphate substrates (dNTPs) are combined with magnesium ions in a system to generate a large amount of magnesium pyrophosphate, so that white precipitates appear, and the amplification products of the pyrophosphate ions can be monitored by a turbidity meter; or the detection can be carried out by gel electrophoresis, or a fluorescent dye of calcein or hydroxynaphthol blue (HNB) and the like is added into the reaction system, and the detection result can be observed and judged by naked eyes according to the color change of the system. The method does not need professional instruments and complex procedures, has the advantages of simple operation, rapidness, high efficiency, strong specificity, high sensitivity and the like, and is a rapid detection technology suitable for basic use. The LAMP technology is widely applied to the rapid detection of various pathogens such as fungi, bacteria, viruses, nematodes and the like, but the LAMP rapid detection method of pythium chimerica has not been reported at home and abroad.

In view of the above, the invention designs the LAMP primer with pythium clotocidum specificity by using the ITS sequence as the target sequence for detection, and establishes the LAMP rapid detection method of pythium clotocidum on the basis.

Disclosure of Invention

Aiming at the problems of long required period, poor specificity and low sensitivity of the detection method in the biological detection method of pythium chimerica in the prior art, the invention aims to provide the LAMP detection primer composition, the kit and the visual detection method for the pythium chimerica.

In order to achieve the above object, the present invention provides the following technical solutions:

an LAMP detection primer composition for pythium bellatus, which comprises the primer composition shown as SEQ ID NO: 1, and a forward outer primer F3 shown as SEQ ID NO: 2, as shown in SEQ ID NO: 3, and a forward inner primer FI shown as SEQ ID NO: 4, and (4) a reverse inner primer BIP.

The invention also provides an LAMP detection kit for pythium bellatus, and the LAMP detection reagent comprises the LAMP detection primer composition.

Preferably, the LAMP detection kit also comprises Bst DNA polymerase, reaction buffer solution and MgSO₄One or more of dNTP, betaine and a staining indicator.

Preferably, the LAMP detection reagent further comprises water.

The invention also provides an LAMP detection method of the pythium bellatus, and the LAMP detection primer is adopted to amplify the DNA of the pythium bellatus.

The LAMP detection method comprises the following reaction systems of 1.6 mu mol/L of FIP and BIP respectively, 0.2 mu mol/L of F3 and B3 respectively, 8UBst DNA polymerase, 2.5 mu L of 10 × reaction buffer solution and 6mmol/L MgSO₄1.4mmol/L dNTPs,0.8mol/L betaine, 180 mu mol/L staining indicator, 2 mu L DNA template and sterile water to 25 mu L.

The reaction temperature of LAMP was 64 ℃ and the reaction time was 60 min.

When the staining indicator is hydroxynaphthol blue, the LAMP detection method comprises the following steps: the color of the reaction solution is purple, the result is negative, and the sample to be detected does not contain pythium clotocaly; the color of the reaction solution is blue, the result is positive, and the sample to be detected contains pythium closterium.

Compared with the prior art, the invention has the advantages and positive effects that:

1. the operation is convenient: the LAMP method for detecting pythium closterium overcomes the problems that the biological detection method for pythium closterium requires a long period, wastes time and labor, is complicated and has poor specificity in the prior art, and the PCR detection technology requires a thermal cycler and cannot detect the pythium closterium rapidly. The detection method can accurately, quickly and efficiently detect the pythium clotocet in 60min under the isothermal condition of 64 ℃, has simple requirements on experimental places, does not need other complex instruments, can better meet the field detection of the pythium clotocet, and is suitable for popularization and use in basic departments.

2. The accuracy is high: the traditional detection method for pythium closterium is mainly characterized by culturing pathogenic bacteria and observing and identifying, but the separation and culture of pythium are difficult, the shapes of hypha, sporangium and oospore among related species are very similar, and the caused disease symptoms are similar to other plant diseases and are difficult to distinguish, so that the identification of the pythium closterium is very difficult. According to the genome sequence of the pythium closterium, Megalign software is used for comparing the pythium closterium genome with other pythium genomes, and a gene with a larger difference between the pythium closterium and other strains is selected as a target gene to design a specific LAMP primer. The LAMP reaction specifically recognizes 6 independent regions on the rake sequence through 4 primers (FIP, BIP, F3, B3), and the specificity is high. In addition, the invention adds the dyeing indicator in advance in the LAMP reaction system, so that the LAMP detection result can be judged by visual observation, and the possibility of uncovering pollution is avoided.

3. The sensitivity is high: the LAMP detection method of pythium closterium established by the invention has very high sensitivity, amplification products can be detected when the DNA concentration is between 10 ng/mu L and 1 pg/mu L, and the system color is changed into blue of different degrees, which indicates that the detection method is enough to accurately and rapidly detect the pythium closterium under the condition of lower DNA concentration.

Drawings

FIG. 1 Pythium closterium colony morphology;

FIG. 2 specific detection of Pythium closterium LAMP primers; wherein (a): detecting the result by 2% agarose gel electrophoresis; (b) the method comprises the following steps The Hydroxyl Naphthol Blue (HNB) visualization color development detection result shows that only pythium closterium can observe blue positive reaction, and the color development result of other pathogenic bacteria is purple negative reaction;

FIG. 3 comparison of sensitivity of conventional PCR and LAMP detection methods for Pythium closterium; wherein (a): detecting the result of 2% agarose gel electrophoresis of the conventional PCR amplification product; (b) the method comprises the following steps Detecting the result of 2% agarose gel electrophoresis of the LAMP amplification product; (c) the method comprises the following steps A Hydroxyl Naphthol Blue (HNB) visual color development detection result of the LAMP amplification product; the DNA concentration range is 10 ng/mu L-1 fg/mu L, and the result shows that the lowest detection limit of the conventional PCR of the pythium closterium is 100 pg/mu L, and the lowest detection limit of the LAMP detection system of the pythium closterium is 1 pg/mu L;

FIG. 4 shows diseased roots of ramie infected with different Pythium species;

FIG. 5 LAMP detection of Boehmeria nivea roots infected with different Pythium species; wherein (a): detecting the result by 2% agarose gel electrophoresis; (b) the method comprises the following steps The result of the visible color development detection of Hydroxy Naphthol Blue (HNB) shows that the ramie root DNA system infected by pythium closterium is produced with a strip, and the color development result of the LAMP detection system is a blue positive reaction; and the DNA system of the ramie root infected by the other pythium does not generate a strip, and the color development result of the LAMP detection system is a purple negative reaction.

Detailed Description

The invention discloses an LAMP detection primer composition for pythium chimerica, a detection kit and a visual detection method thereof, and a person skilled in the art can realize the LAMP detection primer composition and the detection kit by appropriately improving process parameters by referring to the contents in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The LAMP detection primer, detection reagent and detection method for the pythium closterium provided by the invention are all commercially available. The colony morphology of the pythium closterium isolated by the invention is shown in figure 1.

The invention is further illustrated by the following examples:

example 1 detection of Pythium closterium by LAMP method

LAMP primer composition for detecting pythium closterium: forward outer primer F3 is set forth in SEQ ID NO: 1, and a reverse outer primer B3 is shown as SEQ ID NO: 2, the forward inner primer FI is shown as SEQ ID NO: 3, the reverse inner primer BIP is shown as SEQID NO: 4, respectively.

The concentration of each reagent in the LAMP kit for detecting the pythium closterium is 1.6 mu mol/L of FIP and BIP respectively, 0.2 mu mol/L of F3 and B3 respectively, 8U of Bst DNA polymerase, 2.5 mu L of 10 × reaction buffer solution and 6mmol/L of MgSO₄1.4mmol/L dNTPs,0.8mol/L betaine, 180 mu mol/L staining indicator, 2 mu L DNA template and sterile water to 25 mu L.

The LAMP detection method comprises the following steps: extracting DNA of the microorganism to be detected, taking 2 mu L of DNA template, adding 23 mu L of detection solution in an LAMP kit for LAMP detection, and reacting at 64 ℃ for 60 min. And observing the color change of the product, wherein the color of the reaction liquid is purple, which indicates that no pythium clotocet exists in the sample to be detected, and the color of the reaction liquid is blue, which indicates that the pythium clotocet exists in the sample to be detected.

In order to verify the specificity of the LAMP detection method, 2 pythium clotocidum strains, 13 pythium kintosum kindred species and 5 other pathogenic fungi are taken as test materials. The species, hosts, sources and amounts of the test strains are detailed in Table 1. The LAMP detection result shows that blue positive reaction can be observed by pythium chimerica, and the other pathogenic bacteria have purple negative reaction as the color development result (figure 2).

TABLE 1 test strains and sources for LAMP detection

Example 2 detection of sensitivity of Pythium closterium LAMP reaction

In order to determine the sensitivity of the LAMP detection method, the extracted Pythium closterium DNA was diluted 10-fold with a NanoDrop 2000 ultramicro spectrophotometer to a concentration ranging from 10 ng/. mu.L to 1 fg/. mu.L. Performing LAMP amplification reaction respectively as templates, setting negative control, observing color change according to HNB visual detection method after reaction, judging the lowest detection limit of LAMP reaction, and verifying by 2% agarose gel electrophoresis. The results show that: the detection of the DNA concentration by the conventional PCR can detect an amplification product between 10 ng/mu L and 100 pg/mu L, but when the DNA concentration is diluted to 100 pg/mu L, the electrophoresis strip brightness of the gel detection is extremely weak, and when the DNA concentration is continuously diluted to 10 pg/mu L, the amplification product can not be detected, so that the minimum detection limit of the pythium closterium conventional PCR is proved to be 100 pg/mu L [ FIG. 3(a) ]. When the sensitivity of the LAMP detection method is verified, the amplification products can be detected when the DNA concentration is between 10 ng/mu L and 1 pg/mu L, the system color is changed into blue with different degrees, and a trapezoidal band appears in the gel electrophoresis detection, which is a positive result; when the concentration is diluted to 100 fg/. mu.L, the color of the system is not changed, the system is purple negative, and no electrophoresis band exists, so that 1 pg/. mu.L is the lowest detection limit of the LAMP detection system for Pythium closterium [ FIGS. 3(b), (c) ]. The results prove that the sensitivity of the Pythium closterium LAMP detection system established by the invention is at least 100 times of that of the conventional PCR.

Example 3 Strain Back-grafting and Ramie tissue LAMP detection

Asexual propagation of ramie: cutting stems with terminal buds of proper length from plants which grow vigorously and are too high in the ramie cultivated in the soil, selecting ramie stems which are moderate in thickness and do not bend, inserting the ramie stems into a clean 100mL conical flask, adding a proper amount of water, placing the ramie stems into a greenhouse for cultivation at 25 ℃, and selecting healthy plants with similar growth conditions for a back grafting experiment after fibrous roots grow out.

And (3) bacterial strain tieback: activating and culturing 15 pythium species in the test strains shown in the table 1 on a PDA culture medium for 3-4 days, beating fungus cakes by using a puncher, respectively returning the fungus cakes to white fibrous roots growing out from water culture ramie, enabling hyphae to be close to the fibrous roots, inoculating 4 fungus cakes of the same pythium species to the roots of each ramie, culturing for 7-10 days at 25 ℃ in a greenhouse, collecting fibrous roots of diseased parts of each plant, and respectively extracting DNA of the diseased roots.

An LAMP reaction system is constructed by taking diseased plant tissue DNA as a template, and amplification products of the LAMP reaction system are detected by 2 methods of HNB visual color development detection and 2% agarose gel electrophoresis detection, so that the accuracy of the LAMP reaction system of the pythium closterium is further verified.

After 15 days of culture, longer white fibrous roots are grown from water cultured ramie, fungus cakes of activated test strains are attached to densely grown fibrous roots, after 10 days of culture at 25 ℃, the fibrous roots of the ramie infected by different strains present different disease states, the diseased roots are changed from normal white to brown yellow with different degrees, and the fibrous roots of a control group are still white (figure 4).

The ramie roots which are attacked around the fungus cake are cut into pieces and put into a centrifuge tube, the centrifuge tube is subpackaged for 2 tubes, the fibrous root weight of each tube is about 0.2g, DNA of the ramie roots is extracted, the DNA is used as a template to construct an LAMP reaction system, and the accuracy of the reaction is detected. After the reaction is finished, the LAMP system using the 2 pythium campanulatum infected ramie root DNAs as templates is blue positive and the system containing the rest ramie root DNAs is purple negative by observing the system color (FIG. 5 (b)). The electrophoresis detection also shows the same result, except for the pythium closterium, no strip is generated in the ramie root DNA system infected by the pythium aphanidermatum [ figure 5(a) ], which shows that the LAMP amplification reaction carried out by using the primer can more accurately detect the pythium closterium at the root of the ramie and can be used for detecting the pythium closterium primordium of a field ramie root brown rot diseased plant.

Comparative examples 1 to 8

The primer sequences of comparative examples 1-8 are as follows:

TABLE 2 LAMP primers for Pythium closterium of comparative examples 1 to 8

Test example 1 verification of specificity of different primer sets

PCR amplification is carried out on 20 fungus ITS regions listed in Table 1, 9 groups of primers (specifically seen in the primer group sequences of example 1 and comparative examples 1-8) are designed by using on-line primer design software PrimeExplorer V5 for the screened Pythium jarosicum ITS sequences after sequencing and comparison, and each group comprises one pair of outer primers F3 and B3 and one pair of inner primers FIP and BIP, namely 4 LAMP specific primers.

Establishing LAMP reaction system comprising outer primers F3 and B3 of 0.2 mu mol/L, inner primers FIP and BIP of 1.6 mu mol/L, and 2.5 mu L10 × Isotermal Amplification Buffer containing 20mmol/L Tris-HCl (pH8.8) and 10mmol/L (NH)₄)₂SO₄，50mmol/L KCL，0.1％Tween-20]， 1.4mmol/L dNTPs，6mmol/L MgSO₄0.8mol/L betaine, 8U Bst DNA polymerase, 180. mu. mol/L hydroxynaphthol blue (HNB), 2. mu.L DNA template, made up to 25. mu.L with sterile water. Hydroxyl naphthol blue in the system is used as an indicator, purple is a negative reaction, and blue is a positive reaction.

Constructing the system by taking pythium closterium DNA as a template, and carrying out water bath for 60min at 64 ℃ to verify the primers; after being taken out, the color of the system is observed to change, if the color is changed from purple to blue, the primer is proved to successfully amplify the pythium bellatus DNA, and further optimization can be carried out; if no color change occurs, the primer is proved to be unusable. It can also be verified by electrophoresis on a 2.0% agarose gel, where primers are available if trapezoidal bands appear, and not otherwise.

The results show that: the primer group of the example 1 has the highest specificity, electrophoresis detection shows that no band is generated in other pathogenic bacteria except for pythium clotocidum, LAMP results show that only pythium clotocidum can observe blue positive reaction, and the color development results of other pathogenic bacteria are purple negative reactions (figure 2).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Claims

1. An LAMP detection primer composition for pythium bellatus, which is characterized in that the sequence of the primer is as follows:

forward outer primer F3: the sequence is shown as SEQ ID NO: 1 is shown in the specification;

reverse outer primer B3: the sequence is shown as SEQ ID NO: 2 is shown in the specification;

forward inner primer FIP: the sequence is shown as SEQ ID NO: 3 is shown in the specification;

reverse inner primer BIP: the sequence is shown as SEQ ID NO: 4, respectively.

2. An LAMP detection kit for Pythium closterium, which is characterized in that the LAMP detection reagent comprises the LAMP detection primer composition of claim 1.

3. The LAMP detection kit according to claim 2, wherein the LAMP detection reagent further comprises BstDNA polymerase, reaction buffer, MgSO₄One or more of dNTP, betaine and a staining indicator.

4. The LAMP detection kit according to claim 3, characterized in that the LAMP detection reagent further comprises water.

5. An LAMP detection method of Pythium closterium, characterized in that DNA of Pythium closterium is amplified by using the LAMP detection primer composition according to any one of claims 1 to 4.

6. The LAMP detection method according to claim 5, wherein the LAMP detection method comprises the reaction system of 1.6. mu. mol/L for FIP and BIP, 0.2. mu. mol/L for F3 and B3, 8U BstDNA polymerase, 2.5. mu.L 10 × reaction buffer, 6mmol/L MgSO₄1.4mmol/L dNTPs,0.8mol/L betaine, 180 mu mol/L staining indicator, 2 mu L DNA template and sterile water to 25 mu L.

7. The LAMP detection method according to claim 6, wherein the LAMP has a reaction temperature of 64 ℃ and a reaction time of 60 min.

8. The LAMP detection method according to claim 7, wherein the staining indicator is hydroxynaphthol blue, and the LAMP detection method is characterized in that:

the color of the reaction solution is purple, the result is negative, and the sample to be detected does not contain pythium clotocaly;

the color of the reaction solution is blue, the result is positive, and the sample to be detected contains pythium closterium.