CN114164293A

CN114164293A - LAMP (loop-mediated isothermal amplification) combined detection primer, detection kit and detection method for rhizome traditional Chinese medicinal materials

Info

Publication number: CN114164293A
Application number: CN202111474674.1A
Authority: CN
Inventors: 李明珠; 刘兢哲; 郭倩; 梁梦怡; 强毅; 崔浪军; 闫亚平
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2022-03-11
Anticipated expiration: 2041-12-03
Also published as: CN114164293B

Abstract

The invention discloses a LAMP combination detection primer, a detection kit and a detection method for rhizome traditional Chinese medicinal materials. The LAMP combined detection primer comprises: a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP, a forward loop primer LF and a reverse loop primer LB; the detection kit comprises LAMP detection primers and LAMP reaction liquid. The detection method provided by the invention has the advantages of high accuracy, high sensitivity and convenience in operation, realizes constant-temperature amplification, and provides a new technical platform for detection of common root rot and southern blight pathogenic bacteria. The kit can be used for high-sensitivity rapid detection of common root rot and southern blight pathogenic bacteria of traditional Chinese medicinal materials in production practice, can detect the pathogenic bacteria at the early stage of disease infection plants, and can detect the pathogenic bacteria in field soil. The invention provides reliable technical and theoretical basis for preventing and treating common root rot and southern blight of rhizome traditional Chinese medicinal materials (such as salvia miltiorrhiza, monkshood, rhizoma corydalis and the like).

Description

LAMP (loop-mediated isothermal amplification) combined detection primer, detection kit and detection method for rhizome traditional Chinese medicinal materials

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a LAMP combination detection primer, a detection kit and a detection method for rhizome traditional Chinese medicinal materials.

Background

Fusarium oxysporum (Fusarium oxysporum) belongs to the kingdom fungi (Eumycetes), Ascomycota (Ascomycota), Ascomycota (Sordariomycetes), Hypocrea (Hypocrea), Chitosaceae (Nectriaceae), Fusarium (Fusarium). Is a kind of facultative parasitic fungus which can infect plants and survive in soil, and is a pathogenic bacterium of common root rot of Chinese medicinal materials (aconite, rhizoma corydalis, salvia miltiorrhiza, etc.). And the plant blight caused by the infection is a worldwide soil-borne fungal disease. The host range is wide, and the wilt disease of more than 100 plants such as melons, solanaceae, bananas, cotton, leguminous plants, flowers and the like can be caused. The germs damage plants from roots, cause vascular bundle diseases, cause withering of the plants, can occur in the whole growth period of the plants, and cause huge loss on production. The establishment of a rapid molecular detection technology of Fusarium oxysporum has important significance for early control of diseases.

Fusarium solani (also known as Fusarium solani) belongs to the kingdom fungi (Eumycetes), Ascomycota (Ascomycota), Ascomycota (Sordariomycetes), Hypocrea (Hypocrea), Chitosaceae (Nectriaceae), Fusarium (Fusarium). Parasitism or saprophytosis, which is often distributed in roots, stems, fruits, seeds or soil of morbid plants, is the pathogenic bacteria of root rot of aconite. Root rot of salvia miltiorrhiza and rhizoma corydalis can also be caused. And the host range of the fusarium solani is wide, so that the fusarium solani not only causes root rot and stem rot of various plants, but also can infect fruit rot of coffee, peach, banana and the like, and causes leaf spot and blight of mango, tea, erythrina and the like. The establishment of the molecular rapid detection technology of Fusarium solani has important significance for early disease control.

Mucor circinelloides belongs to the kingdoma (Eumycetes), Mucor (Mucor), Mucor class (Mucor), Mucor order (Mucoramycetes), Mucor order (Mucorales), Mucor family (Mucoraceae), Mucor genus (Mucor). Mucor circinelloides is a soil-borne pathogenic fungus, spreads quickly, and is a pathogenic bacterium of radix aconiti carmichaeli chlorosis. At present, no effective detection and control measures are available for the disease, so that the establishment of the rapid molecular detection of the Mucor circinelloides has important significance for early control of the disease caused by the Mucor circinelloides.

The species Mycobacterium tuberculosis (Athalia rolfsii) belongs to the kingdom fungi (Eumycetes), the phylum Basidiomycota (Basidiomycota), the class Agaricales (Agaricamycetes), the order Athaliales (Athaliales), the family Athaliaceae (Athaliaceae), the genus Athalia (Athalia). The sclerotinia rolfsii is a soil-borne pathogenic bacterium, has strong saprophytic property and wide host range, and can damage various plants such as crops, flowers, agriculture and forestry plants and the like to cause southern blight. Comprises commercial crops such as potatoes, tomatoes, Chinese chives, trigone and the like and traditional Chinese medicinal plants such as bighead atractylodes rhizome, houttuynia cordata, rhizoma corydalis, salvia miltiorrhiza, monkshood and the like. And has been distributed worldwide and become one of the important pathogenic bacteria of many crop seedling diseases. At present, no effective detection and control measures are available for the disease, so that the establishment of the rapid molecular detection technology of Athellia rolfsii has important significance for early control of the disease.

At present, the molecular rapid detection technology applicable to the field is lacked aiming at common disease pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athelia rolfsii) of the root-stem traditional Chinese medicinal materials. The traditional detection methods are mainly a plate separation method and a bait method, and the traditional detection methods are identified according to morphological characteristics. The strain growth is affected by temperature, the shape is unstable, the time consumption is long, and the sensitivity is low, so that great difficulty is brought to detection. With the development of molecular biology, especially the popularization of PCR technology, more and more molecular biology technologies are applied to the detection of root rot and southern blight pathogenic bacteria. Although conventional PCR and quantitative PCR methods have been successfully used for detecting common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athellia rolfsii) of rhizome traditional Chinese medicinal materials, the detection process is complex and long in time, and the requirement of rapid detection cannot be met.

Since the first report of Loop-mediated isothermal amplification (LAMP) in 2000, LAMP has been widely used for detection and research of pathogenic bacteria such as viruses, bacteria, parasites, and fungi. The technology has the characteristics of strong specificity, isothermy, high efficiency, simple and convenient operation, short time consumption, easy detection of products and the like, so the technology has extremely wide application prospect. It designs 6 specific primers (two outer primers and two inner primers and two loop primers) aiming at 6 regions of a target gene, utilizes the action of strand displacement DNA polymerase (Bst DNA polymerase) to cause self-circulation strand displacement reaction, and generates white magnesium pyrophosphate precipitate as a byproduct while synthesizing a large amount of target DNA within 80min at the temperature of 60-68 ℃. Although the technology has been developed for nearly 20 years, detection reports of plant pathogenic bacteria are few, and detection of common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides, Athelia rolfsii) of rhizomatic traditional Chinese medicinal materials is not reported at home and abroad.

Disclosure of Invention

The invention aims to provide LAMP combination detection primers, a detection kit and a detection method for rhizomatic traditional Chinese medicinal materials, aims at solving the problems that in the prior art, the detection and identification of pathogenic bacteria of root rot and southern blight of the rhizomatic traditional Chinese medicinal materials are mainly based on morphological characteristics, the method is long in time consumption, tedious in procedure, strong in technical performance, low in accuracy and difficult to timely monitor and control the spread and prevalence of the pathogenic bacteria, and the problems that the existing PCR molecular detection needs expensive instruments such as an amplification instrument and the like, the detection time is long and the like, and provides a rapid molecular detection method for the pathogenic bacteria of the root rot and southern blight of the rhizomatic traditional Chinese medicinal materials, the pathogenic bacteria are detected, the detection period is short, the LAMP accuracy is high, the sensitivity is high, and the detection result is observed by naked eyes.

In order to achieve the purpose of the invention, the invention adopts the technical scheme that:

an LAMP combined detection primer for rhizome traditional Chinese medicinal materials comprises:

LAMP primers for Fusarium oxysporum: a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP and a forward loop primer LF;

LAMP primers for Fusarium solani: a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP, a forward loop primer LF and a reverse loop primer LB;

LAMP primers for Mucor circinelloides: a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP, a forward loop primer LF and a reverse loop primer LB;

LAMP primers for Athelia rolfsii: a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP and a reverse inner primer BIP;

specific gene sequences of the primers are shown in Table 2.

The LAMP combined detection primer for the rhizome traditional Chinese medicinal materials is applied to preparation of a kit for detecting root rot and southern blight of the rhizome traditional Chinese medicinal materials.

A LAMP combination detection kit for rhizome traditional Chinese medicinal materials comprises the LAMP combination detection primer.

Optionally, the method includes:

LAMP detection solution of Fusarium oxysporum: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 0.8. mu.M forward loop primer LF, 1.4mM dNTPs, 1 × LAMP Buffer, 1 × xylenol orange, 0.8M betaine, 8mM MgS0₄，0.32U/μL Bst DNA polymerase；

LAMP detection solution of Fusarium solani: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 0.8. mu.M forward loop primer LF, 0.8. mu.M reverse loop primer LB, 1.4mM dNTPs, 1 × LAMP Buffer, 1 × xylenol orange, 0.8M betaine, 8mM MgS0₄，0.32U/μL Bst DNA polymerase；

LAMP detection solution of Mucor circinelloides: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 1.6. mu.M forward loop primer LF, 0.8. mu.M reverse loop primer LB, 1.4mM dNTPs, 1 × LAMP Buffer, 1 × xylenol orange, 0.8M betaine, 8mM MgS0₄，0.32U/μL Bst DNA polymerase；

LAMP assay solution of Athelia rolfsii: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 1.4mM dNTPs, 1 XPLAMP Buffer, 1 Xxylenol orange, 0.8M betaine, 8mM MgS0₄，0.32U/μL Bst DNA polymerase。

Optionally, the method includes:

extracting DNA of Fusarium oxysporum to be detected, taking 1 mu L of DNA solution, adding 24 mu L of LAMP detection solution of LFusarum oxysporum to carry out LAMP reaction, wherein the LAMP reaction program comprises the following steps: observing the color change of the amplification product at 70 ℃ for 1min and 68 ℃ for 60 min;

secondly, extracting DNA of the Fusarium solani to be detected, taking 1 mu L of DNA solution, adding 24 mu L of LAMP detection solution of the Fusarium solani to carry out LAMP reaction, wherein the LAMP reaction program is as follows: observing the color change of the amplification product at 70 ℃ for 1min and 67 ℃ for 60 min;

extracting DNA of the Mucor circinelloides to be detected, taking 1 mu L of DNA solution, adding 24 mu L of LAMP detection solution of the Mucor circinelloides to carry out LAMP reaction, wherein the LAMP reaction program is as follows: observing the color change of the amplification product at 70 ℃ for 1min and 60 ℃ for 60 min;

extracting DNA of Athelia rolfsii to be detected, taking 1 mu L of DNA solution, adding 24 mu L of LAMP detection solution of Athelia rolfsii to carry out LAMP reaction, wherein the LAMP reaction program is as follows: the color change of the amplification product was observed at 70 ℃ for 1min and 62 ℃ for 60 min.

Optionally, the method includes:

detection of Fusarium oxysporum: extracting DNA of a microorganism to be detected, taking the extracted DNA as a template, detecting by using the LAMP primer of the Fusarium oxysporum, wherein a color developing agent is xylenol orange, yellow indicates that the detection is positive, and the Fusarium oxysporum exists; the purplish red indicates that the detection result is negative and Fusarium oxysporum does not exist;

detection of Fusarium solani: extracting DNA of a microorganism to be detected, taking the extracted DNA as a template, detecting by using the LAMP primer of the Fusarium solani, wherein a color developing agent is xylenol orange, yellow indicates that the detection is positive, and the Fusarium solani exists; the purple color indicates that the detection result is negative, and Fusarium solani does not exist;

detection of Mucor circinelloides: extracting DNA of a microorganism to be detected, taking the extracted DNA as a template, detecting by using an LAMP primer of the Mucor circinelloides, wherein a color developing agent is xylenol orange, yellow indicates that the detection is positive, and Mucor circinelloides exist; the purple red indicates that the detection result is negative, and no Mucor circinelloides exist;

detection of Athelia rolfsii: extracting DNA of a microorganism to be detected, taking the extracted DNA as a template, detecting by using the LAMP primer of Athellia rolfsii, wherein a color developing agent is xylenol orange, yellow indicates that the detection is positive, and Athellia rolfsiii exists; the purple color indicated that the test was negative and Athellia rolfsii was absent.

A LAMP detection method for rhizome traditional Chinese medicinal materials comprises the following steps:

detection of Fusarium oxysporum: taking genome DNA of a microorganism to be detected as a template, carrying out LAMP reaction by using the LAMP primer of the Fusarium oxysporum, and observing the color change of an amplification product, wherein the color change of the amplification product is xylenol orange, if the color is changed from purple to yellow, the pathogenic bacterium Fusarium oxysporum exists in the object to be detected, and if the color is not changed, the pathogenic bacterium Fusarium oxysporum does not exist in the object to be detected;

detection of Fusarium solani: taking genome DNA of a microorganism to be detected as a template, carrying out LAMP reaction by using the LAMP primer of the Fusarium solani, wherein a color developing agent is xylenol orange, then observing the color change of an amplification product, if the color is changed from purple to yellow, indicating that pathogenic bacteria Fusarium solani exists in the object to be detected, and if the color is not changed, still indicating that pathogenic bacteria Fusarium solani does not exist in the object to be detected;

detection of Mucor circinelloides: performing LAMP reaction by using genome DNA of a microorganism to be detected as a template and using the LAMP primer of the Mucor circinelloides, wherein a color developing agent is xylenol orange, and then observing the color change of an amplification product, wherein if the color is changed from purple to yellow, the pathogenic bacteria Mucor circinelloides exist in the object to be detected, and if the color is not changed, the pathogenic bacteria Mucor circinelloides do not exist in the object to be detected;

detection of Athelia rolfsii: the genomic DNA of a microorganism to be detected is used as a template, LAMP reaction is carried out by using the LAMP primer of Athellia rolfsii, the color developing agent is xylenol orange, then the color change of an amplification product is observed, if the color is changed from purple to yellow, the pathogenic bacterium Athellia rolfsii exists in the object to be detected, and if the color is not changed, the pathogenic bacterium Athellia rolfsii does not exist in the object to be detected.

Optionally, the method includes:

Optionally, the method for extracting the DNA of the microorganism to be detected comprises:

mixing 0.3g of soil and 0.2g of magnetic glass beads with the diameter of 1mm, adding 250 mu L of extracting solution, and carrying out vortex oscillation at 4000 r/min for 1 min;

adding 150mL benzyl chloride, oscillating, mixing uniformly, and carrying out metal bath at 60 ℃ for 15min to obtain a suspension;

③ adding 150 mu L of 3mol/L sodium acetate into the suspension, slightly shaking and mixing, and placing on ice for 15 min; centrifuging at 15000r/min for 10min, and collecting supernatant;

and fourthly, purifying the obtained supernatant to obtain DNA, and storing the DNA at the temperature of minus 80 ℃ for later use.

Optionally, the detection solution comprises:

Compared with the prior art, the invention has the advantages that:

(1) high accuracy: because the traditional detection technology aiming at common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athalia rolfsii) of the rhizome traditional Chinese medicinal materials only determines detection objects according to morphological characteristics, the interference of human factors cannot be eliminated, morphological similar species are difficult to distinguish, and the detection accuracy is only 60-80%. According to the Cox1 gene sequence (NMDC: N0000Q8H) of the Fusarium oxysporum strain ACCC30927, the Cox1 gene sequence of the Fusarium oxysporum and sequences of other strains are subjected to permutation analysis by using Bioedit software, and a specific LAMP primer is designed by selecting a section of sequence specific to the Fusarium oxysporum. According to an EF 1-alpha gene sequence (NMDC: N0000Q8S) of a Fusarium solani ACCC30119 strain, a Bioedit software is utilized to carry out permutation analysis on the EF 1-alpha gene sequence of the Fusarium solani and sequences of other strains, and a specific LAMP primer is designed by selecting a section of sequence specific to the Fusarium solani. According to the invention, the ADH1 gene sequence (NMDC: N0000Q8K) of the Mucor circinelloides FZCG190609 strain is utilized to carry out arrangement analysis on the ADH1 gene sequence of the Mucor circinelloides and the sequences of other strains by using Bioedit software, and a specific LAMP primer is designed by selecting a section of sequence specific to the Mucor circinelloides. According to the ITS gene sequence (GenBank: OK275401) of the sclerotium rolfsii FZCG190612 strain, the ITS gene sequence of the sclerotium rolfsii and the sequences of other strains are arranged and analyzed by using Bioedit software, and a specific LAMP primer is designed by selecting a section of sequence specific to the sclerotium rolfsii. The LAMP reaction specifically recognizes 6 independent regions on a target sequence through 6 primers (a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF and a reverse loop primer LB), and the specificity and the sensitivity are high. In addition, the forward loop primer LF can improve the reaction rate, and together with other four primers, under the condition of ensuring the reaction accuracy, the method can quickly detect common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athellia rolfsii) of the rhizome traditional Chinese medicinal materials.

(2) The operation is convenient: the LAMP method for detecting common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athellia rolfsii) of the rhizome traditional Chinese medicinal materials overcomes the problems that in the prior art, a biological detection method for common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athellia rolfsii) of the rhizome traditional Chinese medicinal materials needs a long period, wastes time and labor, is cumbersome and has poor specificity, and a PCR detection technology needs a thermal cycler and cannot quickly detect common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athellia rolfsii) of the rhizome traditional Chinese medicinal materials. The detection method can quickly, conveniently, efficiently, highly specifically and sensitively detect common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athalia rolfsii) of the rhizomatic traditional Chinese medicinal materials under the isothermal condition of 60-68 ℃, does not need a complex instrument, and can better meet the field detection of the common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athalia rolii) of the rhizomatic traditional Chinese medicinal materials.

(3) Constant temperature amplification is realized, thermal cycle is not required to be carried out in a PCR method, so that dependence on a thermal cycle instrument is eliminated, LAMP reaction can be carried out as long as a stable heat source exists, the application range of LAMP is greatly expanded, and the LAMP can be carried out under the constant heat source because betaine is added into LAMP reaction liquid, double-stranded DNA is in the dynamic equilibrium of melting, and amplification is realized under the action of Bst DNA polymerase.

(4) The invention provides a new technical platform for detecting common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athellia rolfsii) of the rhizome traditional Chinese medicinal materials, can be used for high-sensitivity rapid detection of the common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athellia rolfsii) of the rhizome traditional Chinese medicinal materials, identifies the pathogenic bacteria at the initial stage of disease infection, and can also detect pathogens in field soil. The invention also has important significance for reducing blind use of pesticides, reducing production cost and reducing environmental pollution of the pesticides.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 shows the LAMP experimental results of six pairs of primers for Fusarium oxysporum;

FIG. 2 shows the LAMP experimental results of three pairs of primers for Fusarium solani;

FIG. 3 shows the LAMP experimental results of two pairs of primers for Mucor circinelloides;

FIG. 4 shows LAMP experimental results of three pairs of primers for sclerotium rolfsii;

FIGS. 5, 6, 7 and 8 are color development charts for detecting specificity of common root rot pathogens (Fusarium oxysporum, Fusarium solani, Mucor circinelloides, Athellia rolfsii) of rhizome type Chinese medicinal materials by LAMP based on the color change judgment of xylenol orange;

in FIG. 5, a color development chart showing specificity of detection of Fusarium oxysporum by LAMP is shown, wherein 1-9 tubes show yellow and positive, and the rest tubes show purple and negative. Wherein 1-9: fusarium oxysporum; 10: botrytis cinerea; 11: fusarium avenaceum; 12: f, culmorum; 13: equiseti; 14: equiseti; 15: g, graminearum; 16: moniliforme; 17: nivale; 18: proliferatum; 19: solani; 20: solani; 21: phytophthora cacorum; 22: cambrivora; 23: iranica; 24: pseudospinosae; 25: pythium Carbonicum; 26: boreale; 27: h₂O。

In FIG. 6, a color development chart showing specificity of detection of Fusarium solani (Fusarium solani) by color determination LAMP is shown, wherein 1 and 2 tubes show yellow and are positive, and the rest tubes show purple and are negative. Wherein 1, 2: fusarium solani; 3: botrytis cinerea; 4: fusarium avenaceum; 5: f, culmorum; 6: equiseti; 7: equiseti; 8: g, graminearum; 9: lactis; 10: moniliforme; 11: nivale; 12: oxysporum; 13: oxysporum; 14: oxysporum; 15: oxysporum; 16: oxysporum; 17: oxysporum; 18: oxysporum; 19: oxysporum; 20: oxysporum; 21: (iii) clonostachys rosea; 22: oxysporum f.sp.pisi; 23: oxysporum f.sp.vasinffectum; 24: proliferatum; 25: phytophthora cacorum; 26: cambrivora; 27: iranica; 28: pseudospinosae; 29: pythium Carbonicum; 30: boreale; 31: h₂O。

Color determination LAMP detection Mucor circinelloides (Mucor circinelloides) specific color development in FIG. 7. In the figure, 1 and 2 tubes show yellowColor, positive, the other tubes purple, negative. Wherein 1, 2: mucor circinelloides; 3: m. guilliermondii; 4: m.hiemalis; 5: m. ifragilis; 6: m.iindicus; 7: m. lucitancius; 8: (iii) clonostachys rosea; 9: racemosus; 10: ramosissimus; 11: m.plumbeus; 12: phytophthora cacorum; 13: cambrivora; 14: iranica; 15: pseudospinosae; 16: pythium Carbonicum; 17: boreale; 18: h₂O。

FIG. 8 is a color-determination LAMP detection of a specific color development map of Mycobacterium tuberculosis (Athellia rolfsii). In the figure, 1 and 2 tubes are yellow and positive, while the remaining tubes are purple and negative. Wherein 1, 2: athelia rolfsii; 3: alternaria alternata; 4: tenuissima; 5: clonostachys rosea; 6: g, graminearum; 7: oxysporum; 8: solani; 9: mucor circinelloides; 10: phytophthora cacorum; 11: cambrivora; 12: iranica; 13: pseudospinosae; 14: plectosphaerella cucumerina; 15: pythium Carbonicum; 16: boreale; 17: rhizopus oryzae; 18: stagonosporas cucurbitaceae; 19: trichoderma longibrachiatum; 20: h₂O。

FIG. 9, FIG. 10, FIG. 11 and FIG. 12 are LAMP sensitivities detected based on agarose gel electrophoresis and xylenol orange color change;

FIG. 9 (a) is an agarose gel electrophoresis chart showing LAMP detection of specificity of Fusarium oxysporum; wherein M is 100bp DNA marker; FIG. 9 (b) is a color-developed diagram showing the sensitivity of color determination of Fusarium oxysporum; the figure shows

tubes

1, 2, 3 and 4 yellow and positive;

tubes

5, 6, 7 and 8 are negative purple; 25 mu L of reaction system respectively contains 1ng of fusarium oxysporum, 100pg, 10pg, 1pg, 100fg, 10fg and 1fg of fusarium oxysporum and negative control, wherein the reaction tube containing 1ng of fusarium oxysporum, 100pg, 10pg and 1pg of fusarium oxysporum DNA shows yellow and shows positive reaction; the DNA of Fusarium oxysporum at 100fg, 10fg and 1fg appeared purple and was negative. The color development result shows that the sensitivity of LAMP reaction reaches 1 pg.

FIG. 10 (a) is an agarose gel electrophoresis chart showing LAMP detection of the specificity of Fusarium solani; wherein M is 100bp DNA marker; FIG. 10 (b) is a color-developed diagram showing the sensitivity of color determination of Fusarium solani; the figure shows

tubes

1, 2, 3, 4 and 5 yellow and positive;

tubes

6, 7 and 8 are negative for purple; 25 mu L of reaction system respectively contains 1ng, 100pg, 10pg, 1pg, 100fg, 10fg and 1fg of Fusarium solani and negative control, wherein the reaction tube containing 1ng, 100pg, 10pg, 1pg and 100fg of Fusarium solani DNA shows yellow and shows positive reaction; the DNA of Fusarium solani at 10fg and 1fg is purple and shows negative reaction. The color development result shows that the sensitivity of LAMP reaction reaches 100 fg.

FIG. 11 (a) is an agarose gel electrophoresis chart showing LAMP detection specificity of Mucor circinelloides; wherein M is 100bp DNA marker; FIG. 11 (b) is a color development chart showing LAMP Mucor circinelloides sensitivity for color determination; the figure shows

tubes

1, 2, 3 and 4 yellow and positive;

tubes

5, 6, 7 and 8 are negative purple; 25 mu L of reaction system respectively contains 1ng, 100pg, 10pg, 1pg, 100fg, 10fg and 1fg of mucor circinelloides and negative control, wherein the reaction tube containing 1ng, 100pg, 10pg and 1pg of mucor circinelloides DNA is yellow and shows positive reaction; the DNA of Mucor circinelloides at 100fg, 10fg and 1fg shows purple color and is a negative reaction. The color development result shows that the sensitivity of LAMP reaction reaches 1 pg.

FIG. 12 (a) is an agarose gel electrophoresis chart showing LAMP detection of the specificity of Sclerotinia sclerotiorum; wherein M is 100bp DNA marker; FIG. 12 (b) is a color development chart showing the sensitivity of LAMP sclerotinia rolfsii by color determination; the figure shows

tubes

1, 2, 3 and 4 yellow and positive;

tubes

5, 6, 7 and 8 are negative purple; the 25 mu L reaction system respectively contains 1ng, 100pg, 10pg, 1pg, 100fg, 10fg and 1fg of sclerotinia rolfsii and negative control, wherein the reaction tube containing 1ng, 100pg, 10pg and 1pg of sclerotinia rolfsii DNA shows yellow and shows positive reaction; the DNA of 100fg, 10fg, 1fg sclerotinia rolfsii is purple and shows a negative reaction. The color development result shows that the sensitivity of LAMP reaction reaches 1 pg;

FIG. 13LAMP detection results in planting soil of Aconiti lateralis, rhizoma corydalis, and Saviae Miltiorrhizae radix.

Detailed Description

In order to make the purpose and technical solution of the present invention more clear, the present invention is further illustrated with reference to the following specific examples, but not to limit the scope of the present invention. The following examples are conducted according to conventional experimental conditions, or according to the protocols described in the published literature, or according to the experimental conditions recommended by the manufacturers.

TABLE 1 fungal and oomycete strains for detection of pathogen specificity

^a,International identification abbreviations:BNCC:BeNa Culture Collection,China；ACCC:Agricultural Culture Collection of China,China；CGMCC:China General Microbiological Culture Collection Center,China；CBS,Westerdijk Fungal Biodiversity Institute,The Netherlands；P,World Phytophthora Genetic Resource Collection,USA；MAFF,Ministry of Agriculture,Forestry and Fisheries,Japan；^b,Specificity of the F.solani-specific,F.oxysporum-specific,M.circinelloides-specific,A.rolfsii-specific markers were checked by LAMP.+,positive；-,negetive.N,not tested.

The LAMP combined detection primer for the rhizome traditional Chinese medicinal materials comprises the following components: consists of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF and a reverse loop primer LB; the primer sequences are specifically shown in Table 2.

TABLE 2 detection primer sequences for common root rot pathogens of rhizome Chinese medicinal materials

Detection solution of Fusarium oxysporumThe method comprises the following steps: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 0.8. mu.M forward loop primer LF, 1.4mM dNTPs, 1 × LAMP Buffer, 1 × xylenol orange, 0.8M betaine, 8mM MgS0₄，0.32U/μL Bst DNA polymerase。

The detection solution for Fusarium solani includes: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 0.8. mu.M forward loop primer LF, 0.8. mu.M reverse loop primer LB, 1.4mM dNTPs, 1 × LAMP Buffer, 1 × xylenol orange, 0.8M betaine, 8mM MgS0₄，0.32U/μL Bst DNA polymerase。

The detection solution of Mucor circinelloides comprises: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 1.6. mu.M forward loop primer LF, 0.8. mu.M reverse loop primer LB, 1.4mM dNTPs, 1 × LAMP Buffer, 1 × xylenol orange, 0.8M betaine, 8mM MgS0₄，0.32U/μL Bst DNA polymerase。

The detection solution for Athelia rolfsii includes: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 1.4mM dNTPs, 1 XPLAMP Buffer, 1 Xxylenol orange, 0.8M betaine, 8mM MgS0₄，0.32U/μL Bst DNA polymerase。

Application of LAMP kit for detecting common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athalia rolfsii) of rhizome traditional Chinese medicinal materials in detection of pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athalia rolfsii).

The LAMP detection method of the rhizome traditional Chinese medicinal materials comprises the following steps:

detection of Fusarium oxysporum: the method comprises the steps of taking the extracted DNA of a microorganism to be detected as a template, and carrying out LAMP by using an LAMP detection primer composition or an LAMP detection kit of Fusarium oxysporum; carrying out agarose gel electrophoresis on the amplification product, detecting the result under ultraviolet light, and if a characteristic step-shaped strip exists, proving that Fusarium oxysporum exists in the detected sample; if no characteristic ladder band exists, the detected sample has no pathogenic bacterium Fusarium oxysporum; or observing the color change of the LAMP reaction solution, wherein yellow indicates that the detection is positive and Fusarium oxysporum exists; purple indicates a negative test result and the absence of Fusarium oxysporum.

Detection of Fusarium solani: the method comprises the steps of taking the extracted DNA of a microorganism to be detected as a template, and carrying out LAMP by using an LAMP detection primer composition or an LAMP detection kit of Fusarium solani; carrying out agarose gel electrophoresis on the amplification product, detecting the result under ultraviolet light, and if a characteristic stepped band exists, proving that Fusarium solani exists in the detected sample; if no characteristic ladder band exists, the detected sample has no pathogenic bacteria Fusarium solani; or observing the color change of the LAMP reaction solution, wherein yellow indicates that the detection is positive and Fusarium solani exists; purple indicates a negative test result, and Fusarium solani is absent.

Detection of Mucor circinelloides: the method comprises the steps of taking the extracted DNA of a microorganism to be detected as a template, and carrying out LAMP by using an LAMP detection primer composition or an LAMP detection kit of Mucor circinelloides; carrying out agarose gel electrophoresis on the amplification product, detecting the result under ultraviolet light, and if a characteristic step-shaped band exists, proving that Mucor circinelloides exists in the detected sample; if no characteristic ladder band exists, the detected sample has no pathogenic bacteria Mucor circinelloides; or observing the color change of the LAMP reaction solution, wherein yellow indicates that the detection is positive and Mucor circinelloides exist; purple indicates that the detection result is negative and no Mucor circinelloides exist.

Detection of Athelia rolfsii: the method comprises the steps of taking the extracted DNA of a microorganism to be detected as a template, and carrying out LAMP by using an LAMP detection primer composition or an LAMP detection kit of Athellia rolfsii; carrying out agarose gel electrophoresis on the amplification product, detecting the result under ultraviolet light, and if a characteristic stepped strip exists, proving that Athelia rolfsii exists in the detected sample; if no characteristic ladder band exists, the detected sample has no pathogenic bacterium Athellia rolfsii; or observing the color change of the LAMP reaction solution, wherein yellow indicates that the detection is positive and Athellia rolfsii exists; purple indicates a negative test result and absence of Athelia rolfsii.

The method comprises the following specific steps:

extracting DNA of Fusarium oxysporum to be detected, taking 1 mu L of DNA solution, adding the DNA solution into a detection solution in a 24 mu L LAMP kit to carry out LAMP, wherein the LAMP reaction program is as follows: and (3) observing the color change of the amplification product at 70 ℃ for 1min and 68 ℃ for 60 min.

Secondly, extracting DNA of the Fusarium solani to be detected, taking 1 mu L of DNA solution, adding the DNA solution into 24 mu L of detection solution in an LAMP kit to carry out LAMP, wherein the LAMP reaction program is as follows: observing the color change of the amplification product at 70 ℃ for 1min and 67 ℃ for 60 min.

Extracting DNA of the Mucor circinelloides to be detected, taking 1 mu L of DNA solution, adding the detection solution in a 24 mu L LAMP kit to carry out LAMP, wherein the LAMP reaction program is as follows: observing color change of amplification product at 70 deg.C for 1min and 60 deg.C for 60min

Extracting DNA of Athellia rolfsii to be detected, taking 1 mu L of DNA solution, adding the DNA solution into a detection solution in a 24 mu L LAMP kit to carry out LAMP, wherein the LAMP reaction program is as follows: observing the color change of the amplification product at 70 ℃ for 1min and 62 ℃ for 60 min.

One of the key technologies of the invention is a primer sequence for efficient and specific amplification of common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athellia rolfsii) of rhizome traditional Chinese medicinal materials and an amplification method thereof. In order to verify the specific primer sequences of pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athalia rolfsii), the invention takes 4 strains of aconite pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athalia rolfsii) and 47 other strains as test materials, and adopts an improved magnetic bead extraction method to extract the DNA of common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athalia rolfsii) in the soil.

The specific operation steps are as follows:

0.3g of soil and 0.2g of magnetic glass beads having a diameter of 1mm were put into a 1.5mL centrifuge tube.

② adding 250 mul of extracting solution (100mmol/L Tris-HCl with pH of 9.0, 40mmol/L EDTA, 10% SDS, 0.8% skim milk) and vortex shaking for 1min at 4000 r/min.

③ adding 150mL benzyl chloride, oscillating and mixing. The metal bath is carried out for 15min at 60 ℃.

And fourthly, adding 150 mu L of 3mol/L sodium acetate into the suspension, slightly shaking and mixing, and placing on ice for 15 min.

Fifthly, after centrifugation for 10min at 15000r/min, the supernatant is transferred into a new 1.5mL centrifugal tube.

Sixthly, purifying DNA according to the operation instruction of the MagExtractor Plant Genome Kit, and storing the extracted DNA at-80 ℃ for later use.

When LAMP amplification reaction occurs, as shown by a color reaction result of xylenol orange, reaction tubes of common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athellia rolfsii) of traditional Chinese medicinal materials are yellow, the reaction tubes are positive results, reactions of other pythium, phytophthora, fungi and negative control bacteria are purple, and the negative results prove that the designed LAMP specific primer has species specificity. Meanwhile, the reaction product is subjected to 2% agarose gel electrophoresis, and the amplified reaction product is observed through imaging, a typical step-shaped strip appears in reaction liquid in a reaction tube of common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athellia rolfsii) of the traditional Chinese medicinal materials, and a trapezoidal strip does not appear in other pythium species, phytophthora, fungi and negative control reaction tubes. This shows that the primer set can be used for rapid and reliable detection and identification of common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides, Athellia rolfsii) of Chinese medicinal materials in pathogenic tissues and soil in production practice.

Example 1: screening of primers

TABLE 3 Fusarium oxysporum detection primer sequences

In FIG. 1a, a color determination primer set1 LAMP-detected Fusarium oxysporum (Fusarium oxysporum) specific color development chart shows that 1-8 tubes show purple color and are negative. Wherein 1-3: fusarium oxysporum; 4: equiseti; 5: g, graminearum; 6: moniliforme; 7: nivale; 8: h₂O。

In FIG. 1b, the color determination primer set2 shows a specific color development chart for LAMP detection of Fusarium oxysporum (Fusarium oxysporum), and 1-8 tubes show purple color and are negative. Wherein 1-3: fusarium oxysporum; 4: equiseti; 5: g, graminearum; 6: moniliforme; 7: nivale; 8: h₂O。

In FIG. 1c, the color determination primer set3 shows a specific color development chart for LAMP detection of Fusarium oxysporum (Fusarium oxysporum), and 1-8 tubes show purple color and are negative. Wherein 1-3: fusarium oxysporum; 4: equiseti; 5: g, graminearum; 6: moniliforme; 7: nivale; 8: h₂O。

In FIG. 1d, the color determination primer set4 shows a specific color development chart for LAMP detection of Fusarium oxysporum (Fusarium oxysporum), and 1-8 tubes show purple color and are negative. Wherein 1-3: fusarium oxysporum; 4: equiseti; 5: g, graminearum; 6: moniliforme; 7: nivale; 8: h₂O。

FIG. 1e shows the LAMP detection of Fusarium oxysporum (Fusarium oxysporum) specific color development of the color determination primer set5, wherein

tubes

1, 2, 3, 5 and 6 show yellow and positive; 4.

tubes

7 and 8 are purple and negative. Wherein 1-3: fusarium oxysporum; 4: equiseti; 5: g, graminearum; 6: moniliforme; 7: nivale; 8: h₂O。

FIG. 1f shows a specific color development chart of LAMP detection of Fusarium oxysporum (Fusarium oxysporum) by using a color determination primer set6, wherein 1-3 tubes show yellow and positive; the 4-8 tubes showed purple color and were negative. Wherein 1-3: fusarium oxysporum; 4: equiseti; 5: g, graminearum; 6: moniliforme; 7: nivale; 8: h₂O。

And (4) conclusion: the first, second, third and fourth primer pairs can not be amplified; the fifth pair of primers can amplify Fusarium oxysporum (f.equiseti) and Fusarium nivale (f.nivale) at the same time, so that the specificity of the primers is insufficient; the sixth pair of primers only amplified from Fusarium oxysporum and had strong primer specificity, so primer set6 was selected as the LAMP detection primer for Fusarium oxysporum.

TABLE 4 Fusarium solani detection primer sequences

In FIG. 2a, a color determination primer set1 LAMP detection Fusarium solani (Fusarium solani) specific color development picture shows that 1-9 tubes show purple and are negative. Wherein 1-2: fusarium solani; 3: f, culmorum; 4: equiseti; 5: g, graminearum; 6: moniliforme; 7: nivale; 8: lactis; 9: h₂O。

FIG. 2b shows the specific color development of the primer set2 for LAMP detection of Fusarium solani (Fusarium solani), wherein

tubes

1, 2 and 6 show yellow and positive; 3.

tubes

4, 5, 7, 8, and 9 showed purple color and were negative. Wherein 1-2: fusarium solani; 3: f, culmorum; 4: equiseti; 5: g, graminearum; 6: moniliforme; 7: nivale; 8: lactis; 9: h₂O。

FIG. 2c shows the specific color development of the primer set3 for LAMP detection of Fusarium solani (Fusarium solani), wherein

tubes

1 and 2 are yellow and positive; the 3-9 tubes showed purple color and were negative. Wherein 1-2: fusarium solani; 3: f, culmorum; 4: equiseti; 5: g, graminearum; 6: moniliforme; 7: nivale; 8: lactis; 9: h₂O。

And (4) conclusion: the first pair of primers cannot amplify; the second pair of primers can amplify Fusarium solani (Fusarium solani) and simultaneously amplify Fusarium moniliforme (F.moniliforme), so that the specificity of the primers is insufficient; the third pair of primers only amplified by Fusarium solani, and the specificity of the primers is strong, so the primer set3 is selected as the LAMP detection primer of Fusarium solani.

TABLE 5 primer sequences for mucor circinelloides detection

FIG. 3a shows a color development pattern specific to Mucor circinelloides (Mucor circinelloides) detected by LAMP as a color determination primer set 1. In the figure, 1, 2 and 10 tubes are yellow and positive, while the other tubes are purple and negative. Wherein 1, 2: mucor circinelloides; 3: m. guilliermondii; 4: m.hiemalis; 5: m. ifragilis; 6: m.iindicus; 7: m. lucitancius; 8: racemosus; 9: ramosissimus; 10: m.plumbeus; 11: h₂O。

FIG. 3b shows the color development pattern specific to Mucor circinelloides (Mucor circinelloides) detected by the LAMP as the color determination primer set 2. In the figure, 1 and 2 tubes are yellow and positive, while the remaining tubes are purple and negative. Wherein 1, 2: mucor circinelloides; 3: m. guilliermondii; 4: m.hiemalis; 5: m. ifragilis; 6: m.iindicus; 7: m. lucitancius; 8: racemosus; 9: ramosissimus; 10: m.plumbeus; 11: h₂O。

And (4) conclusion: the first pair of primers can amplify Mucor circinelloides (Mucor circinelloides) and also amplify Mucor pusillus (M.plumbeus), so the specificity of the primers is insufficient; the second pair of primers only has amplification of mucor circinelloides, and the specificity of the primers is strong, so that the primer set2 is selected as the LAMP detection primer of mucor circinelloides.

TABLE 6 primer sequences for detecting sclerotium rolfsii

The color determination primer set1 in FIG. 4a is a LAMP detection specific color development map of Mycobacterium tuberculosis (Athellia rolfsii). In the figure, 1-10 tubes are purple and negative. Wherein 1, 2: athelia rolfsii; 3: alternaria alternata; 4: tenuissima; 5: clonostachys rosea; 6: g, graminearum; 7: oxysporum; 8: solani; 9: mucor circinelloides；10：H₂O。

FIG. 4b shows a color-determination primer set2 LAMP-specific chromogenic map for detecting Mycobacterium tuberculosis (Athellia rolfsii). In the figure, 1-10 tubes are purple and negative. Wherein 1, 2: athelia rolfsii; 3: alternaria alternata; 4: tenuissima; 5: clonostachys rosea; 6: g, graminearum; 7: oxysporum; 8: solani; 9: mucor circinelloides; 10: h₂O。

FIG. 4c shows a color determination primer set 3LAMP detection of a chromogen specific for Mycobacterium tuberculosis (Athellia rolfsii). In the figure, 1-2 tubes are yellow and positive; 3-10 tubes appeared purple and negative. Wherein 1, 2: athelia rolfsii; 3: alternaria alternata; 4: tenuissima; 5: clonostachys rosea; 6: g, graminearum; 7: oxysporum; 8: solani; 9: mucor circinelloides; 10: h₂O。

And (4) conclusion: the first and the second primer pairs can not be amplified; the third pair of primers only amplified from sclerotinia rolfsii (Athelia rolfsii), and had strong primer specificity, so primer set3 was selected as the LAMP detection primer for sclerotinia rolfsii.

FIGS. 1, 2, 3 and 4 are color development charts for detecting the specificity of common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides, Athelix rolfsii) of rhizome type Chinese medicinal materials by LAMP based on the color change determination of xylenol orange.

Example 2: an LAMP detection kit suitable for detecting common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides and Athellia rolfsii) of rhizome traditional Chinese medicinal materials (such as salvia miltiorrhiza, monkshood, rhizoma corydalis and the like):

LAMP primer composition of Fusarium oxysporum: comprises 25 μ L of a detection solution comprising: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 0.8. mu.M forward loop primer LF, 1.4mM dNTPs, 1 × LAMP Buffer, 1 × xylenol orange, 0.8M betaine, 8mM MgS04, 0.32U/. mu.L Bst DNA polymerase.

LAMP primer composition of Fusarium solani: comprises 25 μ L of a detection solution comprising: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 0.8. mu.M forward loop primer LF, 0.8. mu.M reverse loop primer LB, 1.4mM dNTPs, 1 × LAMP Buffer, 1 × xylenol orange, 0.8M betaine, 8mM MgS04, 0.32U/. mu.L Bst DNA polymerase.

LAMP primer composition of Mucor circinelloides: comprises 25 μ L of a detection solution comprising: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 1.6. mu.M forward loop primer LF, 0.8. mu.M reverse loop primer LB, 1.4mM dNTPs, 1 × LAMP Buffer, 1 × xylenol orange, 0.8M betaine, 8mM MgS04, 0.32U/. mu.L Bst DNA polymerase.

LAMP primer composition of Athelia rolfsii: comprises 25 μ L of a detection solution comprising: 0.2. mu.M forward outer primer F3, 0.2. mu.M reverse outer primer B3, 1.6. mu.M forward inner primer FIP, 1.6. mu.M reverse inner primer BIP, 1.4mM dNTPs, 1 XPLAMP Buffer, 1 Xxylenol orange, 0.8M betaine, 8mM MgS04, 0.32U/. mu.L Bst DNA polymerase.

The primer sequences are specifically shown in Table 2.

Example 3: specificity test of LAMP reaction of common root rot and sclerotium rolfsii of rhizome Chinese medicinal materials (Fusarium oxysporum, Fusarium solani, Mucor circinelloides, Athellia rolfsii)

In order to verify the specific primer sequences of Fusarium oxysporum, 9 Fusarium oxysporum strains and 17 other strains were used as test materials (table 1), and the LAMP detection results showed that the 9 Fusarium oxysporum strains all observed a yellow color change and a LAMP step-like band after electrophoresis. Selecting 10 strains of bacteria (Fusarium avenaceum, F. culmorum, F. equiseti, F. graminearum, F. moniliforme, F. nivale, F. proliferum, F. solani, F. solaani) different from Fusarium oxysporum and 7 strains of bacteria (Borris cinerea, Phytophthora cactorum, P. cambrivora, P. iranica, P. psuedosporidae, Pyrhiium carbonicum, Py. Boreale.) of different genera as templates, taking 1 μ L of DNA solution, adding 24 μ L of detection solution to perform LAMP reaction at 70 ℃ 1min and 68 ℃ 60 min. Based on the reaction system agarose gel electrophoresis and color reaction as the result judgment criteria, the result showed that the color was yellow when Fusarium oxysporum was used as the template, and that the DNA template and the negative control of the bacterium of the same species and genus as Fusarium oxysporum were amplified to be purple (fig. 5).

In order to verify the specific primer sequences of Fusarium solani, 2 Fusarium solani strains and 28 other strains are used as test materials (table 1), and the LAMP detection result shows that 2 Fusarium solani strains can observe yellow color change and LAMP ladder-shaped bands can be observed after electrophoresis. 20 different species of bacteria from Fusarium solani (Fusarium avenaceum, F. culmorum, F. equiseti, F. graminearum, F. lactis, F. moniliforme, F. nivale, F. oxysporum, F.oxysporum) and 8 different species of bacteria from Fusarium (Bacillus cinerea, Clonospora, Physiophygmin, Physiophyceae) were selected as well as a reaction solution, and subjected to a detection at a temperature of 1. mu.L, a temperature of a reaction, a detection at a temperature of 70. mu.L.70. detect DNA. Based on the reaction system agarose gel electrophoresis and color reaction as the result judgment criteria, the result showed that the color was yellow when Fusarium solani was used as the template, while the DNA template and negative control of the bacterium of the same species and genus as Fusarium solani were amplified to be purple (fig. 6).

In order to verify the specific primer sequences of the Mucor circinelloides, 2 strains of Mucor circinelloides and 15 other strains were used as test materials (Table 1), and the LAMP detection result showed that 2 strains of Mucor circinelloides all observed a yellow color change and a LAMP-like band after electrophoresis. DNA of 8 strains (M.guilliermondii, M.hiemalis, M.ifragilias, M.iindicus, M.lucitancis, M.racemosus, M.ramosissimus, M.plumbeus) different from that of Mucor circinelloides and 7 strains (lonostachys rosea, Phytophthora calstrum, P.cambrivora, P.iranica, P.pseudosyringae, Pyrocarcinonicaum, Pyth. Boreale) different from that of Mucor circinelloides were selected as templates, 1. mu.L of the DNA solution was taken, 24. mu.L of the detection solution was added to the DNA solution to carry out LAMP reaction at 70 ℃ for 1min and 60 ℃ for 60 min. Based on the reaction system, agarose gel electrophoresis and color reaction were used as the result judgment criteria, and the results showed that the color was yellow when Mucor circinelloides were used as the template, and that the DNA template and the negative control, which amplified the bacteria of the same species and genus as the Mucor circinelloides, were purple (FIG. 7).

In order to verify the specific primer sequences of Athelia rolfsii, 2 Athelia rolfsii strains and 18 other strains were used as test materials (Table 1), and LAMP detection results showed that 2 Athelia rolfsii strains all observed a yellow color change and a LAMP ladder-like band after electrophoresis. 2 strains of a bacterium (Alternaria alternata, A. tenuissima) different from Athalia rolfsii and 18 strains of a bacterium (Clinostasys rosea, F. graminearum, F. oxysporum, F. solalani, Mucor circinelloides, Phytophthora cactorum, P. cambrivora, P. iranica, P. pseudosyringae, Plectosphaeraella cuumerina, Pyrhium carbonicum, Py. Boreale, Rhizopus oryzae, Stagonosporus cucurbiaum, Trichoderma longibrachiatum) of a different genus were selected as templates, 1. mu.L of DNA solution was taken, 24. mu.L of detection solution was added for reaction, and the reaction program was LAMP 1min at 70 ℃ and LAMP 1min at 60 ℃. Based on the reaction system agarose gel electrophoresis and color reaction as the result judgment criteria, the result showed that the color was yellow when Athelia rolfsii was used as the template, while the DNA template and the negative control, which amplified a bacterium of a different species and genus from Athelia rolfsii, were purple (FIG. 8).

Example 4 LAMP reaction sensitivity test of common root rot and southern blight pathogenic bacteria (Fusarium oxysporum, Fusarium solani, Mucor circinelloides, Athellia rolfsii) of rhizome type Chinese medicinal materials.

To determine the sensitivity of the LAMP detection method, the extracted DNA of Fusarium oxysporum was diluted 10-fold with a measured concentration (1 ng/. mu.L) followed by DEPC water. mu.L of each DNA dilution of 10-fold dilution was used as a template, and added to 24. mu.L of the detection solution to carry out LAMP reaction. The LAMP reaction program of Fusarium oxysporum is 70 ℃ for 1min and 68 ℃ for 60 min. The result of sampling 8. mu.L of the amplified product shows that agarose gel electrophoresis and xylenol orange color reaction indicate that the sensitivity of LAMP reaction reaches 1pg of DNA of Fusarium oxysporum (FIG. 9).

To determine the sensitivity of the LAMP detection method, the extracted DNA of Fusarium solani was diluted 10-fold with DEPC water at a measured concentration (1 ng/. mu.L). mu.L of each DNA dilution of 10-fold dilution was used as a template, and added to 24. mu.L of the detection solution to carry out LAMP reaction. The LAMP reaction program of Fusarium solani is that the Fusarium solani is preserved at 70 ℃ for 1min and at 67 ℃ for 60 min. The result of sampling 8. mu.L of the amplified product showed that agarose gel electrophoresis and xylenol orange color reaction showed that the sensitivity of LAMP reaction reached 100fg of DNA of Fusarium solani (FIG. 10).

To determine the sensitivity of the LAMP detection method, the extracted DNA of Mucor circinelloides was diluted 10-fold with a assay concentration (1 ng/. mu.L) followed by DEPC water. mu.L of each DNA dilution of 10-fold dilution was used as a template, and added to 24. mu.L of the detection solution to carry out LAMP reaction. The LAMP reaction program of the Mucor circinelloides is 1min at 70 ℃ and 60min at 60 ℃. The result of sampling 8. mu.L of the amplified product showed that agarose gel electrophoresis and xylenol orange color reaction showed that the sensitivity of LAMP reaction reached 1pg of DNA of Mucor circinelloides (FIG. 11).

To determine the sensitivity of the LAMP detection method, the extracted DNA of Athellia rolfsii was diluted 10-fold with DEPC water at a measured concentration (1 ng/. mu.L). mu.L of each DNA dilution of 10-fold dilution was used as a template, and added to 24. mu.L of the detection solution to carry out LAMP reaction. The LAMP reaction program of Athellia rolfsii is 70 ℃ for 1min and 62 ℃ for 60 min. The result of sampling 8. mu.L of the amplified product showed that agarose gel electrophoresis and xylenol orange color reaction showed that the sensitivity of LAMP reaction reached 1pg of Athelia rolfsii DNA (FIG. 12).

Example 5 detection of common root rot and southern blight pathogens (Fusarium oxysporum, Fusarium solani, Mucor circinelloides, Athellia rolfsii) in rhizomatic plants from soil samples with bacteria

Extracting genome DNA of the bacteria-bearing soil by adopting an improved magnetic bead method, taking 2 mu L of DNA solution according to the LAMP detection method of Fusarium oxysporum, adding 23 mu L of detection solution in an LAMP kit to carry out LAMP, wherein the LAMP reaction program comprises the following steps: 1min at 70 ℃ and 60min at 68 ℃. When LAMP amplification reaction occurs, the DNA reaction tubes of the bacteria-carrying soil are all yellow as shown by the color reaction result of xylenol orange, and the result is a positive result; the negative control groups are purple and are negative results, which prove that the designed LAMP specific primer can be used for detecting whether the soil contains Fusarium oxysporum.

Extracting genome DNA of the bacteria-bearing soil by adopting an improved paramagnetic particle method, taking 2 mu L of DNA solution according to the LAMP detection method of Fusarium solani, adding 23 mu L of detection solution in an LAMP kit to carry out LAMP, wherein the LAMP reaction program comprises the following steps: 1min at 70 ℃ and 60min at 67 ℃. When LAMP amplification reaction occurs, the DNA reaction tubes of the bacteria-carrying soil are all yellow as shown by the color reaction result of xylenol orange, and the result is a positive result; the negative control groups are purple and are negative results, and the result proves that the designed LAMP specific primer can be used for detecting whether the soil contains Fusarium solani.

Extracting genome DNA of the bacteria-carrying soil by adopting an improved paramagnetic particle method, taking 2 mu L of DNA solution according to the LAMP detection method of Mucor circinelloides, adding 23 mu L of detection solution in an LAMP kit to carry out LAMP, wherein the LAMP reaction program comprises the following steps: 1min at 70 ℃ and 60min at 60 ℃. When LAMP amplification reaction occurs, the DNA reaction tubes of the bacteria-carrying soil are all yellow as shown by the color reaction result of xylenol orange, and the result is a positive result; the negative control groups are purple and are negative results, which prove that the designed LAMP specific primer can be used for detecting whether the soil contains the Mucor circinelloides.

Extracting genome DNA of the bacteria-bearing soil by adopting an improved magnetic bead method, taking 2 mu L of DNA solution according to the LAMP detection method of Athellia rolfsii, adding 23 mu L of detection solution in an LAMP kit to carry out LAMP, wherein the LAMP reaction program comprises the following steps: 1min at 70 ℃ and 60min at 62 ℃. When LAMP amplification reaction occurs, the DNA reaction tubes of the bacteria-carrying soil are all yellow as shown by the color reaction result of xylenol orange, and the result is a positive result; the negative control groups are purple and are negative results, which prove that the designed LAMP specific primer can be used for detecting whether the soil contains Athellia rolfsii.

FIG. 13 is a color development chart of LAMP detection of Fusarium oxysporum, Fusarium solani, Mucor and Sclerotinia sclerotiorum in a soil sample with bacteria; in the figure 13(a), LAMP is used for detecting the color development of fusarium oxysporum in the soil of the planting fields of monkshood, rhizoma corydalis and salvia miltiorrhiza. In the figure, 1-2 tubes of the soil for the planting field of monkshood are shown to be yellow and positive; 3-4 tubes of the rhizoma corydalis planting soil show yellow and positive; 5-6 tubes of the root of red-rooted salvia are soil of the planting field, and the root of red-rooted salvia is yellow and positive; 7 tubes are negative control, purple; 8 tubes are positive control, yellow, positive.

In the figure 13(b), LAMP is used for detecting the color development of Fusarium solani in the soil of the planting fields of monkshood, rhizoma corydalis and Salvia miltiorrhiza. In the figure, 1-2 tubes of the soil for the planting field of monkshood are shown to be yellow and positive; 3-4 tubes of the rhizoma corydalis planting soil show yellow and positive; 5-6 tubes of the root of red-rooted salvia are soil of the planting field, and the root of red-rooted salvia is yellow and positive; 7 tubes are negative control, purple; 8 tubes are positive control, yellow, positive.

In the figure, 13(c) LAMP is used for detecting the chromogenic picture of mucor in the soil of the planting fields of monkshood, rhizoma corydalis and salvia miltiorrhiza. In the figure, 1-2 tubes of the soil for the planting field of monkshood are shown to be yellow and positive; 3-4 tubes of the rhizoma corydalis planting soil show purple and are negative; 5-6 tubes of the root of red-rooted salvia are soil of the planting field, purple is shown, and the root of red-rooted salvia is negative; 7 tubes are negative control, purple; 8 tubes are positive control, yellow, positive.

In the figure 13(d), LAMP is used for detecting the color development of sclerotium rolfsii in the soil of the planting fields of monkshood, rhizoma corydalis and salvia miltiorrhiza. In the figure, 1-2 tubes of the soil for the planting field of monkshood are shown to be yellow and positive; 3-4 tubes of the rhizoma corydalis planting soil show purple and are negative; 5-6 tubes of the root of red-rooted salvia are soil of the planting field, and the root of red-rooted salvia is yellow and positive; 7 tubes are negative control, purple; 8 tubes are positive control, yellow, positive.

The method can be used for rapidly detecting the diseases in the production process of the traditional Chinese medicinal materials, is simple and easy, can effectively improve the detection technical level of the diseases of the traditional Chinese medicinal materials, and provides a comprehensive disease control technology for scientific planting of the traditional Chinese medicinal materials.

The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Claims

1. A LAMP combined detection primer for rhizome traditional Chinese medicinal materials is characterized by comprising the following components:

specific gene sequences of the primers are shown in Table 2.

2. The application of the LAMP combination detection primer for rhizome traditional Chinese medicinal materials in claim 1 in preparing a kit for detecting root rot and southern blight of the rhizome traditional Chinese medicinal materials.

3. A LAMP combination detection kit for rhizome traditional Chinese medicinal materials is characterized by comprising the LAMP combination detection primer of claim 1.

4. The LAMP combination detection kit for rhizome traditional Chinese medicinal materials according to claim 3, which is characterized by comprising:

5. The LAMP combination detection kit for rhizome traditional Chinese medicinal materials according to claim 4, which is characterized by comprising a detection system:

6. The LAMP combination detection kit for rhizome traditional Chinese medicinal materials according to claim 3, which is characterized by comprising:

7. A LAMP detection method for rhizome traditional Chinese medicinal materials is characterized by comprising the following steps:

detection of Fusarium oxysporum: performing LAMP reaction by using genome DNA of a microorganism to be detected as a template and the LAMP primer of the Fusarium oxysporum of claim 1, wherein a color developing agent is xylenol orange, and then observing the color change of an amplification product, wherein if the color changes from purple to yellow, the pathogenic bacterium Fusarium oxysporum exists in the object to be detected, and if the color does not change, the pathogenic bacterium Fusarium oxysporum does not exist in the object to be detected;

detection of Fusarium solani: performing LAMP reaction by using genome DNA of a microorganism to be detected as a template and the LAMP primer of Fusarium solani as claimed in claim 1, wherein a color developing agent is xylenol orange, then observing the color change of an amplification product, if the color changes from purple to yellow, indicating that pathogenic bacteria Fusarium solani exists in the object to be detected, and if the color does not change, still changing to purple, indicating that pathogenic bacteria Fusarium solani does not exist in the object to be detected;

detection of Mucor circinelloides: performing LAMP reaction by using genome DNA of a microorganism to be detected as a template and the LAMP primer of the Mucor circinelloides as defined in claim 1, wherein a color developing agent is xylenol orange, and then observing the color change of an amplification product, wherein if the color changes from purple to yellow, the pathogenic bacteria Mucor circinelloides exist in the object to be detected, and if the color does not change, the pathogenic bacteria Mucor circinelloides do not exist in the object to be detected;

detection of Athelia rolfsii: performing LAMP reaction by using genome DNA of a microorganism to be detected as a template and the LAMP primer of Athellia rolfsii as defined in claim 1, and observing the color change of the amplified product, wherein if the color changes from purple to yellow, the color change indicates the presence of pathogenic bacteria Athellia rolfsii in the sample, and if the color does not change, the color change indicates the absence of pathogenic bacteria Athellia rolfsii in the sample.

8. The LAMP detection method for rhizome traditional Chinese medicinal materials according to claim 7, which is characterized by comprising the following steps:

9. The LAMP detection method of rhizome-type traditional Chinese medicinal materials according to claim 7 or 8, wherein the method for extracting the DNA of the microorganism to be detected comprises the following steps:

10. The LAMP detection method for rhizome traditional Chinese medicinal materials according to claim 8, characterized in that the detection solution comprises: