CN111041120B - LAMP primer and kit for detecting Lasiodipia thailandica - Google Patents

Abstract

The present invention provides a set of LAMP primers and kits for detecting blueberry branch blight (Lasiodiplodia thailandica). The LAMP primers include outer forward and reverse primers and inner forward and reverse primers, and their nucleotide sequences are shown in SEQ ID NOs: 1-4. The invention uses the LAMP primer constant-temperature amplification technology for the rapid detection of Lasiodiplodia thailandica pathogenic bacteria, and can accurately detect the pathogenic bacteria from the complex pathogenic bacteria environment in diseased plant tissues and seedlings. The specificity, sensitivity and reproducibility of this method are higher than that of conventional PCR methods, and it is of great significance for early warning of Lasiodiplodia thailandica and pathogen monitoring in epidemic areas.

Description

LAMP primers and kits for detection of Lasiodiplodia thailandica

technical field

The invention belongs to the technical field of microorganism detection, in particular to a LAMP primer and a kit for detecting Lasiodiplodiathailandica.

Background technique

Blueberry Stem Blight caused by the infection of Botryosphaericeae has been reported in major blueberry producing areas in my country, which has brought serious harm to the healthy development of China's blueberry industry. The fungus infects blueberry plants from wounds or natural orifices, causing cortex and phloem to die, causing blueberry shoots withered, xylem necrosis and plant death in the field. At present, many species of Botryosphaeria cortices, Botryosphaeria dothidea, Fusicoccum aesculin, Lasiodiplodia chinesis, Lasiodiplodia paraphysoides, Lasiodiplodia thailandica, Lasiodiplodia theobromae, Lasiodiplodia vaccinii, Neofusicoccum arbuti, Neofusicoccum austral, Neofusicoccum parvum, Neofusicoccum ribis), of which Lasiodiplodia thailandica is a strong pathogen, the cortical tissue of the diseased part becomes soft and black, easy to peel, and can form reddish-brown to black lesions on the surface of blueberry branches, tissue necrosis, and oval to irregular shape can be seen Brown canker lesions spread to the entire branch, causing pruning and wilting and death. The fungus mainly overwintered in the branch lesions, produced conidia under suitable conditions, and invaded field plants through natural orifices or mechanical wounds. The hyphae rapidly expand into the vascular tissue, develop laterally through the intercellular space, and eventually, colonize various cell types such as callus, cortex, xylem, tracheids, or vessels. The fungus has the characteristics of latent infection, and usually exists as saprophytic fungi in soil or endophyte in healthy plants. Therefore, timely monitoring of the initial infection source of this type of pathogen is of great significance for the prevention and control of the disease. Traditional disease control strategies mainly rely on control measures such as varieties, cultivation, chemical control and ecological regulation. The measures are mainly implemented when the disease outbreaks or even cause obvious harm, neglecting to take comprehensive prevention and control and efficient control measures in the early stage of the initial infection source, so the results are twice the result with half the effort, the prevention effect is very small, and it is ultimately difficult to control the occurrence and prevalence of the disease.

Ordinary PCR technology requires precise temperature-changing equipment and advanced and complex analytical instruments, or requires relatively high proficiency and professional level of operators, and has a long reaction time, which is not conducive to grass-roots promotion. Since the establishment of loop-mediated isothermal amplification (LAMP) technology, this technology has been widely used in the detection and research of viruses, bacteria, parasites, fungi and other pathogens. As a constant temperature nucleic acid amplification technology, LAMP technology has the greatest advantages of fast reaction speed, simple equipment, and easy identification of results, especially suitable for primary inspection and quarantine institutions and medical institutions. There is no relevant report on the detection of blueberry branch blight (Lasiodiplodia thailandica) by LAMP technology.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a LAMP primer and a kit for detecting Lasiodiplodia thailandica.

Another object of the present invention is to provide a method for detecting Lasiodiplodiathailandica based on LAMP technology.

In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a LAMP primer for detecting blueberry branch blight (Lasiodiplodia thailandica), the LAMP primer is (SEQ ID NO: 1-4):

Outer forward primer F3: 5'-CAAGACCAAGTCCACCGG-3;

Outer reverse primer B3: 5'-CATTCTAGTTCGACGCCGTT-3;

Inside forward primer FIP (F1C+F2): 5'-TAGACGTCCTGGAGGGGCAGGAGGCCATCGACAGCATC-3';

Inner reverse primer BIP (B1C+B2): 5'-GCCGTGTGGAAACTGGCGTCGAGCGTAACC CTCACACG-3'.

In a second aspect, the present invention provides detection reagents or kits containing the LAMP primers shown in SEQ ID NOs: 1-4.

In a third aspect, the present invention provides a blueberry branch blight (Lasiodiplodia thailandica) detection kit, the kit includes the LAMP primers shown in SEQ ID NOs: 1-4, and also includes dNTPs, BstDNA polymerase, reaction buffer, At least one of standard positive templates and the like.

In a fourth aspect, the present invention provides the use of the LAMP primers shown in SEQ ID NOs: 1-4, and the detection reagent or kit containing the LAMP primers in the detection of Lasiodiplodia thailandica.

A fifth aspect, the present invention provides a blueberry branch blight (Lasiodiplodia thailandica) detection method, comprising the following steps:

1) Extract the DNA in the sample to be tested;

2) using the DNA extracted in step 1) as a template, using the LAMP primers shown in SEQ ID NOs: 1-4 to carry out a LAMP amplification reaction (LAMP-PCR);

3) Determination of amplification results.

Wherein, step 2) used reaction system is:

Wherein, in the reaction system, primers FIP and BIP are added in equal amounts, primers F3 and B3 are added in equal amounts, and the total mass ratio of primers FIP and BIP to primers F3 and B3 is 8:1.

The following reaction system is preferably used:

The reaction conditions used in step 2) are: 61-65° C. for 50-90 minutes. The following reaction conditions are preferably used: 60 minutes at 63°C and 2 minutes at 80°C.

Step 3) Any one of the following methods ① to ③ can be used to determine the amplification result:

①Fluorescence staining method: Add the dye SYBR Green I to the amplified product to carry out a color reaction. If the reaction system changes from orange (orange) to green, it indicates that the sample to be tested contains Lasiodiplodiathailandica; or , before the amplification reaction, add Calcein (Calcein) to the reaction system, after the amplification reaction, the reaction system shows fluorescent green under the irradiation of ultraviolet lamp, indicating that the sample to be tested contains blueberry branch blight bacteria (Lasiodiplodia thailandica); Alternatively, add hydroxynaphthol blue (HNB) to the reaction system before the amplification reaction. After the amplification reaction, if the reaction system changes from purple to sky blue, it indicates that the sample to be tested contains Lasiodiplodia thailandica );

②Agarose gel electrophoresis method: If the amplified product shows a characteristic ladder-like band on the agarose gel, it indicates that the sample to be tested contains Lasiodiplodia thailandica;

③Magnesium pyrophosphate turbidity detection method: judge whether the LAMP amplification reaction has occurred by observing the turbidity (or producing milky white precipitate) after the reaction with the naked eye, or use a turbidimeter to detect its absorbance at 400nm to achieve real-time quantitative detection .

By the above-mentioned technical scheme, the present invention at least has the following advantages and beneficial effects:

The invention uses the LAMP primer constant-temperature amplification technology for the rapid detection of Lasiodiplodia thailandica pathogenic bacteria, and can accurately detect the pathogenic bacteria from the complex pathogenic bacteria environment in diseased plant tissues and seedlings. The specificity, sensitivity and reproducibility of this method are higher than those of conventional PCR methods, and the detection sensitivity reaches 664 fg/mL. ; At the same time, it can avoid expensive equipment investment, which is convenient for grass-roots promotion and use.

Description of drawings

FIG. 1 is the result of the gene sequence alignment of the EF-1α region of fungi involved in Table 1 in Example 2 of the present invention.

FIG. 2 is the analysis results of specificity and sensitivity of LAMP primers in Examples 2 and 3 of the present invention.

Wherein, A, B: Visual chromogenic detection and agarose gel electrophoresis for specific detection of primer set 1. 1 to 8 are Lasiodiplodia thailandica, Lasiodiplodia chinensis, Lasiodiplodia henanica, Lasiodiplodia paraphysoides, Botryosphaeria sinensia, Neofusicoccum algeriense, Alternaria alternata, NC (negative control).

C, D: Sensitivity of primer set 1 by visual chromogenic detection and agarose gel electrophoresis. 1 to 8 are Lasiodiplodia thailandica DNA samples with dilution times of 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , and 10 ⁸ , respectively.

E, F: Visual chromogenic detection and agarose gel electrophoresis for specific detection of primer set 2. 1 to 8 are Lasiodiplodia thailandica, Lasiodiplodia chinensis, Lasiodiplodia henanica, Lasiodiplodia paraphysoides, Botryosphaeria sinensia, Neofusicoccum algeriense, Alternaria alternata, NC (negative control).

G, H: Sensitivity of primer set 2 by visual chromogenic detection and agarose gel electrophoresis. 1 to 8 are Lasiodiplodia thailandica DNA samples with dilution times of 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , and 10 ⁸ , respectively.

I, J: Visual chromogenic detection and agarose gel electrophoresis for specific detection of primer set 3. 1 to 8 are Lasiodiplodia thailandica, Lasiodiplodia chinensis, Lasiodiplodia henanica, Lasiodiplodia paraphysoides, Botryosphaeria sinensia, Neofusicoccum algeriense, Alternaria alternata, NC (negative control).

K, L: Sensitivity of primer set 3 by visual chromogenic detection and agarose gel electrophoresis. 1 to 8 are Lasiodiplodia thailandica DNA samples with dilution times of 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , and 10 ⁸ , respectively.

M, N: Visual chromogenic detection and agarose gel electrophoresis for specific detection of primer set 4. 1 to 8 are Lasiodiplodia thailandica, Lasiodiplodia chinensis, Lasiodiplodia henanica, Lasiodiplodia paraphysoides, Botryosphaeria sinensia, Neofusicoccum algeriense, Alternaria alternata, NC (negative control).

Among them, M in the agarose gel electrophoresis is DL 2000 DNA Marker.

FIG. 3 is a field disease detection experiment of LAMP primer set 1 in Example 4 of the present invention. Among them, A: diseased spot of diseased plant; B: detection result of LAMP primer set 1 (1 means positive reaction result, 2 means negative reaction result).

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the examples are in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

Example 1 Design and synthesis of LAMP primers for detection of blueberry branch blight (Lasiodiplodia thailandica)

For Lasiodiplodia, the elongation factors (EF) gene has a higher mutation rate, so the EF gene was selected for species-level identification and molecular phylogenetic studies.

According to the gene sequence of the EF-1α region of Lasiodiplodia thailandica (Figure 1), three sets of LAMP primers for the detection of Lasiodiplodia thailandica were designed, including:

Primer set 1:

Outer forward primer F3: 5'-AAGTCCACCGGCAAGACC-3';

Outer reverse primer B3: 5'-ATTCTAGTTCGACGCCGTTA-3';

Inside forward primer FIP: 5'-AGACGTCCTGGAGGGGCAGACGAGGCCATCGACAGCAT-3';

Inside reverse primer BIP: 5'-CGTCGGCCGTGTGGAAACTGAGCGTAACCCTCACAACG-3'.

Primer set 2:

Outer forward primer F3: 5'-ATCGAGGCCTCCACCAAC-3';

Outer reverse primer B3: 5'-CGTAACCCTCACAACGGAG-3';

Inside forward primer FIP: 5'-TGTCGATGGCCTCGAGGAGGCAAGGGCTGGGAGAAGGA-3';

Inside reverse primer BIP: 5'-AGGACGTCTACAAGATTGGCGGTGACGCCAGTTTCCACAC-3'.

Primer set 3:

Outer forward primer F3: 5'-CAAGACCAAGTCCACCGG-3';

Outer reverse primer B3: 5'-CATTCTAGTTCGACGCCGTT-3';

Inside forward primer FIP: 5'-TAGACGTCCTGGAGGGGCAGGAGGCCATCGACAGCATC-3';

Inside reverse primer BIP: 5'-GCCGTGTGGAAACTGGCGTCGAGCGTAACCCTCACAACG-3'.

Primer synthesis was completed by Shenzhen Huada Gene Technology Co., Ltd.

Example 2 Specificity analysis of LAMP primers for detection of Lasiodiplodia thailandica

1.1 Reagents and equipment

LAMP-PCR kit was purchased from Guangzhou Huafeng Company.

1.2 Sample source

Lasiodiplodia thailandica, Lasiodiplodia chinensis, Lasiodiplodia henanica, Lasiodiplodia paraphysoides, Botryosphaeria sinensia, Neofusicoccum algeriense, Alternaria alternata, etc. (Table 1) used in this example are stored in the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. These strains are publicly available and do not need to be deposited.

The results of the gene sequence alignment of the EF-1α region involving fungi in Table 1 are shown in Figure 1 .

Table 1 Sample sources for LAMP-PCR detection

1.3 DNA extraction

Plant tissue DNA was extracted using CTAB Plant Genomic DNA Rapid Extraction Kit (Beijing Aidelai Biotechnology Co., Ltd.), and DNA from healthy blueberry stems was used as a control.

The tested strains were cultured on MEA medium at 25°C for 3-5 days, the mycelial DNA was extracted by CTAB method, and the DNA was stored at -20°C for future use.

1.4 LAMP-PCR reaction

Reaction system (25 μl):

The LAMP-PCR reaction conditions were: 63°C for 60 minutes and 80°C for 2 minutes.

1.5 Color reaction

After the reaction, 6 μl of 1000×SYBR Green I was added to the reaction system obtained in 1.4 to carry out a color reaction. According to the color change of the reaction system, it was determined whether the sample to be tested contained Lasiodiplodiathailandica.

1.6 Results

Fig. 2I is LAMP primer set 3 thermostatic amplification reaction system naked eye visual inspection effect, tube 1 is Lasiodiplodiathailandica bacteria, reaction system shows fluorescent green, tube 2-7 are respectively Lasiodiplodia chinensis, Lasiodiplodia henanica, Lasiodiplodia paraphysoides, Botryosphaeria sinensia, Neofusicoccum algeriense, Alternaria alternata, the reaction systems all showed orange. Tube NC is a negative control. This result shows that the specificity of the primer set of the present invention is strong.

Agarose gel electrophoresis was performed on the above amplification products, and Figure 2J is the isothermal amplification result of LAMP primer set 3 of the present invention; wherein, lane 1 is Lasiodiplodia thailandica, and a ladder-shaped band is amplified, and lanes 2-7 are Lasiodiplodia chinensis, Lasiodiplodia henanica, Lasiodiplodia paraphysoides, Botryosphaeria sinensia, Neofusicoccum algeriense, Alternaria alternata, other species of the same genus and other fungi of the Botrypsaceae family did not produce bands, and NC was the negative control.

Comparing the specificity detection results of primer set 1 (Figure 2A and Figure 2B) and primer set 2 (Figure 2E and Figure 2F), the comparison shows that primer sets 2 and 3 have the highest specificity and the best detection effect.

Example 3 Sensitivity analysis of LAMP primer set for detection of Lasiodiplodia thailandica

1.1 DNA sample concentration:

The DNA concentration of the Lasiodiplodiathailandica sample extracted in Example 2 was detected by NanoDrop (Thermo Fisher Scientific Corporation), and it was 8.3 μg/ml.

1.2 LAMP primer set sensitivity detection:

The DNA samples were serially diluted 10 times, and 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , and 10 ⁸ times diluted DNA samples were used for LAMP isothermal amplification reaction. The reaction system and reaction conditions are the same as those in Example 2.

1.3 Results:

Figure 2K shows the visual effect of the constant temperature amplification reaction system of LAMP primer set 3, and the reaction tubes 1-8 are respectively Lasiodiplodia thailandica bacteria from 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ For the diluted samples, the reaction systems of reaction tubes 1-3 show fluorescent green, and the reaction systems of reaction tubes 4-8 show orange. Fig. 2M is the repeated experiment of the macroscopic effect of the constant temperature amplification reaction system of LAMP primer set 3 of the present invention, wherein the reaction systems of reaction tubes 1-3 show fluorescent green, and the reaction systems of reaction tubes 4-8 show orange. The experimental results are stable. This result indicates that the primer set of the present invention can directly detect DNA diluted to 10 ³ times.

The amplification product was subjected to agarose gel electrophoresis, and the electrophoresis detection result is shown in Figure 2L. LAMP can detect DNA samples diluted 10 ³ times. When DNA samples are diluted more than 10 ³ times, detection cannot be guaranteed. Therefore, the sensitivity of this primer set can reach 664fg/mL, which can detect about 16600fg of Lasiodiplodia thailandica DNA in the sample. FIG. 2N is the repeated experiment result of agarose gel electrophoresis of the amplification product obtained by the constant temperature amplification reaction of the LAMP primer set 1 of the present invention.

Comparing the sensitivity detection results of primer set 1 (Figure 2C and Figure 2D) and primer set 2 (Figure 2G and Figure 2H), the comparison shows that primer set 3 has the highest sensitivity and the best detection effect.

Example 4 Detection of diseased tissue infected with Lasiodiplodia thailandica using LAMP primer set 3

1.1 DNA extraction from blueberry branch blight diseased tissue

The test Lasiodiplodia thailandica was transferred to the MEA medium plate, and after culturing in the dark at 25°C for 2-3 days, the colony block (1cm×1cm) was taken from the edge of the colony with a hole punch, and acupuncture was used to inoculate the stem of blueberry (four-year-old). , 7 days after inoculation, the onset effect is shown in Figure 3A. Then cut the diseased tissue, use the CTAB Plant Genome DNA Rapid Extraction Kit (Beijing Aidelai Biotechnology Co., Ltd.) to extract the diseased tissue DNA: Take an appropriate amount of diseased stem, fully grind it into powder by freezing in liquid nitrogen, and then use the CTAB plant genome DNA rapid extraction kit extracts DNA from diseased plant lesions and diseased-healthy junction tissue. The extracted DNA was used for LAMP-PCR amplification.

DNA from healthy blueberry stems was extracted by the same method as blank control.

1.2 LAMP primer set for detection of Lasiodiplodia thailandica backlink tissue

The LAMP-PCR reaction system, reaction conditions, and detection method of reaction results are the same as those in Example 2.

1.3 Results

The visual effect of the color reaction of the isothermal amplification reaction system of the LAMP primer group is shown in Figure 3B. Tube 1 is a sample of diseased plants infected with Lasiodiplodia thailandica, and the reaction system shows fluorescent green, indicating that the reaction is positive; tube 2 is a healthy plant control , showing orange, indicating that the reaction is negative. The results show that the primer set of the present invention has strong specificity and can be directly used for detection of field diseases.

Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

sequence listing

<110> Beijing Forestry University

<120> LAMP primers and kits for the detection of Lasiodiplodia thailandica

<130> PI201960604

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Claims (8)

1. The LAMP primer group for detecting the blueberry branch blight bacteria Lasiodipia thailandica is characterized by comprising the following components in parts by weight:

outer forward primer F3: 5'-CAAGACCAAGTCCACCGG-3', respectively;

outer reverse primer B3: 5'-CATTCTAGTTCGACGCCGTT-3';

inner forward primer FIP: 5'-TAGACGTCCTGGAGGGGCAGGAGGCCATCGACAGCATC-3', respectively;

inner reverse primer BIP: 5'-GCCGTGTGGAAACTGGCGTCGAGCGTAACCCTCACAACG-3' are provided.

2. A detection reagent or kit comprising the LAMP primer set of claim 1.

3. The detection kit for detecting the blueberry branch blight pathogen Lasiodipia thailandica is characterized by comprising the LAMP primer group of claim 1 and at least one of dNTPs, BstDNA polymerase, reaction buffer and standard positive template.

4. The LAMP primer group of claim 1, the detection reagent or kit of claim 2, or the application of the detection kit of claim 3 in detecting Lasiodipoides thailandica.

5. The detection method of the blueberry branch blight pathogen Lasiodipia thailandica is characterized by comprising the following steps of:

1) extracting DNA in a sample to be detected;

2) performing LAMP amplification reaction by using the DNA extracted in the step 1) as a template and using the LAMP primer group of claim 1;

3) and (5) judging an amplification result.

6. The method as claimed in claim 5, wherein the reaction system used in step 2) is:

ddH₂o is complemented to 25 mu l;

wherein, in the reaction system, the FIP and the BIP are added in equal amount, the F3 and the B3 are added in equal amount, and the total mass ratio of the FIP and the BIP to the F3 and the B3 is 8: 1.

7. The method of claim 5, wherein the reaction conditions used in step 2) are: 50-90 minutes at 61-65 ℃.

8. The method according to any one of claims 5 to 7, wherein the amplification result is determined in step 3) by any one of the following (i) to (iii):

a fluorescent staining method: adding dye SYBR Green I into the amplification product, carrying out a color development reaction, and if the reaction system is changed from orange to Green, indicating that the sample to be detected contains blueberry branch blight pathogen Lasiodipia thailandica; or adding calcein into the reaction system before the amplification reaction, and after the amplification reaction is finished, displaying fluorescent green in the reaction system under the irradiation of an ultraviolet lamp, which indicates that the sample to be detected contains the blueberry branch blight pathogen lasiodendrodia thailandica; or adding hydroxy naphthol blue into the reaction system before the amplification reaction, and after the amplification reaction is finished, if the reaction system is changed from purple to sky blue, indicating that the sample to be detected contains blueberry branch blight pathogen Lasiodipia thailandica;

② agarose gel electrophoresis method: if the amplification product presents a characteristic ladder-shaped strip on the agarose gel, the blueberry branch blight pathogen Lasiodipia thailandica is contained in the sample to be detected;

③ turbidity detection of magnesium pyrophosphate: whether LAMP amplification reaction occurs or not is judged by observing the turbid condition after reaction by naked eyes, or the absorbance of the LAMP amplification reaction at 400nm is detected by a turbidimeter, so that real-time quantitative detection is realized.

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