CN111041123A - LAMP primer and kit for detecting Botryosphaeria sinensia - Google Patents
LAMP primer and kit for detecting Botryosphaeria sinensia Download PDFInfo
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Abstract
The invention provides a set of LAMP primers and a kit for detecting bacterial canker of blueberry stems (Botryosphaeria sinensia). The LAMP primers comprise an outer forward primer, an outer reverse primer, an inner forward primer and an inner reverse primer, and the nucleotide sequences of the primers are shown as SEQ ID NO: 1-4. The LAMP primer isothermal amplification technology is used for rapid detection of Botryosphaeria sinensia pathogen, and the pathogen can be accurately detected from complex pathogenic bacteria environments in diseased plant tissues and nursery stocks. The specificity, sensitivity and repeatability of the method are higher than those of the conventional PCR method, and the method has important significance on aspects such as Botryosphaeria sinensia early warning, pathogen monitoring of epidemic areas and the like; meanwhile, the high instrument investment can be avoided, and the popularization and the use of the basic level are facilitated.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to an LAMP primer and a kit for detecting Botryosphaeriasinensis.
Background
Blueberry stem canker (blueberries disease) caused by the infection of botryococcaceae fungi (Botryosphaericeae) is reported in main blueberry production areas in China at present, and brings serious harm to the health development of the blueberry industry in China. The strain is infected from a wound or a natural orifice of a blueberry plant, so that the withering of cortex and phloem in the field causes blueberry branch withering, xylem necrosis, plant death and other symptoms. At present, there are many diseases which can be caused in the family of Staphylocomiaceae (Botryosphaeria cordices, Botryosphaeria dothidea, Botryosphaeria sinenia, Fusicoccum aesculin, Lasiodelodia chinensis, Lasiodelodia theobroma, Lasiodelodia vaccinii, Neopractical cortex arbuti, Neopractical cortex australia, Neopractical cortex parvum, Neopractical cortex ribis), wherein Botryosphaeria siness is a strong pathogenic bacterium, the cortex tissue of the diseased part is soft and black, easily peeled, reddish brown to black lesions and tissue necrosis can be formed on the surfaces of blueberry branches, and elliptic to irregular brown ulcer lesions can be seen and spread to the whole branches to cause the whole branches to wither and die, the strain mainly overwinter in branch scabs, conidia are generated under proper conditions, the strain invades field plants through natural orifices or mechanical wounds, hyphae rapidly expand to vascular tissues after infection, transversely develop through intercellular spaces, and finally, colonize various types of cells such as callus, cortex, xylem, tracheid or ductus. The bacterium has the characteristic of latent infection, is usually taken as saprophytic bacteria to exist in soil or endophytes of plants to exist in healthy plants, and the plants begin to be infected when the bacterium meets adverse conditions such as high temperature, water accumulation, wind damage and the like. Therefore, the early-stage monitoring of the primary infection source of the pathogenic bacteria has important significance for the prevention and control of the disease, the traditional disease prevention and control strategy mainly depends on prevention and control measures such as variety, cultivation, chemical prevention and ecological control, the prevention and control measures are mainly implemented when the disease outbreak and even obvious harm occurs, comprehensive prevention and control and high-efficiency treatment measures for the early-stage of the primary infection source are ignored, and therefore the method has double effects and little prevention effect, and finally the occurrence and the popularity of the disease are difficult to control.
The common PCR technology needs precise temperature-changing equipment and advanced and complex analytical instruments, or has high requirements on the proficiency and the professional level of operators, and long reaction time, which is not beneficial to basic popularization. Since loop-mediated isothermal amplification (LAMP), the technology has been widely used for detection and research of pathogenic bacteria such as viruses, bacteria, parasites, and bacteria. As a constant-temperature nucleic acid amplification technology, the LAMP technology has the greatest advantages of high reaction speed, simple equipment and easy result identification, and is particularly suitable for basic inspection and quarantine organizations and medical institutions. At present, no relevant report of detection of bacterial canker of blueberry stem (Botryosphaeria sinensia) by using LAMP technology is found.
Disclosure of Invention
The invention aims to provide an LAMP primer and a kit for detecting blueberry stem canker pathogen (Botryosphaeria sinensia).
The invention also aims to provide a detection method of blueberry stem canker pathogenic bacteria (Botryosphaeriasensis) based on LAMP technology.
In order to achieve the object, in a first aspect, the invention provides a LAMP primer for detecting blueberry stem canker pathogenic bacteria (Botryosphaeria sinensis), wherein the LAMP primer is (SEQ ID NO: 1-4):
outer forward primer F3: 5'-AAGATCGACCGCCGTACC-3', respectively;
outer reverse primer B3: 5'-GCGACAGTTTGCCTTGGAA-3', respectively;
inner forward primer FIP (F1C + F2): 5'-CACATGGGCTTGGAGGGAATCATCCCCAAGTTCATCAAGTCCG-3', respectively;
inside reverse primer BIP (B1C + B2): 5'-CTCTTGGCCGTTTCGCCGTAGCACGATGAAGTTCGGAG-3' are provided.
In a second aspect, the present invention provides a polypeptide comprising SEQ ID NO: 1-4.
In a third aspect, the present invention provides a blueberry stem canker pathogen (Botryosphaeria sinensis) detection kit comprising SEQ ID NO: 1-4, and further comprises at least one of dNTPs, BstDNA polymerase, reaction buffer, standard positive template, and the like.
In a fourth aspect, the invention provides SEQ ID NO: 1-4, and a detection reagent or kit containing the LAMP primer in the detection of blueberry stem canker pathogen (Botryosphaeria sinensia).
In a fifth aspect, the invention provides a method for detecting bacterial canker of blueberry stem (Botryosphaeria sinensis), which comprises the following steps:
1) extracting DNA in a sample to be detected;
2) using the DNA extracted in step 1) as a template, and using the DNA sequence shown in SEQ ID NO: 1-4 to carry out LAMP amplification reaction (LAMP-PCR);
3) and (5) judging an amplification result.
Wherein, the reaction system used in the step 2) is as follows:
ddH2make up to 25. mu.l of O.
Wherein, in the reaction system, the primers FIP and BIP are added in equal amount, the primers F3 and B3 are added in equal amount, and the total mass ratio of the primers FIP and BIP to the primers F3 and B3 is 8: 1.
The following reaction system is preferably employed:
the reaction conditions used in step 2) are: 50-90 minutes at 61-65 ℃. The following reaction conditions are preferably employed: 60 minutes at 63 ℃ and 2 minutes at 80 ℃.
Step 3) may be carried out by any one of the following methods ① to ③:
① fluorescent staining method, adding dye SYBR Green I into the amplification product to perform color reaction, if the reaction system changes from orange (orange) to Green, indicating that the sample to be detected contains blueberry stem canker pathogenic bacteria (Botryosphaeriensis), or adding Calcein (Calcein) into the reaction system before the amplification reaction, after the amplification reaction is finished, the reaction system shows fluorescent Green under the irradiation of an ultraviolet lamp, indicating that the sample to be detected contains blueberry stem canker pathogenic bacteria (Botryosphaeria sinensis), or adding hydroxynaphthol blue (HNB) into the reaction system before the amplification reaction, after the amplification reaction is finished, if the reaction system changes from purple to sky blue, indicating that the sample to be detected contains blueberry stem canker pathogenic bacteria (Botryosphaeria sinensis);
② agarose gel electrophoresis method, wherein if the amplification product presents a characteristic ladder-shaped band on agarose gel, the result shows that the sample to be detected contains blueberry stem canker pathogen (Botryosphaeria sinensia);
③ turbidity detection method of magnesium pyrophosphate, which comprises visually observing the turbidity (or milky precipitate) after reaction to determine whether LAMP amplification reaction occurs, or detecting the absorbance at 400nm with a turbidity meter to realize real-time quantitative detection.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the LAMP primer isothermal amplification technology is used for rapid detection of Botryosphaeria sinensia pathogen, and the pathogen can be accurately detected from complex pathogenic bacteria environments in diseased plant tissues and nursery stocks. The specificity, sensitivity and repeatability of the method are higher than those of the conventional PCR method, the detection sensitivity reaches 7.52fg/mL, and the method has important significance in the aspects of early warning of blueberry stem canker pathogen (Botryosphaeria sinensia), pathogen monitoring of epidemic areas and the like; meanwhile, the high instrument investment can be avoided, and the popularization and the use of the basic level are facilitated.
Drawings
FIG. 1 is an alignment of the gene sequences of EF-1 α region of fungus related to Table 1 in example 2 of the present invention.
FIG. 2 shows the results of analysis of the specificity and sensitivity of LAMP primers in examples 2 and 3 of the present invention.
Wherein, A, B: visual color development test for specific detection of primer set 1 and agarose gel electrophoresis. 1 to 8 are Botryosphaeria sinensis, Botryosphaeria facerciana, Botryosphaeria rosaceae, Neofuscoccum illiciii, Neofuscoccum sinense, Lasiodipia chinensis, Alternaria alternata, NC (negative control), respectively.
C, D: sensitivity of primer set 1 visual detection of color development and agarose gel electrophoresis. 1 to 8 are respectively dilution times of 101、102、103、104、105、106、107、108Double Botryosphaeria sinensia DNA samples.
E, F: visual color development test and agarose gel electrophoresis for specific detection of primer set 2. 1 to 8 are Botryosphaeria sinensis, Botryosphaeria facerciana, Botryosphaeria rosaceae, Neofuscoccum illiciii, Neofuscoccum sinense, Lasiodipia chinensis, Alternaria alternata, NC (negative control), respectively.
G, H: sensitivity of primer set 2 visual detection of color development and agarose gel electrophoresis. 1 to 8 are respectively dilution times of 101、102、103、104、105、106、107、108Double Botryosphaeria sinensia DNA samples.
I, J: visual color development test for specific detection of primer set 3 and agarose gel electrophoresis. 1 to 8 are Botryosphaeria sinensis, Botryosphaeria facerciana, Botryosphaeria rosaceae, Neofuscoccum illiciii, Neofuscoccum sinense, Lasiodipia chinensis, Alternaria alternata, NC (negative control), respectively.
K, L: sensitivity of primer set 3 visual detection of color development and agarose gel electrophoresis. 1 to 8 are respectively dilution times of 101、102、103、104、105、106、107、108Double Botryosphaeria sinensia DNA samples.
M, N: visual color development test for specific detection of primer set 4 and agarose gel electrophoresis. 1 to 8 are Botryosphaeria sinensis, Botryosphaeria facerciana, Botryosphaeria rosaceae, Neofuscoccum illiciii, Neofuscoccum sinense, Lasiodipia chinensis, Alternaria alternata, NC (negative control), respectively.
Wherein M in the agarose gel electrophoresis picture is DL 2000 DNA Marker.
FIG. 3 is a field diseased plant detection experiment of the LAMP primer set 1 in example 4 of the present invention. Wherein, A: diseased plant spots; b: the detection result of the LAMP primer group 1 (1 is positive reaction result, and 2 is negative reaction result).
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the molecular cloning handbook, Sambrook et al (Sambrook J & Russell DW, molecular cloning: aLaboratoria Manual, 2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 design and Synthesis of LAMP primers for detection of blueberry Stem canker pathogen (Botryosphaeria sinensia)
For Botryosphaeria, the translation Elongation Factors (EF) gene has a high mutation rate, and therefore the EF gene is selected for species level identification and molecular phylogenetic studies.
Based on the gene sequence of EF-1 α region of blueberry stem canker pathogen (Botryosphaeria sinensia) (FIG. 1), three groups of LAMP primers for detecting Botryosphaeria sinensia were designed respectively, including:
primer set 1:
outer forward primer F3: 5'-AAGATCGACCGCCGTACC-3', respectively;
outer reverse primer B3: 5'-GCGACAGTTTGCCTTGGAA-3', respectively;
inner forward primer FIP: 5'-CACATGGGCTTGGAGGGAATCATCCCCAAGTTCATCAAGTCCG-3', respectively;
inner reverse primer BIP: 5'-CTCTTGGCCGTTTCGCCGTAGCACGATGAAGTTCGGAG-3' are provided.
Primer set 2:
outer forward primer F3: 5'-GTCCATTGAGAACAACCCCA-3', respectively;
outer reverse primer B3: 5'-GGCGACAGTTTGCCTTGG-3', respectively;
inner forward primer FIP: 5'-GCACATGGGCTTGGAGGGAAGTTCATCAAGTCCGGTGAC-3', respectively;
inner reverse primer BIP: 5'-TACCCCCCTCTTGGCCGTTTGCACGATGAAGTTCGGAGAA-3' are provided.
Primer set 3:
outer forward primer F3: 5'-AAGATCGACCGCCGTACC-3', respectively;
outer reverse primer B3: 5'-GCGACAGTTTGCCTTGGAA-3', respectively;
inner forward primer FIP: 5'-CACATGGGCTTGGAGGGAATCACCCCAAGTTCATCAAGTCCG-3', respectively;
inner reverse primer BIP: 5'-TACCCCCCTCTTGGCCGTTTAGCACGATGAAGTTCGGAG-3' are provided.
The primer synthesis is completed by Shenzhen Hua Dagen science and technology Limited.
Example 2 LAMP primer detection specificity assay for Botryosphaeria sinensia
1.1 reagents and devices
The LAMP-PCR kit was purchased from Guangzhou Huafeng company.
1.2 sample sources
Botryosphaeria sinensis, Botryosphaeria facericiana, Botryosphaeria rosaceae, Neofuscoccum illiciii, Neofuscoccum sinense, Lasiodipia chinensis, Alternaria alternata, etc. (Table 1) used in this example were stored in the national stress laboratory of microbiology of the institute of sciences, China. These species are publicly available and do not require preservation.
The results of the alignment of the gene sequences of the EF-1 α region of the fungus involved in Table 1 are shown in FIG. 1.
TABLE 1 sample sources for LAMP-PCR detection
1.3 DNA extraction
A CTAB plant genome DNA rapid extraction kit (Beijing Ederly Biotech limited) is used for extracting plant tissue DNA, and DNA extracted from healthy blueberry stems is used as a control.
Culturing the test strain on MEA culture medium at 25 deg.C for 3-5 days, extracting mycelium DNA by CTAB method, and storing at-20 deg.C.
1.4 LAMP-PCR reaction
Reaction system (25. mu.l):
the LAMP-PCR reaction conditions are as follows: 60 minutes at 63 ℃ and 2 minutes at 80 ℃.
1.5 color reaction
After the reaction is finished, 6 mu l of 1000 XSYBR Green I is added into the reaction system obtained from 1.4 for color reaction, and whether the sample to be detected contains blueberry stem canker pathogen (Botryosphaeria sinensia) is judged according to the color change of the reaction system.
1.6 results
FIG. 2A shows the visual observation effect of LAMP primer set 1 in isothermal amplification reaction system, tube 1 is Botryosphaeriensis germ, the reaction system shows fluorescent green, tubes 2 to 7 are Botryosphaeria facicularia, Botryosphaeria rosaceae, Neofuscoccum illiciii, Neofuscoccum sine, Lasiodipia chinensis, Alternaria altena, and the reaction system shows orange color. Tube NC was negative control. The results show that the primer set of the present invention has strong specificity.
Performing agarose gel electrophoresis on the amplification product, wherein FIG. 2B shows the isothermal amplification result of the LAMP primer group 1 of the invention; wherein lane 1 is Botryosphaeria sinensis, amplified ladder-like bands, lanes 2-7 are Botryosphaeria fabericiana, Botryosphaeria rosae, Neofusicoccum illiciii, Neofusicoccum sinense, Lasiodipia chinensis, Alternaria alternata, other species of the same genus and other fungi of the family Botryosphaeaceae do not produce bands, and NC is a negative control.
Comparing the specificity detection results of the primer group 2 (fig. 2E and fig. 2F) and the primer group 3 (fig. 2I and fig. 2J), the comparison shows that the specificity of the three primer groups is high and the detection effect is good.
Example 3 sensitivity assay for LAMP primer set detection in Botryosphaeria sinensia
1.1 DNA sample concentration:
the DNA concentration of the Botryosphaeriensis sample extracted in example 2 was measured to be 9.4. mu.g/ml using NanoDrop (Seimer Feishale science).
1.2 LAMP primer group sensitivity detection:
the DNA sample is diluted by 10 times of gradient, 10 are taken1、102、103、104、105、106、107、108And (3) carrying out LAMP isothermal amplification reaction on the doubly diluted DNA sample. The reaction system and reaction conditions were the same as in example 2.
1.3 results:
FIG. 2C shows the macroscopic observation effect of the isothermal amplification reaction system of LAMP primer set 1, and reaction tubes 1-8 are Botryosphaeria sinensia bacteria from 101、102、103、104、105、106、107、108For the diluted sample, the reaction system of the reaction tube 1-5 shows fluorescent green, and the reaction system of the reaction tube 6-8 shows orange. FIG. 2M is a repeated experiment of visual observation effect of the LAMP primer group 1 constant temperature amplification reaction system of the present invention with naked eyes, wherein the reaction system of the reaction tubes 1-5 shows fluorescent green, and the reaction system of the reaction tubes 6-8 shows orange. The experimental result is stable. The results show that the primer set of the present invention can be directly detected to be diluted to 105Double DNA.
The amplification products were subjected to agarose gel electrophoresis, and the results of the electrophoresis are shown in FIG. 2D, where LAMP detected 10 dilution5Double DNA sample. When the DNA sample is diluted to 105If the number is more than twice, detection cannot be ensured. Therefore, the sensitivity of the primer group can reach 7.52fg/mL, and about 188fg of Botryosphaeria sinensia DNA in a sample can be detected. FIG. 2N shows the result of repeated agarose gel electrophoresis of the amplification product obtained by isothermal amplification reaction of LAMP primer set 1 of the present invention.
Comparison of the sensitivity detection results of the primer set 2 (FIGS. 2G and 2H) and the primer set 3 (FIGS. 2K and 2L) shows that the sensitivity of the primer set 1 is the highest and the detection effect is the best.
Example 4 detection of diseased tissue infected with Botryosphaeria sinensia Using LAMP primer set 1
1.1 extraction of DNA from diseased tissue of blueberry stem ulcer disease
Botryosphaeria sinensia bacteria to be tested are transferred to an MEA culture medium plate, after dark culture is carried out for 2-3 days at 25 ℃, a colony block (1cm multiplied by 1cm) is taken from the edge of the colony by a puncher, the colony block is inoculated to stems of blueberries (four-year-old) through needle punching, and after inoculation is carried out for 7 days, the morbidity effect is shown in figure 3A. Then, diseased tissues were excised, and DNA of the diseased tissues was extracted using a CTAB plant genomic DNA rapid extraction kit (beijing edlely biotechnology limited): taking a section of appropriate diseased stem, freezing by liquid nitrogen, fully grinding into powder, and then extracting diseased plant lesion and diseased key junction tissue DNA by using a CTAB plant genome DNA rapid extraction kit. The extracted DNA is used for LAMP-PCR amplification.
DNA of healthy blueberry stems was extracted as a blank control by the same method.
1.2 LAMP primer set for Botryosphaeria sinensia germ reissue tissue detection
The LAMP-PCR reaction system, reaction conditions, and reaction result detection method were the same as in example 2.
1.3 results
The visual observation effect of the LAMP primer group isothermal amplification reaction system in the color reaction is shown in FIG. 3B, a tube 1 is a diseased strain sample infected with Botryosphaeria sinensia, the reaction system is in fluorescent green, and the reaction is positive; tube 2 is a healthy plant control, appearing orange, showing a negative response. The result shows that the primer group has strong specificity and can be directly used for detecting field diseases.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (8)
1. The LAMP primer for detecting the bacterial canker of blueberry stem (Botryosphaeria sinensis) is characterized by comprising the following components in parts by weight:
outer forward primer F3: 5'-AAGATCGACCGCCGTACC-3', respectively;
outer reverse primer B3: 5'-GCGACAGTTTGCCTTGGAA-3', respectively;
inner forward primer FIP: 5'-CACATGGGCTTGGAGGGAATCATCCCCAAGTTCATCAAGTCCG-3', respectively;
inner reverse primer BIP: 5'-CTCTTGGCCGTTTCGCCGTAGCACGATGAAGTTCGGAG-3' are provided.
2. A detection reagent or kit comprising the LAMP primer according to claim 1.
3. A blueberry stem canker pathogen (Botryosphaeria sinensis) detection kit, which is characterized by comprising the LAMP primer as claimed in claim 1, and further comprising at least one of dNTPs, BstDNA polymerase, reaction buffer and standard positive template.
4. The LAMP primer as set forth in claim 1, the detection reagent or kit as set forth in claim 2, or the detection kit as set forth in claim 3, for detecting bacterial canker of blueberry stem (Botryosphaeria sinensis).
5. The detection method of the bacterial canker of blueberry stem canker (Botryosphaeria sinensis) 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 according to 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 primers FIP and BIP are added in equal amount, the primers F3 and B3 are added in equal amount, and the total mass ratio of the primers FIP and BIP to the primers F3 and 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 determination of the amplification result in step 3) is carried out by any one of the following methods ① to ③:
① fluorescent staining method, adding dye SYBR Green I into the amplification product to perform color reaction, if the reaction system changes from orange to Green, indicating that the sample to be detected contains blueberry stem canker pathogenic bacteria (Botryosphaeria sinensis), or adding calcein into the reaction system before the amplification reaction, after the amplification reaction, the reaction system shows fluorescent Green under the irradiation of an ultraviolet lamp, indicating that the sample to be detected contains the blueberry stem canker pathogenic bacteria (Botryosphaeria sinensis), or adding hydroxynaphthol blue into the reaction system before the amplification reaction, after the amplification reaction, if the reaction system changes from purple to sky blue, indicating that the sample to be detected contains the blueberry stem canker pathogenic bacteria (Botryosphaeria sinensis);
② agarose gel electrophoresis method, wherein if the amplification product presents a characteristic ladder-shaped band on agarose gel, the result shows that the sample to be detected contains blueberry stem canker pathogen (Botryosphaeria sinensia);
③ turbidity detection method of magnesium pyrophosphate, which comprises visually observing turbidity after reaction to determine whether LAMP amplification reaction occurs, or detecting absorbance at 400nm with nephelometer to realize real-time quantitative detection.
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