CN111041119A - LAMP primer and kit for detecting Neofusicoccum macroclavatum - Google Patents
LAMP primer and kit for detecting Neofusicoccum macroclavatum Download PDFInfo
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Abstract
The invention provides a set of LAMP primers and a kit for detecting blueberry branch blight bacteria (Neofusicoccum macroclavatum). 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 rapidly detecting Neofuscoccum macroclavatum pathogen, and the pathogen can be accurately detected from complex pathogenic bacteria environments in pathogenic 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 in the aspects of Neofuscum macrocaulatum 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 Neofusicoccumsubclavatum.
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. Currently, there are several diseases in the family of staphylococci that can cause this disease (Botryosphaeria cortices, Botryosphaeria dothidea, fusacoccus aesculin, lasiodipia chinensis, lasiodipia paradoxoides, lasiodipia theobroma, lasiodipia vaccinii, neofusicum arbuti, neofusicocume, neofusicocus arbuti, neofusicocus maculopatula, which is a strong type of lesion on the surface of a blueberry shoot, causing necrosis of vascular bundles and vascular death, which is mainly responsible for overwintering in the diseased tissue, producing vascular hyphae under suitable conditions, spreading laterally into the vascular cortex, through the opening of the field, through the opening of the duct, or the like, and finally spreading into the tissue of the duct, through the opening of the duct, after mechanical invasion of the tissue, or other types of pathogenic bacteria. 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. The LAMP technology is used as a constant-temperature nucleic acid amplification 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 organizations. At present, no relevant report of detecting the bacterial wilt of blueberries (Neofusicoccum macrocovatum) by using LAMP technology is found.
Disclosure of Invention
The invention aims to provide an LAMP primer and a kit for detecting blueberry branch blight bacteria (Neofusicoccum macroclavatum).
The invention also aims to provide a blueberry branch blight bacterium (Neofusicoccumsubclavaptatum) detection method based on LAMP technology.
In order to achieve the purpose of the invention, in a first aspect, the invention provides a LAMP primer for detecting blueberry branch blight bacteria (Neofusicoccum macroclavatum), wherein the LAMP primer is (SEQ ID NO: 1-4):
outer forward primer F3: 5'-AAAGTTTTTCCTTCCGCTGC-3', respectively;
outer reverse primer B3: 5'-TGGGACGGCCGTTAGC-3', respectively;
inner forward primer FIP (F1C + F2): 5'-CGACCCCACCACCAAAATGCCTGGGTTCCCGCACTCA-3', respectively;
inside reverse primer BIP (B1C + B2): 5'-GGCTCGGCAAAATCTCCGCATCCATGCGTGGCGTGTCTG-3'
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 branch blight pathogen (Neofusicoccum macroclavatum) detection kit, which comprises 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 branch blight bacteria (Neofusicoccum macroclavatum).
In a fifth aspect, the invention provides a method for detecting blueberry branch blight pathogen (Neofusicoccum macroclavatum), 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:
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 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 branch blight bacteria (Neofusicoccobaclavatum), or adding Calcein (Calcein) into the reaction system before the amplification reaction, after the amplification reaction is finished, the reaction system displays fluorescent Green under the irradiation of an ultraviolet lamp to indicate that the sample to be detected contains the blueberry branch blight bacteria (Neofusicoccum macroclavum), or adding Hydroxy Naphthol 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 the blueberry branch blight bacteria (Neofusicoccum macroclavum);
② agarose gel electrophoresis, wherein if the amplification product presents a characteristic ladder-shaped strip on the agarose gel, the amplification product shows that the sample to be detected contains blueberry branch blight bacteria (Neofusicoccum macroclavatum);
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 rapidly detecting Neofuscoccum macroclavatum pathogen, and the pathogen can be accurately detected from complex pathogenic bacteria environments in pathogenic 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 0.808fg/mL, and the method has important significance in the aspects of early warning of blueberry branch blight bacteria (Neofusicoccum macrocovatum), 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 Neofusicoccum macroclavatum, Neofusicoccum illiciii, Neofusicoccum sinense, Botryosphaeria sinensia, Botryosphaeria rosaceae, 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、108A multiplied Neofusicoccum macrocaulum DNA sample.
E, F: visual color development test and agarose gel electrophoresis for specific detection of primer set 2. 1 to 8 are Neofusicoccum macroclavatum, Neofusicoccum illiciii, Neofusicoccum sinense, Botryosphaeria sinensia, Botryosphaeria rosaceae, 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、108A multiplied Neofusicoccum macrocaulum DNA sample.
I, J: visual color development test for specific detection of primer set 3 and agarose gel electrophoresis. 1 to 8 are Neofusicoccum macroclavatum, Neofusicoccum illiciii, Neofusicoccum sinense, Botryosphaeria sinensia, Botryosphaeria rosaceae, 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、108A multiplied Neofusicoccum macrocaulum DNA sample.
M, N: visual color development test for specific detection of primer set 4 and agarose gel electrophoresis. 1 to 8 are Neofusicoccum macroclavatum, Neofusicoccum illiciii, Neofusicoccum sinense, Botryosphaeria sinensia, Botryosphaeria rosaceae, Lasiodipia chinensis, Alternaria alternata, NC (negative control), respectively.
Wherein M in the agarose gel electrophoresis picture is DL 2000DNA 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 primer for detecting blueberry Branch blight bacterium (Neofusicoccum macroclavatum)
For Neofusicoccum, the translation Elongation Factors (EF) gene has a high mutation rate, so that the EF gene is selected for species level identification and molecular systematics research.
According to the gene sequence (figure 1) of EF-1 α region of blueberry branch blight bacteria (Neofusicoccum macrocephalatum), three groups of LAMP primers for detecting the Neofusicoccum macrocephalatum are respectively designed, and the three groups of LAMP primers comprise:
primer set 1:
outer forward primer F3: 5'-GCTGCGACTCCTTCAACG-3', respectively;
outer reverse primer B3: 5'-GGCGTCACCAGACTTGATG-3', respectively;
inner forward primer FIP: 5'-CAGTCCAGGACGGGAGCGTACCCAGGTCATCGTCCTCA-3', respectively;
inner forward primer BIP: 5'-GCTTGCAAGTTCTCTGAGCTGCCTTGGGGCTGTTCTCAATGG-3' are provided.
Primer set 2:
outer forward primer F3: 5'-AAAGTTTTTCCTTCCGCTGC-3', respectively;
outer reverse primer B3: 5'-TGGGACGGCCGTTAGC-3', respectively;
inner forward primer FIP: 5'-CGACCCCACCACCAAAATGCCTGGGTTCCCGCACTCA-3', respectively;
inner forward primer BIP: 5'-GGCTCGGCAAAATCTCCGCATCCATGCGTGGCGTGTCTG-3' are provided.
Primer set 3:
outer forward primer F3: 5'-TCCTGGACTGCCACACTG-3', respectively;
outer reverse primer B3: 5'-GAGGGTACTCGGTGAAAGC-3', respectively;
inner forward primer FIP: 5'-TCTCAATGGACTTGCCGGTACGTGCTTGCAAGTTCTCTGAGC-3', respectively;
inner forward primer BIP: 5'-TTCATCAAGTCTGGTGACGCCGTCAACGCACATGGGCTTG-3' are provided.
The primer synthesis is completed by Shenzhen Hua Dagen science and technology Limited.
Example 2LAMP primer detection specificity assay for Neofusicoccum macroclavatum
1.1 reagents and devices
The LAMP-PCR kit was purchased from Guangzhou Huafeng company.
1.2 sample sources
Neofusicoccum macroclavatum, Neofusicoccum illiciii, Neofusicoccum sinense, Botryosphaeria sinensia, Botryosphaeria rosaceae, Lasiodipia chinensis, Alternaria alternata, etc. (Table 1) used in this example were stored in national stress laboratories of institute of microbiology, China academy of sciences. 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.
1.4LAMP-PCR reaction
Reaction system (25. mu.l):
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 branch blight bacteria (Neofusicoccumcroclatavasum) 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 Neofusicoccumascolatum, the reaction system shows fluorescent green, tubes 2-7 are Neofusicoccumallii, Neofusicoccumasinense, Botryosphaeria rosaceae, Lasiodipidia chinensis, Alternaria alternata, and the reaction system shows orange yellow. 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 Neofuscoccum macroclavatum germ, a ladder-shaped band is amplified, lanes 2-7 are Neofuscoccum illiciii, Neofuscoccum sine, Botryosphaeria rosaceae, Lasiodipidia chinensis, Alternaria alternata, other species of the same genus and other fungi of the family Botryosphaeaceae do not generate a band, and NC is a negative control.
Comparison of the results of the specificity detection of the primer set 2 (FIGS. 2E and 2F) and the primer set 3 (FIGS. 2I and 2J) shows that the primer set 1 has the highest specificity and the best detection effect.
Example 3 sensitivity analysis of LAMP primer set for detection of Neofusicoccum macroclavatum
1.1DNA sample concentration:
the DNA concentration of the Neofusicoccumaminoclatsheet sample extracted in example 2 was measured at 10.4. mu.g/ml with a NanoDrop (Seimer Feishell science Co.).
1.2LAMP 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 LAMP primer set 1 isothermal amplification reaction system, and the reaction tubes 1-8 are respectively Neofusicocum macroclavatum 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 1-6 shows fluorescent green, and the reaction system of the reaction tubes 7-8 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 106Double 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 dilution6Double DNA sample. When the DNA sample is diluted to 106If the number is more than twice, detection cannot be ensured. Therefore, the sensitivity of the primer group can reach 0.808fg/mL, and about 20.2fg of Neofusicocume macrocaulovatum 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 Neofusicoccum macroclavatum Using LAMP primer set 1
1.1 extraction of DNA from diseased tissue of blueberry stem ulcer disease
Neofusicoccum macroclavatum to be tested is transferred to an MEA culture medium plate, after dark culture is carried out for 2-3 days at 25 ℃, a puncher is used for taking a colony block (1cm multiplied by 1cm) from the edge of the colony, the colony block is inoculated to stems of blueberries (four-year-old) through needle punching, and after inoculation 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.
1.2LAMP primer group for Neofusicocum macroclavatum germ tieback 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 constant-temperature amplification reaction system in the color reaction is shown in figure 3B, a tube 1 is a diseased plant sample infected with Neofusicocum macroclavatum, 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 blueberry branch blight pathogen (Neofusicoccum macroclavatum) is characterized by comprising the following components in parts by weight:
outer forward primer F3: 5'-AAAGTTTTTCCTTCCGCTGC-3', respectively;
outer reverse primer B3: 5'-TGGGACGGCCGTTAGC-3', respectively;
inner forward primer FIP: 5'-CGACCCCACCACCAAAATGCCTGGGTTCCCGCACTCA-3', respectively;
inner reverse primer BIP: 5'-GGCTCGGCAAAATCTCCGCATCCATGCGTGGCGTGTCTG-3' are provided.
2. A detection reagent or kit comprising the LAMP primer according to claim 1.
3. A blueberry branch blight pathogen (Neofusicoccum macroclavatum) detection kit, which is characterized by comprising the LAMP primer as claimed in claim 1, and at least one of dNTPs, BstDNA polymerase, reaction buffer and standard positive template.
4. The LAMP primer of claim 1, the detection reagent or kit of claim 2, or the application of the detection kit of claim 3 in detecting blueberry branch blight bacteria (Neofusicoccum macrocovatum).
5. The detection method of the blueberry branch blight pathogen (Neofusicoccum macroclavatum) 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:
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 branch blight germ (Neofusicoccum macroclavum), or adding 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 branch blight germ (Neofusicoccum macroclavum), or adding hydroxynaphthol blue 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 branch blight germ (Neofusicoccum macroclavum);
② agarose gel electrophoresis, wherein if the amplification product presents a characteristic ladder-shaped strip on the agarose gel, the amplification product shows that the sample to be detected contains blueberry branch blight bacteria (Neofusicoccum macroclavatum);
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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| CN107557446A (en) * | 2017-10-31 | 2018-01-09 | 青岛农业大学 | For detecting the nucleic acid, kit and method of four kinds of disease pathogens of blueberry simultaneously |
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| CN107557446A (en) * | 2017-10-31 | 2018-01-09 | 青岛农业大学 | For detecting the nucleic acid, kit and method of four kinds of disease pathogens of blueberry simultaneously |
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| WO2019213624A1 (en) * | 2018-05-04 | 2019-11-07 | The Regents Of The University Of California | Spiked primers for enrichment of pathogen nucleic acids among background of nucleic acids |
| CN109182591A (en) * | 2018-11-06 | 2019-01-11 | 福建省农业科学院植物保护研究所 | A kind of sword-leaved cymbidium Pathogen LAMP detection primer group and its rapid detection method |
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