CN111041125A - LAMP primer and kit for detecting Macrophomina vaccini - Google Patents
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Abstract
The invention provides a set of LAMP primers and a kit for detecting blueberry branch blight bacteria (macrophosina vaccini). 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 the macrophosina vaccini 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 early warning of the macrophosina vaccini, pathogen monitoring of an epidemic area 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 macrophormonadacini.
Background
Blueberry branch Blight (blueberries light) caused by infection of botryococcaceae fungi (botryosphaeriae) is reported in main blueberry production areas in China at present, and serious harm is brought to the healthy 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 many diseases (Botryosphaeria cortices, Botryosphaeria dothidea, fusacoccus aesculin, lasiodipa chinensis, lasiodipia theobroma, lasiodipia vaccinii, macrophosmin vaccini, neococcus arbuti, neococcus australis, neococcus cumparvum, neococcus verticillium, etc.) which are caused by this disease in the plasmodiomycetaceae family, wherein macrophosta vaccini is a strong pathogenic bacterium which forms red spots on the surface of blueberry branches, causing vascular bundle necrosis and death of whole branches, which overwinter alia in diseased branch spots, produces conidia under suitable conditions, and after infection in the field, rapidly spreads into the vascular cortex by natural or mechanical wounds, develops into the vascular cortex, and finally into various types of vascular ducts, colonial cells, or ducts. 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 report related to detection of blueberry branch blight bacteria (macrophosina vaccini) 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 (macrophosina vaccini).
The invention also aims to provide a LAMP technology-based blueberry branch blight bacterium (macrophormonadacini) detection method.
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 (macrophosina vaccini), wherein the LAMP primer is (SEQ ID NO: 1-4):
outer forward primer F3: 5'-CGTTGAAGGAGTCGCAGC-3', respectively;
outer reverse primer B3: 5'-TGGTCGTTACCTTCGCTCC-3', respectively;
inner forward primer FIP (F1C + F2): 5' -TCGGCTTCAACGTCAAGAACGTGGGGGTCGTTCTTGGAGT-3;
inside reverse primer BIP (B1C + B2): 5'-CGTTGTCACCGGGAACACCCTCACCACCGAGGTCAAGT-3' are provided.
In a second aspect, the present invention provides a polypeptide comprising SEQ ID NO: 1-4.
In a third aspect, the invention provides a blueberry branch blight pathogen (macrophosmin vaccini) 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 application of a detection reagent or kit containing the LAMP primer in detection of blueberry branch blight bacteria (macrophosina vaccini).
In a fifth aspect, the invention provides a method for detecting blueberry branch blight bacteria (macrophosina vaccini), 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 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 bacteria (macrophormonadacini), 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 (macrophorina vaccini), 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 the blueberry branch blight bacteria (macrophorina vaccini);
② agarose gel electrophoresis method, 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 (macrophosina vaccini);
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 the macrophosina vaccini 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 99.2fg/mL, and the method has important significance in the aspects of early warning of blueberry branch blight bacteria (Macrophomina vaccini), 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 macrophostina vaccini, lasiodipia henanica, lasiodipia vaccinii, Botryosphaeria facericiana, Botryosphaeria sinensia, Neofusicoccum algenerensis, 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 Macrophomina vaccini DNA sample.
E, F: visual color development test and agarose gel electrophoresis for specific detection of primer set 2. 1 to 8 are macrophostina vaccini, lasiodipia henanica, lasiodipia vaccinii, Botryosphaeria facericiana, Botryosphaeria sinensia, Neofusicoccum algenerensis, 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 Macrophomina vaccini DNA sample.
I, J: visual color development test for specific detection of primer set 3 and agarose gel electrophoresis. 1 to 8 are macrophostina vaccini, lasiodipia henanica, lasiodipia vaccinii, Botryosphaeria facericiana, Botryosphaeria sinensia, Neofusicoccum algenerensis, 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 Macrophomina vaccini DNA sample.
M, N: sensitivity of primer set 1 repeated experiments of visual color detection and agarose gel electrophoresis. 1 to 8 are respectively dilution times of 101、102、103、104、105、106、107、108A multiplied Macrophomina vaccini DNA sample.
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 detection of blueberry Branch blight fungus (Macrophomina vaccini)
In the case of macrophostina, the translation Elongation Factors (EF) gene has a high rate of variation, and therefore the EF gene was selected for species-level identification and molecular phylogenetic studies.
According to the gene sequence (figure 1) of EF-1 α region of blueberry branch blight bacteria (macrophosmin vaccini), three groups of LAMP primers for detecting the macrophosmin vaccini are respectively designed, and comprise:
primer set 1:
outer forward primer F3: 5'-CGTTGAAGGAGTCGCAGC-3', respectively;
outer reverse primer B3: 5'-TGGTCGTTACCTTCGCTCC-3', respectively;
inner forward primer FIP: 5'-TCGGCTTCAACGTCAAGAACGTGGGGGTCGTTCTTGGAGT-3', respectively;
inner reverse primer BIP: 5'-CGTTGTCACCGGGAACACCCTCACCACCGAGGTCAAGT-3' are provided.
Primer set 2:
outer forward primer F3: 5'-GATGGACTTGCCGGTACG-3', respectively;
outer reverse primer B3: 5'-TCAAGGAGATCCGCCGTG-3', respectively;
inner forward primer FIP: 5'-GGCTACGCTCCTGTCCTGGAGCAGCTCAGAGAACTTGCA-3', respectively;
inner reverse primer BIP: 5'-ACCTGACCGGGGTGGTTGAGAAGAACGACCCCCCCAAG-3' are provided.
Primer set 3:
outer forward primer F3: 5'-TCCAGGACAGGAGCGTAG-3', respectively;
outer reverse primer B3: 5'-GAGATGCACCACGAGCAG-3', respectively;
inner forward primer FIP: 5'-TCCAAGAACGACCCCCCCAAGGGGTGGTTGAGGACGAT-3', respectively;
inner reverse primer BIP: 5'-CCACGGCGGATCTCCTTGACGTGTTCCCGGTGACAACG-3' are provided.
The primer synthesis is completed by Shenzhen Hua Dagen science and technology Limited.
Example 2 LAMP primer detection specificity assay for Macrophomina vaccini
1.1 reagents and devices
The LAMP-PCR kit was purchased from Guangzhou Huafeng company.
1.2 sample sources
Macrophomina vaccini, Lasiodipia henanica, Lasiodipia vaccinii, Botryosphaeria facerciana, Botryosphaeria sinensia, Neofuscum algeneriense, Alternaria alterna, etc. (Table 1) used in this example were stored in national stress laboratories of the 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 IAMP-PCR assays
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 mul of 1000 XSYBR Green I is added into the reaction system obtained by 1.4 for color reaction, and whether the blueberry branch blight bacteria (macrophormonadacini) exists in the sample to be detected 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 Macrophominacci, the reaction system shows fluorescent green, tubes 2-7 are Lacidophilia heniana, Lacidophilia vaccinii, Botryosphaeria faceciana, Botryosphaeria sinensia, Neofusicoccum algeriense, Alternaria, 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 Macrophomina vaccini pathogen, ladder-shaped bands are amplified, Lane 2-7 are Lasiodipia henanica, Lasiodipia vaccinii, Botryosphaeria faricina, Botryosphaeria sinensia, Neofuscoccum algeriense, Alternaria alterna, and other species of the same genus do not generate bands, and NC is 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 sets 1 and 3 have the highest specificity and the best detection effect.
Example 3 sensitivity assay for LAMP primer set detection of Macrophomina vaccini
1.1 DNA sample concentration:
the DNA concentration of the macrophosina vaccini sample extracted in example 2 was measured at 19.7. 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 LAMP primer set 1 isothermal amplification reaction system, and the reaction tubes 1-8 are respectively Macrophomina vaccini from 101、102、103、104、105、106、107、108For the diluted sample, the reaction system of the reaction tube 1-6 shows fluorescent green, and the reaction system of the reaction tube 7-8 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 systems of the reaction tubes 7 and 8 show orange color. 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 FIGS. 2D and 2N, and LAMP detected dilution 106Double 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 39.4 fg/ml.
Comparison of the sensitivity detection results of the primer set (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 Microphomina vaccini-infected diseased tissue Using LAMP primer set 1
1.1 extraction of DNA from diseased tissue of blueberry stem ulcer disease
The test macrophosmin vaccini was transferred to an MEA medium plate, cultured in the dark at 25 ℃ for 2-3 days, and then a colony block (1 cm. times.1 cm) was picked up from the edge of the colony by a punch and needle-inoculated to the stem of a blueberry (four-year-old), and after 7 days of inoculation, the onset effect was as shown in FIG. 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 detection of Macrophomina vaccini pathogen rejoined tissue
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 macrophosina vaccini, the reaction system is 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 bacteria (macrophosina vaccini) is characterized by comprising the following components in parts by weight:
outer forward primer F3: 5'-CGTTGAAGGAGTCGCAGC-3', respectively;
outer reverse primer B3: 5'-TGGTCGTTACCTTCGCTCC-3', respectively;
inner forward primer FIP: 5'-TCGGCTTCAACGTCAAGAACGTGGGGGTCGTTCTTGGAGT-3', respectively;
inner reverse primer BIP: 5'-CGTTGTCACCGGGAACACCCTCACCACCGAGGTCAAGT-3' are provided.
2. A detection reagent or kit comprising the LAMP primer according to claim 1.
3. The blueberry branch blight pathogen (macrophosina vaccini) detection kit 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 blueberry branch blight disease bacteria (macrophosina vaccini).
5. The detection method of blueberry branch blight bacteria (macrophosina vaccini) 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 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 (macrophosina vaccini), 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 the blueberry branch blight bacteria (macrophosina vaccini), 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 the blueberry branch blight bacteria (macrophosina vaccini);
② agarose gel electrophoresis method, 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 (macrophosina vaccini);
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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