CN111041119B - LAMP primer and kit for detecting Neofusicoccum macroclavatum - Google Patents

LAMP primer and kit for detecting Neofusicoccum macroclavatum Download PDF

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CN111041119B
CN111041119B CN202010013015.7A CN202010013015A CN111041119B CN 111041119 B CN111041119 B CN 111041119B CN 202010013015 A CN202010013015 A CN 202010013015A CN 111041119 B CN111041119 B CN 111041119B
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primer
lamp
neofusicoccum
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macroclavatum
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CN111041119A (en
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张英
王宇
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Beijing Forestry University
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6844—Nucleic acid amplification reactions
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Abstract

The invention provides a set of LAMP primers and a kit for detecting bacterial wilt of blueberries (Neofusicoccum macrocephalum). 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

LAMP primer and kit for detecting Neofusicoccum macroclavatum
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to an LAMP primer and a kit for detecting Neofusicoccum macroclavatum.
Background
Blueberry Stem canker (blue Stem canker) caused by infection of botryosphaeriaceae fungi (Botryosphaeiceae) is reported in main Blueberry producing areas in China at present, and brings serious damage to the health development of the Blueberry industry in China. The strain is infected from wounds or natural orifices of blueberry plants, so that cortex and phloem wither in fields to cause blueberry branch withering, xylem necrosis, plant death and other symptoms. Currently, there are many diseases that can be caused in the family gluconoraceae (Botryosphaeria cortices, Botryosphaeria dothidea, fusacoccus aesculin, lasiodipa chinensis, lasiodipa paraphyoides, lasiodipia theobroma, lasiodipia vaccinii, neousage coccus arbuti, neousage coccus armatrial, neousage maculosum mactivum, neousage coccus ribis), wherein a pathogen forms a reddish brown lesion on the surface of a blueberry shoot, causing vascular death and blast death, which is a disease causing bacterial growth mainly in the area of the stem, cross-wound, lateral invasion of the plant through the meristematic cortex, lateral opening of the stem, transverse wound, or other areas, and finally develops through various types of vascular ducts, or other tissue after entering the tissue in the field. The bacterium has the characteristic of latent infection, usually exists in soil as saprophytic bacteria or plant endophytes 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 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 implemented when the disease is outbreak and even generates obvious harm, comprehensive prevention and control and high-efficiency treatment measures are not taken for the early stage of the primary infection source in time, so that the work is doubled, the prevention effect is very little, 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 disease germ (Neofusicoccum macroclavatum) 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'-GCTGCGACTCCTTCAACG-3', respectively;
outer reverse primer B3: 5'-GGCGTCACCAGACTTGATG-3', respectively;
inner forward primer FIP (F1C + F2): 5'-CAGTCCAGGACGGGAGCGTACCCAGGTCATCGTCCTCA-3', respectively;
inside reverse primer BIP (B1C + B2): 5'-GCTTGCAAGTTCTCTGAGCTGCCTTGGGGCTGTTCTCAATGG-3' are provided.
In a second aspect, the present invention provides a polypeptide comprising SEQ ID NO: 1-4, or a kit for detecting a LAMP primer.
In a third aspect, the present invention provides a blueberry branch blight pathogen (Neofusicoccum macroclavatum) 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 the application of the LAMP primer, the detection reagent containing the LAMP primer or the kit in detecting 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:
Figure BDA0002357847060000021
Figure BDA0002357847060000031
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:
Figure BDA0002357847060000032
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) can adopt any one of the following methods to judge the amplification result:
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 (orange) to Green, indicating that the sample to be detected contains blueberry branch blight pathogen (Neofusicoccum macroclavatum); or Calcein (Calcein) is added into the reaction system before the amplification reaction, and after the amplification reaction is finished, the reaction system shows fluorescent green under the irradiation of an ultraviolet lamp, which indicates that the sample to be detected contains blueberry branch blight bacteria (Neofusicoccum macroclavatum); or, Hydroxyl Naphthol Blue (HNB) is added 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, the blueberry branch blight bacteria (Neofusicoccum macroclavatum) are contained in the sample to be detected;
② agarose gel electrophoresis method: if the amplification product presents a characteristic ladder-shaped strip on the agarose gel, the blueberry branch blight germ (Neofusicoccum macroclavatum) is indicated to be 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 (or generating milky white precipitate) after reaction through naked eyes, or the absorbance of the LAMP amplification reaction at 400nm is detected by using a nephelometer, so that real-time quantitative detection is realized.
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 method has higher specificity, sensitivity and repeatability than 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 macrocephalavatum), 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. alpha. region of fungi 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 detection and agarose gel electrophoresis for specific detection of primer set 1.1 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 color detection 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 detection and agarose gel electrophoresis for specific detection of primer set 3. 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 illicii, Neofusicoccum sinense, Botryosphaeria sinensia, Botryosphaeria rosaceae, Lasiodipia chinensis, Alternaria alternata, and NC (negative control), respectively.
Wherein M in the agarose gel electrophoresis picture is DL 2000DNA Marker.
FIG. 3 is a detection experiment of the LAMP primer group 1 of example 4 of the invention on diseased plants in the field. Wherein, A: disease spots of diseased plants; 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 bacterial wilt of blueberry (Neofusicoccum macrocephalavatum)
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 alpha region of blueberry branch blight bacteria (Neofusicoccum macrocephalavatum), three groups of LAMP primers for detecting the Neofusicoccum macrocephalavatum 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';
inner forward primer FIP: 5'-CAGTCCAGGACGGGAGCGTACCCAGGTCATCGTCCT CA-3', respectively;
inner forward primer BIP: 5'-GCTTGCAAGTTCTCTGAGCTGCCTTGGGGCTGTTCT CAATGG-3' is added.
Primer set 2:
outer forward primer F3: 5'-AAAGTTTTTCCTTCCGCTGC-3', respectively;
outer reverse primer B3: 5'-TGGGACGGCCGTTAGC-3', respectively;
inner forward primer FIP: 5'-CGACCCCACCACCAAAATGCCTGGGTTCCCGCACTC A-3', respectively;
inner forward primer BIP: 5'-GGCTCGGCAAAATCTCCGCATCCATGCGTGGCGTGT CTG-3' are provided.
Primer set 3:
outer forward primer F3: 5'-TCCTGGACTGCCACACTG-3', respectively;
outer reverse primer B3: 5'-GAGGGTACTCGGTGAAAGC-3';
inner forward primer FIP: 5'-TCTCAATGGACTTGCCGGTACGTGCTTGCAAGTTCT CTGAGC-3', respectively;
inner forward primer BIP: 5'-TTCATCAAGTCTGGTGACGCCGTCAACGCACATGGG CTTG-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. alpha. region of fungi involved in Table 1 are shown in FIG. 1.
TABLE 1 sample sources for LAMP-PCR detection
Figure BDA0002357847060000061
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):
Figure BDA0002357847060000071
1.5 color reaction
After the reaction is finished, 6 μ l of 1000 × SYBR 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 (Neofusicoccum macroclavitum) is judged according to the change of the color of the reaction system.
1.6 results
FIG. 2A shows the visual observation effect of LAMP primer set 1 in the isothermal amplification reaction system, tube 1 is Neofusicoccum macroclavatum, the reaction system shows green fluorescence, tubes 2 to 7 are Neofusicoccum illinii, Neofusicoccum sinense, Botryosphaeria sinensia, Botryosphaeria rosae, Lasiodipidia chinensis, Alternaria alternata, 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 result of isothermal amplification of the LAMP primer group 1 according to 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 of Staphylocodiomycetaceae do not generate a band, 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 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 Neofusicoccum macrocaulatum sample extracted in example 2 was measured at 10.4. mu.g/ml with a NanoDrop (Seimerfell science Co., Ltd.).
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 diluted DNA sample. The reaction system and reaction conditions were the same as in example 2.
1.3 results:
FIG. 2C shows the macroscopic effect of the LAMP primer set 1 isothermal amplification reaction system, and the reaction tubes 1-8 are respectively Neofusicoccum 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. Drawing (A)2M is a repeated experiment of visual observation effect of the LAMP primer group 1 constant-temperature amplification reaction system, 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 primer set 2 (FIGS. 2G and 2H) and primer set 3 (FIGS. 2K and 2L) shows that primer set 1 has the highest sensitivity and the best detection effect.
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 punch is used for taking a colony block (1cm multiplied by 1cm) from the edge of the colony, the colony block is pricked and inoculated to the stem of blueberry (four-year-old), and after inoculation for 7 days, the disease onset effect is shown in figure 3A. Then, the diseased tissue was excised, and DNA of the diseased tissue was extracted using a CTAB plant genome DNA rapid extraction kit (beijing edlele biotechnology limited): taking a section of appropriate diseased stem, freezing by liquid nitrogen, fully grinding into powder, and extracting the DNA of diseased spots and tissues at the junction of diseased spots and diseased keys 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. An LAMP primer group for detecting Neofusicoccum macroclavatum of blueberry branch blight bacteria is characterized in that the LAMP primer group is as follows:
outer forward primer F3: 5'-GCTGCGACTCCTTCAACG-3';
outer reverse primer B3: 5'-GGCGTCACCAGACTTGATG-3', respectively;
inner forward primer FIP: 5'-CAGTCCAGGACGGGAGCGTACCCAGGTCATCGTCCTCA-3', respectively;
inner reverse primer BIP: 5'-GCTTGCAAGTTCTCTGAGCTGCCTTGGGGCTGTTCTCAATGG-3' are provided.
2. A detection reagent or kit comprising the LAMP primer set according to claim 1.
3. The detection kit for detecting Neofuscoccum macroclavatum of blueberry branch blight bacteria, which 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 Neofusiccum macroclavatum of blueberry Sclerotinia sclerotiorum.
5. The detection method of the blueberry branch blight bacterium Neofusicoccum macroclavatum is characterized by comprising the following steps:
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 judging the amplification result.
6. The method as claimed in claim 5, wherein the reaction system used in step 2) is:
template DNA 2. mu.l
10mM dNTPs 1.25μl
20 mu M primer FIP 0.2-0.8 mu l
20 mu M primer BIP 0.2-0.8 mu l
10 mu M of primer F30.05-0.2 mu l
10 mu M of primer B30.05-0.2 mu l
8U/. mu.L Bst DNA polymerase 1. mu.l
10 × reaction buffer 12.5 μ l
ddH2O is added 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 (r) - (c):
a fluorescent staining method: adding a 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 the blueberry branch blight bacterium Neofusicoccum macrocephalavatum; 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 bacterial wilt of blueberry Neofusicoccum macrocephalavatum; 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 the blueberry branch blight bacterium Neofusicoccum macrocarpavatum;
② agarose gel electrophoresis method: if the amplification product presents a characteristic ladder-shaped strip on the agarose gel, the blueberry branch blight bacterium Neofusicoccum macrocyllavatum 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.
CN202010013015.7A 2020-01-07 2020-01-07 LAMP primer and kit for detecting Neofusicoccum macroclavatum Expired - Fee Related CN111041119B (en)

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