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LAMP primer for detecting peach venturia and detection kit

A kit and the technology of black star bacteria, applied in the direction of microorganism-based methods, microorganisms, recombinant DNA technology, etc., can solve the problems of long detection cycle, heavy workload, high technical requirements, etc., and achieve the elimination of instrument investment, high sensitivity and specificity The effect of high sex and sensitivity

Active Publication Date: 2019-04-19
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to overcome the shortcomings of the existing methods, such as long detection period, heavy workload, need for aseptic conditions, and high technical requirements for instruments and personnel in PCR technology, and provide LAMP primers and detection kits for detecting P. And on this basis, a convenient, sensitive, and intuitive result judgment method based on the molecular level is established, which is easy to promote and use at the grassroots level.

Method used

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  • LAMP primer for detecting peach venturia and detection kit
  • LAMP primer for detecting peach venturia and detection kit
  • LAMP primer for detecting peach venturia and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 is used to detect the design and synthesis of the LAMP primers of Pyreum spp.

[0043] Alignment of the ITS1 / ITS4 sequences of Myovicia and its close relatives (S. apples, S. pears) (primers used to amplify the ITS1 / ITS4 sequences of Myovicia ITS1:5'-TCCGTAGGTGAACCTGCGG -3' and ITS4:5'-TCCTCCGCTTATTGATATGC-3'), the results showed that the site 147-163 of the amplified fragment of P. peach smut ITS1 / ITS4 was a specific recognition site different from other pathogenic bacteria ( Figure 9 ). On this basis, 10 groups of LAMP primers (Table 1) were designed, and the results showed that only when the 8th group of primers (SEQ ID NO:1-4) was used to amplify the DNA of Pythum nigrum, a ladder-like band could be produced ( Figure 11 ), in order to realize the detection of Pyreum spp. For the design site of group 8 LAMP primers, see Figure 10 .

[0044] LAMP primers for detection of Peach nigella, including (SEQ ID NO:1-4):

[0045] Outer forward primer VC-F3: 5'-...

Embodiment 2

[0055] Embodiment 2 Utilizes LAMP primer to detect the specificity analysis of peach scurvy

[0056] 1. Reagents and equipment

[0057] Water bath, pipette, etc.

[0058] 2. Sample

[0059] The plant pathogens used in this embodiment include peach scab (Venturia carpophila), apple scab (Venturia inaequalis), pear scab (Venturia nashicola), peach canker fungus (Phomopsisamygdali), peach gum disease fungus (Botryosphaeria dothidea) , Alternaria tenuissima, Colletotrichum acutatum, Xanthomonascampetris.

[0060] 3. DNA extraction

[0061] Pick a small amount of mycelium from the lesion and put it in 50 μL of 10×TE lysate (100 mM Tris-HCL, 10 mM EDTA, pH 8.0), boil it for 2 minutes.

[0062] 4. LAMP reaction and detection

[0063] LAMP reaction system (25μl): 4U Bst DNA polymerase, 2.5μL 10×ThermoPol Buffer, 5mM MgSO 4 , 0.8mM dNTPs, 1.2mM VC-FIP, 1.2mM VC-BIP, 0.4mM VC-F3, 0.4mM VC-B3, 0.8M betaine, 1 μL DNA template (the lysate of the treated hyphae above).

[0064] React...

Embodiment 3

[0069] The optimization of embodiment 3LAMP reaction system and reaction conditions

[0070] 1. Mg 2+ Concentration optimization

[0071] Adopt the reaction system and reaction condition of embodiment 2, only change Mg 2+ concentration.

[0072] For the color development of the amplified product after adding SYBR Green I dye, see figure 2 . Among them, 1-8 corresponds to Mg 2+ The final concentrations were 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, respectively. The results showed that 5mM Mg 2+ The corresponding color rendering effect is the best.

[0073] 2. Optimization of dNTPs concentration

[0074] Using the reaction system and reaction conditions of Example 2, only the concentration of dNTPs was changed.

[0075] For the color development of the amplified product after adding SYBR Green I dye, see image 3 . Among them, the final concentrations of dNTPs corresponding to 1-8 are 0.4mM, 0.8mM, 1.0mM, 1.2mM, 1.4mM, 1.6mM, 2.0mM, 2.4mM, respectively. The results s...

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PUM

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Abstract

The invention provides an LAMP primer for detecting peach venturia and a detection kit. The sequence of the LAMP primer is shown as Seq ID No.1-4. According to the LAMP primer and the detection kit, aloop-mediated isothermal amplification technology is adopted to rapidly detect the peach venturia and can accurately detect the peach venturia from complex pathogenic bacterium environments such as pathogenic plant tissue. The LAMP primer can detect propagules in various forms, such as hyphae and spores, of the peach venturia, has great significance on the aspects such as early warning of epidemic situation and pathogenic surveillance of affected areas of the peach venturia, can also avoid high investment in instruments and is convenient to apply and popularize in grass-roots units.

Description

technical field [0001] The invention belongs to the technical field of detection of plant pathogenic bacteria, and in particular relates to a LAMP primer and a detection kit for detection of Myocarpus spp. Background technique [0002] Peach scab caused by Venturia carpophila (Venturia carpophila, also known as Carpophila, belonging to Ascomycota subphylum Sclerotia fungus) is one of the important diseases in peach producing areas, the occurrence of peach scab Seriously affected the quality and value of peaches. The disease produces dark brown to black dots on the fruit surface. Symptoms are easily confused with peach punch, black spot, canker and early anthracnose. It is difficult for grassroots agricultural technology extension personnel and fruit farmers to distinguish, and cases of disease diagnosis errors often occur; The incubation period of peach scurvy on the host is long, and it is difficult to isolate, grow slowly, and easily contaminate in laboratory pure culture...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12Q1/6844C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 罗朝喜周扬范飞尹良芬阴伟晓王丽罗梅谭钦
Owner HUAZHONG AGRI UNIV
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