Loop-mediated isothermal amplification primer set for identifying three kinds of pratylenchus pratensis on sugarcane and kit comprising loop-mediated isothermal amplification primer set

A technology of ring-mediated isothermal and short-bodied nematodes, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems affecting the yield and quality of sugarcane, and achieve good promotion and application value. Realize the effect of visualization and guarantee safety

Active Publication Date: 2017-02-15
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are no reports at home and abroad on the identification and detection of short-bodied nematodes by the loop-mediated isothermal amplification method
Due t

Method used

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  • Loop-mediated isothermal amplification primer set for identifying three kinds of pratylenchus pratensis on sugarcane and kit comprising loop-mediated isothermal amplification primer set
  • Loop-mediated isothermal amplification primer set for identifying three kinds of pratylenchus pratensis on sugarcane and kit comprising loop-mediated isothermal amplification primer set
  • Loop-mediated isothermal amplification primer set for identifying three kinds of pratylenchus pratensis on sugarcane and kit comprising loop-mediated isothermal amplification primer set

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Experimental program
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Example Embodiment

[0035] Example 1 Primer design

[0036] 1. According to the mitochondrial DNA of Brachymeria maize, Brachyura maize, Brachyura shortesti, and other Brachymycidae in NCBI COI Sequence design primers were designed using PRIMEREXPLORER v.4 software (http: / / primerexplorer.jp).

[0037] (1) The designed primer set and sequence of Brachymeria maize are as follows:

[0038] PZF3 (as shown in SEQ ID NO.1):

[0039] 5’-GGTTTAGATCTTGATTCTCGG-3’

[0040] PZB3 (as shown in SEQ ID NO.2):

[0041] 5’-GTAAAAATAAATCCAAAGTGGCA-3’

[0042] PZFIP (as shown in SEQ ID NO.3):

[0043] 5’-ACCAGGTAAAAACCTTAATTCCGGggatccGCTTATTTTAGGGGGGCT-3’

[0044] PZBIP (as shown in SEQ ID NO.4):

[0045] 5’-TATTCGTTTTGGTCCGGTTTTTTTaaaaaaCAAAATAACCCCAGAGCAAC-3’

[0046] (2) The designed primer set and sequence of P. maize nematode are as follows:

[0047] PPF3 (as shown in SEQ ID NO.5):

[0048] 5’-GGWTTTATTGGTTGTATGGTRTG-3’

[0049] PPB3 (as shown in SEQ ID NO.6):

[0050] 5’-GAGAAACCCCCCACAGTA-3’

[0051] PPFIP (as shown in SEQ ID...

Example Embodiment

[0064] Example 2 Optimization of primer reaction conditions

[0065] 1. Optimization of reaction temperature (primer annealing temperature)

[0066] The DNA of Brachymeria maize, Brachyura maize and Brachyura shortesta were used as templates to optimize the annealing temperature of the primers.

[0067] (1) Extract DNA

[0068] The DNA extraction is performed according to conventional methods in the art. This example is carried out using the method described by Subbotin et al. (2008), specifically as follows: collect nematodes by centrifugation or pick one nematode under a stereoscope, and then add 16 μL of sterilized double-distilled water, 2 μL 10 ×PCR buffer (without Mg 2+ ) (Purchased from Takara) and 2 μL of proteinase K (600 mAnson U / mL) (purchased from Takara), followed by cutting the nematodes with a needle, and then placing the mixture at 65°C for 1 h and 95°C for 15 min.

[0069] (2) Loop-mediated isothermal PCR reaction

[0070] The loop-mediated isothermal PCR reaction sy...

Example Embodiment

[0079] Example 3 LAMP Visual detection and sensitivity detection of reaction

[0080] 1. To sum up, the loop-mediated isothermal amplification methods established by the present invention for rapid detection of Brachymia maize, Brachyura maize and Brachyura shortesti, respectively, are as follows:

[0081] The loop-mediated isothermal PCR reaction system is: 1 μL DNA, inner primer PZFIP / PZBIP or PPFIP / PPBIP or PBFIP / PBBIP each 1.6 μM, outer primer PZF3 / PZB3 or PPF3 / PPB3 or PBF3 / PBB3 each 0.2 μM, 3.5 μL dNTPS (10 mM), 2.5 μL 10× BST 2.0 DNA polymerase buffer, 4 μL betaine (5 M), 1.5 μL MgSO 4 (100 mM), 1 μL BST 2.0 DNA polymerase, margin ddH 2 O make up, a total of 25 μL.

[0082] The reaction conditions of the three kinds of short-body nematode loop-mediated isothermal amplification reactions are: 64°C for 60 min.

[0083] 2. Carry out the LAMP reaction according to the above reaction system and optimized reaction conditions. After the reaction, it can be detected not only ...

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Abstract

The invention discloses a loop-mediated isothermal amplification primer set for identifying three kinds of pratylenchus pratensis on sugarcane and a kit comprising the loop-mediated isothermal amplification primer set. The loop-mediated isothermal amplification primer set comprises three groups of primers, namely, an outer primer pair PZF3/PZB3 and an inner primer pair PZFIP/PZBIP, an outer primer pair PPF3/PPB3 and an inner primer pair PPFIP/PPBIP, as well as an outer primer pair PBF3/PBB3 and an inner primer pair PBFIP/PBBIP, wherein the nucleotide sequences of the primers are respectively shown as SEQ ID NO.1-12. The three kinds of pratylenchus pratensis are pratylenchus zeae, P. parazeae and P. brachyurus. The loop-mediated isothermal amplification primer set and the kit comprising the loop-mediated isothermal amplification primer set have low requirements on instrument and equipment, are simple, rapid and safe, and are good in specificity and high in sensitivity, and a technical support is provided for the detection for pratylenchus zeae, P. parazeae and P. brachyurus, especially, the rapid identification and distinguishing for the three kinds of pratylenchus pratensis of the grassroots units.

Description

Technical field [0001] The invention belongs to the technical field of plant pathogen detection. More specifically, it relates to a set of loop-mediated isothermal amplification primers and kits for identifying three kinds of Brachyteoma on sugarcane. Background technique [0002] Brachytematodes, also known as root rot nematodes, are an important migratory endoparasitic nematode, which consists of more than 70 effective species. The movement, puncture and feeding of the worms in the plant roots cause the root tissues to form necrotic spots and cavities, and then necrosis of the root tissues. Due to the necrosis of plant roots, plants harmed by Brachymeria nematodes will show symptoms of water shortage and nutrient insufficiency, which is one of the three types of plant nematodes that cause the greatest economic loss. [0003] The corn worms, the corn worms, and the shortest brambles can all harm sugarcane, and they will infect the sugarcane. In addition, these three kinds of bra...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2600/16C12Q2537/143C12Q2531/119C12Q2563/107
Inventor 卓侃刘星彤林柏荣王宏洪廖金铃
Owner SOUTH CHINA AGRI UNIV
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