A set of loop-mediated isothermal amplification primers and their kits for the identification of three species of Brachybody nematodes on sugarcane

A loop-mediated isothermal, short-bodied nematode technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems affecting sugarcane yield and quality, and achieve good popularization and application value Strong primer specificity and good reproducibility

Active Publication Date: 2019-06-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 to the infestation of the roots of sugarcane by Brachybody zea, Brachybody zea pseudomaize and Brachypoda zea, the yield and quality of sugarcane are seriously affected.

Method used

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  • A set of loop-mediated isothermal amplification primers and their kits for the identification of three species of Brachybody nematodes on sugarcane
  • A set of loop-mediated isothermal amplification primers and their kits for the identification of three species of Brachybody nematodes on sugarcane
  • A set of loop-mediated isothermal amplification primers and their kits for the identification of three species of Brachybody nematodes on sugarcane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Primer Design

[0036] 1. According to the mitochondrial DNA of Brachybody zea zea, Brachybody maize pseudomaize, Brachybody shortest tail and other short-body nematodes in NCBI COI Primers were designed for the sequence, and the primer design was performed using the software PRIMEREXPLORER v.4 software (http: / / primerexplorer.jp).

[0037] (1) The primer set and sequence of the designed B. maize nematode 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'-ACCAGGTAAAAAACCTTAATTCCGGggatccGCTTATTTTAGGGGGGCT-3'

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

[0045] 5'-TATTCGTTTTGGTCCGGTTTTTTTaaaaaaCAAAATAACCCCAGAGCAAC-3'

[0046] (2) The primer set and sequence of the designed Brachybody zea zea is as follows:

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

[0048] 5'-GGWTTTATTGGTTGTATGGTRT...

Embodiment 2

[0064] Embodiment 2 primer reaction condition optimization

[0065] 1. Reaction temperature (primer annealing temperature) optimization

[0066] The annealing temperature of the above primers was optimized by using the DNA of Brachybody zea zea, Brachybody zea pseudonym and Brachybody maximala as templates respectively.

[0067] (1) DNA extraction

[0068] The extraction of the DNA is carried out according to conventional methods in the art. This example is carried out using the method described in Subbotin et al. (2008), specifically as follows: collect nematodes by centrifugation or pick 1 nematode under a stereoscope, then add 16 μL sterilized double-distilled water, 2 μL 10 ×PCR buffer (Mg-free 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 at 95°C for 15 min.

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

[0070] The...

Embodiment 3

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

[0080] 1. In summary, the loop-mediated isothermal amplification method for rapid detection of Brachybody zea zea, Brachybody zea pseudomaize and the shortest-tailed Brachybody worm established by the present invention is as follows:

[0081] The reaction system of loop-mediated isothermal PCR is: 1 μL DNA, 1.6 μM each of inner primer PZFIP / PZBIP or PPFIP / PPBIP or PBFIP / PBBIP, 0.2 μM each of outer primer PZF3 / PZB3 or PPF3 / PPB3 or PBF3 / PBB3, 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, balance ddH 2 O make up to a total of 25 μL.

[0082] The reaction conditions of the three brevis nematode ring-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, not only c...

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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 a kit thereof for identifying three species of short-bodied nematodes on sugarcane. Background technique [0002] Brachybody nematodes, also known as root-rot nematodes, are an important migratory endoparasitic nematode consisting of more than 70 valid species. The short-bodied nematodes move, puncture and feed in the roots of plants, causing the formation of necrotic spots and cavities in the root tissue, and then necrosis of the root tissue. Due to the necrosis of plant roots, plants harmed by short-body nematodes will show symptoms of water shortage and nutritional deficiency. Short-body nematodes are one of the three types of plant nematodes that cause the greatest economic losses. [0003] Brachybody maize nematode, Brachybody pseudomaize and Brachybody pseudomaize can all damage s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
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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