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Loop-mediated isothermal amplification primer for rapidly detecting rotylenchulus reniformis and application of loop-mediated isothermal amplification primer

A ring-mediated isothermal, reniform nematode technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. The effect of detecting a wide range of species, ensuring safety and low cost

Inactive Publication Date: 2019-03-08
广东生态工程职业学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the metagenome contains the genes of a variety of microorganisms in the environment, it is necessary to manually count the number of reniform reniform nematodes and other nematodes, and the efficiency is low
[0005] The above methods are not conducive to the promotion and use in grassroots testing units, therefore, a simple, fast and accurate method for identifying reniform nematodes is needed

Method used

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  • Loop-mediated isothermal amplification primer for rapidly detecting rotylenchulus reniformis and application of loop-mediated isothermal amplification primer
  • Loop-mediated isothermal amplification primer for rapidly detecting rotylenchulus reniformis and application of loop-mediated isothermal amplification primer
  • Loop-mediated isothermal amplification primer for rapidly detecting rotylenchulus reniformis and application of loop-mediated isothermal amplification primer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The design of embodiment 1 primer

[0034] Primers were designed according to the 28S rDNA sequence (28SrDNA-D2D3 region) of reniform reniform nematode in NCBI. Multiple sets of primer sets were designed, and after preliminary screening, the following five sets of primers were obtained: primer pair RrF3 / RrB3 and primer pair RrFIP / RrBIP, primer pair 1-F3 / 1-B3 and primer pair 1-FIP / 1-BIP, primer pair Pair 4-F3 / 4-RrB3 and primer pair 4-RrFIP / 4-RrBIP, primer pair 17-F3 / 17-B3 and primer pair 17-FIP / 17-BIP, primer pair 22-F3 / 22-B3 and primer There are 5 sets of primers for 22-FIP / 22-BIP.

[0035] The sequences of the five primer sets are as follows:

[0036] Primer RrF3 (as shown in SEQ ID NO.1):

[0037] GCGCAATGAAAGTGAAGGTC.

[0038] Primer RrB3 (as shown in SEQ ID NO.2):

[0039] CGGACTTCCACCAGAGTTTC.

[0040] Primer RrFIP (as shown in SEQ ID NO.3):

[0041] GGACGGGACCATGTTGCTCCggatccCTTGTGGAGCTGATGTGTGA.

[0042] Primer RrBIP (as shown in SEQ ID NO.4):

[0043] G...

Embodiment 2

[0077] Embodiment 2 primer reaction condition optimization

[0078] 1. Reaction temperature (primer temperature) optimization

[0079] The annealing temperature of the above primers was optimized using reniform nematode DNA as a template.

[0080] (1) DNA extraction

[0081] The extraction of the DNA is carried out 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 sterilized double-distilled water, 2 μL 10×PCRbuffer ( No Mg 2+ ) (purchased from Takara), 2 μL proteinase K (600mAnson U / mL) (purchased from Takara) in the lysate, then cut the nematodes with a needle, and then put the mixture at 65°C for 1h and 95°C for 15min.

[0082] (2) Loop-mediated isothermal PCR reaction

[0083] The PCR reaction system is: 1 μL DNA, each 1.6 μM outer primer pair (such as primer RrFIP / RrBIP)...

Embodiment 3

[0094] Visual detection and detection sensitivity of embodiment 3LAMP reaction

[0095] 1. In summary, the loop-mediated isothermal amplification method for the rapid detection of reniform reniform nematode established by the present invention is as follows:

[0096] The PCR reaction system is: 1 μL DNA, primers RrFIP / RrBIP each 1.6 μM, primers RrF3 / RrB3 each 0.2 μM, dNTP 0.35 μM, 2.5 μL 10×BST2.0 DNA polymerase buffer (BST2.0 DNA polymerase buffer), 0.8M betaine, 1.5 μL MgSO 4 (100mM), 1μL BST2.0DNA polymerase (BST2.0DNApolymerase), the balance ddH 2 O make up, a total of 25 μL.

[0097] Reaction conditions: react at 66°C for 54 minutes.

[0098] 2. Carry out LAMP reaction according to the above reaction system and optimized reaction conditions. After the reaction, it can not only be detected by agarose gel electrophoresis, but also can be detected visually by adding SYBR Green I dye or calcein. The result judgment method is more accurate. Flexible and more convenient.

...

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Abstract

The invention discloses a loop-mediated isothermal amplification primer for rapidly detecting rotylenchulus reniformis and an application of the loop-mediated isothermal amplification primer. A loop-mediated isothermal amplification primer group is designed with a 28S rDNA-D2D3 region as a target sequence and comprises a primer pair RrF3 / RrB3 and a primer pair RrFIP / RrBIP; the primer sequences areshown in SEQ ID NO. 1-4; a loop-mediated isothermal amplification detection method is further established based on the primer group. The detection method has high specificity, high sensitivity, goodrepeatability and wide detection population range for rotylenchulus reniformis, has low requirements for instruments and detection personnel, has visual reaction result, is accurate, reliable, safe, simple, rapid and low in cost, provides technical support for detection of rotylenchulus reniformis, especially inspection and quarantine work, is very suitable for rapid detection at the grass-roots level, and has quite good popularization and application value.

Description

technical field [0001] The invention belongs to the technical field of plant pathogen detection. More specifically, it relates to a loop-mediated isothermal amplification primer for rapid detection of reniform reniform nematode and its application. Background technique [0002] Rotylenchulus reniformis is the model species of the genus Reniformis, and it is also an economically important agricultural nematode in the genus Rotylenchulus, which seriously harms agricultural production. It mainly occurs in tropical, subtropical and warm and humid areas. It was first found in the roots of soybeans. Today, its host range is widely distributed all over the world. More than 300 species of its hosts have been found in various vegetables such as Cucurbitaceae, Solanaceae and Legumes. crops, as well as ornamental plants such as Asteraceae, Palmaceae and Kapokceae, and various other economic crops. my country has successively found the distribution of reniform reniform nematodes in Gu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2531/119C12Q2565/125C12Q2563/107
Inventor 廖金铃孙丹丹林柏荣王宏洪卓侃
Owner 广东生态工程职业学院
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