Loop isothermal amplification primer for quickly detecting tylenchulus semipenetrans and application of loop isothermal amplification primer

A ring isothermal amplification, citrus technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Use and other issues to achieve good detection effect, good sensitivity, and visualization.

Active Publication Date: 2015-07-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, at present, semi-piercing nematodes are mainly identified through morphological identification, but morphological identification requires careful observation of the shape of nematodes and measurement of the size of different structures of nematodes. This requires the identification personnel to have many years of experience in nematode identification, and the professional requirements are very high.
Liu Guokun first developed a method to identify citrus hemipuncture nematodes by PCR method; however, this method requires the use of expensive PCR equipment, and also requires electrophoresis detection, which requires high technical requirements for operators and is also not conducive to the grassroots. Promotion and use in testing units

Method used

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  • Loop isothermal amplification primer for quickly detecting tylenchulus semipenetrans and application of loop isothermal amplification primer
  • Loop isothermal amplification primer for quickly detecting tylenchulus semipenetrans and application of loop isothermal amplification primer
  • Loop isothermal amplification primer for quickly detecting tylenchulus semipenetrans and application of loop isothermal amplification primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Design of primers

[0032] Primers were designed according to the ITS DNA sequence of the citrus puncture nematode in NCBI (gene accession numbers: FJ969705, GU433381 JN112270, JN112269, GU433403, GU433405), and the primer design was carried out using the software PRIMEREXPLORER v.4 software (http: / / primerexplorer.jp) .

[0033] The designed primers and their sequences are as follows:

[0034] TSF3 (as shown in SEQ ID NO.1):

[0035] GCATCTGGCGAGTCTGTG.

[0036] TSB3 (as shown in SEQ ID NO.2):

[0037] GCACCGAATCTGGAACTCAT.

[0038] TSFIP (as shown in SEQ ID NO.3):

[0039] CRGGTAAGAGCCGAgaAGGACAggatccGTCATACTTCCTCTgcCGCT.

[0040] TSBIP (as shown in SEQ ID NO.4):

[0041] TGTAACGCTGAGCgaCTGTTGAttttttGCGACATGTGGAGAAGGC.

Embodiment 2

[0042] Example 2 Optimization of primer reaction conditions

[0043] 1. Reaction temperature (primer annealing temperature) optimization

[0044] The annealing temperature of the above primers was optimized using Nematode citrus hemipuncture DNA as a template.

[0045] (1) DNA extraction

[0046] 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), then the nematodes were cut with a needle, and then the mixture was placed at 65°C for 1h and 95°C for 15min.

[0047] (2) PCR reaction

[0048] PCR reaction system: 1 μL DNA, 1.6 μM primers TSFIP / TSBIP, 0.2 μM primers TSF3 / TSB3, 0.35 μM dNTP...

Embodiment 3

[0057] Visual detection and detection sensitivity of embodiment 3LAMP reaction

[0058] 1. In summary, the ring isothermal amplification method for the rapid detection of citrus hemipuncture nematode established by the present invention is as follows:

[0059] PCR reaction system: 1 μL DNA, 1.6 μM primers TSFIP / TSBIP, 0.2 μM primers TSF3 / TSB3, 0.35 μM dNTP, 2.5 μL 10× BST 2.0 DNA polymerase buffer ( BST 2.0 DNA polymerase buffer), 0.8M betaine, 1.5 μL MgSO 4 (100 mM), 1 μL BST 2.0 DNA polymerase ( BST 2.0 DNA polymerase), balance ddH 2 O make up to a total of 25 μL.

[0060] Reaction conditions: react at 65°C for 60 minutes.

[0061] 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.

[006...

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Abstract

The invention discloses a loop isothermal amplification primer for quickly detecting tylenchulus semipenetrans and application of the loop isothermal amplification primer. The loop isothermal amplification primer comprises a primer pair TSF3 / TSB3 and a primer pair TSFIP / TSBIP, and the sequences of the primer pairs are shown as SEQ ID NO. 1 to 4 respectively. A loop isothermal amplification method is established through the loop isothermal amplification primer, loop isothermal amplification is performed through taking a sample DNA as a template, and results can be judged in two ways after the end of reaction, wherein the first way is that an amplification primer is subjected to electrophoresis, and a sample of a specific ladder-shaped strip, which occurs, is judged to be positive; the second way is that through adding SYBR green I into a reaction system, a sample of which the color is changed is observed with naked eyes to be positive. The loop isothermal amplification method is low in requirement for instruments and equipment, quick, safe, and high in specificity and sensitivity; a technical support is provided for the detection of the tylenchulus semipenetrans, in particular to the quick detection work of a grass-root quarantine unit for the tylenchulus semipenetrans, and the popularization and application values are very high.

Description

technical field [0001] The invention belongs to the technical field of plant pathogen detection. More specifically, it relates to a circular isothermal amplification primer for rapid detection of citrus hemipuncture nematode and its application. Background technique [0002] There are many kinds of plant-parasitic nematodes in the root circle of citrus, but only a few of them can affect the yield of citrus. Among them, citrus semi-piercing nematode is the most harmful nematode, which causes 10-30% loss of citrus yield every year. Citrus semipiercing nematodes ( Tylenchulus semipenetrans ) is widely distributed and is currently found in all major citrus growing regions of the world. In the United States, up to 60 to 90 percent of citrus orchards are infested with this nematode. In China, this kind of nematode damage is also very common. For example, the incidence rate of citrus semi-puncture nematode in citrus orchards in Sichuan Province reached 87.14%, and the incidence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/119
Inventor 林柏荣廖金铃卓侃王宏洪
Owner SOUTH CHINA AGRI UNIV
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