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Detection of Phytophthora cambivora(Petri)Buisman with loop-mediated isothermal amplification (LAMP) technique based on color differentiation

A Phytophthora and blackwater technology, applied in the biological field, can solve the problems of inability to quickly detect Chestnut Phytophthora blackwater bacteria, time-consuming, laborious, poor specificity, etc., and achieve the effect of expanding the scope of use, time-consuming, and high accuracy

Inactive Publication Date: 2017-05-31
中华人民共和国昆山出入境检验检疫局
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0014] The technical problem to be solved by the present invention is to solve the required period of the detection method of Phytophthora chestnutii blackwater pathogen in the prior art, time-consuming and laborious, loaded down with trivial details, poor specificity and PCR detection technology need thermal cycle instrument, cost is high, can't detect chestnut rapidly. The problem of Phytophthora blackwater pathogen, thus providing a new molecular detection method for Chestnut Phytophthora blackwater pathogen, LAMP detection of Chestnut Phytophthora blackwater pathogen, this method has a short detection cycle (only 80min), strong specificity, and sensitivity High, can observe the test results with naked eyes and does not require expensive instruments, low cost, easy to operate

Method used

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  • Detection of Phytophthora cambivora(Petri)Buisman with loop-mediated isothermal amplification (LAMP) technique based on color differentiation
  • Detection of Phytophthora cambivora(Petri)Buisman with loop-mediated isothermal amplification (LAMP) technique based on color differentiation
  • Detection of Phytophthora cambivora(Petri)Buisman with loop-mediated isothermal amplification (LAMP) technique based on color differentiation

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Selecting novel target genes, designing and screening LAMP primers for detecting Chestnut Phytophthora blackwater pathogen, and establishing a LAMP detection system

[0040] (1) Selection of novel target genes and design and screening of primers

[0041] The selection of new target genes and the screening of primers are the key factors for LAMP detection. At present, molecular detection is widely used based on the ribosomal transcriptional spacer (ITS) target, but because the ITS sequence does not have enough variation sites to distinguish all Phytophthora species, it is necessary to discover new molecular detection targets. First, we searched relevant literature and selected available targets such as tRNA (transfer RNA) EF1α (translation elongation factor), Ypt1 (guanosine triphosphate-binding protein gene), and beta tubulin gene. Taking the target Ypt1 as an example, download the Ypt1 gene sequence of Phytophthora chestnutii and the Ypt1 gene sequence of o...

Embodiment 2

[0053] Embodiment 2: Preparation of DNA template

[0054] The DNA extracted from the sample is used as a template for the LAMP reaction. The specific process is as follows:

[0055] (1) Bacterial strain culture and preparation of mycelium powder

[0056]The Phytophthora strains for testing are transferred to LBA (Lima bean medium (Zheng Xiaobo. Phytophthora and its research technology. China Agricultural Press. 1997)) plate, and other bacterial strains are transferred to PDA (Potato Dextrose Solid Medium (Erwin , D.C.; Ribeiro, O.K. Phytophthora lateralis. In: Phytophthora disease worldwide. American Phytopathological Society, St.Paul (US), 1996, pp 365-367)) plate, 25 ℃ dark culture for 3 days and cut 10 pieces from the edge of the colony 2× 2mm hyphae block, the bacterial strain of Phytophthora is transferred to V8 liquid medium (Zheng Xiaobo. Phytophthora and its research technology. China Agricultural Press. 1997), other bacterial strains are transferred to PDB (potato de...

Embodiment 3

[0059] Example 3: Detection of specificity and sensitivity of LAMP primers

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Abstract

The invention discloses a method for molecular detection of Phytophthora cambivora(Petri)Buisman with the loop-mediated isothermal amplification (LAMP) technique based on color differentiation, and primers. The sequences of the primers are shown from SEQ ID NO.1 to SEQ ID NO.6. According to the detection system, under the isothermal condition of 62 DEG C, Phytophthora cambivora(Petri)Buisman can be detected quickly, conveniently and efficiently with high specificity and sensitivity, no complicated instrument is needed, a new technical platform is provided for detection of Phytophthora cambivora(Petri)Buisman, and the requirement for field detection of Phytophthora cambivora(Petri)Buisman can be well met. The detection system is suitable for inspection and quarantine of entry / exit plants and plant products, and investigation, rapid diagnosis and monitoring of diseases, and is of great importance in preventing Phytophthora cambivora(Petri)Buisman from entering China. Meanwhile, the establishment of the system provides technical guidance and theoretical basis for detection of other pathogenic bacteria.

Description

technical field [0001] The invention relates to a LAMP primer for detecting Phytophthora lateralis Tucker et Mibrath and a molecular detection method for detecting Phytophthora lateralis Tucker et Mibrath, belonging to the field of biotechnology. Background technique [0002] Phytophthora chestnut blackwater fungus is an important destructive plant pathogen that can cause severe chestnut blackwater disease and root and stem rot of various fruit trees [1] . Due to the serious damage and rapid spread of the disease, and the extremely difficult control and eradication, the pathogen has attracted great attention from many countries such as the European Union, and measures have been taken to prevent its introduction. In recent years, due to the increasingly frequent transfer of international seedlings and plant packaging materials, the pathogen is likely to be introduced into my country. Once introduced, it will pose a major threat to my country's agriculture, forestry, and ecol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 纪睿曾丹丹廖太林郑小波张正光周锐吴军
Owner 中华人民共和国昆山出入境检验检疫局
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