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Quick molecular detection kit for colletotrichum kahawae and application thereof

An anthracnose bacteria and kit technology, which is used in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effects of simple operation, strong specificity and high sensitivity

Inactive Publication Date: 2015-01-14
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, based on the limitations of key technologies, especially the design of primers and the technical problems of detection reagents related to primers, there is no relevant technical report on the application of LAMP to detect the pathogenic fungus of the genus Neptonia

Method used

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  • Quick molecular detection kit for colletotrichum kahawae and application thereof
  • Quick molecular detection kit for colletotrichum kahawae and application thereof
  • Quick molecular detection kit for colletotrichum kahawae and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, LAMP reaction plug detects coffee berry anthracnose bacteria (Colletotrichum kahawae)

[0045] 1. Preparation of loop-mediated isothermal amplification primer set for detection of coffee berry anthracnose (C. kahawae)

[0046] In this example, the loop-mediated isothermal amplification primer set for detecting coffee berry anthracnose (C. kahawae) consists of primer 1, primer 2, primer 3 and primer 4. These four primers were prepared according to the following method: According to the Apn2 / Mat gene sequence, use ClustalX 1.81 and BioEdit 7.0 software to compare and analyze these gene sequences, find polymorphic rich regions, use the online software Primers-Primer Explore (Fujitsu Ltd., Tokyo, Japan) (http: / / primer explorer.jp / e / ), combined with the basic principles of general primer design, designed and artificially synthesized a set of specific LAMP detection primers, including F3, B3, FIP and BIP, the nucleotide sequence of the primers (5'-3 ') are de...

Embodiment 2

[0060] Example 2. Optimization of reaction temperature conditions for LAMP detection of coffee berry anthracnose (Colletotrichum kahawae)

[0061] In this example, the primer set prepared in step 1 was used to insert the plasmid DNA of coffee berry anthracnose bacteria (C. Eiken Chemical Co., Ltd., Tokyo, Japan, Lot No.18001) was used as the experimental material, and the LAMP reaction was carried out at different temperatures (61°C, 63°C, 65°C) to optimize the important parameter affecting the LAMP reaction - temperature. The specific operation is as follows:

[0062] 1. Using the CTAB method (Yang and Liu. 2005. Dactylella coccinella sp. nov., an anamorphic species. Mycotaxon, 91: 127-132.) to extract the genomic DNA template, the specific operation is the same as Step 2 1 in Example 1.

[0063] 2. Using the Apn2 / Mat gene as the target gene, using the primer set prepared in step 1 in Example 1, perform a LAMP reaction on the genomic DNA template of the sample to be tested pre...

Embodiment 3

[0066] Embodiment 3, LAMP detects the specificity analysis of coffee berry anthracnose bacteria (Colletotrichum kahawae)

[0067] In this example, the primer set prepared in step 1 was used to use coffee berry anthracnose (C. kahawae) IMI319418, coffee berry anthracnose (C. 1) LAMP reaction is carried out for the experimental material, and the specificity of the primer set provided by the present invention is analyzed. The specific operation is as follows:

[0068] 1. Using the CTAB method (Yang and Liu. 2005. Dactylella coccinella sp. nov., an anamorphic species. Mycotaxon, 91: 127-132.) to extract the genomic DNA template, the specific operation is the same as Step 2 1 in Example 1. The precise quantification by Biospec-NANO (UV-VIS Spectrophotometers, Shimadzu, Japan) instrument was 20-30 ng / uL.

[0069] 2. Using the Apn2 / Mat gene as the target gene, using the primer set prepared in step 1 in Example 1, perform a LAMP reaction on the genomic DNA template of the sample to ...

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Abstract

The invention discloses a specific primer group and loop-mediated isothermal amplification kit for quickly detecting colletotrichum kahawae (C.kahawae) and applications thereof. The primer group provided by the invention comprises a primer 1, a primer 2, a primer 3 and a primer 4, wherein nucleotide sequences of the primer 1, primer 2, primer 3 and primer 4 are sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table respectively. The kit also comprises strand displacement type DNA polymerase, Mg<2+>, betaine, dNTPs and a fluorescence color developing agent. The kit provided by the invention is simple to operate, has strong specificity and high sensitivity and is used for detecting the C.kahawae genome DNA with sensitivity up to 0.08pg / mu L.

Description

technical field [0001] The invention relates to a specific primer set for rapid detection of coffee berry anthracnose bacteria (Colletotrichum kahawae), a loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) kit and applications thereof. Background technique [0002] The fungi of the genus Colletotrichum are a group of important economic and academic value, many of which can cause plant diseases of some commercial crops and flower plants, namely anthracnose. Coffee berry anthracnose (C. kahawae) can cause serious lesions of coffee berries, and it is the only phytopathogenic fungus quarantine object of the genus C. It was once treated as a species complex of C. gloeosporioides until it was reconfirmed as a new species in 1993 (Waller, J.M., Bridge, P.D., et.al.1993.Characterization of the coffee berry disease pathogen, Colletotrichum kahawae sp. nov. Mycol Res 97, 989-994.). But so far, a fast, simple and accurate method and technology platf...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 陶刚蔡磊
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI