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Method for detecting areca-nut yellow leaf disease phytoplasma pathogen and special reagent kit therefor

A technology for phytoplasma and yellowing disease, applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the real-time fluorescent PCR detection technology for phytoplasmic pathogens of betel nut yellowing disease, etc. problem, to achieve the effect of eliminating false positive pollution, simple operation, and simple detection process

Active Publication Date: 2009-07-08
ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Application Information

AI Technical Summary

Problems solved by technology

[0008] There is no real-time fluorescent PCR detection technology for the phytoplasma pathogen of betel nut yellowing disease

Method used

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  • Method for detecting areca-nut yellow leaf disease phytoplasma pathogen and special reagent kit therefor
  • Method for detecting areca-nut yellow leaf disease phytoplasma pathogen and special reagent kit therefor
  • Method for detecting areca-nut yellow leaf disease phytoplasma pathogen and special reagent kit therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Acquisition of the 16SrDNA sequence of the phytoplasma of betel nut yellowing disease and determination of its taxonomic status

[0027] 1. Extraction of total DNA from betel nut flower buds

[0028] Collect samples of betel nut yellowing plants showing typical yellowing symptoms. The yellowing disease of betel nut is based on Luo Quanquan et al. , 2001, 22 (2): 43-46.) said method identification, wherein, collected from Hainan Tunchang (3 strains): Dc1, Dc2, Dc5; Ding'an (3 strains): Da2,, Da3, Da5; Qiong Hai (3 strains): Qh21, Qh22, Qh23; Wanning (3 strains): Wn1, Wn2, Wn3; Sanya (3 strains): Sy15, Sy21, Sy22; healthy samples were collected from Danzhou, Hainan. Refer to Lee et al. (Lee I M, Davis R E, Hiruki C. Genetic interrelatedness among cloverproliferation mycoplasmalike organisms (MLOs) and other MLOs investigated by nucleic acid hybridization and restriction fragment length polymorphism analyses. Appllied and environmental Microbiology: 35-6 (191) ...

Embodiment 2

[0042] Embodiment 2, the present invention detects the preparation of the probe of betel nut yellowing disease phytoplasma pathogen, kit and their effect verification

[0043] 1. The present invention detects the design of the probe and primer and the preparation of the kit for detecting the phytoplasma pathogen of betel nut yellowing disease

[0044] In this study, phytoplasma 16SrDNA was used as the target gene to design primers and probes, and the 16SrDNA sequences of all known phytoplasmas were downloaded from NCBI, and compared with ClustalX1. The region with a stable mutation in the sequence, and then use Primer Real-time fluorescent PCR primers / TaqMan MGB probes were designed with version3.0 software, and the forward and reverse primers were compared using the Blast program in NCBI (http: / / blast.ncbi.nlm.nih.gov / Blast). No species other than the 16Sr I group could pair with this pair of primers and cause amplification was found. The sequence looks like this:

[004...

Embodiment 3

[0089] Example 3, the test kit for detecting the phytoplasma pathogen of betel nut yellowing disease of the present invention is verified for the detection accuracy effect of sampling from different parts of betel nut

[0090] Cutting 30 plants infected with betel nut yellowing disease on the east and south lines of Hainan Island, according to Luo Quanquan et al. Acta Crops Sinica, 2001, 22 (2): 43-46.) said method identification, get flower bud, heart leaf, blade respectively (get the vein of the 2nd, 3rd, 5th leaf successively from the bottom leaf of lower betel nut plant) ), root as the detection site, respectively extracting total DNA as a template sample, all using the kit prepared in step 1 of embodiment 2, and detecting according to the real-time fluorescent PCR detection method described in step 3 of embodiment 2.

[0091] The test results are as follows:

[0092] (1) Test results of flower bud samples

[0093] The total DNA extracted from the unexpanded betel nut bu...

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Abstract

The present invention discloses a method for detecting betelnut yellow phytoplasma pathogen and its special kit. The kit includes a probe whose nucleotide sequence is the sequence 1 of the sequence table, and a primer pair composed of an DNA fragment whose nucleotide sequence is the sequence 2 of the sequence table and an DNA fragment whose nucleotide sequence is the sequence 3 of the sequence table. The method includes: extracting the DNA of the betelnut to be tested as a template, using the above kit to perform real-time fluorescence PCR, if the fluorescence signal is enhanced in the PCR process, the plant infects phytoplasma. Proved by experiment, the sensitivity of the inventive method detecting the betelnut phytoplasma infection is same with that of the traditional nested PCR electrophoresis detection, while the invention has simple operation and closed tube operation, processes automation control in the entire testing, eliminates the PCR false-positive contamination opportunities, does not need post-PCR handling, is suitable for bulk samples, and has simple, rapid, and accurate features.

Description

technical field [0001] The invention relates to a method for detecting the phytoplasma pathogen of betel nut yellowing disease and a special kit thereof, which are suitable for use in quarantine, agricultural production, plant protection and other departments. Background technique [0002] Arecacathecu L. is one of the important southern medicine resources in my country. According to statistics from the Hainan Provincial Department of Agriculture, by the end of 2006, the betel nut planting area in Hainan Province reached 53,100 hectares, the harvested area was 23,300 hectares, and the total output was 74,800 tons. More than 1.8 billion yuan. [0003] Betel nut yellowing is a devastating disease that first occurred in central Kerala, India. By the 1960s, betel nut yellowing had generally occurred in Quilon, Kerala state, India. In 1981, betel nut yellowing disease was first discovered in the medicinal materials field (Tunchang County) of Hainan Province in my country; in the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 车海彦罗大全
Owner ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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