Method for detecting pine wood nematode based on pseudomonas fluorescent flagellum protein
A technology for Pseudomonas fluorescens and pine wood nematode disease, applied in fluorescence/phosphorescence, biochemical equipment and methods, material excitation analysis, etc., can solve the problems of high cost, time-consuming, expensive equipment, etc., and achieve fast detection speed , the effect of low detection cost
Inactive Publication Date: 2008-12-31
QINGDAO UNIV +1
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Some methods are time consuming, others are expensive and require expensive equipment
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Embodiment 1
[0024] 1. Cultivation of Pseudomonas fluorescens carried by pine xylophilus and purification of flagellin
[0025] Two loops of lawn of Pseudomonas fluorescens GcM5-1A strain cultured on the slant were inoculated in 30 mL LB liquid medium, cultured at 30 °C for 24 h, transferred to 1 L LB medium, and continued to culture for 24 h under the same conditions.
[0026] Centrifuge the bacterial culture solution at 4°C and 15,000rpm for 20min, take the supernatant for ammonium sulfate fractional precipitation, take the 20% saturated precipitate and dissolve it with 20ml TE (20mM Tris-Cl, pH8.0, 5mM EDTA), and dialyze the 2LTE solution overnight Afterwards, use AmiconUltra ultrafiltration concentration tube (5kDa) to concentrate, take 3mL concentrated solution on Superdex-75 gel filtration column (1.6×60cm) for molecular sieve chromatography, eluent is TE solution, flow rate 1.0mL / min, step by step Collected, each step was analyzed and detected by SDS-PAGE, and the eluate containing ...
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Abstract
The invention relates to a method for detecting the pine wood nematode disease based on a fluorescent pseudomonas flagellin that is carried by pine wood nematodes, which comprises following steps: a fluorescent pseudomonas strain GcM5-1A (preservation number is CCTCC No: M2040 65) that is carried by the pine wood nematodes is cultivated in vitro, and the purified flagellin is separated from a zymotic fluid of the pseudomonas strain and taken as an antigen for immunizing rarebits, thus preparing an anti-flagellin antiserum of the rarebits; a lauryl sodium sulfate solution is used for respectively extracting a holoprotein of a health Lodgepole pine tissue and a Lodgepole pine tissue that is infected with the pine wood nematode disease, little extracted solution is point sampled on a polyvinylidene fluoride film, the prepared anti-flagellin antiserum of the rarebits is used for detecting the tissue protein of the Lodgepole pines according to the Western blot method, the protein sample that is extracted from the Lodgepole pine infected with the pine wood nematode disease is positive, and the extract of the health Lodgepole pine tissue is negative. The detecting method has quick detecting speed and low detecting cost, is suitable for large-scale investigation of the pine wood nematode disease, and is the beneficial supplement to the existing detecting methods of the pine wood nematode disease.
Description
Technical field: [0001] The invention relates to a method for detecting pine wood nematode disease based on Pseudomonas fluorescens flagellin carried by pine wood nematode, and belongs to the field of forestry biotechnology. Background technique: [0002] Pine wood nematode (Bursaphelenchus xylophilus) is a destructive disease of pine species, mainly transmitted by Monochamus alternatus, which can harm black pine (Pinus thunbergii), masson pine (P.massoniana) and other species pine. At present, the disease has occurred in Japan, the United States, Canada, China, South Korea, Nigeria, Portugal and other countries, among which Japan and China are the most severely affected. Since the first discovery of pine wood nematode disease in Nanjing Zhongshanling Scenic Area in the autumn of 1982 in my country, the disease has occurred in Jiangsu, Anhui, Zhejiang, Shandong, Guangdong, Hubei, Taiwan and Hong Kong, and the epidemic area has continued to expand the trend of. Pine wood ne...
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IPC IPC(8): G01N21/78G01N21/64C12Q1/04
Inventor 李荣贵郭道森赵博光
Owner QINGDAO UNIV