Method for storing monochamus alternatus hope sample and method for fast detecting bursaphelenchus xylophilus therein
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A pine brown longhorn beetle, detection method technology, applied in the preservation of human or animal body, biochemical equipment and methods, microbial determination/inspection, etc. problems, to achieve the effect of improving detection efficiency, improving accuracy, and speeding up detection
Inactive Publication Date: 2010-11-10
SOUTH CHINA AGRI UNIV
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[0019] An object of the present invention is to overcome the deficiencies in the specimen preservation technology in the existing pine wood nematode detection technology, provide a kind of method for preserving the monochamus monochamus (Monochacha alternifera) sample, and solve the problem of pine wood nematode detection for pine wood nematode The number of pine wood nematodes in the live monochamus was lost during the transportation or storage of cattle samples, and the test results could not accurately reflect the actual number of pine wood nematodes carried by monochamus in the forest
[0020] Another object of th...
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Embodiment 1
[0049] A sample of Monochamus alternatus was collected from Nanhu Amusement Park in Guangzhou and stored for 5 days for identification. The expected result was obtained, the inspection task was completed, and the inspection results are shown in the attachment Image 6 And Figure 7 . Specific steps are as follows:
[0050] (1) Preparation of PCR primers:
[0051] One week earlier than the sampling time, a pair of specific PCR amplification primers were synthesized by Beijing Cypress Bioengineering Company:
[0052] P155: 5’-CTACGTGCTGTTGTTGAGTTGGC-3’;
[0053] P538: 5'-TGGTGCCTAACATTGCGCGA-3'.
[0054] The PCR amplification primers are stored in a refrigerator at -20°C.
[0055] The results of PCR-specific amplification of pine wood nematode are attached figure 1 As shown, where M represents DNAMarkDL2000; 1 represents the genomic DNA of Bursaphelenchus xylophilus; 2 represents the genomic DNA of Bursaphelenchus xylophilus; 3 represents the genomic DNA mixture of Bursaphelenchus xylop...
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Abstract
The invention discloses a method for storing a monochamus alternatus hope sample and a method for fast detecting bursaphelenchus xylophilus therein. The method comprises the following step of putting the caught monochamus alternatus hope sample into 75% ethyl alcohol or a refrigeratory with -70 DEG C to be stored, thereby furthest preserving the amount of the bursaphelenchus xylophilus carried bythe monochamus alternatus hope. The invention creates a production technology of the bursaphelenchus xylophilus DNA template in the monochamus alternatus hope with the storing state, directly freezesand thaws lyses and centrifuges the middle and back thoracic tracheal tissues of the adult monochamus alternatus hop for PCR amplification, has the amplification rate of 100%, fastens the detecting speed, improves the detecting accuracy, and achieves the aim of convenient, fast and accurate detection.
Description
Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a preservation technology of Monochamus alternatus samples carrying pine wood nematodes, and a rapid detection method of Monochamus alternatus samples in the body. Background technique [0002] Pine wood nematode disease of Monochamus alternatus is an important quarantine disease. Since pine wood nematode disease was discovered in Nanjing Zhongshan Mausoleum in 1982, pine wood nematode disease has spread to Jiangsu, Shandong, Anhui, Zhejiang, Fujian, Hubei, Hunan, Guangdong, Guangxi, Yunnan, Guizhou, Sichuan, Hong Kong, Macau, And Taiwan and other provinces, cities, regions and other places, the country’s occurrence area reached 87,000 hm 2 More than 35 million dead pine trees accumulatively. The diseased wood in the epidemic area not only cannot be used normally, but also affects the circulation of other agricultural and sideline products and forest products, causing direct ec...
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