LAMP detection method of Arceuthobium sichuanense

A technique for spruce dwarf mistletoe, detection method, applied to spruce dwarf mistletoe. field, to achieve the effect of simple operation, simple operation and short detection cycle

Inactive Publication Date: 2016-03-30
BEIJING FORESTRY UNIVERSITY
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  • Abstract
  • Description
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  • LAMP detection method of Arceuthobium sichuanense
  • LAMP detection method of Arceuthobium sichuanense
  • LAMP detection method of Arceuthobium sichuanense

Examples

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Embodiment 1

[0040] Example 1 Primer Design

[0041] According to the analysis of the nucleotide sequence information of the plastid gene clpP of the spruce dwarf mistletoe and its host spruce, select the specific region of the spruce dwarf mistletoe to design multiple pairs of LAMP-specific primers. The length of the primers is generally as follows. :

[0042] F3: 5'-CAAGGGGCTGATAGTGAA-3';

[0043] B3: 5'-GGATAAAAGATCCCATTGAGG-3';

[0044] FIP: 5'-TCCGCCAGGAGAGTTTATAAAAAAAGAATCAACTTATTAGTCTGATGGT-3';

[0045] BIP: 5'-GGTCATCCCAGGAGTCGCTACTAAAGCCTATACATATAGTCTGTAC-3'.

[0046] The above primers were synthesized by Beijing Sanbo Polygala Biotechnology Co., Ltd.

Embodiment 2

[0047] Example 2 extracts total DNA

[0048] 1. Take out 1.0-3.0g of the collected spruce dwarf mistletoe and healthy spruce from the ultra-low temperature refrigerator, immediately put them in a pre-cooled mortar, add liquid nitrogen to quickly grind them into powder;

[0049] 2. Use the plant genome extraction kit (Beijing Quanshijin Biotechnology Co., Ltd.) to extract the genomic DNA of the above samples, as follows:

[0050] 2-1. Add 250 μl RB1 solution and 15 μl RnaseA to two 2ml centrifuge tubes respectively, take 0.1 g of each of the above two ground powders, add them to the corresponding centrifuge tubes, and mix well;

[0051] 2-2. Place the centrifuge tube in a constant temperature water bath and incubate at 55°C for 15 minutes;

[0052] 2-3. Centrifuge the centrifuge tube, centrifuge at 4°C, 12000rpm for 5min, gently suck the supernatant into 4 clean centrifuge tubes to obtain the supernatant;

[0053] 2-4. Add 100μl solution PB1 to 2 supernatant centrifuge tubes ...

Embodiment 3

[0060] Example 3: Spruce Dwarf Mistletoe LAMP Amplification Reaction

[0061] The specific implementation steps of LAMP amplification are as follows:

[0062] 1) Add the following reagents to the PCR tube so that the total reaction volume is 25 μl:

[0063]

[0064] Among them, BstDNA polymerase is produced by New England Biotechnology (Beijing) Co., Ltd., dNTP

[0065] Produced for Beijing Quanshijin Biotechnology Co., Ltd.

[0066] 2) LAMP reaction conditions are as follows:

[0067] 60℃45min

[0068] 85℃10min

[0069] That is, mix well according to the above system, and incubate at 60°C for 45 minutes; at 85°C, 10min to inactivate the stDNA polymerase.

[0070] 3) LAMP result analysis

[0071] After the LAMP amplification system was reacted for 45 minutes, the reaction system was detected by 1% agarose gel electrophoresis, in which ddH 2 O is a blank control. Test results such as figure 1 As shown, those with expanded bands are positive, indicating that the samp...

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Abstract

The invention relates to molecular detection of Arceuthobium sichuanense and belongs to the field of biotechnology. Based on sequence differences between clpP genes of Arceuthobium sichuanense and its host dragon spruce and by comparing sequence differences between Arceuthobium sichuanense and its host dragon spruce, a group of specific primers (4 primers) are designed. Meanwhile, the invention provides a group of LAMP primer sequences for detecting Arceuthobium sichuanense and also further provides a detection method for detecting Arceuthobium sichuanense. Primers specificity is good. The detection method is fast and simple, has high accuracy and provides an effective technical support for early diagnosis and disease control of Arceuthobium sichuanense.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a set of LAMP primers, which can quickly detect whether there is latently infected spruce dwarf mistletoe in a host. Background technique [0002] Natural spruce forests have irreplaceable ecological value in the ecologically fragile areas of western my country. They play important ecological functions such as soil and water conservation and water conservation. They are an important protection barrier for the ecological environment security in western my country and even the whole country. Picea dwarf mistletoe damage is one of the most devastating disasters in my country's spruce forests. It has caused serious diseases of spruce natural forests and secondary forests in the Sanjiangyuan area, and brought huge economic and ecological losses. In 2003 alone, 11,000 cubic meters of spruce were cut down at Xianmi Forest Farm in Menyuan County, Qinghai Province due to spruce dw...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 陈磊田呈明赵瑛瑛
Owner BEIJING FORESTRY UNIVERSITY
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