Detection method of rape Leptosphaeria maculans

A technology based on ulcers and rape, applied in the field of microbial detection, can solve the problems of complex operation, limited applicability, and long time consumption, and achieve the effects of simple operation, low requirements for equipment, and personal and environmental safety

Inactive Publication Date: 2015-08-05
舟山出入境检验检疫局综合技术服务中心 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the detection of L.maculans is mainly based on the polymerase chain reaction (PCR) method, but the PCR detection operation is complicated and time-consuming, and require

Method used

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  • Detection method of rape Leptosphaeria maculans
  • Detection method of rape Leptosphaeria maculans
  • Detection method of rape Leptosphaeria maculans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: the method establishment of LAMP-LFD technique detection canker canker bacterium of rape

[0043] 1.1 Materials and methods

[0044] 1.1.1 Materials

[0045] Bst DNA polymerase, dNTPs were purchased from TaKaRa Company, and LFD detection strips were purchased from Milenia Biotec GmbH (Milenia GenLine HybriDetect MGHD1, Germany).

[0046] 1.1.2 Method

[0047] 1.1.2.1 Design of LAMP primers

[0048] The following 3 pairs of primers were designed and screened according to the IST gene of Rape canker sores, and the primer sequences are as follows:

[0049] Lep-F3:5′-TCTCTTGGTTCTGGCATCGA-3′

[0050] Lep-B3:5′-AAATGTGCTGCGCTCCA-3′

[0051] Lep-FIP:

[0052] 5′-AAGGGGCGCAATGTGCGTT-ACGCAGCGAAATGCGATA-3′

[0053] Lep-BIP:

[0054] 5′-TACCCTCAAGCTCTGCTTGGTG-GCCAATTGTTTCAAGGCGAG-3′

[0055] Lep-LF:5′-CACTGAATTCTGCAATTCACACTAC-3′

[0056] Lep-LB: 5′-TTGGGTGTTTGTTCCACTTGG-3′

[0057] Wherein Lep-FIP is a 5' end biotin-labeled primer, and Lep-LF is a 5' end ...

Embodiment 2

[0066] Determination of Sensitivity of Using LAMP-LFD Technology of the Present Invention to Detect Rape Stem Canker Pathogen

[0067] 1. With reference to the genomic DNA of X. canker sores extracted by the DNA extraction kit, the original concentration (11.4ng / ul) of the extracted X. cankers genomic DNA template was diluted by 10-fold gradients to make templates respectively.

[0068] 2. Using LAMP-AGE, LAMP-LFD and PCR to compare the sensitivity of detection of canker canker of rapeseed.

[0069] 2.1 According to the three pairs of primers of LAMP combined with LFD technology provided by the present invention, the genomic DNA after the above gradient dilution is used as a template for the LAMP reaction to amplify, and the LFD method provided by the present invention is used to analyze the LAMP reaction. The results are shown in image 3 .

[0070] 2.2 Use the above-mentioned serially diluted genomic DNA as a template for LAMP-AGE for agarose gel electrophoresis test, the r...

Embodiment 3

[0074] Specific Detection of Rapeseed Canker Pathogens Using the LAMP-LFD Technology of the Present Invention

[0075] Select Alternaria sp, Lasiodiplodia theobromae, Fusarium sp, Diaporthe phaseolorum caulivora, Diaporthe phaseolorum Var meridionalis ) 5 strains of pathogenic bacteria were used as LAMP-LFD specific test strains, and double distilled water was used as negative control. The DNA of the bacteria was extracted, and the amplified products were observed by LFD test strips and 1% agarose gel electrophoresis respectively. See the test results Figure 4-6 , indicating that the LAMP-LFD detection method for canker canker of rapeseed provided by the present invention can ensure the specific detection of canker canker of rape, and does not cross-react with other related bacteria.

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Abstract

The invention provides a LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection method of rape Leptosphaeria maculans, which comprises the following steps: designing three pairs of LAMP primers FIP, BIP, LF, LB, F3 and B3, wherein FIP is a 5'-terminal biotin labeled primer, and LF is a 5'-terminal fluorescein isothiocyanate (FITC) labeled probe; and preparing an LAMP reaction system containing the three pairs of primers and a sample template, amplifying the LAMP reaction system, carrying out LFD detection, and carrying out specificity and sensitivity evaluation. The result shows that the lowest detection line of the LAMP-LFD method for the L.maculans genome DNA (deoxyribonucleic acid) is 114fg/mu L. In the specificity verification experiment, when five related phytopathogens, including Alternaria, Lasiodiplodia theobromae, fusarium and Diaporthe phaseolorum var. caulivora, are used and Diaporthe phaseolorum var. meridionalis and L.maculans are used as specific test strains, the LAMP-LFD detection method only has positive reaction for the L.maculans.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular relates to a detection method of rapeseed canker bacterium Leptosphaeria maculans. Background technique [0002] Rapeseed stem canker (phom a stem canker) is one of the most serious fungal diseases on rapeseed, and its pathogen is Leptosphaeria maculans of the genus Leptosphaeria maculans. L. maculans is very pathogenic and can cause canker at the base of rapeseed, which is the main reason for the serious prevalence of canker at the base of rapeseed and the loss of rapeseed yield. It is estimated that the oilseed rape economic loss caused by the disease in the world exceeds 300 million euros every year. The pathogen is widely distributed in Europe, Australia, Canada, and the United States, and has not been reported in my country. However, the climate of rapeseed production areas in my country is similar to that of Europe, the United States, and Australia. The p...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q2531/119
Inventor 周圆李孝军单长林李雪松杨赛军叶露飞朱鹏范建忠黄海龙章礼平
Owner 舟山出入境检验检疫局综合技术服务中心
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