A kind of detection method of rapeseed canker bacterium
A technology based on ulcers and rapeseed, applied in the field of microbial detection, can solve problems such as time-consuming, complicated operation, and large applicability limitations, and achieve the effects of personal and environmental safety, simple operation, and low requirements for equipment
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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 test 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 fluor...
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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