Real-time fluorescence quantitative PCR method for detecting sugarcane ratoon stunning disease

A real-time fluorescence quantitative and dwarf disease technology, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of laborious, inability to quantify the number of pathogenic bacteria, and time-consuming

Inactive Publication Date: 2012-01-11
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology

[0004] The purpose of the present invention is to provide a real-time fluorescent quantitative PCR method for detecting sugarcane perennial dwarf pathogens in order to solve the problems of inability to quantify the number of pathogens, poor accuracy, time-consuming and laborious problems in the existing methods for detecting dwarf pathogens of sugarcane

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  • Real-time fluorescence quantitative PCR method for detecting sugarcane ratoon stunning disease
  • Real-time fluorescence quantitative PCR method for detecting sugarcane ratoon stunning disease

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Embodiment 1

[0022] Extraction of pathogenic bacteria of dwarfism in perennial roots by CTAB method Leifsonia xyli subsp .xyli (Preserved in the Sugarcane Comprehensive Research Institute of Fujian Agriculture and Forestry University. The method for isolating the pathogenic bacteria of sugarcane perennial dwarf disease is provided by Y.-B. Pan). Using the DNA template, qualitative PCR amplification was performed using the above primers. Eppendorf Mastercycler EP PCR Thermal Cycler Model 5333 Gene Amplifier. 25 μL PCR reaction system composition: 10×PCR Buffer (Mg 2+ ) 2.5 μL, dNTP (2.5 mmol / L) 2 μL, forward and reverse primers (10 μmol / L) 1 μL each, ExTaq enzyme (5U / μL) 0.125 μL, template DNA (100 ng / μL) 1 μL, add Sterilize double distilled water to bring the total volume to 17.375. PCR amplification conditions: 95°C for 5 min; 35 cycles of 95°C for 30 S, 56°C for 30 S, and 72°C for 30 S; finally extend at 72°C for 7 min and store at 4°C for later use. After the reaction, the obtained...

Embodiment 2

[0057] Sugarcane juice DNA was extracted from sugarcane in the Ninth National Sugarcane Regional Experiment in Fujian Agriculture and Forestry University in 2011. The 11 different varieties of sugarcane are as follows: YA05-179, Liucheng LC1, YG35, YG34, ROC20, FN36, YZ05-5, ROC22, RC16, Y06-407, LC03-1137. According to the traditional nucleic acid extraction method CTAB method, the purified genomic DNA was used as a template, and the 7500 real-time fluorescent quantitative PCR instrument of ABI Company was used to perform PCR amplification with the specific primer pair and TaqMan probe of the present invention. Reaction system includes: TaqMan Universal Master Mix (2×) 12.5 μL; forward and reverse primers (10 μmol / L) 0.75 μL each; TaqMan probe (10 μmol / L) 0.40 μL; DNA template 1.0 μL; , each sample was repeated 3 times. Reaction conditions: 50 ℃, 2 min, pre-denaturation 95 ℃, 10 min; 95 ℃, 15 s; 60 ℃, annealing extension, 1 min, 40 cycles, single-point fluorescence detection...

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Abstract

The invention firstly provides primers and a probe for detecting sugarcane ratoon stunning disease with a real-time fluorescence quantitative PCR method. The primers comprise a primer pair represented by the following sequences: the forward primer sequence is 5'-GGTTCCATTGCTTACCGATT-3', the reverse prime sequence is 5'-CAAGTTTCGACAGGAACAGC-3', and the probe sequence is FAM-5-CCACGGCTACGTCAATTCGGG-3-TAMRA. The primer pair and probe are used for detecting ratoon stunning disease Pat1 gene infected by sugarcane in a real-time fluorescence quantitative PCR instrument. By Ct values generated in the process of PCR amplification, Ct values are converted into DNA copy number of the initial reaction by using the established standard curve, so that the quantitative determination of the quantity of sugarcane which is infected by ratoon stunning disease can be realized. The method can be used for the fields of the detection of sugarcane health seedling and identification of the resistance of sugarcane.

Description

technical field [0001] The invention relates to the technical field of molecular biology for sugarcane healthy seedlings and sugarcane resistance identification, in particular to a detection primer and a quantitative PCR detection method for sugarcane perennial root dwarf pathogens. Background technique [0002] Sugarcane ratoon stunting disease (RSD) is a bacterial disease that commonly occurs in sugarcane growing areas around the world. Corynebacterium Leifsonia xyli subsp .xyli It is caused by parasitizing in the vascular bundles of sugarcane plants, and is mainly transmitted between diseased sugarcane seeds and cutting tools during harvest, and partly through soil within a certain period of time. Studies by Davis et al. have shown that the bacterium is not easy to isolate and culture, and it is a Gram-positive rod-shaped bacterium. Since the disease has no obvious external and internal symptoms, it is difficult to identify it only from the appearance. When it is parti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/01
Inventor 王恒波郭晋隆许莉萍高三基陈如凯陈平华
Owner FUJIAN AGRI & FORESTRY UNIV
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