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Method and application for quantitatively detecting quantity of late blight bacteria in soil

A quantitative detection, soil technology, applied in the field of agricultural biology, can solve the problems of long detection cycle, low sensitivity, poor specificity, etc.

Inactive Publication Date: 2015-04-22
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the main methods and technologies for detecting the amount of P. infestans in soil and seed potatoes are: ①Field detection method. This method uses the observation of the main tissue symptoms caused by potato infestation as evidence of detection and identification. It can only be carried out when obvious symptoms occur in potato tissues. Detection, poor accuracy, time-consuming and laborious (5.1.1.2 in GB7331-2003); ② laboratory detection method, this method utilizes the physiological and biochemical characteristics of the main potato pathogenic bacteria, through microscope, pathogenic bacteria resuscitation culture and biochemical and other means to detect detection
The accuracy is higher than that of the field detection method, but the detection cycle is long, and the prevention detection of late blight lags behind (GB6682)
③Molecular biology methods (Judelson and Tooley, 2000; Niepold and Schober–Butin, 1995; Tooley., 1997; Trout et al., 1997), these methods use the genomic DNA sequence of P.infestans to design primers to detect potato The content of P. infestans in leaves, tubers, etc., such as Niepold and other designed primers to detect potato tubers and leaves inoculated with P. infestans, but this method can only detect P. Butin., 1995); Trout et al. (Trout et al., 1997) used primers ITS5 and PINF to detect Phytophthora infestans in natural tubers, with high sensitivity but poor specificity
[0006] Based on the above analysis, it can be seen that the existing technology cannot quickly, accurately, efficiently and specifically quantify the number of soil potato infestation pathogens, and thus cannot effectively track potato growth The occurrence process of late blight in the process, and accordingly put forward effective disease prevention measures

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  • Method and application for quantitatively detecting quantity of late blight bacteria in soil

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Experimental program
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Embodiment 1

[0032] The construction process of embodiment 1 standard curve

[0033] Dilute the standard strain (P.infestans) DNA solution (concentration: 100ng / μl) 10 times to 10 2 ng / μl, 10ng / μl, 1ng / μl, 10 -1 ng / μl, 10 -2 ng / μl, 10 -3 ng / μl, 10 -4 ng / μl; 1 μl of DNA template was added to the Real Time PCR amplification tube (concentrations were 10 -1 ng / μl, 10 -2 ng / μl, 10 -3 ng / μl, 10 -4 ng / μl, 10 -5 ng / μl), add 10 μl of SYBR-Green fluorescent labeling reagent mixture (SYBR green PCR Master Mix, Applied Biosystems), add 0.5 μl each of specific primers PIF / PIR (10 μM), and sterile deionized water to a total volume of 20 μl. Three parallel experiments were performed for each reaction. The setting program of the real-time fluorescent quantitative PCR instrument is: 50°C-5min, 95°C-10min; then 95°C-20s, 60°C-1min, cycle 35 times; finally keep at 72°C for 5min. After the entire cycle is completed, the sample is ramped from 60°C to 95°C under a 35°C gradient at 0.03°C / s. After the...

Embodiment 2

[0034] Example 2 Quantitative Detection of Infestation Phytophthora Bacteria Carrier Amount in Soil

[0035] 1. Take 0.5g each of the three potato planting soils (sandy soil, clay clay and humus soil Muck), and add DNA extraction solution (100mmol / L, Tris-HCl, 100mmol / L EDTA, 100mmol / L sodium phosphate, 1.5mol / L NaCl, 2% CTAB, pH8.0) 1.2ml, extract DNA according to CTAB method, measure the quality of extracted DNA by ultraviolet spectrophotometry, the standard is OD260 / 280 is about 1.8.

[0036] 2. Add 2μl (50ng) of DNA template to the amplification tube, add 10×Tris-HCl buffer (pH8.3, 500mM KCl, 15mM MgCl 2 ) 3μl; dNTP (2.5mM) 2μl; each specific primer (PIF / PIR, 10μM) 1μl; Taq enzyme (5U / μl) 0.2μl and 2% Blotto (10% skimmed milk powder and 0.2% NaN 3 ), add sterile deionized water to a total volume of 25 μl.

[0037] 3. Carry out ordinary PCR amplification on the above reaction system, the specific procedure is: 95°C-4min; then cycle 40 times at 95°C-55s, 60°C-1min, 72°C-45...

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Abstract

The invention provides a method and application for quantitatively detecting the quantity of potato late blight bacteria in soil, in particular to a method for quantitatively detecting the quantity of live late blight bacteria in potato growing soil by using the common PCR (Polymerase Chain Reaction) technology and the Real Time PCR technology and through specific primers 5'-CGGCGGCTGCTGGCTTTAT-3' and 5'-GCTCAGACCGAAGTCCAAACG-3'. According to the method provided by the invention, the quantity of potato late blight bacteria in soil can be quantitatively detected.

Description

technical field [0001] The invention relates to a method for quantitatively detecting the carrier amount of potato infestans in soil and its application, which can quantitatively and specifically detect potato infestation in soil, and belongs to the field of agricultural biotechnology. Background technique [0002] Potato late blight is a devastating disease of potato, which has been listed as the first major disease of food crops in the world. Late blight commonly occurs in potato growing areas, with field yield losses of 20% to 50%, cellar losses of 5% to 30%, and even crop failures in pandemic years. In recent years, the frequency and prevalence of late blight have gradually increased, becoming an important obstacle to potato production. Potato is the fourth largest food crop in the world (Reader, 2009), and the loss caused by late blight of potato in the world has reached 6.7 billion U.S. dollars (Haverkort et al., 2008). China is already the largest potato production c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
Inventor 万东石邵旭平李永泉
Owner LANZHOU UNIVERSITY