PCR (polymerase chain reaction) primer for detecting tobacco soil-borne fungal pathogens as well as application and method of PCR primer

A technology for soil-borne fungi and pathogens, applied in the direction of microbe-based methods, biochemical equipment and methods, microbiological measurement/testing, etc., can solve problems such as multiple PCR that have not yet been seen, and achieve simple and fast detection and accurate detection results High, high-sensitivity effects

Active Publication Date: 2014-12-24
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no multiplex PCR technology for simultaneous det

Method used

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  • PCR (polymerase chain reaction) primer for detecting tobacco soil-borne fungal pathogens as well as application and method of PCR primer
  • PCR (polymerase chain reaction) primer for detecting tobacco soil-borne fungal pathogens as well as application and method of PCR primer
  • PCR (polymerase chain reaction) primer for detecting tobacco soil-borne fungal pathogens as well as application and method of PCR primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1 Preparation of DNA template

[0098] The DNA of the four pathogen samples was extracted as a template for the PCR reaction. The specific process is as follows:

[0099] 1. Mycelia collection

[0100] Transfer the strains of black shank and damping-off pathogens to NA solid medium plates, transfer the strains of root black rot and blight pathogens to PDA plates, and cut them out at the edge of the colony after 3 days of dark cultivation at 28°C Small colonies, root black rot and blight pathogens were transferred to PDB, and other bacterial strains were transferred to tomato juice liquid medium, and after 7 days of shaking culture at 28°C, the mycelia were collected by filtration.

[0101] 2. Extraction of sample DNA

[0102] Grind 50-100 mg of mycelium into powder with liquid nitrogen and add it to a 1.5 ml centrifuge tube. Add 400 μL Buffer Digestion and 4 μL β-mercaptoethanol, shake and mix. Water bath at 65°C for 1 h until the cells were completely l...

Embodiment 2

[0103] Example 2 Establishment of PCR reaction system

[0104] 1. Establish a single-plex PCR reaction system for 4 pathogens

[0105] The total volume of the PCR reaction mixture is 25 μL, including 0.2 μL TaKaRa La Taq with a concentration of 5 U / μL; 2.5 μL 10×LA PCR Buffer II (Mg 2+ Free); 2.5 μL of 25 mM MgCl 2 ; 4 μL of dNTP Mixture with a concentration of 2.5 mM each; 0.5 μL of upstream and downstream primers with a concentration of 20 μM corresponding to the four pathogens; 1 μL of DNA template; make up the volume with sterile water. All reagents were purchased from TaKaR Company, and the amplification reaction was performed on an ABI Veriti gradient PCR instrument. The reaction program was pre-denaturation at 94°C for 4 min, denaturation at 94°C for 40 s, annealing at 56°C for 40 s, extension at 72°C for 45 s, 30 cycles, and finally extension at 72°C for 7 min. Take 10 μL of PCR product and go through 1.5% agarose gel electrophoresis, detect and take pictures on the...

Embodiment 3

[0108] Example 3 Specificity and Sensitivity of Multiplex PCR Detection

[0109] 1. Specific detection

[0110] Genomic DNA of 4 kinds of pathogens was randomly combined as a template, and the specific primers P1~P8 designed and screened by the present invention were used to amplify with the optimized reaction system, and the random combination of pathogens could be specifically detected, which verified the Specificity of multiplex PCR.

[0111] 2. 4-fold PCR sensitivity test

[0112] The sensitivity of the established 4-fold PCR detection system was measured in a 25 μL reaction system. The DNA samples of the four pathogens with concentrations of 227ng / μL, 179ng / μL, 190ng / μL and 254 ng / μL were divided into 10 0 -10 5 Gradient dilution ratio, and using the 4-fold PCR reaction system and reaction program designed in the present invention, can stably amplify 4 specific bands from 0.01ng / μL genome template figure 2 ).

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Abstract

The invention discloses a PCR (polymerase chain reaction) primer for detecting tobacco soil-borne fungal pathogens as well as application and a method of the PCR primer. The PCR primer comprises a tobacco rhizoctonia solani primer, a phytophthora nicotianae primer, a thielaviopsis basicola primer and a pythium aphanidermatum primer; the application is application of the PCR primer to simultaneous detection of four tobacco soil-borne fungal pathogens, and four soil-borne fungi are tobacco rhizoctonia solani, phytophthora nicotianae, thielaviopsis basicola and pythium aphanidermatum; the method comprises the steps of pre-treating, PCR reaction and electrophoretic detection; the detection operation is simple, convenient, quick, high in sensitivity and accurate; a sample is prepared and detected within 4 hours; the four pathogens of tobacco rhizoctonia solani, phytophthora nicotianae, thielaviopsis basicola and pythium aphanidermatum are simultaneously detected; a DNA (deoxyribonucleic acid) sample of the pathogen of 0.01 ng can be detected; the accuracy of a detection result is higher than that in a conventional separation identification and symptom identification method.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis of plant diseases, and in particular relates to a PCR primer for detecting tobacco soil-borne fungal pathogens and its application and method. Background technique [0002] Tobacco soil-borne fungal diseases include black shank, root black rot, damping-off and blight, etc. These diseases are important diseases in tobacco production and pose a serious threat to tobacco production. In recent years, due to changes in the tobacco environment, the occurrence of these diseases has been complicated. The main and secondary diseases have varied in local areas, and often occur in combination. There are many factors such as time-consuming, low sensitivity, and being easily interfered by human and environmental factors, so that the results cannot be obtained quickly and accurately. Early molecular diagnostic techniques developed in recent years are mainly single disease detection techniques, such...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/686C12Q1/6895C12Q2537/143
Inventor 方敦煌刘萍花童治军肖炳光杨大海黄昌军
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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