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PCR quantitative detection method and kit for corn southern rust pathogenic bacteria in incubation period

A technology for southern rust and pathogenic bacteria, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of early molecular quantitative detection of southern corn rust and quantitative detection of southern corn rust , predicting future illnesses, etc.

Pending Publication Date: 2022-01-28
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Crouch&Szabo (2011) also designed specific real-time qPCR primers and probes based on the ITS sequence of Puccinia multicumulus to distinguish between southern corn rust and common rust in the United States, but this method cannot be used for southern corn rust. Quantitative detection, so it is impossible to carry out early molecular quantitative detection and prediction of future disease of maize southern rust
There is no report on the qPCR early molecular quantitative detection system of maize southern rust fungus in China

Method used

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  • PCR quantitative detection method and kit for corn southern rust pathogenic bacteria in incubation period
  • PCR quantitative detection method and kit for corn southern rust pathogenic bacteria in incubation period
  • PCR quantitative detection method and kit for corn southern rust pathogenic bacteria in incubation period

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Primer design and performance testing

[0039] According to the ITS sequence of Puccinia multicumulus, the DNAMAN software was used to compare the ITS sequences of other control pathogenic bacteria, and the sequence segments with large differences were selected, and the primer PpoF (5'-ACCTGTTTGAGTGTCATG-3', SEQ ID NO.1) / PpoR (5'-CCTCAATGTGATCTCAGTTA-3', SEQ ID NO.2) and probe PpoP (HEX-ACACATCAAGTCAATCCACTCCATCT-BHQ1, SEQ ID NO.3). According to the Actin2 gene sequence of maize, use Beacon Designer 8.2 software to design primer ZmF (5'-CCTGATGAAGATCCTTACTG-3', SEQ ID NO.4) / ZmR (5'-CTGCACCGATTGTGATAA-3', SEQ ID NO.5) and probe ZmP (FAM-CTACGACTGCCGAGCGAGAA-BHQ1, SEQ ID NO. 6). Primers and probes were synthesized by Bao Biological Engineering (Dalian) Co., Ltd.

[0040] Using the same concentration of Puccinia multicumulus DNA, DNA of 11 other pathogenic bacteria and DNA of healthy maize leaves as templates, qPCR amplification was performed, with 3 replicates...

Embodiment 2

[0045] Quantitative analysis of embodiment 2 incubation period Puccinia multiheap

[0046] qPCR standard curve establishment:

[0047] In order to carry out quantitative analysis on Puccinia multicumensis during the incubation period, it is necessary to establish the qPCR standard curves of Puccinia multicumensis and maize respectively. The gradient dilution of Puccinia multicumulus DNA extracted in the "Main Materials, Reagents and Methods" section was 100, 10, 1, 10 -1 、10 -2 、10 -3 ng / μL with a total of 6 concentrations, used as a template for qCR amplification, and ddH 2 O was used as a negative control, and each concentration was repeated 3 times to obtain respective cycle thresholds (threshold cycle, Ct), and the relationship between the Ct value and the known DNA concentration of Puccinia spp. Make a standard curve for the ordinate and establish a linear equation. Dilute the corn DNA extracted in the "Main Materials, Reagents and Methods" section to 100, 10, 1, 10 ...

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Abstract

The invention relates to a real-time fluorescent quantitative PCR technology-based detection method and a kit for corn southern rust pathogenic bacteria puccinia polypoda. Specific primers and TaqMan probes are designed according to ITS of corn southern rust and Actin2 gene sequences of corn, and a PCR quantitative detection system for the incubation period of the corn southern rust is established on the basis of the two pairs of primers and probes. The primers and the probes for the corn southern rust fungus and the corn have the characteristics of high specificity and sensitivity. The PCR quantitative detection system for the puccinia multiflora incubation period, established by the invention, not only can detect the puccinia multiflora in the incubation period, but also can detect the infection amount at different time after infection, and the system can be used for early detection and quantitative detection of the puccinia multiflora. The research method provided by the invention can be effectively applied to early molecular detection of puccinia polypoda, and has important significance in the aspects of early prediction, forecast, comprehensive treatment and the like of the corn southern rust disease.

Description

technical field [0001] The invention relates to the field of molecular biology and early monitoring of plant diseases, in particular, the application provides a quantitative detection technology and a corresponding kit for the pathogenic bacteria of southern corn rust, Puccinia solani based on real-time fluorescence quantitative PCR technology. Background technique [0002] Maize southern rust caused by Puccinia polysora is a devastating airborne disease worldwide, and it is also a serious maize disease in my country in recent years. The fungus mainly harms corn leaves and leaf sheaths. In severe cases, it can infect all the green tissues in the shoots of corn, causing the plants to dry up or even die. Since 1998, the disease has broken out in many provinces in my country. In 2015, it was the most serious year for southern corn rust in my country in recent years. It became a big epidemic in the Huanghuaihai summer corn area and other places, and the national occurrence area ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/11
CPCC12Q1/6895C12Q1/6851C12Q2600/158C12Q2531/113
Inventor 马占鸿张克瑜孙秋玉李磊福黄莉群董佳玉高建孟
Owner CHINA AGRI UNIV
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