Polymerase chain reaction (PCR) detection method and kit for anti-amyotrophic lateral sclerosis (ALS) inhibitor herbicide beckmannia syzigachne

A technology of herbicides and inhibitors, which is applied in the field of PCR detection methods and kits for anti-ALS inhibitor herbicides, can solve the problems of not being able to detect in the current season, heavy workload, and large land occupation, and meet the requirements of fast Diagnosis, practical effect

Active Publication Date: 2014-09-10
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can objectively evaluate the resistance of a certain weed to the corresponding herbicide, but it also has its limitations, mainly in the following two aspects: one is that it cannot be tested in the same season and at that time, and the other is that it takes up a lot of land and time long, heavy workload

Method used

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  • Polymerase chain reaction (PCR) detection method and kit for anti-amyotrophic lateral sclerosis (ALS) inhibitor herbicide beckmannia syzigachne
  • Polymerase chain reaction (PCR) detection method and kit for anti-amyotrophic lateral sclerosis (ALS) inhibitor herbicide beckmannia syzigachne
  • Polymerase chain reaction (PCR) detection method and kit for anti-amyotrophic lateral sclerosis (ALS) inhibitor herbicide beckmannia syzigachne

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 is used to detect the synthesis of the PCR primers of anti-ALS inhibitor class herbicide Smilax

[0036]According to the ALS gene sequence of Alopecurus (Apera spica-venti (GenBank: JN646110.1), Alopecurus aequalis (GenBank: JQ743908.1) and Alopecurus aequalis (GenBank: JQ743908.1) The primers were designed for the conserved region of the ALS gene sequence in ), and obtained by segmental amplification, the nucleotide sequence is shown in Seq ID No.5), and the specific primer pair WC-ALS-IF / WC-ALS-IR and WC -ALS-ⅡF / WC-ALS-IIR is used to specifically amplify the ALS gene of Sagegrass, and the relative position of each primer on the ALS gene of Smilax is shown in figure 1 , the ALS fragment amplified by these two pairs of primers contains all 8 SNP sites currently found in other plants resistant to ALS inhibitor herbicides ( image 3 and Figure 4 ). The primer sequences are as follows:

[0037] Forward primer WC-ALS-IF: 5'-CGCCTTACCCAAACCTACT-3'(Seq ID No.1)...

Embodiment 2

[0042] Example 2 The PCR detection method of the anti-ALS inhibitor herbicide Smilax

[0043] Experimental material: Herbs with resistance and sensitivity.

[0044] experimental method:

[0045] 1. The preparation of the DNA of Saffron

[0046] Take 200 mg of fresh leaves of Smilax, grind with liquid nitrogen, and extract the DNA of each Smilax leaf by conventional CTAB method.

[0047] 2. Specific primers for detecting the ALS mutation site in Smilax chinensis, see Example 1 for the primer sequence.

[0048] 3. PCR reaction system for detection of ALS mutation site in Smilax

[0049] PCR reaction system, in which 10×PCR reaction buffer 2μL, 15mM MgCl 2 0.5 μL, 1 μL of 2.5 mM dNTPs, 0.5 μL each of 10 μM forward and reverse primers, 1 U of Taq DNA polymerase, 1 μL of DNA template, and sterilized double distilled water for the rest, the final volume is 20 μL.

[0050] 4. PCR amplification program for detecting the ALS mutation site in Smilax

[0051] Pre-denaturation at 94...

Embodiment 3

[0058] Embodiment 3PCR method detects the field anti-ALS inhibitor's Smilax

[0059] The detection method is as follows:

[0060] 1. Sample collection and DNA preparation

[0061] In order to verify the feasibility of the PCR detection method, we collected the Smilax plants in the farmland where resistance was suspected to occur, and after proper moisturizing treatment, they were mailed to the laboratory. Take 200 mg of fresh leaves of Smilax, grind with liquid nitrogen, and extract the DNA of each Smilax leaf by conventional CTAB method.

[0062] 2. Specific primers for detecting the ALS mutation site in Smilax chinensis, see Example 1 for the primer sequence.

[0063] 3. PCR reaction system for detection of ALS mutation site in Smilax

[0064] With embodiment 2.

[0065] 4. PCR amplification program for detecting the ALS mutation site in Smilax

[0066] With embodiment 2.

[0067] 5. Identification of PCR products

[0068] With embodiment 2.

[0069] 6. PCR product s...

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Abstract

The invention provides a specific polymerase chain reaction (PCR) primer combination for detecting an anti-amyotrophic lateral sclerosis (ALS) inhibitor herbicide beckmannia syzigachne. The combination comprises primer pairs (Seq ID No.1-4) for specific amplification of a beckmannia syzigachne ALS gene. The PCR detection method of adopting the primer combination has excellent specificity and sensitivity. The specificity test proves that the ALS gene of the beckmannia syzigachne can be amplified. The PCR method is simple and fast to operate, sampling detection can be carried out in the season on the basis of detection of nucleotide, the time from sampling to result just is 2-4 days, and rapid identification of a suspected anti-ALS inhibitor beckmannia syzigachne in a field and verification of a mutation site can be achieved by the sensitivity and the specificity, so as to guide scientific management of resistant weeds in production practice.

Description

technical field [0001] The invention relates to a detection technology for weeds resistant to herbicides, in particular to a PCR detection method and a kit for the resistance to ALS inhibitor herbicides, weeds. Background technique [0002] Acetolactate synthase (ALS for short), also known as acetohydroxyacid synthase (AHAS for short), is a biosynthetic process that induces valine, leucine, and isoleucine in plants and microorganisms. A key enzyme and an important herbicide target. The development and use of ALS inhibitor herbicides began in the early 1980s. Due to the obvious advantages of such herbicides, such as super high activity, low toxicity to humans and animals, and environmental friendliness, they have attracted widespread attention. At present, 50 Multiple varieties were developed and used. Since the 1980s, the middle and lower reaches of the Yangtze River in my country have successively promoted the use of chlorsulfuron, metsulfuron-methyl, iodosulfuron, acesul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2531/113
Inventor 崔海兰韩玉皎李香菊张宏军
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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