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Positive standard molecule for detecting diaporthe phaseolorum (Cooke etEll) Sacc. var. caulivora Athow et Caldwell (DPC) and diaporthe phaseolorum (Cooke etEll) Sacc. var. meridionalis F.A.Fernandex (DPM) as well as preparation method and detection method thereof

A technology of stem canker and positive standard, which is applied in the field of positive standard molecules for detection of soybean stem canker, can solve the problem that the stability of genomic DNA cannot meet long-term and frequent use, etc., and achieves high copy number, strong specificity and good quality. Effects of uniformity and stability

Inactive Publication Date: 2012-03-14
SHENZHEN AUDAQUE DATA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The stability of the prepared genomic DNA can not meet the needs of long-term and frequent use

Method used

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  • Positive standard molecule for detecting diaporthe phaseolorum (Cooke etEll) Sacc. var. caulivora Athow et Caldwell (DPC) and diaporthe phaseolorum (Cooke etEll) Sacc. var. meridionalis F.A.Fernandex (DPM) as well as preparation method and detection method thereof
  • Positive standard molecule for detecting diaporthe phaseolorum (Cooke etEll) Sacc. var. caulivora Athow et Caldwell (DPC) and diaporthe phaseolorum (Cooke etEll) Sacc. var. meridionalis F.A.Fernandex (DPM) as well as preparation method and detection method thereof
  • Positive standard molecule for detecting diaporthe phaseolorum (Cooke etEll) Sacc. var. caulivora Athow et Caldwell (DPC) and diaporthe phaseolorum (Cooke etEll) Sacc. var. meridionalis F.A.Fernandex (DPM) as well as preparation method and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Construction of standard molecules

[0062] 1. Experimental reagents

[0063] Restriction enzymes XbaI, KpnI and restriction enzyme buffer, pMD18-T vector, T4 DNA ligase and its buffer, dNTPs, Taq DNA polymerase and its buffer, DL2000Marker, Agarose Gel DNA Purification Kit and MiniBEST Plasmid Purification Kit was purchased from Dalian Bao Biological Engineering Co., Ltd. DH5α competent cells were purchased from Beijing Tiangen Biological Company. TaqMan probes and primers were synthesized by Shanghai Chaoshi Biotechnology Co., Ltd.

[0064] Other biochemical reagents are imported aliquots or domestically produced analytical grades.

[0065] Two, experimental equipment

[0066] DY-8 nucleic acid electrophoresis instrument (Beijing Liuyi Instrument Co., Ltd.), Biometra Grident PCR instrument, Syngene gel imaging system, other instruments include: centrifuge, constant temperature water bath, incubator, balance.

[0067] 3. Test method and process

[0068] 1. Search for...

Embodiment 2

[0087] Example 2: Evaluation of molecular performance of standard plasmids

[0088] 1. Experimental reagents

[0089] Universal Real-time Master Mix was purchased from ABI Company.

[0090] The primers and probes were synthesized by Shanghai Chaoshi Biotechnology Co., Ltd.

[0091] Two, experimental equipment

[0092] ABI 7700 real-time fluorescent PCR machine.

[0093] 3. Test method and process and results

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Abstract

The invention discloses a positive standard molecule for detecting diaporthe phaseolorum (Cooke etEll) Sacc. var. caulivora Athow et Caldwell (DPC) and diaporthe phaseolorum (Cooke etEll) Sacc. var. meridionalis F.A.Fernandex (DPM). The positive standard molecule is a plasmid molecule capable of autonomously replicating, and a plasmid contains a deoxyribose nucleic acid (DNA) sequence of a riboseinternal transcribed spacer of the DPC and the DPM. The invention further discloses a preparation method and a detection method of the positive standard molecule. The positive standard molecule constructed in the invention can be used for replacing positive DNAs of the DPC and the DPM, and the standard plasmid molecule is suitable for analyzing and detecting the DPC and the DPM through qualitative polymerase chain reaction (PCR) and quantitative PCR. The standard molecule is suitable for being used in the port inspection and quarantine department, the agricultural production department, the plant protection department and the like.

Description

Technical field [0001] The invention relates to a standard molecule for detection in the field of bioengineering, in particular to a positive standard molecule for detection of soybean stem canker, a preparation method and a detection method thereof. Background technique [0002] Soybean (Glycine max) is an important oil crop and food crop in my country, as well as the world's main source of edible oil and vegetable protein, and it occupies an important position in my country's national economy. At present, my country's soybean output ranks fourth in the world, but it still cannot meet the needs of domestic vegetable oil and feed protein processing. As the number of imported soybeans in my country increases year by year, the risk of the introduction of various diseases that harm soybean production is also gradually increasing. [0003] Soybean Stem Canker (SSC) is an important disease in soybean production. The pathogenic bacteria are Diaporthe phaseolorum (Cooke et Ell) Sacc.var....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/11C12Q1/68C12Q1/04G01N21/64
Inventor 章桂明缪建锟王颖程颖慧向才玉
Owner SHENZHEN AUDAQUE DATA TECH
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