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Double real-time fluorescence PCR identification method for indica-japonica subspecies rice based on chloroplast DNA difference

A technology of real-time fluorescence and japonica subspecies, applied in the direction of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of unidentification and difficulty, and achieve the effect of maintaining the order of the grain market

Inactive Publication Date: 2020-05-08
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the perspective of rice morphology, there is a certain difference between the two subspecies, but after processing into rice, it is difficult to accurately identify it. After the rice is crushed and processed into rice products, it is impossible to identify it from the morphology.

Method used

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  • Double real-time fluorescence PCR identification method for indica-japonica subspecies rice based on chloroplast DNA difference
  • Double real-time fluorescence PCR identification method for indica-japonica subspecies rice based on chloroplast DNA difference
  • Double real-time fluorescence PCR identification method for indica-japonica subspecies rice based on chloroplast DNA difference

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1 test material

[0023] Rice seeds were collected by our laboratory.

[0024] 2 main reagents

[0025] CTAB lysate [weigh 4.00 g CTAB, 16.38 g NaCl, 2.42 g Tris, 1.50 g Na 2 EDTA, dissolve with appropriate amount of water, adjust pH to 8.0, set volume to 200 mL, autoclave], CTAB precipitation solution [weigh 1.00 g CTAB, 0.50 gNaCl, dissolve with appropriate amount of water, adjust pH to 8.0, set volume to 200mL, autoclaved] self-preparation; Premix Ex Taq TM (Probe qPCR) is a product of Bao Biological Engineering (Dalian) Co., Ltd. (Dalian TaKaRa Company).

[0026] 3 main instruments and equipment

[0027] Grinding instrument, vortex instrument, water bath, desktop centrifuge, large refrigerated centrifuge, real-time fluorescent quantitative PCR instrument (US BIO-RAD company, CFX96).

[0028] 4 Primers, probes

[0029] According to the standard SN / T 2584-2010 "Real-time fluorescent PCR detection method of genetically modified components in rice and its products...

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Abstract

The invention provides a double real-time fluorescence PCR rapid identification method for indica-japonica subspecies rice based on chloroplast DNA sequence difference. On the basis of a pair of disclosed primers, a probe 5'-HEX-ATG CAA TAG AGA GCG AGT GG-BHQ1-3' is designed in the invention. The invention establishes the double real-time fluorescence PCR identification method for indica-japonicasubspecies rice by utilizing primers and probes which is used for real-time fluorescence PCR detection of endogenous gos9 genes of rice in published standards. Only one fluorescence amplification curve of the endogenous gos9 gene of the rice appears in the PCR amplification result of cpDNA-deleted indica rice; and two fluorescence amplification curves of the endogenous gos9 gene of the rice and the specific cpDNA of the japonica rice appears in the PCR amplification result of the cpDNA-not-deleted japonica rice at the same time, and the difference of the curves is used for effectively identifying the variety of the rice to be detected.

Description

technical field [0001] The invention relates to a double real-time fluorescence PCR rapid identification method for indica-japonica subspecies rice species based on chloroplast DNA (chloroplast DNA, cpDNA) differences. Background technique [0002] Rice is the staple food in most countries, it is also the staple food in southern my country, and it is also the main raw material of important processed food. There are two main subspecies, namely the indica rice (rice) subspecies ( Hsien rice, also known internationally Indica rice) and japonica rice (rice) subspecies ( Keng rice, also known internationally Japonica rice), due to the different types of rice, its commodity value is not the same, therefore, it is necessary to distinguish and identify. From the perspective of rice morphology, there is a certain difference between the two subspecies, but after processing into rice, it is difficult to accurately identify it. After the rice is crushed and processed into rice ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/13C12Q2600/16C12Q2561/113C12Q2563/107C12Q2537/143
Inventor 邵碧英陈文炳缪婷玉彭娟
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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