Isothermal amplification method for detecting cry1Ac-transfected sugarcane

Abstract

The invention relates to an isothermal amplification method for detecting cry1Ac-transfected sugarcane. The method comprises the steps of extracting sugarcane template DNA (Deoxyribonucleic Acid), establishing an isothermal amplification system and identifying an isothermal amplification product. According to the isothermal amplification method for detecting the cry1Ac-transfected sugarcane, four specific primers are designed aiming at base sequences of an exogenous target gene cry1Ac of stem-borer-resistant transgenic sugarcane, and a chain-displacement amplification reaction is carried out under the action of polymerase Bst, so that the specificity is high; meanwhile, the amplification reaction can be completed by only a water bath kettle and a normal-temperature low-speed centrifuge capable of completing instant centrifugation, so that instruments and equipment required are simple, the amplification cost is relatively lower compared with that of the conventional PCR (Polymerase Chain Reaction) technology, which needs a gel scanning system and a PCR (or real-time PCR) instrument, which are expensive, and the like, the amplification time is short, the amplification efficiency is high, and the result viewing is visual and convenient due to visual color reaction; and after the system is established, extracted DNA of a sugarcane genome directly serves as a template, so that the method is applicable to the cry1Ac-transfected sugarcane screening of laboratories and fields and the detection and tracking of cry1Ac ingredients and has the advantages of low cost, rapidness, sensitivity, simplicity and accuracy.

Description

technical field [0001] The invention relates to a method for detecting a plant transgene, in particular to a method for detecting a transgene cry1Ac The invention relates to a constant temperature amplification method of genetic sugarcane, which belongs to the field of biotechnology. Background technique [0002] Sugarcane borer, belonging to the order Lepidoptera of Insecta, is a common and serious type of borer pest that harms sugarcane. There are many kinds of borers, there are 65 kinds of sugarcane borers in the world, and five kinds are common in my country, namely the barborer ( Chilo venosatus ), two point borer ( C. infuscatellus ) and white borer ( Scirpopophaga excerpalis ); the yellow borer ( Aryroploce schistaceana ); S. inferens ). Sugarcane is originally produced in the tropics. After domestication, it is mainly planted in Guangxi, Yunnan, Fujian and other places in my country. Its processed product sucrose accounts for about 92% of my country's tota...

AI Technical Summary

Problems solved by technology

The nucleic acid level detection methods mainly include PCR method, Southern hybridization and dot blot method, etc. Among them, PCR method is widely used because of its mature technology, rapid amplification, strong specificity, high sensitivity, and low requirement for template purity. , which is also the preferred technology of the transgenic detection laboratories of the Ministry of Agriculture in my country. However, conventional PCR detection methods require expensive PCR amplification instruments in the target sequence amplification process, and expensive gel scanning imaging systems are required for the detection and judgment of amplified products. The picture is collected, and the embedded chromogenic agent EB commonly used in electrophoresis detection is a carcinogen, etc., which has certain limitations for rapid detection and detection at the grassroots level such as test stations.

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