Internal-standard-based apple scar skin viroid RT-PCR (reverse transcription-polymerase chain reaction) detection technique

Abstract

The invention relates to an internal-standard-based apple scar skin viroid RT-PCR (reverse transcription-polymerase chain reaction) detection technique. The technique comprises the following steps: by using a 0-5cm cortical tissue on the top end of an apple annual branch with leaf buds as a test piece, extracting total tissue RNA (ribonucleic acid) by an RNA extraction improved process; by using the total RNA as a template, carrying out RT-PCR reaction by using apple scar skin viroid specific primers and apple endogenous gene primers; and judging whether the apple tissue has apple scar skin viroid according to the existence and quantity of the amplification product in agarose gel electrophoresis. The judgment standards are as follows: no existence of amplified endogenous gene specific segments indicates an aborted experiment; the existence of amplified endogenous gene specific segments indicates an effective experiment; the existence of amplified endogenous gene specific segments and apple scar skin viroid specific segments indicates that the apple tissue has the apple scar skin viroid; and the existence of amplified endogenous gene specific segments and no existence of amplified apple scar skin viroid specific segments indicate that the tissue does not have the apple scar skin viroid.

Description

technical field [0001] The invention relates to an internal standard-based RT-PCR detection technology for apple rust virus. Background technique [0002] Apple rust disease, also known as flower face disease or fruit cracking disease, is one of the non-occult viruses that are more harmful to apples. It is also an important quarantine target for apple viruses in China. It is widely distributed in apple-producing areas in Northeast, Northwest, and North China. The rate of diseased plants in serious orchards is as high as 30%, and there is a tendency to expand and spread; under the current technical level, there is no effective control agent, and the use of virus-free methods to propagate virus-free seedlings is currently the most effective control method, which is accurate, sensitive and fast Advanced detection technology is the key link and basic guarantee for the non-toxic cultivation of fruit trees; however, due to the low content of the virus in the tree, uneven distribu...

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Problems solved by technology

[0002] Apple rust disease, also known as flower face disease or fruit cracking disease, is one of the non-occult viruses that do serious damage to apples, and it is also an important quarantine target for apple viruses in China , are widely distributed in the northeast, northwest, and north China apple producing areas, and the rate of diseased plants in orchards with severe disease is as high as 30%, and there is a tendency to expand and spread; under the current technical level, there is no effective control agent, and the virus-free method is used to reproduce without Virus seedlings are currently the most effective means of prevention and control. Accurate, sensitive and rapid detection technology is the key link and basic guarantee for the non-toxic cultivation of fruit trees; It is rich in polysaccharides, polyphenols and other characteristics, and it is prone to false negatives in the detection process. At present, there are reports on ASSVd PCR detection both at home and abroad, but there are no reports on the ASSVd RT-PCR detection technology based on internal standards; the present invention Developed RT-PCR detection technology for apple rust viroid containing nad 5 internal standard gene, which successfully overcomes the false negative phenomenon and lays the foundation for accurate detection of the virus and efficient breeding of non-toxic seedlings. To provide a theoretical basis for the effective control of apple virus diseases in the future

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