Method for identifying homozygotic type or heterozygous type NK603 on basis of digital PCR (polymerase chain reaction)

Abstract

The invention discloses a method for identifying homozygotic type or heterozygous type NK603 on the basis of digital PCR (polymerase chain reaction), and relates to the technical field of molecular biology and transgenic test. The method comprises steps as follows: 1), collection: NK603 specificity transformation events verified by European Union and a corn internal standard gene adh1 quantitative determination method are adopted; 2), digital PCR amplification: a DNA (deoxyribonucleic acid) of NK603 is taken as a research template; 3), data analysis: if 95% of confidence intervals of target molecular number calculated values of NK603 specificity transformation events and corn internal standard genes adh1are overlapped, NK603 is judged as homozygotic type NK603; and if two times of 95% of confidence intervals of target molecular number calculated values of NK603 specificity transformation events and 95% of confidence intervals corn internal standard genes adh1are overlapped, NK603 is judged as heterozygous type NK603. The method can rapidly, accurately and reliably identify absolute quantification of homozygotic type and heterozygous type herbicide-resistant genetically-modified corn strain NK603.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and transgene detection, in particular to a method for identifying homozygous or heterozygous NK603 based on digital PCR. Background technique [0002] In recent years, genetically modified crops such as corn, soybean, cotton, rapeseed and tomato have been approved for planting and production in many countries. As of 2013, there are 27 crops worldwide involving 336 transformants in 36 different countries and regions Approved to be planted or used as raw materials for processing, the planted area has reached 175 million hectares, of which 27% are planted with gene stacked varieties including multiple transformation events (James, 2013, International Agricultural Biotechnology Application Service Organization). With the increasing number of genetically modified organisms, it brings more and more challenges to the safety supervision of genetically modified organisms in various countries. ...

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Problems solved by technology

At the same time, studies have found that using traditional breeding methods requires many years of self-crossing to obtain homozygous transgenic materials, which is time-consuming, time-consuming and laborious; and the real-time fluorescent quantitative PCR method itself has a large error, and the homozygosity identification of transgenic crops has a large error. It is relatively large, which brings great difficulties to the judgment of the results

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