Two step genome comparison method for designing quantitative PCR specific primer of guizhouense NJAU 4742

Abstract

The invention discloses a two step genome comparison method for designing a quantitative PCR specific primer of guizhouense NJAU 4742. At first, full genome sequence of NJAU 4742 is compared in NCBI database and JGI database; genome segments, which are not matched and greater than 500 bp, are picked out; then the screened segments are compared with the full genome of a guizhouense strain 916; anda primer sequence with unique bases is designed. At the same time, three pairs of primers, which are prepared by inserting labeled segments into a gfp labeled strain (gfp-NJAU 4742), are used to examine the specificity of the primers designed based on the genome. By examining the amplification specificity of strains of same genus and different species, re-detecting the soil addition, and actuallydetecting the potting soil, an optimal quantitative PCR primer is screened out. The provided primer design method and design strain specific primer can be applied to quantitative PCR specific primer design of other strains and quantifying of guizhouense NJAU 4742.

Description

technical field [0001] The invention belongs to the field of agricultural microorganisms, and relates to the design and application of specific primers for the quantitative PCR technology of a Guizhou Trichoderma NJAU 4742. Background technique [0002] In the past ten years, a large number of functional microbial agents have been directly inoculated into the soil or combined with organic fertilizers as soil improvers in the study of beneficial growth of target plants. Therefore, the survival of the inoculant strain in the new environment plays a key role in its functional performance, as efficient colonization is the fundamental prerequisite for the functional strain to stimulate plant growth or induce beneficial effects on the soil microbial ecosystem. [0003] Traditionally, selective media are mostly used to detect or isolate beneficial growth-promoting bacteria in the environment. However, this method has low sensitivity and time-consuming, and it is difficult to accura...

AI Technical Summary

Problems solved by technology

The currently commonly used primer design must first find a specific sequence (the usual practice is to obtain the species-specific gene from the literature, and determine its generality according to its conservative range), however, the so-called " "Species-specific genes" often have relatively lagging information and have not been updated based on the ever-increasing genome data, resulting in the fact that the primers obtained based on the gene sequence may not be able to ensure their specificity in practical applications

Primers were designed for Trichoderma based on the internal transcriptome spacer (ITS) shared by Trichoderma and detected and quantified in different soils; however, this was not specific enough for Trichoderma species discrimination

Some researchers have also used SCAR marker technology to accurately identify and monitor species. However, this molecular method is based on the analysis of RAPD sequences, which is a very time-consuming and laborious process.

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