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.