Development Of Extraction Kit About The Complex Soil Microbial DNA

In the process of PCR amplification for the detection of target microorganisms in soil samples,the most crucial prerequisite is the extraction of high-quality total DNA of the soil microbial genomic.However,the wide variety and relatively complex composition of soil samples,and the fact that microbial cells are often tightly adsorbed on the surface of soil particles,often lead to incomplete lysis of the bacterium and diffuse DNA bands during DNA extraction.Besides,a large amount of humic acid and other inhibitors present in the soil may remain in the DNA solution,resulting in poor purity of extracted DNA and inhibiting downstream PCR amplification.Though commercial kits from foreign brands such as MP have been available to solve these problems,the monopolistic position in the market for a long time and the high price increase the cost of research.To tackle this problem,in this article,a rapid,simple,economical,and efficient DNA extraction kit for complex soil microorganisms was developed,instead of imported brands.Some optimization was explored for PCR amplification of humic acid-containing inhibitors.1.Based on the characteristics of soil samples,a high yield and high purity soil microbial DNA extraction kit were developed.In comparison with three mainstream commercial kits（MP,Tiangen,and Omega）,the overall extraction performance is comparable to the imported MP Fast DNA^?Spin Kit for Soil,and in extracting some samples（dust and silt）the yield is better than Tiangen Biochemical’s Soil Genomic DNA Extraction Kit and Omega’s Soil DNA Small Volume Extraction Kit.The cost of the Soil DNA Extraction Kit developed in this study is only a quarter of the cost of the imported MP kits,hence very suitable for commercial application.2.Small amounts of contaminants such as humic acid that may be present in extracted soil microbial DNA,to eliminate the inhibitory effect on PCR amplification,a comparison of human serum albumin（HSA）,equine serum albumin（ESA）,bovine serum albumin（BSA）,chicken serum albumin（CSA）and porcine serum albumin（PSA）in enhancing PCR resistance to inhibition was investigated.The results showed that all five serum albumins enhanced PCR resistance to inhibition.HSA showed the strongest resistance to humic acid,requiring only 1ng/μL in the PCR system to remove the inhibition caused by 2 ng/μL of humic acid,while the minimum concentrations required to remove the same humic acid inhibition were 100 ng/μL,100 ng/μL,10 ng/μL,1μg/μL for ESA,PSA,BSA and CSA,respectively.The stability test results proved that only HSA had the ability to resist humic acid inhibition after being stored at37℃for 21 days,and the stability was higher than that of BSA（14 days）,PSA（14 days）,CSA（7 days）and ESA（7 days）.Therefore,HSA can replace the traditional optimizer BSA as a new optimizer to enhance the inhibition resistance of PCR.3.Apart from the five serum albumins mentioned above,the optimization of PCR by eight animal-derived proteins was further explored,namely IgG,Histone,Mucoprotein,Hemoglobin,Trypsin,Actin,Avidin,and Collagen.The test results demonstrated that only IgG and Mucoprotein had enhanced PCR resistance inhibition at concentrations of 10 ng/μL and above（≥10 ng/μL）,and the remaining six proteins were not found effective in optimizing PCR.On this basis,we also developed a composite protein additive that can greatly enhance PCR inhibition resistance.The mixture of HSA,IgG,and Mucoprotein at equal concentrations（20μg/μL）can resist humic acid interference up to 6 ng/μL,providing a favorable solution to the problem of PCR amplification inhibition by residual humic acid in soil DNA solution.