| Soil plays important for human, it bears about 90% pollutants from environment. Requirements of soil quality from people is focused on pesticide residues, heavy metals and organic pollution. The study of pathogenic microorganisms in soil is not much concerned. Pathogenic microorganisms in the soil can enter surface or within the organization vegetables in a certain way, contaminate vegetables, and then endanger human health. Pathogen of vegetables mainly comes from soil contaminated by feces, including Escherichia.coli and Salmonella. Nowdays pathogens is mainly detected by traditional culture methods, time-consuming and labor-intensive operations. This way requires 5 to 6 days to complete.and its sensitivity of detection is relatively low. Recently, as immunology and molecular biology developing, the new pathogen detection methods have been established, especially abroad some production is being used, however, in domestic it is in the exploratory stage.To establish an effective method for quantifying detection of pathogenic microorgani-sms on the basis of a large number of reference, we use a MPN-PCR approach to quantify detection of pathogenic microorganisms. The key idea of this method is the use of PCR method to determine the positive results, instead of traditional method, which is determined by biochemical identification and serological identification. Then we can reduce the tedious steps in national standard method to improve the detection efficiency. Two pairs of specific primers were designed according to the gene phoA of Escherichia coli and the gene invA of Salmonella spp. We optimize the method of PCR template preparation, establish a quantitative method of detection of pathogenic microorganisms and assesse its sensitivity. Finally, on this basis a quantitative detection kit was developed. The results are the following:1,Used Escherichia coli and Salmonella.spp as an research object, we conducted a regression analysis on the same sample using MPN count method and plate count method. we establish a quantitative relationship between the logarithm of this two method. Escherichia coli:Regression equation is y=0.9566x. R2=0.9941; Salmonella.spp: Regression equation is y=1.0144x, R2=0.9957. The variance analysis of regression coefficients showed significant. the MPN count method has the same accuracy with the plate count method. So as to pathogenic microorganisms, even functional microbial counts, this provide more accurate quantitative results.2,we designe specific primers (invA-1 and invA-2) to detecte salmonella spp according to lots of references. and another specific primers(phoA-1 and phoA-2)was designed to detecte Escherichia coli, the target gene fragment amplified by PCR respectively was 284bp and 684bp. We compare three different methods of PCR template preparation, a pyrolysis method was used as PCR template preparation in the end. The sensitivity of quantitative detection methods was assessed by adding pure culture to sterile soil. The sensitivity of quantitative detection for Escherichia coli was 25CFU per gram of soil,and Salmonella spp was 250CFU per gram of soil.3,On the basis of estabilishing the quantitative detection methods, we further develop a MPN-PCR quantitative detection kit, and assess its sensitivity and shelf. The sensitivity of quantitative detection for Escherichia coli is 25CFU per gram of soil, and salmonella spp is 250CFU per gram of soil. The shelf are both 3 months.4,Using developed quantitative detection kit, we detect 13 samples from zhangjiagang and beijing, The results show Salmonella spp does not exist and six samples have Escherichia coli, the numerber was respectively 350,500,950,700,800,1700 CFU per gram of soil. |
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