Establish a Quantitative Method and Its Kit for Detection of Escherichia Coli and Salmonella.

Spp
Author :DongLiNing
Tutor:CaoHui
School :Nanjing Agricultural College
CLC :S154.3
TYPE :Masters thesis
Download the PDF Full Text:http://www.topresearch.org/showinfo-153-665303-0.html
Year:2011
Abstract:
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 3505009507008001700 CFU per gram of
soil.