الفهرس 
Cover Page Title in EnglishOnly 14 pages are availabe for public view
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Abstract
Salmonella Enteritidis Considered one of the most important bacterial diseases affecting man and animals, several Salmonella Enteritidis considered studies were conducted to find out the best possible ways to diagnose of Salmonella Enteritidis, one of the most important one is PCR method enabling laboratories to make a rapid and reliable identification. In this study 3 reference bacterial isolates (Salmonella Enteritidis) were used in this study. The three isolates were confirmed by traditional methods. The three samples were passed in validation by operating one run of the three samples against each other by Real time and comparing results. Only one bacterial isolate was valid for carrying PCR (Reference strain of salmonella Enteritidis from Food hygiene Unit in Animal Health Research Institute, Dokki, Giza , Egypt). The valid sample subjected to ten folds serial dilution into two groups one spiked in meat samples (6 samples, at a concentration from 106CFU/ml to 101 CFU/ml) and others non spiked in meat and ten folds serial diluted ( crude samples) (9 samples a concentration from 109CFU/ml to 100CFU/ml). A total of 15 ten folds serial diluted samples both crud and spiked in meat samples were subjected to the study. The ten fold serial diluted valid samples were only both non spiked meat ten fold diluted groups and spiked meat ten fold serial diluted, firstly were subjected to DNA extraction By two different extraction kits one for non spiked in meat samples and others for spiked in meat samples. Designed two primers for S. Enteritidis were subjected to validation against reference primer for general Salmonella. Primer (SEFA139, SEFA 141) where validated only one of them (SefA (sefa2 and Sef a 4).Cyber green (bioline U.K) was successively against cyber green reference (Teingen Taiwan). The diluted samples were run by a protocol of PCR mentioned in material and methods in details, by both Convention and Real time PCR methods. All samples of non spiked ten fold diluted samples by Real time PCR gave a product curve (positive result ) but we cannot specify a two high concentrated samples (107 CFU/ml -108 CFU/ml). because of high dilution other product are conflict on the curve and make raising of melting point although Salmonella Enteritidis product were surely present but not a target ( so can not be dependent in diagnosis at this level of high dilution)In the other hand all sample of spiked meat samples by Real time PCR gave a product curve (Positive result) from (106CFU/ml to 101 CFU/ml).Highly concentrated samples resulted in other non specific product (may due to attach of primer with non specific region of gene with high concentration.).The tested cyber green is highly sensitive for detection of PCR product of S. Enteritidis from (108CFU/ml) until (100CFU/ml) in non spiked meat samples (Sensitivity until one colony)and less selective in high concentrated (From108CFU/ml) until (107CFU/ml) samples (Selectivity was not more than (106 CFU/ml).While the sensitivity of spiked meat was from 106CFU/ml to 101CFU/ml. from the beginning we can not obtain a concentration of 107CFU/ml, 108 CFU/ml and 100CFU/ml for spiked in meat sample because of CFU/ml from haemogezinates spiked in meat is different when obtained from a solid colony from non spiked one. So the sensitivity in spiked in meat ten fold diluted samples cannot exceed 106 CFU/ml, and not more than 101CFU/ml. Although non spiked ten fold diluted sample could obtained a dilution from 108 CFU/ml to 100CFU/ml. PCR product by Conventional PCR A. from non spiked in meat samples four samples ten fold serial diluted non spiked in meat from 108CFU/ml to-105CFU/ml) gave positive results by distinguished definite bands at 310 bp so highly selective product but after those dilution (104 CFU/ml to 100 CFU/ml) ten fold serial diluted non spiked in meat gave no product although surly that Salmonella Enteritidis product were present , but by low concentration can not be distinguished in gel (less sensitive).B. Ten fold serial diluted spiked in meat samples gave negative results although surly of presence of Salmonella Enteritidis product and this owing to presence of inhibitory substance in meat that may lead to interfere Conventional completely (.less sensitive). Real time PCR is highly sensitive as they give a products from 108 CFU/ml to 100 CFU/ml) but it gave other non specific product cannot be separated from each other. So it is highly sensitive and less specific. But in Conventional PCR .Obtained from non spiked meat samples four samples ten fold serial diluted non spiked meat from 108CFU/ml to-105CFU/ml) gave positive results by distinguished definite bands at 310 bp (SO it gives highly selective product) ,but after those dilution (104 CFU to 100 CFU) ten fold serial diluted non spiked in meat gave no product although , surly that Salmonella Enteritidis product are present by low concentration but can not be distinguished in gel (less sensitive).but highly specific this is showed in two sample (107CFU/ml-108 CFU/ml) where other non specific bands product are present but separately from target band (310bp).Real time PCR could detect all entire 6 samples in ten fold diluted meat samples from 106 CFU/ml to101 CFU/ml. The conventional fail to distinguish any product due to inhibitory effect of spiked meat.