[Efficacy of RT-PCR for the detection of E. coli in drinking water]

Escherichia coli [E. coli] is an indicator of potential human fecal contamination. Polymerase Chain Reaction [PCR] as an ideal detection method for detecting E. coli has some advantages like rapidity, high sensitivity and accuracy, easy performance, ability to run in high numbers and inexpensiveness. On the other hand, the disadvantage of PCR is possibility of its false positive results. In this study, reverse transcriptase polymerase chain reaction [RT-PCR] method was used to overcome the problem, and the results were compared to most probable number [MPN]. 16srRNA forward [SF] and 16srRNA reverse [SR] primers were designed using E. coli 16srRNA sequence. After preparing different diluted samples of E. coli in distilled water, the bacteria were separated by FHLP and HAWP filters and its 16srRNA was propagated using mentioned primers. To confirm the sensitivity of the RTPCR method compared to MPN one, samples obtained from 15 water sources in Arak city were examined. The number of bacteria in dilutions were confirmed with culture. RT-PCR data showed that FHLP compared to HAWP filters have a higher capability in separating of bacteria in different dilutions. Also there was a higher sensitivity of RT-PCR compared to RT-PCR and MPN. RT-PCR can detect the bacteria in lower dilutions of bacterial suspension. Hydrophobic filters [e.g. FHLP] compared to hydrophilic filters [e.g. HAWP] have higher capability in separating bacteria. To detect all coliform bacteria RT-PCR amplifications achieved by cells concentrated with hydrophobic filters are recommended

Improvement of PCR in detection of coliform in water pollution

In molecular diagnosis of microbial agent, purification of chromosome is very important step. In this study, after cell destruction, DNA replication was done by increasing the denaturation time, without DNA purification. In this experimental study eight different dilution of E.coli [8/100, 4/100, 2/100, 1/100, 1/200, 1/400, 1/800 and 1/1600] solution were madce in D.W, Bacteria were separated by filtration. Polymerase chain reaction method was used to propagate 162 rRNA gene by design primers without DNA Purification. In order to confirme sensitivity of PCR, contamination of 15 different sources of Arak well water wafer was compared by MPN method. For confirmed sensitivity of PCR, 15 sources of water in Arak were examined and compared with MPN method. Present of bacteria in diution soution were confirmed by culture. Polymerale Chain reaction [PCR] data were shown this method is able to recognize bacteria in above dilutions after filtration. This study showed high sensitivity of PCR method in compare to MPN method. Results were shown without stages of extraction of DNA, PCR were done without losing chromosome. Therefore false negative results were decrease and avoided from difficult phases

[Expression of recombinant streptolysin O by escherichia coli]

Streptolysin O is an antigenic protein that is secreted by Streptococcus pyogenes. Streptococcal infections are diagnosed with anti streptolysin O. At present, streptolysin O is produced by vectors that have fusion protein. In this study we produced streptolysin O without fusion protein vectors. In this experimental study, we amplified Streptolysin O gene by Polymerase chain reaction [PCR] method and subcloned it to prokaryotic expression vector pET28a. Escherichia coli BL21-DE3-plySs were transformed with pET28a-SLO and gene expression was induced by IPTG. Then it was purified by Ni-NTA kit. The concentration of SLO was assayed by Bradford method. To confirm recombinant SLO Western Blot was used. The sequencing result was confirmed by Sanger method and was the same as SLO gene. Escherichia coli BL21 [DE3] pLysS was transformed with pET28a-SLO and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography by Ni-NTA resin. The concentration of purified protein was 100microg/ml. The integrity of product was confirmed by Western Blot analysis using a mouse anti streptolysin O. Our data showed that recombinant SLO protein can be produced by pET28a in Escherichia coli. This protein maintains its antigenic effect very well. Therefore, recombinant SLO has same epitopes with natural form of this antigen

[Specificity of recombinant P39 protein in detection of anti-brucella antibody in patients with brucellosis]

In many studies, immunogenicity of Brucella proteins such as P39 in animals is investigated. In this study, we evaluated antigenicity of recombinant P39 from Brucella abortus in patients with Brucellosis. In this experimental study, at first recombinant P39 was produced in Escherichia coli. Sera reactivity of six infected individuals against the recombinant P39 protein was analysed by Western Blot. Data indicated that P39 protein from Brucella abortus was recognized by patients' sera antibodies. Our data showed that recombinant P39 protein can be detected as an antigen by sera in infected human. Therefore, recombinant P39 have same epitopes with natural form of this antigen

[Detection of helicobacter pylori-specific genes in the oral yeast]

Until today human stomach is the only recognized habitat of H. pylori. However, recruitment of DNA-based methods has made possible the detection of H. pylori in water and oral cavity, thus suggesting faecal-oral and oral-oral routes for transmission of H. pylori, respectively. In this study yeast has been proposed as a common vector for transmission of H. pylori. Thus designed primers were recruited to target 16S rDNA and cagA genes in the oral yeasts by PCR. Eighteen yeasts were examined microscopically for the presence of bacterial-like bodies. DNAs were extracted from oral yeasts using phenol-chloroform method. Amplification conditions were optimized as 33 cycles and annealing temperatures of 63°C for 16S rDNA and 51°C and 52°C for cagA gene which was targetted in two steps. DNAs of H. pylori and saccharomyces cerevisiae were used as controls. PCR products of two genes from one yeast and from H. pylori were cloned in pCAP and subsequently subcloned in pSK+ and sequenced. Bacterial-like bodies were observed in all oral yeasts. The amplified products of 16S rDNA from all oral yeasts were homologous in size with those of H. pylori. 15/18 [83%] yeasts contained cagA gene, homologous to H. pylori. CagA was not amplified from three yeasts and S. cerevisiae. Analysis of sequenced products of 16S rDNA and cagA from one oral yeast showed 98% homology with those of H. pylori. The presence of H. pylori inside the yeast was indicated by light microscopy and PCR. It appears that yeasts which are abundant in nature and thrive the mucosal surfaces of human might serve as reservoirs and vehicles of H. pylori