Detection of sea, seb, sec, seq genes in staphylococcus aureus isolated from nasal carriers in Tehran province, Iran; by multiplex PCR

Staphylococcus[S.] aureus produces different extra-cellular protein toxins and virulence factors. One of the most important extra-cellular proteins is an enterotoxin which causes staphylococcal food poisoning [SFP] due to their enterotoxins. Different methods have been used to detect this toxin, each of which has advantages and disadvantages. DNA amplification methods, however, can show the presence of enterotoxigenic strains of S. aureus before the expression of enterotoxins on the basis of specific gene sequences. In this study, 150 S. aureus strains isolated from nasal carriers were confirmed by biochemical testing. PCR was used to amplify the staphylococcal enterotoxin A, B, C and Q genes, as well as the staphylococcal nuclease gene. Among the 150 healthy human isolates from the nasal carrier, 95 were confirmed as S. aureus. Only 58.9% of the isolates were diagnosed as sea, b, c, q positive. There were 24 [25.3%] isolates associated with the sea gene, 15.8% isolates associated with the seb gene, 9.5% of the isolates were associated with the sec gene, and 8.4% of the isolates associated with the seq gene. Of these isolates, 41% might be possessing additional se genes but they were not see [178 bp] and sed [319 bp] genes. The nuc gene, which encodes thermo nuclease, was used as a target DNA to identify S. aureus. Additionally, one of these enterotoxigenic isolates carried more than one toxin gene

Cloning and Expression of N-terminal Region of IpaD from Shigella dysenteriae in E. coli

Genus Shigella is one of the important members of the family Enterobacteriaceae. There are numerous antigens in Shigella carrying by a 220 kb plasmid. Among them, IpaD is the key virulence factor of S. flexneri. Apart from having effectors function that is essential for host cell invasion and intracellular survival, this protein also controls the secretion and translocation of other effector proteins into eukaryotic host cells. In the present study, we have cloned and expressed the ipaD in E. coli. The ipaD gene was amplified by PCR. Prokaryote expression vector pET-28a [+] - ipaD was constructed, and used to transform E. coli BL21DE3 plySs. The expression of recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot were used to determine immunoreactivity of IpaD-His by a rabbit monoclonal antibodies against his-tag. SDS-PAGE demonstrated that the constructed prokaryotic expression efficiently produced IpaD at the 1 mmol/L of IPTG. IpaD protein was able to react with the rabbit monoclonal antibody against His-tag. IpaD is essential for Shigella spp invasion. N-terminal region is most significant functional fragment of IpaD. Purification of IpaD from the wild type of Shigella is difficult furthermore profound study on a specific domain on the N-terminal of IpaD by using the wild type of purified IpaD is not feasible

Simultaneous molecular detection of Neisseria Meningitidis and Haemophilus Influenzae

Bacterial meningitis is a dangerous and sometimes fatal infection that affects the central nervous system. Because some antibiotics can prevent some types of these Bacteria and suppress them from spreading and infecting, therefore it is important to know what type of virus or bacterium is causing meningitis. Haemophilus influenzae and Neisseria meningitides are the two main pathogens causing acute bacterial meningitis. Different methods are used for the detection of H. influenzae and N. meningitidis but they are of low sensitivity, taking long time and difficult to perform . Therefore, complementary methods are necessary for more sensitive detection of these agents. In this study, a muliplex polymerase chain reation [mPCR] assay was developed for detection of H. influenzae and N. meningitidis. These strains were confirmed by biochemical methods. Two specific primer pairs were designed for lic-1 and opa genes of H. influenzae and N. meningitidis respectively. DNA amplification product fragments were 150 bp and 320 bp for H. influenzae and N. meningitidis, respectively. Streptococcus pneumoniae used as a negative control and did not yield a PCR product. The result of this study indicated that PCR is a useful complementary diagnostic technique, especially when Gram stain, culture, or antigenic detection is negative or inconclusive

[Detection of staphylococcus aureus enterotoxin type C isolated from healthy carriers by PCR]

Staphylococcus aureus is a pathogen which can cause food poisoning under certain conditions though growth in nutrients and producing enterotoxin. Only some strains are capable of producing enterotoxin and causing food poisoning and their presence can be detected by DNA amplification and gene sequence specification. Therefore, this research was conducted to detect type C enterotoxinproducing staphylococcus aureus. 95 staphylococcus aureus strains were isolated from 150 nasal carriers using sterilized swabs and were confirmed by biochemical tests. Then primers were designed and the PCR was used to amplify amplify the staphylococcal enterotoxin C gene [sec] in order to detect type C enterotoxogenic strains. DNA amplification fragments of 397 bp for staphylococcal nuclease and those of 271 bp for type C gene were confirmed by enzymatic digestion. Only 9.5% of the isolated strains contained sec gene. Specificity and sensitivity were also evaluated and its sensitivity was found to be 125 cells. This technique is a rapid, sensitive, specific, inexpensive and different alternative to conventional biochemical and serologic assays and it can be used to detect the agent producing type C staphylococcal enterotoxin