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Development of SYBR Green Real time PCR for identification methicillin-resistance Staphylococcus aureus (MRSA)
الكشف عن البكتريا العنقودية الذهبية المقاومة للمثسلين Methicillin-Resistant Staphylococcus aureus باستخدام طريقة التضاعف التسلسلي المزدوج SYBR Green Real time PCR

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Abstract

The increasing resistance of staphylococci to ß -lactam antibiotics has become a major clinical problem. Development of rapid and sensitive techniques for detection of MRSA is an important aim for public health. A duplex PCR were established for specific identification of methicillin-resistance Staphylococcus aureus (MRSA) in clinical samples. In this work a duplex SYBR Green real time PCR was developed for rapid identification of MRSA in local methicillin-resistance S. aureus isolates. Twenty methicillin-resistance S. aureus isolates, as determined by disc diffusion method, were subjected to DNA extraction and PCR amplification. Two genes were amplified successfully, mecA (533bp) and femA (314bp), as targets for methicillin-resistance and specific identification of S. aureus, respectively using conventional PCR. Sensitivity of the duplex PCR showed that the minimum concentration of DNA that gave positive results for the two genes was 30ng/µl. In order to develop rapid and sensitive test for identification of MRSA, serial dilutions of purified DNA were amplified gradually according to their concentrations using SYBR Green real time PCR. These results indicated that the SYBR Green real time PCR can be used for identification of methicillin-resistance S. aureus (MRSA) in clinical

تعد صفة المقاومة لدى بكتريا الـS. aureus لمضادات البيتا لاكتام من المشاكل الطبية المنتشرة في العالم.لذلك يعد تشخيص بكتريا S.aureus المقاومة للمثسيلين من خلال تطوير طريقة سريعة و حساسة لهذا التشخيص من اهم الاهداف للصحة العامة . استخدمت طريقة التضاعف التسلسلي المزدوج لعينات البكتريا السريرية ، حيث تم تطوير طريقة الـReal-Time PCR ببادئات مزدوجة للتشخيص السريع لعينات البكتريا المحلية . استخدمت عشرون عزلة بعد تشخيصها بواسطة الطريقة التقليدية ( disk diffusion method) وبعد ان تم استخلاص الدنا منها و اجراء عملية التضاعف التسلسلي لها باستخدام بادئان بنجاح احدهما متخصص لصفة المقاومه mecA بحجم 533 قاعدة نايتروجينية و اخر متخصص لتعريف هذا النوع femA بحجم 318 قاعدة نايتروجينية . ان حساسية هذا الاختبار قد وصلت الى تركيز 30مايكرو غرام /مل . ولغرض ايجاد طريقة سريعة وحساسة في الوقت نفسه للتعرف على هذه البكتريا ، اجريت عدة تخافيف متسلسلة على الدنا المستخلص وتم اجراء فحص التضاعف التسلسلي بطريقه الـ Real-Time باستخدام صبغة سايبر الخضراء . واثبتت النتائج امكانية استخدام هذه الطريقة لتعريف البكتريا المقاومة للمثيسلين في العينات السريرية .


Article
Molecular Study of Virulence Genes of Staphylococcus aureus Isolates from Various Clinical Origins by PCR

Authors: Zahraa Mohammed Al-Taee --- Ghazi Munem Aziz --- Ali Hussein Al-Marzoqi --- Lena Fadhil Hamza
Journal: Medical Journal of Babylon مجلة بابل الطبية ISSN: 1812156X 23126760 Year: 2015 Volume: 12 Issue: 3 Pages: 677-688
Publisher: Babylon University جامعة بابل

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Abstract

Staphylococci pathogen was blamable for a huge scale of infections in human because of their invention of secreted and other cell-surface related virulence factors that regulate by various genes. This study was carrying out to realize the frequency of Staphylococcus species in Hillah city using specific genes for that purpose.PCR technique was applied to amplify the staphylococcal virulence genes like sea, see, eta, tst, coa, nuc, seb, femA, mecA, etb, cna, fib and fnbA, and they determined either multiplex or uniplex PCRs depending on the sizes of the PCR products amplicons.Out of 229 collected samples from different clinical sources, only 100Staphylococcus aureus (43.7%) were revealed. According to phenotypic and biochemical tests, the isolates can be distributed as follow; Urine 22 (9.6%), Pus 19 (8.3 %), Sputum 12 (5.2%), CSF 4 (1.7%), Blood 32 (14%), Pericardial fluid 9 (3.9%) and Peritoneal fluid 2 (0.9%). From the 13 examined genes, most abundant gene was fib (27 cases) followed by femA (25 cases). While tst gene reported as the least frequently detected gene. S. aureus isolated from Blood infection formed the highest ratio among genes reveals in this study (194; 38.6%) followed by pus infections isolates (120; 23.9%), urine infections isolates (102; 20.3%), sputum isolates (32; 6.4%), Pericardial fluid (28; 5.6%), CSF (19; 3.8%) and finally Peritoneal fluid (8; 1.6%).

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