ارزیابی تشخیص سریع و بررسی حضور ژن‌های حدت اپرون‌ spv در جدایه‌های سالمونلا با استفاده از روش‌های مولکولی simplexPCR و multiplexPCR

نوع مقاله : مقاله علمی پژوهشی

نویسندگان

1 دانش‌آموخته کارشناسی ارشد رشته ژنتیک، دانشکده علوم پایه، واحد زنجان، دانشگاه آزاد اسلامی، زنجان، ایران.

2 استادیار گروه پاتوبیولوژی، دانشکده دامپزشکی، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.؛ مرکز تحقیقات بیوتکنولوژی، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.

3 استادیار گروه زیست‌شناسی، دانشکده علوم پایه، واحد زنجان، دانشگاه آزاد اسلامی، زنجان، ایران.

چکیده

امروزه بـرای شناسـایی و تشخیص سروتیپ­های سالمونلا در نمونه­های بالینی و مواد غذائی از روش‌های سنتی تشخیص این باکتری استفاده مـی‌گـردد که وقت­گیر و گاهی مشکل­ساز می‌باشند، اما با کشف روش‌های سریع تشخیص مولکولی، این مشکلات تا حد زیادی مرتفع گردیده ­است. مطالعه حاضر، با هدف تشخیص سریع جدایه­های مختلف سالمونلا بر اساس جسجوی ژن کروموزومی invA و نیز شناسائی جدایه­های حاد حاوی ژن­های حدت اپرون ­­ spv­انجام گردید. بدین منظور تعداد 20 جدایه انسانی سالمونلا از بیمارستان­های شهر تبریز و 20 جدایه این باکتری که از پنیرهای سنتی عرضه­شده در بازار مصرف تبریز جدا شده بود، تهیه گردید. ابتدا با استفاده از پرایمر­های اختصاصی ژن­invA ، تائید مولکولی جدایه­ها با استفاده از روشsimplex PCR  ارزیابی گردید. در ادامه برای بررسی سویه­های حاد باکتری مذکور براساس حضور ژن­های اپرون ­spv، با استفاده از پرایمرهای اختصاصی مربوطه، به روشmultiplex PCR  حضور ژن­های R، C، B وspvA بررسی شد. یافته­ها اولاً نشان­دهنده تائید مولکولی همه جدایه­ها بود. ثانیاً همه 40 جدایه مورد آزمایش دارای 3 ژن R، C وspvA بوده ولی هیچ­کدام از آن­ها دارای ژنspv­B  نبودند. به نظر می­رسد که با توجه به محدودیت­ها و مشکلات موجود در روش بررسی آزمایشگاهی سنتی سالمونلاها، می­توان ازPCR  به عنوان روشی سریع در تشخیص آلودگی به باکتری سالمونلا استفاده کرد. همچنین بایستی وجود 3 ژن از 4 ژن حدت اپرون ­spv در جدایه­های مختلف سالمونلا در منطقه تبریز را یافته­ای نامطلوب تلقی کرد که لزوم رعایت بیشتر اصول کنترل و پیشگیری در جوامع دامی و انسانی را تاکید می­کند.

کلیدواژه‌ها


عنوان مقاله [English]

Evaluation of rapid detection and investigation of the presence of spv operon virulence genes in Salmonella isolates using simplex PCR and multiplex PCR molecular methods

نویسندگان [English]

  • Somayeh Yazdi-Amirkhiz 1
  • Younes Anzabi 2
  • Sanaz Mahmazi 3
1 MSc Graduate in Genetics, Faculty of Basic Sciences, Zanjan Branch, Islamic Azad University, Zanjan, Iran. Zanjan, Iran.
2 Assistant Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Tabriz Branch, Islamic Azad University, Tabriz. Iran.؛ Biotechnology Research Center, Tabriz Branch, Islamic Azad University, Tabriz. Iran.
3 Assistant Professor, Department of Biology, Faculty of Basic Sciences, Zanjan Branch, Islamic Azad University, Zanjan, Iran.
چکیده [English]

The traditional methods of diagnosing Salmonella which are time-consuming and sometimes problematic, are still used to identify Salmonella serotypes in clinical and food samples, but with the invention of rapid molecular detection methods, these problems have been largely eliminated. The present study aimed to rapidly detect different Salmonella isolates based on invA chromosomal gene search and also to identify acute isolates containing spv operon virulence genes. To this end, 20 human isolates of Salmonella were obtained from hospitals in Tabriz and 20 isolates of this bacterium were isolated from traditional cheese available on Tabriz consumer market. The molecular confirmation of isolates was first evaluated using specific primers of invA gene by simplex PCR method. Then, in order to evaluate the acute strains of the bacterium based on the presence of operon spv, the presence of spvA, B, C and R genes was examined by multiplex PCR using the relevant specific primers. The results showed that firstly, all isolates had molecular confirmation. Secondly, all 40 tested isolates had 3 spvA, C and R genes, but none of them had spvB gene. It seems that due to the limitations and problems in the traditional laboratory examination of Salmonella, PCR can be used as a rapid method to detect Salmonella infection. Also, the presence of 3 out of 4 virulence genes of opron spv in different Salmonella isolates in Tabriz region should be considered an undesirable finding, which emphasizes the need to further observe principles of control and prevention in animal and human communities.

کلیدواژه‌ها [English]

  • Salmonella
  • Rapid diagnosis
  • invA
  • spv
  • PCR
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