ردیابی و شناسایی ویروس موزاییک یونجه (AMV) به وسیله آزمون تکثیر هم دمای وابسته به حلقه

نوع مقاله: علمی پژوهشی

نویسنده

دانشگاه آزاد اسلامی، واحد تهران شمال، باشگاه پژوهشگران جوان و نخبگان، تهران، ایران.

چکیده

ویروس موزاییک یونجه (Alfalfa mosaic virus, AMV) در ایران که سبب بیماری در محصول یونجه (Medicago sativa) می‌ شود، بر اساس خصوصیات مورفولوژیکی و بیان علایم شناسایی تشخیص داده می‌شود. روش های متعددی جهت تشخیص این ویروس وجود دارند که می توان به آزمون های سرولوژیک و روش‌های مولکولی اشاره کرد. آزمون نسخه برداری معکوس تکثیر هم دمای وابسته به حلقه (revers transcription loop mediated isothermal amplification) روش جدیدی جهت تکثیر RNA تحت شرایط هم دما، با اختصاصیت، حساسیت، سرعت و کارایی بالا است که در این پژوهش جهت تشخیص ویروس موزاییک یونجه به کار گرفته شد. به منظور انجام آزمون سرولوژیک، نمونه های برگی (100 نمونه) با علائم مشابه به ویروس موزاییک یونجه از سطح استان های همدان و کردستان جمع آوری شدند. چهار آغازگر واکنش RT-LAMP (شامل F3، B3، FIP و BIP) به همراه آغازگرهای واکنش RT-PCR (شامل F و B) بر اساس توالی بسیار حفاظت شده ای از ژن پوشش پروتئین (CP) طراحی شدند. جهت انجام آزمون های مولکولی، RNA کل استخراج و واکنش های مولکولی انجام شدند و در نهایت شش نمونه مثبت تشخیص داده شد. از مزایای این روش جدید در مقایسه با سایر روش های پیشین می‌توان به اختصاصیت بالا، حساسیت بالا، سرعت بالا، کارآیی بالا، ایمنی، سهولت، عدم نیاز به تجهیزات پرهزینه جهت تکثیر، عدم نیاز به کارهای تشخیصی بعد از تکثیر، تشخیص مشاهده ای و کاربردوستی آن اشاره کرد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Tracking and identification of Alfalfa Mosaic Virus (AMV) by Loop mediated isothermal amplification assay

نویسنده [English]

  • Mohammad Amin Almasi
Young Researchers and Elites Club, North Tehran Branch, Islamic Azad University, Tehran, Iran
چکیده [English]

Alfalfa mosaic virus (AMV) causing a disease in Alfalfa (Medicago sativa) crop in Iran has been identified on the basis of determination of symptom expression and morphological properties. There are several techniques to detect the virus including serological test and molecular methods. Reverse transcription Loop-mediated isothermal amplification (RT-LAMP) assay is a novel technique for amplifying RNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency that is used in this study for detection of Alfalfa mosaic virus. Leaf samples (100 samples) with symptoms similar to AMV were collected from Hamedan and Kurdestan provinces and were subjected to a serological test. All four RT-LAMP reaction primers (i.e. F3, B3, FIP and BIP) together with RT-PCR reaction primers were designed on the basis of the highly conserved sequence of coat protein (CP) gene. Total RNA was extracted and molecular reactions were carried out and finally was detected six positive sampled The advantages of this new method in compare to other methods include the high specificity, high sensitivity, high rapidity, high efficiency, safety, fascinatingly, no requirement of expensive and tools for amplification, no post-amplification treatment of the amplicons, visual detection and user friendly.

کلیدواژه‌ها [English]

  • DAS- ELISA
  • RT-PCR
  • RT-LAMP
  • Alfalfa mosaic virus
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