ارزیابی آزمون تکثیر هم‌دمای وابسته به حلقه در شناسایی ویروس موزاییک کاهو (LMV)

نوع مقاله : علمی پژوهشی

نویسندگان

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

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

چکیده

شایع‌ترین ویروس تأثیرگذار بر کاهو در سراسر مزارع جهان، ویروس موزاییک کاهو (Lettuce mosaic virus; LMV) متعلق به خانواده Potyviridea و جنس Potyvirus است. روش‌های متعددی شامل آزمون‌های سرولوژیکی و مولکولی جهت تشخیص این ویروس وجود دارند، اما این روش‌ها زمان‌بر بوده و نیازمند ابزارهای پیچیده‌ای هستند. هدف از این پژوهش، کاهش زمان موردنیاز جهت تشخیص LMV با استفاده از روش تکثیر هم‌دمای وابسته به حلقه (LAMP) بود. نمونه‌های برگی (38 نمونه) با علایم مشابه به ویروس موزاییک کاهو از سطح استان‌ کردستان جمع‌آوری و برای آزمون سرولوژیکی (DAS-ELISA) به‌کارگرفته شدند و در نهایت چهار نمونه مثبت تشخیص داده شد. سپس RNA کل استخراج و واکنش رونویسی معکوس‌ LAMP به صورت یک مرحله‌ای، تحت شرایط هم‌دما انجام شد و نتایج به وسیله الکتروفورز بر روی ژل آگارز و رنگ هیدروکسی‌نفتول‌بلو (HNB) ارزیابی شد. نتیجه مثبت استفاده از رنگ HNB تغییر رنگ مخلوط واکنش از بنفش به آبی آسمانی بود. علاوه بر این، در اینجا آزمون جدید IC-RT-LAMPجهت تشخیص سریع و آسانLMV (بدون نیاز به استخراج RNA) توسعه داده شد و کارآیی آن با آزمون‌های دیگر مقایسه شد. از مزایای روش RT-LAMP در مقایسه با سایر روش‌های پیشین می‌توان به اختصاصیت بالا، حساسیت بالا، سرعت بالا، کارآیی بالا، ایمنی، سهولت، عدم نیاز به تجهیزات پرهزینه جهت تکثیر، عدم نیاز به استخراج RNA (در آزمون IC-RT-LAMP)، عدم نیاز به کارهای تشخیصی بعد از تکثیر، تشخیص مشاهده‌ای و کاربردوستی آن اشاره کرد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Evaluation of loop mediated isothermal amplification assay for identification of Lettuce Mosaic Virus (LMV)

نویسندگان [English]

  • Mohammad Amin Almasi 1
  • Galavizh Almasi 2
1 Young Researchers and Elites Club, North Tehran Branch, Islamic Azad University, Tehran, Iran
2 Young Researchers and Elites Club, North Tehran Branch, Islamic Azad University, Tehran, Iran
چکیده [English]

The most common virus affecting lettuce (Lactuca sativa L.) in the field worldwide is Lettuce mosaic virus (LMV), belonging to the family Potyviridea, genius Potyvirus. There are several techniques to detect LMV including serological and molecular assays, but these methods take a long time and requiring sophisticated tools. The aim of this study was to reduce the time required to detect LMV, using loop-mediated isothermal amplification (LAMP) technique. Leaf samples (38 samples) with symptoms similar to LMV were collected from Kurdestan province and were subjected to a serological (DAS-ELISA) test and lastly, four samples were detected as the positive samples. Then, total RNA was extracted and one-step reverse transcription (RT)-LAMP was carried out under isothermal conditions and results were evaluated by agarose gel electrophoresis and hydroxynaphthol blue (HNB) dye. A positive result using the HNB dye was a color change in master-mix from the violet to sky blue. In addition, a novel immunocapture (IC)-RT-LAMP assay for rapid and easy detection (without RNA extraction) of LMV was developed here and its potential compared with other assays. The advantages of RT-LAMP 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 need to RNA extraction (in IC-RT-LAMP assay), no post-amplification treatment of the amplicons, visual detection and user friendly.

کلیدواژه‌ها [English]

  • DAS- ELISA
  • IC-RT-LAMP
  • Lettuce mosaic virus
  • RT-LAMP

مراجع

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