بهینه سازی روش پی سی آر آشیانه ای در ردیابی باکتری سخت کشت Xylella fastidiosa
محورهای موضوعی : باکتری شناسیجبرئیل بهمنی 1 , نادر حسن زاده 2 , مریم غایب زمهریر 3 , بهروز حریقی 4
1 - دانشجوی دکتری، گروه گیاه¬پزشکی، واحد علوم و تحقیقات تهران، دانشگاه آزاد اسلامی، تهران، ایران
2 - دانشیار، گروه گیاه¬پزشکی، واحد علوم و تحقیقات تهران، دانشگاه آزاد اسلامی، تهران، ایران
3 - دانشیار، بخش بیماریهای گیاهی، سازمان تحقیقات، آموزش و ترویج کشاورزی، مؤسسه تحقیقات گیاهپزشکی کشور، تهران، ایران
4 - استاد، گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه کردستان، سنندج، ایران
کلید واژه: پیرس انگور, باکتری سخت¬کشت, PCR استاندارد, PCR آشیانه¬ای, RNA polymerase gene 70S sigma, Xylella fastidiosa,
چکیده مقاله :
باکتری Xylella fastidiosa، گونه ای سختکشت و محدود به آوند چوبی و عامل بروز بیماریهای جدی در گیاهان مختلف میباشد. تاکنون، اپیدمیهای وسیع و ریشهکن کننده در نقاط مختلف دنیا از این باکتری گزارش شده است. در دهه اخیر، علائم بیماریهای ناشی از X. fastidiosa در برخی از مناطق ایران روی درختان میوه از جمله بادام و انگور مشاهده شده است. میزان حساسیت، به صرفه بودن، مؤثر و قابل اعتماد بودن روشهای ردیابی X. fastidiosa در گیاهان و حشرات ناقل بهویژه در گیاهان چند ساله مانند انگور و پسته که جمعیت باکتری در آنها کم است، از نظر قرنطینه و مدیریت بیماری بسیار ضروری است. علیرغم دقت بالای ردیابی به روش PCR روی یاختههای کشت شده، کشت باکتری X. fastidiosa بسیار پرهزینه، زمانبر و با درصد موفقیت پایین گزارش شده است. بنابراین، به منظور بهینهسازی روشی سریع، کم هزینه و با دقت برای ردیابی باکتری سخترشد آوندی X. fastidiosa بهطور مستقیم از گیاه میزبان، PCR آشیانهای دو مرحلهای، برای تکثیر ژنRNA polymerase gene 70S sigma طراحی شد. طبق نتایج بهدست آمده از این روش، از 2% کل نمونههای استخراج یافته، به 7% در نمونههای استخراج یافته بهبود یافت. بنابراین، استفاده از روش PCR آشیانهای میتواند روش جایگزین سریع، بهصرفه و مؤثر برای ردیابی مستقیم این باکتری از میزبان باشد.
Xylella fastidiosa is a difficult-to-cultivate, xylem-limited bacterium that causes sever diseases in various plants. Extensive and devastating epidemics of this bacterium have been reported in different parts of the world. In the last decade, symptoms of diseases caused by X. fastidiosa have been observed in some regions of Iran on fruit trees, including almonds and grapes. Sensitivity, cost effectiveness, and reliability of methods for detecting X. fastidiosa in plants and insects, especially in perennial plants such as grapes and pistachios, where the bacterial population is low, are essential in terms of quarantine and disease management. Detection by PCR on cultured bacterial cells is highly accurate, but the cultivation of X .fastidiosa is very expensive, time-consuming, and has been reported to have a low success rate. Therefore, in order to optimize a rapid, low-cost, and accurate method for detecting the hard-to-grow vascular bacterium X. fastidiosa directly from the host plant, a two-step nested PCR was designed for gene amplification (RNA polymerase gene 70S sigma). The results obtained from this method improved from 2% of the total extracted samples to 7% of the extracted samples. Therefore, the use of nested PCR method can be a rapid, cost-effective and effective alternative method for direct detection of this bacterium from the host.
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