مقایسه حساسیت دو روش RT-PCR و RRT-PCR برای تشخیص ویروس بیماری نیوکاسل در طیور

نویسندگان

1 گروه علوم دامی، دانشکده کشاورزی، دانشگاه رازی

2 گروه علوم دامی، دانشکده کشاورزی دانشگاه رازی، کرمانشاه-ایران

3 بخش پژوهشهای بیوتکنولوژی، موسسه تحقیقات علوم دامی کشور، کرج-ایران

4 پژوهشکده بیوتکنولوژی کشاورزی ایران، کرج - ایران

چکیده

زمینه مطالعه: بیماری نیوکاسل یکی از مخرب ترین بیماریهای ویروسی طیور در سرتاسر جهان است. هدف: با توجه به اینکه روش‌های سنتی قابلیت محدودی در کنترل این بیماری دارند، انجام این مطالعه به منظور استفاده از تکنولوژی‌های نوین برای تشخیص به موقع بیماری جهت کاهش خسارت پیش روی صنعت طیور می‌باشد. روش کار: استخراج RNA با استفاده از کیت RNease mini و بر اساس دستورالعمل شرکت سازنده انجام شد. RNA استخراج شده با 109×23/68 رونوشت اولیه به صورت سری رقت‌های µL‌100، برای انجام واکنش  RT-PCRو RRT-PCR تهیه شد. واکنش RT-PCR با استفاده از کیت RNease mini و واکنش RRT-PCR با استفاده از کیت تجاری (شرکت Genekam Biotechnology ،آلمان) انجام شد. نتایج: برای روش RRT-PCR تا سری رقت تهیه شده 34-10 تکثیر صورت گرفت و برای روش RT-PCR تا سری رقت تهیه شده 20- 10 بر روی ژل آگارز 5/1٪ باند مشاهده شد. براساس نتایج مشاهده شده روش RRT-PCR قادر به تشخیص 34-10×1رونوشت و روش RT-PCR قادر به تشخیص 20-10×1 رونوشت از نمونه اولیه است. نتیجه‌گیری‌نهایی: حساسیت روش RRT-PCR تقریباً دو برابر روش RT-PCR است و در مقایسه با روش RT-PCR، قادر به تشخیص ویروس بیماریزای نیوکاسل در نمونه‌های آلوده‌ای با 10000 برابر رونوشت کمتر از RNA ویروسی می‌باشد. 

کلیدواژه‌ها

عنوان مقاله [English]

A comparison of sensitivity analysis of RRT-PCR and RT-PCR techniques for diagnosis of avian Newcastle disease virus

نویسندگان [English]

  • Somayeh Satari 1
  • Sheida Varkoohi 2
  • Mohammad hosein Banabazi 3
  • Meisam Tabatabaei Pezhveh 4
1 Department of Animal Science, Faculty of Agriculture, Razi University
2 Department of Animal Sciences, Faculty of Agriculture, Razi University, Kermanshah-Iran
3 Biotechnology Research Center, Research Institute of Animal Sciences, Karaj-Iran
4 Agricultural Biotechnology Research Institute, Karaj, Iran
چکیده [English]

BACKGROUND: Newcastle disease is one of the most serious viral diseases in the poultry worldwide. OBJECTIVES: Since the traditional strategies have been hardly effective in controlling the disease, the purpose of this study was to introduce new methods for early and rapid diagnosis of Newcastle. The present study helps to reduce further damage to the poultry industry. METHODS: RNA extraction was performed, using RNease mini kit, according to the manufacturer’s instructions. Extracted RNA with 68.23×109 copy numbers was prepared as serial dilutions of 100 μL for RT-PCR and RRT-PCR reactions. RRT-PCR and RT-PCR were performed, using commercial kit and RNease mini kit, respectively.  RESULTS: Results showed that amplification was done according to prepared dilution equal 10-34 for RRT-PCR reaction and a visible band observed on 1.5% Agarose gel up to 10-20 for RT-PCR reaction. Based on the results observed, RRT-PCR and RT-PCR reactions are able to detect 10-34 and 10-20 copy numbers of primary sample, respectively. CONCLUSIONS: The sensitivity of RRT-PCR reaction is almost twice compared with RT-PCR reaction, also RRT-PCR reaction is able to diagnose Newcastle disease virus in infected samples with 10,000 copy numbers of the RNA virus less than RT-PCR.

کلیدواژه‌ها [English]

  • Diagnosis
  • Newcastle disease virus (NDV)
  • RRT-PCR
  • RT-PCR
  • Sensitivity

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