شناسایی و تعیین سویه مایکوباکتریوم پاراتوبرکلوزیس (MAP) به روش PCR و REA بر اساس قطعات درون جایگیر IS900 و IS1311

نوع مقاله : مقاله پژوهشی

نویسندگان

1 2دانشیار، گروه پژوهشی بیوتکنولوژی کشاورزی و دامی، پژوهشکده فناوری زیستی، دانشگاه فردوسی مشهد1دانشیار، گروه علوم دامی، دانشکده کشاورزی، دانشگاه فردوسی مشهد

2 3دانشجوی مقطع دکتری، گروه علوم دامی، دانشگاه فردوسی مشهد

3 دانشجوی مقطع دکتری، گروه علوم دامی، دانشگاه فردوسی مشهد

4 جوی مقطع دکتری، گروه علوم دامی، دانشگاه فردوسی مشهد

چکیده

هدف از این مطالعه شناسایی مایکوباکتریوم آویوم زیرگونه پاراتوبرکلوزیس (M. avium paratuberculosis)، بررسی درصد شیوع آلودگی به آن و همچنین بهینه سازی یک روش سریع، دقیق و ساده به کمک PCR برای تشخیص این زیرگونه و جدایه های آن بود. مایکوباکتریوم آویوم زیرگونه پاراتوبرکلوزیس (MAP)، از لحاظ اقتصادی عامل یکی از مهمترین بیماری ها در صنعت دامپروری به نام بیماری یون (Johne's Disease) می باشد، که یکی از عفونت های مزمن نشخوارکنندگان به شمار می رود. به منظور شناسایی این زیرگونه 243 نمونه مدفوع و 56 نمونه شیر خام از دام های مشکوک گاوداری های اطراف مشهد جمع آوری شد. پس از استخراج DNA به منظور شناسایی نمونه های آلوده، قطعه درون جایگیر اختصاصی MAP به نام IS900 به طول 413 جفت باز، با استفاده از پرایمرهای P90 و P91 تکثیر شد. سپس به منظور تعیین جدایه گاوی و گوسفندی MAP، نسبت به تکثیر قطعه درون جایگیر IS1311 با طول 268 جفت باز به کمک دو پرایمر M56 و M94  اقدام شد. قطعات حاصله به کمک آنزیم HinfI هضم شدند. تعداد 107 نمونه از 243 نمونه مدفوع (44%) و 10 نمونه از 56 نمونه شیر خام (18%) مورد مطالعه، آلوده به MAP بودند. از بین 56 نمونه شیر و مدفوع که از دام های یکسانی گرفته شده بودند 19 نمونه مدفوع و 10 نمونه شیر، آلوده شناسایی شدند (18% در مقابل 34%). نتایج هضم آنزیمی نیز نشان داد که تمامی MAP های شناسایی شده از جدایه گاوی می باشند.
 

کلیدواژه‌ها

عنوان مقاله [English]

Identification and strain determination of M. paratuberculosis (MAP) by PCR and REA methods based on IS900 and IS1311 insertion segments

نویسندگان [English]

  • Mohammadreza Nassiri 1
  • Mohammad hassan Jahandar 2
  • Mehdi Soltani 3
  • Morteza Mahdavi 4
  • Mohammad Doosti 3
1
2
3
4
چکیده [English]

The purpose of this study was to identifying the M. avium paratuberculosis (MAP), investigating its outbreaks and optimizing a fast, accurate and simple method based on PCR and RFLP for detection of this subspecies and their strains. This pathogen cause a chronic infection called Johne's disease. Johne's disease a ruminant chronic infection and economically, is one of the most important disease in livestock industry. 243 fecal samples and 56 raw milk samples of suspected cattle, from some farms of Mashhad were gathered. After DNA extraction, in order to identifying contaminated samples, a specific MAP insertion segment called IS900, with length of 413bp was amplified with PCR using P90 and P91 primers. Then, in order to determining the cow and sheep strains of MAP, 268bp fragment of insertion segment IS1311  was amplified with PCR using M56 and M94 primers. The resulting fragments were digested with HinfI restriction enzyme. 107 of 243 stool samples (44%) and 10 of 56 raw milk samples (18%) were identified as infected with MAP. Among 56 Milk and feces samples taken from the same animal, 19 stools and 10 milk samples were identified as infected (18% vs. 34%). Results of enzymatic digestion also showed that all detected MAP were from bovine isolates.

کلیدواژه‌ها [English]

  • M. paratuberculosis
  • Johne's disease
  • IS900
  • IS1311
  • REA-PCR

اصل مقاله

M. paratuberculosis ,Johne's disease,  IS900, IS1311, REA-PCR.

 

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مجله بیوتکنولوژی کشاورزی
دوره 4، شماره 1 - شماره پیاپی 1
سال 4 شماره 1 بهارو تابستان 1391
شهریور 1391
صفحه 83-96

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