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تشخیص سریع کوی هرپس ویروس (CyHV-3) به روش تکثیر همدما به واسطه حلقه (LAMP) و مقایسه با PCR PCR | ||
| شیلات | ||
| دوره 75، شماره 4، آذر 1401، صفحه 565-580 اصل مقاله (985.23 K) | ||
| نوع مقاله: مقاله پژوهشی | ||
| شناسه دیجیتال (DOI): 10.22059/jfisheries.2022.345426.1337 | ||
| نویسندگان | ||
| میثم باورصاد1؛ امیر عابد علم دوست* 2؛ محمد رضا تابنده3؛ حمید فرحمند4؛ علیرضا میرواقفی4؛ مجتبی علیشاهی5 | ||
| 1دانشجوی دکتری گروه شیلات، دانشکده منابع طبیعی، دانشگاه تهران، کرج، ایران | ||
| 2استادیار گروه شیلات، دانشکده منابع طبیعی، دانشگاه تهران، کرج، ایران | ||
| 3استادیارگروه علوم پایه، دانشکده دامپزشکی، دانشگاه شهید چمران اهواز، اهواز، ایران | ||
| 4. استاد گروه شیلات، دانشکده منابع طبیعی، دانشگاه تهران، کرج، ایران | ||
| 5استاد گروه علوم درمانگاهی، دانشکده دامپزشکی، دانشگاه شهید چمران اهواز، اهواز، ایران | ||
| چکیده | ||
| هرپس ویروس نوع 3 کپورماهیان (CyHV-3) یا کوی هرپسویروس (KHV)، یک ویروس نوظهور به شدت مسری و با حدت بالا است که سبب ایجاد تلفات سنگین و ناگهانی در ماهی کپور معمولی و کوی و هیبریدهای آنها میشود. روش LAMP یک تکنیک مولکولی مبتنی بر تکثیر بسیار اختصاصی اسید نوکلئیک اجرام پاتوژن در شرایط همدما و بدون نیاز به تجهیزات گرانقیمت میباشد که در سال‌های اخیر به منظور تشخیص پاتوژن‌های انسان و حیوانات توسعه یافته است. با توجه به اهمیت تشخیص سریع بیماری کوی هرپسویروس در مزارع مشکوک به آلودگی و پایش دایمی مزارع پرورش کپورماهیان کشور، این مطالعه با هدف ارزیابی روش تلفیقی LAMP با پروب‌های نانوطلا در تشخیص سریع این ویروس صورت گرفت. در این مطالعه با استفاده از 6 آغازگر اختصاصی، بخشی از ژن تیمیدین کیناز (TK) کوی هرپسویروس با روش بهینه شده LAMP، تکثیر و برای تشخیص رقت‌های مختلف DNA ژنومیک و DNA در نمونههای بافتی کپور معمولی، آزمایش اختصاصیت و حساسیت مورد بررسی قرار گرفت و آزمایش سرعت و حساسیت نسبت به روش PCR معمولی نیز مقایسه گردید. نتایج بیانگر اختصاصی بودن صد درصدی و حساسیت تشخیص بر اساس رقت استوک 8-10 معادل 25/1 فمتوگرم در میکرولیتر برای DNA ژنومیک ویروس و 7-10 معادل 2/8 فمتوگرم در میکرولیتر برای DNA ویروسی آمیخته شده در نمونه بافتی بود. همچنین بر اساس نتایج حاصله، روش LAMP پیشنهادی در مقایسه با PCR معمولی سه برابر سریعتر و 8000 برابر دقیق‌تر بود. با توجه به کارایی بالا، روش LAMP میتواند به عنوان یک ابزار قدرتمند و دقیق و سریع جهت شناسایی مولکولی کوی هرپسویروس در آزمایشگاه‌های تشخیصی با تجهیزات اندک مورد استفاده قرار گیرد. | ||
| کلیدواژه‌ها | ||
| هرپس ویروس نوع 3 کپورماهیان؛ LAMP؛ PCR؛ تشخیص سریع؛ تکثیر همدما؛ تیمیدین کیناز | ||
| عنوان مقاله [English] | ||
| Development of Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cyprinid Herpesvirus-3 (CyHV-3) and its comparison to PCR | ||
| نویسندگان [English] | ||
| Meisam Bavarsad1؛ Amirreza ABED-ELMDOUST2؛ Mohammad Reza Tabandeh3؛ Hamid Farahmand4؛ Alireza Mirvaghefi4؛ Mojtaba Alishahi5 | ||
| 1Ph.D student, Department of Fisheries, Faculty of Natural Resources, University of Tehran, Iran | ||
| 2Assistant Professor, Department of Fisheries, Faculty of Natural Resources, University of Tehran, Iran | ||
| 3Department of biochemistry and molecular biology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran | ||
| 4Department of Fisheries and aquaculture sciences, university of Tehran | ||
| 5Professor, Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran | ||
| چکیده [English] | ||
| Koi Herpes Virus (KHV or Cyprinid Herpesvirus 3, CyHV-3) is an etiological agent of an emerging and notifiable disease and one of the mostrisky factors affecting common carp and koi worldwide production with high mortality rates. Molecular methods have been developed and routinely used for the detection of KHV infections. These diagnostic tools are sensitive but still have some drawbacks: they require expensive and bulky apparatus, a large sample volume, and long sample preparation/detection times. Therefore, it is desirable to develop simpler, faster, cheaper and more portable detection methods. Loop-mediated isothermal amplification (LAMP) technique is a novel and next-generation tool which can amplify nucleic acids with high specificity, sensitivity and rapidity under isothermal conditions. In this study, a fragment of the CyHV-3 TK gene was amplified at 65°C in the presence of Bst DNA polymerase and a set of six specially designed primer mixture. The reliability, sensitivity and specificity of this assay for rapid detection of CyHV-3 was investigated and compared to conventional PCR. LAMP assay was successfully developed with the detection of KHV- generated product, and showed 100% specificity and sensitivity of 1.25 fg/µL, that is comparable to the most sensitive method reported to date. The assay was 8000 fold more sensitive and three times faster than conventional PCR. The LAMP assay described in this study revealed a highly sensitive, rapid and reliable diagnostic protocol for detection of CyHV-3. . | ||
| کلیدواژه‌ها [English] | ||
| KHV, CyHV-3, LAMP, PCR, Rapid detection, Isothermal amplification, Thymidine kinase | ||
| مراجع | ||
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