تکثیر هم‌دمایی به واسطه حلقه: روش تشخیص سریع و کم هزینه عوامل عفونی
محورهای موضوعی : میکروب شناسی تشخیصی
اعظم عسکری
1
,
محمد کارگر
2
,
صادق قربانی دالینی
3
,
عباس دوستی
4
1 - دانشگاه آزاد اسلامی، واحد جهرم، گروه میکروبیولوژی
2 - دانشگاه آزاد اسلامی، واحد جهرم، گروه میکروبیولوژی
3 - دانشگاه آزاد اسلامی، واحد جهرم، باشگاه پژوهشگران جوان
4 - دانشگاه آزاد اسلامی، واحد شهرکرد، مرکز تحقیقات بیوتکنولوژی
کلید واژه: بهینه سازی, LAMP, طراحی پرایمر,
چکیده مقاله :
روش تکثیر هم دمایی به واسطه حلقه (LAMP) مبتنی بر تکثیر اسید نوکلئیک در دمای ثابت ˚C 65-62 است. در این روش بدون نیاز به مرحله واسرشت شدن حرارتی، به کمک آنزیم DNA پلی مراز Bst به صورت هم زمان امکان جداسازی و تکثیر دو رشته DNA وجود دارد. بنابراین بر خلاف سایر روش های متداول تکثیری ، بدون نیاز به چرخه های دمایی و دستگاه ترموسایکلر و با استفاده از تجهیزات ارزان قیمتی مانند حمام آب گرم و یا بلوک حرارتی قابل انجام است. برای طراحی واکنش LAMP نیاز به 6 عدد پرایمر وجود دارد که توانایی اتصال کاملاً اختصاصی به 8 ناحیه مجزا از توالی هدف را داشته باشند. در نتیجه واکنش LAMP مقدار بسیار زیادی محصول ایجاد می شود که بدون نیاز به روش پر زحمت الکتروفورز در ژل، به کمک روش های ساده تری مانند مشاهده کدورت و یا تغییر رنگ ناشی از رنگ های فلورسنس در مخلوط واکنش به سادگی قابل تشخیص می باشد. بدین ترتیب روش LAMP با توجه به مزایایی مانند حساسیت و ویژگی مناسب ، کارایی بالا ، عدم نیاز به تجهیزات آزمایشگاهی گران قیمت و سادگی تشخیص، می تواند به عنوان ابزاری ارزشمند برای تشخیص سریع بیماری های عفونی در آزمایشگاه های کلینیکی و بیمارستانی به ویژه در کشورهای درحال توسعه به کار برده شود. این مقاله با هدف معرفی مبانی و کاربردهای روش LAMP در تشخیص عوامل عفونی تدوین گردیده است.
Loop-mediated isothermal amplification (LAMP) is nucleic acid amplification method that amplifies target isothermally at 62-65˚C. In this approach, withouth thermal denaturation step, double stranded DNA is simultaneousely denatured and synthesized by using the Bst (Bacillus stearothrmophilus) DNA polymerase with high strand displacement activity. Thus the reaction can be conducted with a cost-effective quipment such as water bath or heating block, and the thermal-cycling needs of a PCR are avoided. The LAMP reaction uses six primers that specifically recognize eight distinct regions on the target sequence. Moreover, the LAMP reaction produces a large amount of amplified products, resulting in easier detection, such as visual judgment based on the turbidity or colour change result from fluorescent dsDNA intercalating dye in the reaction mixture, so gel electrophoresis is not required. Therefore, the LAMP assay has the advantages of high specificity, sensitivity, amplification efficiency and simple detection, that without the need for expensive equipment could be applicable as valuable tool for rapid diagnosis of infectious diseases in both clinical and hospital laboratories of developing countries. The aim of this article is to introduce the principles and applications of LAMP method for detection of infectious agents.
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