کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
3408368 1593512 2006 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
A long distance RT-PCR able to amplify the Pestivirus genome
کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری ایمنی شناسی و میکروب شناسی ویروس شناسی
پیش نمایش صفحه اول مقاله
A long distance RT-PCR able to amplify the Pestivirus genome
چکیده انگلیسی

A method to amplify long genomic regions (up to ∼12.3 kb) from pestiviruses in one RT-PCR is described. The difficulty in designing conserved Pestivirus primers for the amplification of genomes from highly divergent isolates simply by means of overlapping segments is demonstrated using new bioinformatic tools. An alternative procedure consisting of optimizing the length of the genomic cDNA fragments and their subsequent amplification by polymerase chain reaction (PCR) using a limited set of specific primers is described. The amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs as well as cDNA produced by reverse transcription (RT) has been achieved using this methodology, known as long distance PCR. In the case of viruses, it is necessary to obtain viral particles from infected cells prior to RT procedures. This work provides improvements in four steps of long distance RT-PCR (L-RT-PCR): (i) preparation of a viral stock, (ii) preparation of template RNA, (iii) reverse transcription and (iv) amplification of the cDNA by LD-PCR. The usefulness of L-RT-PCR is discussed in the light of current knowledge on pestivirus diversity. The genomic sequence of Singer_Arg reference strain obtained using this method is presented and characterized.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Virological Methods - Volume 134, Issues 1–2, June 2006, Pages 197–204
نویسندگان
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