Amplification and cloning of infectious bursal disease virus genomic RNA segments by long and accurate PCR
Amplification and cloning of infectious bursal disease virus genomic RNA segments by long and accurate PCR
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DOI：
10.1016/s0166-0934(99)00083-x
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发表时间：
1999-09-01
影响因子：
3.1
通讯作者：
Lin, TL
中科院分区：
文献类型：
--
作者：
Akin, A;Wu, CC;Lin, TL
对传染性法氏囊病病毒（IBDV）变异株E基因组的反转录、聚合酶链反应（PCR）扩增和全长编码区克隆的方法进行了改进。在引物存在下通过加热使IBDV RNA变性并使用缺乏RNase-H活性的逆转录酶产生病毒基因组区段的全长编码区和部分非编码区cDNA拷贝。用RNase-H酶切RNA-cDNA杂交体的RNA组分，然后用一步长的和精确的PCR（LA-PCR）扩增IBDV cDNA，结果合成了IBDV基因组的3182个碱基对（bp）的A节段和2777 bp的B节段cDNA拷贝。成功地克隆了所得扩增子并测序，揭示了它们的IBDV身份。LA-PCR方法可用于扩增和克隆其它IBDV毒株或分离物，并将大大提高序列信息或IBDV感染性cDNA拷贝的可用性。(C)1999 Elsevier Science B. V.保留所有权利。
Improved methods of reverse transcription, polymerase chain reaction (PCR) amplification, and cloning of full-length coding region of both strands of infectious bursal disease virus (IBDV) variant strain E genome were developed. Denaturation of IBDV RNA by heat in the presence of primers and use of a reverse transcriptase lacking RNase-H activity produced full-length coding region and partial non-coding region cDNA copies of the viral genomic segments. Digestion of the RNA component of RNA-cDNA hybrids by RNase-H followed by long and accurate PCR (LA-PCR) amplification of IBDV cDNA in a single step resulted in the synthesis of 3182 base-pairs (bp) of segment A and 2777 bp of segment B cDNA copies of IBDV genome. The resulting amplicons were successfully cloned and sequenced revealing their identity of IBDV. The LA-PCR method can be utilized for the amplification and cloning of the other IBDV strains or isolates and will greatly enhance the availability of sequence information or infectious cDNA copies of IBDV. (C) 1999 Elsevier Science B.V. All rights reserved.