Liu H J, Chen J H, Liao M H, Lin M Y, Chang G N
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Taiwan, ROC.
J Virol Methods. 1999 Aug;81(1-2):83-90. doi: 10.1016/s0166-0934(99)00063-4.
A nested reverse transcription (RT)-polymerase chain reaction with subsequent restriction endonuclease analysis was developed for identification of the sigma C-encoded gene of avian reoviruses (ARV). PCR products derived from the sigma C-encoded gene of all tested ARVs resulted in a specific DNA band of 1023 bp, indicating that there were no apparent insertions or deletions in this region. Amplification with the nested primer pairs S1M-S1N and S1P-S1N generated 330 and 239 bp, respectively. PCR products amplified from the sigma C-encoded of all tested ARVs isolates were further confirmed by Southern blot hybridization and restriction endonuclease analysis. PCR amplified cDNA fragment (1023 bp) cleaved with Pst I generated two fragments of 565 and 458 bp. The amplified sigma C-encoded gene of ARV was subcloned into PQE 32 vector for further study of its antigenicity and immunogenicity. The sensitivity of RT-PCR was examined on nucleic acids from the ARV infected cell cultures. The detection limit was 10(0) to 10(-1) TCID50 of ARV in a ethidium bromide stained gel and could be increased further to 10(-1) to 10(-2) TCID50 of ARV by Southern blot hybridization using a digoxigenin-labeled cDNA probe. The sensitivity increased approximately 10(3) to 10(4) folds when the cDNA was reamplified with two sets of nested primers.
为鉴定禽呼肠孤病毒（ARV）的σC编码基因，开发了一种巢式逆转录（RT）-聚合酶链反应及后续限制性内切酶分析方法。所有测试的ARV的σC编码基因的PCR产物产生了一条1023 bp的特异性DNA条带，表明该区域没有明显的插入或缺失。用巢式引物对S1M-S1N和S1P-S1N扩增分别产生了330和239 bp的片段。从所有测试的ARV分离株的σC编码基因扩增的PCR产物通过Southern印迹杂交和限制性内切酶分析进一步得到证实。用Pst I切割PCR扩增的cDNA片段（1023 bp）产生了565和458 bp的两个片段。将扩增的ARV的σC编码基因亚克隆到PQE 32载体中，以进一步研究其抗原性和免疫原性。在ARV感染的细胞培养物的核酸上检测了RT-PCR的敏感性。在溴化乙锭染色的凝胶中，检测限为ARV的10(0)至10(-1) TCID50，通过使用地高辛标记的cDNA探针的Southern印迹杂交，检测限可进一步提高到ARV的10(-1)至10(-2) TCID50。当用两组巢式引物对cDNA进行再扩增时，敏感性提高了约10(3)至10(4)倍。
