The Specific Detection of Infectious Pancreatic Necrosis Virus by Reverse Transcriptase-Polymerase Chain Reaction and Dot Blot Hybridization

利用反轉錄－聚合酶連鎖反應及點墨雜交反應作感染性胰臟壞死病毒專一性檢測

林奎甫(Kwen-Fu Lin) ； 洪紋玲(Wen-Lin Hong) ； 徐亞莉(Ya-Li Hsu)

《師大生物學報》 31卷1期 (1996/06) Pp. 39-46

https://doi.org/10.29812/BBNTNU.199606.0006

摘要

基於RT-PCR原理，檢測IPNV的快速、靈敏且專一的方法發展出來了。病毒RNA可自細胞培養中抽取，作為合成cDNA的template。依IPNV的VP2 coding region設計並合成、專一性的引子合成cDNA。利用primer B合成之dsDNA為template #2和B、D primers，作PCR，可得單一且專一的約300bp的PCR產物，此產物大小可作血清型的確定。利用此300bp PCR產物作探針，DNA-RNA的點墨雜交反應可檢測到T42G為10到100pg RNA，SP的10ngRNA，EVE的1到10 ngRNA，及AB的10ngRNA。這些方法可以進一步研究而改進。

關鍵字

感染性胰臟壞死病毒 ； 反轉錄－聚合酶連鎖反應 ； 點墨雜交

並列摘要

Based on reverse transcription-polymerase chain reaction (RT-PCR), a rapid, sensitive and specific detection method has been developed to detect virus in cultured fish cells. Fish viral RNA was extracted from cell cultures and used for cDNA synthesis. Specific primers of coding region of VP2 were used for ds cDNA synthesis. Using B, D primers and template #2 (ds cDNA from primer B). a single one 300 bp PCR product could be obtained, and the size of the product can be used as the indicator for serotype determination. By using the probe of this 300 bp PCR product. the DNA-RNA dot blot hybridization assay developed for IPNV detects 10ta 100 pg RNA of T42G 10 ng RNA of SP, 1 to 10ng RNA of EVE, and 10ng RNA of AB, respectively. These methods could be improved by further studies.

並列關鍵字

IPNV ； RT-PCR ； dot blot hybridization