口蹄疫病毒抗原ELISA

1、口蹄疫病毒抗原ELISA、病毒分離及RT-PCR敏感性之比較豬瘟研究組黃郁珳助理研究員摘 要口蹄疫(Foot and mouth disease；FMD)是一種急性偶蹄類動物傳染病，會造成嚴重之經濟損失。口蹄疫病毒(FMDV)是屬於正股單鏈RNA病毒，歸類於小核醣核酸病毒科(Picornaviridae)、口瘡病毒屬(Aphthovirus)。FMDV共有O、A、C、Asia 1、SAT 1、SAT 2、SAT 3等七種血清型，各血清型病毒間無交叉免疫保護作用。而口蹄疫的實驗室診斷以抗原鑑定為主，目前本所使用的口蹄疫抗原鑑定方法包括病毒分離(Virus isolation)、抗原-酵素結合免疫

2、吸附試驗(Antigen-ELISA)、反轉錄聚合酶連鎖反應(RT-PCR)等。其中病毒分離是將試材接種至BHK 21、PK 15或EBK等株化細胞，置於37含5% CO2之細胞培養箱中培養，逐日觀察有無細胞病變作用(CPE)產生。反轉錄聚合酶連鎖反應是利用一對方向相反的寡核苷酸引子，先利用反轉錄酶將待測RNA轉錄成cDNA，再經由DNA聚合酶的作用，反覆進行變性作用、煉合作用及延展作用等三步驟，而將特定的DNA片段大量複製，從而提升檢測病毒核酸之敏感性達百萬倍之多。而抗原-酵素結合免疫吸附試驗則是利用抗原抗體間專一性鍵結的特性，檢測所採取試材中的口蹄疫病毒抗原，且鑑定其血清型別。由於口蹄疫疫

3、情的控制，首重快速而準確的診斷，因此，為了解本所目前使用的三種口蹄疫診斷技術之敏感性，乃進行此一實驗。首先我們選擇一株源自細胞培養的口蹄疫病毒及一株口蹄疫病毒感染的水疱上皮組織10倍乳劑，分別以含2%胎牛血清之細胞培養液，進行10倍連續稀釋至10-8，再分別進行病毒力價回歸試驗、病毒分離、RT-PCR檢測及Antigen-ELISA。病毒力價回歸試驗的結果為病毒力價104.89 TCID50/0.05ml，組織10倍乳劑之病毒力價104.67TCID50/0.05ml；在病毒分離部份，我們使用BHK-21及EBK二種細胞，結果顯示，以EBK細胞分離病毒的敏感性可達10-5，而以BHK 21細胞

4、者只有10-4；在RT-PCR方法中，不論是以TRIZOL或Qiagen之商品化核酸萃取套組進行病毒核酸萃取，其均可檢測至10-6；而Antigen-ELISA方法僅能檢測至10-1。因此，本所目前使用的三種口蹄疫診斷技術中，以RT-PCR的方法最敏感，其次是病毒分離，而以Antigen-ELISA最不敏感。Comparison of the sensitivity of the FMDV antigen ELISA, virus isolation, and RT-PCRYu-Wen HuangAbstractFoot and mouth disease (FMD) is an acute

5、and infectious disease of cloven-hoofed animals. It can cause severe economic loss. FMDV is a positive sense and single stranded RNA virus and it belongs to Picornaviridae, Aphthovirus. FMDV has seven distinct serotypes including type O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3. There is no cross-prote

6、ction between each serotype. The laboratory diagnosis of FMD is based on antigen identification. Currently, FMDV is diagnosed by virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), and antigen enzyme-linked immunosorbent assay (Ag-ELISA). For the virus isolation of FMDV, sampl

7、es were inoculated to BHK-21, PK-15, or EBK cells. Then, the cells were incubated at 37 and 5 CO2 and the cytopathic effect (CPE) was observed under an inverted microscope every day. For RT-PCR, viral RNA was extracted from samples first. The RNA had then been transcribed into cDNA by reverse transc

8、riptase. Then, a specific cDNA fragment was massively replicated by the process of denaturation, annealing, and extension. The Ag-ELISA detection was based on the specific binding between antigen and antibody. It not only detects the FMDV antigen but also identifies the serotypes of the viruses.Sinc

9、e the control of the FMD epidemic requires fast and accurate diagnosis, for this reason, the sensitivities among these three diagnostic techniques currently used have been compared.First of all, a FMDV suspension from cell culture and a 10-times emulsion from infected vesicular epithelium had been p

10、repared. A 10-fold serial dilution had been performed by using medium with 2 fetal calf serum. Then, virus back titration, virus isolation, RT-PCR, and Ag-ELISA were conducted. The results of the virus back titration showed that the titers of the cell-cultured FMDV and 10-times emulsion were 104.89

11、TCID50/0.05ml and 104.67TCID50/0.05ml, respectively. For virus isolation, both of BHK-21 and EBK cells were used. And the results revealed that the sensitivity of the virus isolation in EBK was 10-5 and in BHK-21 was 10-4. In RT-PCR, the sensitivity was 10-6 when the viral RNA was extracted by either Trizol reagent or the Qiagen commercial kit. As for the Ag