兩種小鼠濾過性小病毒多重引子聚合酶鏈鎖反應之建立與台灣實驗小鼠感染之調查

Development of the Multiplex PCR Assays for Mouse Parvovirus (MPV) and Minute Virus of Mice (MVM), and the Surveillance of MPV and MVM Infection in Laboratory Mice in Taiwan

王克偉(Ko-Wei Wang)

指導教授 ： 萬灼華

共同指導教授 ： 闕玲玲(Ling-Ling Chueh)

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02110

摘要

Minute virus of mice (MVM) 與mouse parvovirus (MPV) 兩種小鼠的濾過性小病毒 (mouse parvoviruses) 為實驗小鼠中常見的病原。此病毒常經由污染的試劑與細胞培養液在實驗操作下或經帶原小鼠傳染給實驗動物中心的實驗小鼠。多項研究資料指出小鼠的濾過性小病毒會影響in vitro與 in vivo的免疫、腫瘤以及移植等相關的研究結果。為同時偵測此兩種病毒並顯示檢體樣本DNA品質，本研究針對MPV與MVM基因序列與小鼠的管家基因 (housekeeping gene)，建立了一個多重引子聚合酶鏈鎖反應試劑 (MPV/MVM/Actin Multiplex PCR Assay)。利用此試劑可特異性的增幅出MPV與MVM病毒基因，但不會複製其他的rodent parvoviruses的基因；針對敏感度測試結果顯示本試劑可同時偵測到50個MVM與MPV病毒，此結果顯示本診斷試劑具有高特異性與高敏感度。在模擬MPV與MVM不等量共同感染，即使兩種病毒量相差高達200倍的情況下，本試劑仍維持至少可偵測到50個病毒的敏感度，顯示本試劑偵測效果穩定。利用本試劑於台灣實驗小鼠的濾過性小病毒感染調查中，分別由7個單位蒐集到14種品系，共174隻實驗小鼠，其中MPV的陽性率高達11.5%，但未偵測到MVM的感染。此結果顯示，MPV在台灣實驗小鼠的感染率高，為了維持動物實驗數據的正確與品質，建議應將此病毒列入主要的疾病監控項目。此MPV/MVM/Actin Multiplex PCR Assay可同時且有效的偵測到MPV與MVM兩種病毒，亦可應用於監測其他生物製劑的污染情形，未來可成為實驗動物疾病管理上的一個利器。

關鍵字

小鼠濾過性小病毒 (MPV) ； 小鼠小病毒 (MVM) ； 小鼠的濾過性小病毒多重引子聚合酶鏈鎖反應 ； 台灣小鼠的濾過性小病毒感染率

並列摘要

Mouse parvoviruses are among the highly prevalent infectious pathogens in contemporary mouse colonies. There are two serotypes, minute virus of mice (MVM) and mouse parvovirus (MPV). Mouse parvoviruses have a predilection for mitotically active cells and can interfere with immunology, transplantation, and oncology research through virus-infected rodents, contaminated cells and biological materials. In order to detect these two viruses simultaneously, a multiplex PCR assay that amplifies the VP gene of MPV and MVM, and a mouse housekeeping gene has been developed in this study. The MPV/MVM/Actin Multiplex PCR assay specifically detects MPV and MVM, but doesn’t amplify KRV, RMV-1, RPV-1c, and H-1. The multiplex PCR assay could simultaneously detect both MVM and MPV in as low as 50 copies in the condition of equal-amount dual infection. The sensitivity of this multiplex PCR assay remained to detect at least 50 copies of MPV when the copies of MVM were 200 times higher than the MPV copy number, and vice versa. The prevalence surveillance result revealed that 3 out of 7 laboraotry mouse colonies were contaminated with MPV with the prevalence of 11.5%; however, none of the tested mouse colony was contaminated with MVM. The MPV/MVM/Actin Multiplex PCR assay developed in this study can provide a useful tool in the mouse health monitoring in the future.

並列關鍵字

mouse parvovirus ； minute virus of mice ； MPV/MVM multiplex PCR ； MPV prevalence ； MVM prevalence

參考文獻

廖翊君。台灣新發現之大鼠小病毒的分子特徵。國立台灣大學碩士論文，台北， 2009。

Abschuetz, A., Kehl, T., Geibig, R., Leuchs, B., Rommelaere, J., Régnier-Vigouroux, A., 2006. Oncolytic murine autonomous parvovirus, a candidate vector for glioma gene therapy, is innocuous to normal and immunocompetent mouse glial cells. Cell Tissue Res 325, 423-6.

Baer, P.N. and Kilham, L., 1974. Dental defects in hamsters infected with minute virus of mice. Oral Surg 37, 385-89.

Ball-Goodrich, L.J., Hansen, G., Dhawan, R., Paturzo, F.X., Vivas-Gonzalez, B.E., 2002. Validation of an enzyme-linked immunosorbent assay for detection of mouse parvovirus infection in laboratory mice. Comp Med 52, 160-6.

Ball-Goodrich, L.J. and Johnson, E. 1994. Molecular characterization of a newly recognized mouse parvovirus. J Virol 68, 6476-86.