本文關(guān)鍵詞：水貂阿留申病毒TaqMan-MGB熒光定量PCR檢測(cè)方法的建立與應(yīng)用　出處：《中國預(yù)防獸醫(yī)學(xué)報(bào)》2016年11期 　論文類型：期刊論文

  更多相關(guān)文章： 水貂阿留申病毒 TaqMan-MGB探針 熒光定量PCR

【摘要】：為建立一種快速、敏感檢測(cè)水貂阿留申病毒(AMDV)的方法,本研究根據(jù)AMDV的VP2基因保守區(qū)設(shè)計(jì)特異性引物和MGB探針,并優(yōu)化檢測(cè)反應(yīng)條件,建立了AMDV的TaqMan熒光定量PCR檢測(cè)方法。結(jié)果顯示,以重組質(zhì)粒為標(biāo)準(zhǔn)品建立的標(biāo)準(zhǔn)曲線在1.0×10~2拷貝/μL~1.0×10~8拷貝/μL內(nèi)具有良好的線性關(guān)系,相關(guān)系數(shù)(R~2)為0.998。該方法僅對(duì)AMDV的靶基因擴(kuò)增呈陽性,而對(duì)豬細(xì)小病毒、犬細(xì)小病毒、水貂腸炎細(xì)小病毒等相關(guān)病毒檢測(cè)結(jié)果均為陰性,特異性良好。該方法對(duì)AMDV檢測(cè)的靈敏度為10拷貝/μL,為普通PCR靈敏度的100倍;組內(nèi)和組間重復(fù)性試驗(yàn)表明該方法的組內(nèi)和組間變異系數(shù)均小于2%,具有良好的重復(fù)性。應(yīng)用建立方法和普通PCR方法分別對(duì)40份疑似臨床組織病料樣品進(jìn)行檢測(cè),該方法檢出率比普通PCR高約7.5%。本研究結(jié)果表明建立的AMDV TaqMan-MGB熒光定量PCR方法靈敏、快速、特異性強(qiáng)、重復(fù)性好,適用于對(duì)AMDV的快速定量檢測(cè)。
[Abstract]:In order to establish a rapid and sensitive method for detection of Aleutian virus (AMDV) in mink, specific primers and MGB probes were designed according to the conserved region of VP2 gene of AMDV. The detection reaction conditions were optimized and the TaqMan quantitative PCR detection method for AMDV was established. The standard curve established by using recombinant plasmid as standard material has a good linear relationship in the range of 1.0 脳 10 ~ (2) copy / 渭 L ~ (-1) ~ (-1) 脳 10 ~ (8) copy / 渭 L. The correlation coefficient was 0.998.The method was only positive for target gene amplification of AMDV, but positive for porcine parvovirus and canine parvovirus. The detection results of mink enteritis parvovirus and other related viruses were negative and specific. The sensitivity of this method to AMDV detection was 10 copies / 渭 L, which was 100 times of that of ordinary PCR. The intra-group and inter-group reproducibility tests showed that the coefficient of variation within and between groups of the method was less than 2%. With good reproducibility, 40 samples of suspected clinical tissue samples were detected by the established method and the general PCR method. The detection rate of this method is about 7.5% higher than that of ordinary PCR. The results of this study show that the established AMDV TaqMan-MGB fluorescent quantitative PCR method is sensitive, rapid, specific and reproducible. It is suitable for rapid quantitative detection of AMDV.
【作者單位】： 吉林農(nóng)業(yè)大學(xué);遼寧出入境檢驗(yàn)檢疫局;遼寧省水產(chǎn)技術(shù)推廣總站;四川出入境檢驗(yàn)檢疫局;
【基金】：國家質(zhì)檢總局科研項(xiàng)目(2013IK036) 十二五“國家科技支撐計(jì)劃項(xiàng)目”(2013BAD12B01)
【分類號(hào)】：S852.65
【正文快照】： 水貂阿留申病(Aleutian mink disease,AMD)是由水貂阿留申病病毒(AMDV)引起的一種主要侵害水貂免疫細(xì)胞,導(dǎo)致自身免疫系統(tǒng)紊亂,同時(shí)并發(fā)自身免疫的慢性進(jìn)行性傳染病。該病以漸進(jìn)性消瘦、高免疫蛋白血癥、神經(jīng)癥狀、腎臟腫大等為主要特征[1]。1946年,美國學(xué)者Hartsough首次觀察

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