【摘要】：參照GenBank中犬細小病毒(CPV)VP2基因保守區(qū)域序列設(shè)計合成特殊的內(nèi)、外引物,在擴增反應體系中加入熒光染料SYBR GreenⅠ,通過恒溫熒光檢測儀Deaou-308C擴增檢測,建立了一種快速檢測CPV的實時熒光環(huán)介導等溫擴增(LAMP)方法。結(jié)果顯示,這種方法對CPV具有特異性的擴增反應,對照病毒核酸的擴增結(jié)果均為陰性。靈敏度試驗最低可檢出3.72 copies/μL的病毒核酸。重復性試驗結(jié)果表明,其檢測重復性良好。對30份臨床寵物犬的糞便樣品進行檢測,與免疫膠體金檢測試紙方法進行比較,符合率為90%。將本方法初步應用于大熊貓糞便中犬細小病毒的檢測,結(jié)果表明,從大熊貓基地采集的84份糞便樣品中有16份為細小病毒陽性,陽性率為19.0%。本方法在63℃下恒溫45 min可以完成檢測,操作簡單、靈敏度高、特異性強,通過恒溫實時熒光檢測儀可實時檢測及自動判讀檢測結(jié)果,對大熊貓的早期診斷非常重要。
[Abstract]:Specific internal and external primers were designed and synthesized according to the conservative region sequence of the canine parvovirus (CPV) VP2 gene in GenBank. The fluorescent dye SYBR Green I was added to the amplification reaction system, and a real-time fluorescence ring mediated isothermal amplification (LAMP) method for rapid detection of CPV was established. The results showed that this method was used for rapid detection of CPV. CPV has a specific amplification reaction, and the amplification result of the virus nucleic acid is negative. The sensitivity test can detect the virus nucleic acid of 3.72 copies/ mu L. The repeatability test results show that the detection repeatability is good. The fecal samples of 30 clinical pet dogs are detected and the colloid gold detection test paper method is carried out. The results showed that the method was applied to the detection of canine parvovirus in the feces of pandas by 90%.. The results showed that 16 of the 84 fecal samples collected from the giant panda base were positive for parvovirus, and the positive rate was 19.0%. at the constant temperature of 45 min at 63 C, which was simple, sensitive and specific. The constant temperature real-time fluorescence detector can detect and interpret the test results in real time. It is very important for the early diagnosis of giant pandas.
【作者單位】： 四川農(nóng)業(yè)大學動物醫(yī)學院;東莞出入境檢驗檢疫局;
【基金】：成都大熊貓繁育研究基金會項目(CFP研2012-12、CFP研2012-9)
【分類號】：S852.655

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