本文選題：鼠痘病毒　切入點(diǎn)：HA基因　出處：《實(shí)驗(yàn)動物科學(xué)》2016年02期 　論文類型：期刊論文

【摘要】：目的建立快速、敏感、特異檢測鼠痘病毒的Taq Man MGB探針實(shí)時(shí)熒光定量PCR方法。方法針對鼠痘病毒血凝素HA基因設(shè)計(jì)特異性引物和探針,構(gòu)建含有HA基因的標(biāo)準(zhǔn)質(zhì)粒進(jìn)行定量分析,建立Taq Man MGB探針實(shí)時(shí)熒光定量PCR檢測方法,評價(jià)其敏感性、特異性和穩(wěn)定性。對臨床標(biāo)本中的鼠痘病毒進(jìn)行檢測。結(jié)果研究結(jié)果顯示,建立的鼠痘病毒Taq Man MGB探針實(shí)時(shí)熒光定量PCR檢測方法特異性為100%,與其他正痘病毒屬病毒、非正痘病毒屬病毒、細(xì)菌、真菌、寄生蟲和細(xì)胞均無交叉反應(yīng)。該技術(shù)靈敏度高,能精確定量檢測鼠痘病毒DAN線性范圍達(dá)10個(gè)數(shù)量級(100—109拷貝),最低檢測限度為4拷貝。該方法重復(fù)性非常好,組內(nèi)變異系數(shù)和組間變異系數(shù)均小于3%。測試中相關(guān)系數(shù)、斜率和效率測量線性沒有顯著變化,表明該方法準(zhǔn)確度高、精密度好。將其成功應(yīng)用于臨床標(biāo)本中鼠痘病毒載量的定量檢測,用普通PCR和測序進(jìn)行確證。整個(gè)檢測過程可在2 h內(nèi)完成,可以用作快速診斷方法。結(jié)論本研究新建立的鼠痘病毒Taq Man MGB探針實(shí)時(shí)熒光定量PCR方法,具有快速簡便、特異性強(qiáng)、靈敏度高的特性,適用于動物源性產(chǎn)品中鼠痘病毒檢測、食品和藥品安全檢查、環(huán)境監(jiān)測、流行病毒調(diào)查,為臨床標(biāo)本中鼠痘病毒的快速定量檢測提供了一種特異有效的評價(jià)工具,值得推廣應(yīng)用。
[Abstract]:Objective to establish a rapid, sensitive and specific Taq Man MGB probe real-time quantitative PCR method for the detection of mouse pox virus. Methods specific primers and probes were designed for hemagglutinin HA gene of mouse pox virus. The standard plasmid containing HA gene was constructed for quantitative analysis, and the real-time fluorescent quantitative PCR detection method for Taq Man MGB probe was established to evaluate its sensitivity. Specificity and stability. Mouse pox virus was detected in clinical specimens. The results showed that the specificity of the established real-time fluorescent quantitative PCR detection method of mouse pox virus Taq Man MGB probe was 100%, which was similar to that of other positive poxvirus viruses. There is no cross reaction between viruses, bacteria, fungi, parasites and cells. The linear range of accurate quantitative detection of mouse pox virus DAN is 10 orders of magnitude 100-109 copies and the minimum detection limit is 4 copies. This method has a very good reproducibility, the coefficient of variation within group and the coefficient of variation between groups are less than 3 parts. The linear measurement of slope and efficiency showed that the method had high accuracy and good precision. It was successfully applied to the quantitative detection of rodent pox virus load in clinical samples. The whole detection process can be completed within 2 hours and can be used as a rapid diagnostic method. Conclusion the newly established real-time fluorescent quantitative PCR method for Taq Man MGB probe of mouse pox virus is rapid, simple and specific. High sensitivity, suitable for the detection of mouse pox virus in animal products, food and drug safety inspection, environmental monitoring, epidemic virus investigation, It provides a specific and effective tool for rapid quantitative detection of mouse pox virus in clinical specimens and is worth popularizing and applying.
【作者單位】： 中國食品藥品檢定研究院;
【基金】：國家科技支撐計(jì)劃資助項(xiàng)目(No.2013BAK11B03)
【分類號】：R511;R440

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