豬德?tīng)査跔畈《綬T-PCR檢測(cè)方法的建立及應(yīng)用
發(fā)布時(shí)間：2018-05-19 03:34
本文選題：豬德?tīng)査跔畈《?/strong> + RT-PCR��； 參考：《中國(guó)獸醫(yī)科學(xué)》2017年03期
【摘要】：為建立一種能夠快速、準(zhǔn)確檢測(cè)豬德?tīng)査跔畈《?PD Co V)的方法,根據(jù)G en Bank上已發(fā)表的PDCo V的M基因序列,設(shè)計(jì)并合成1對(duì)特異性引物,擴(kuò)增的目的基因片段的大小為359 bp。通過(guò)優(yōu)化反應(yīng)條件,建立了檢測(cè)PDCo V的RT-PCR方法,并對(duì)其敏感性、特異性和重復(fù)性進(jìn)行了檢測(cè)。結(jié)果顯示,僅PDCo V陽(yáng)性模板可擴(kuò)增得到約359 bp的目的條帶,而豬繁殖與呼吸綜合征病毒、豬瘟病毒、豬流行性腹瀉病毒、豬傳染性胃腸炎病毒、輪狀病毒、牛病毒性腹瀉病毒、豬細(xì)小病毒、豬圓環(huán)病毒2型、偽狂犬病病毒、豬鏈球菌2型、大腸桿菌的擴(kuò)增結(jié)果均為陰性。敏感性試驗(yàn)結(jié)果顯示,該方法能檢測(cè)到的最低核酸質(zhì)量濃度為1 ng/L。應(yīng)用該方法對(duì)110份臨床疑似發(fā)病的腹瀉豬樣品進(jìn)行檢測(cè),結(jié)果檢出13份PDCo V陽(yáng)性,陽(yáng)性檢出率為11.8%。應(yīng)用Blast對(duì)測(cè)序結(jié)果進(jìn)行比較分析,結(jié)果表明檢測(cè)的陽(yáng)性樣品與其他PDCo V序列的同源性為92.2%~100%。M基因的系統(tǒng)進(jìn)化分析表明,陽(yáng)性樣品與PDCo V中國(guó)分離株的親緣關(guān)系較近。以上結(jié)果表明,本研究成功建立了PDCo V的RT-PCR方法,該方法具有良好的特異性、敏感性和重復(fù)性,可用于PDCo V的臨床診斷及流行病學(xué)監(jiān)測(cè)。
[Abstract]:In order to establish a rapid and accurate detection method for porcine Delta coronavirus (PRV) PD Co V, a pair of specific primers were designed and synthesized according to the M gene sequence of PDCo V published on G en Bank. The amplified target gene fragment size was 359bp. By optimizing the reaction conditions, a RT-PCR method for the detection of PDCo V was established, and its sensitivity, specificity and repeatability were determined. The results showed that only PDCo V positive template could amplify about 359bp, while porcine reproductive and respiratory syndrome virus, swine fever virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, rotavirus, bovine viral diarrhea virus were obtained. The amplification results of porcine parvovirus, porcine circovirus 2, pseudorabies virus, Streptococcus suis type 2 and Escherichia coli were all negative. The sensitivity test results show that the minimum mass concentration of nucleic acid detected by this method is 1 ng / L. This method was used to detect PDCo V in 110 suspected diarrhea pigs. The positive rate of PDCo V was 11. 8%. Blast was used to compare the sequencing results. The results showed that the homology between the positive samples and other PDCo V sequences was 92.2and the phylogenetic analysis of 100.M gene showed that the relationship between the positive samples and the Chinese isolates of PDCo V was close. The results show that the RT-PCR method of PDCo V has been successfully established, which has good specificity, sensitivity and reproducibility, and can be used in the clinical diagnosis and epidemiological monitoring of PDCo V.
【作者單位】： 天津市畜牧獸醫(yī)研究所;天津市畜禽健康養(yǎng)殖技術(shù)工程中心;
【基金】：天津市農(nóng)業(yè)科技轉(zhuǎn)化與推廣項(xiàng)目(201301030)
【分類號(hào)】：S852.651
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