Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry
Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry
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DOI：
10.17221/1882-vetmed
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发表时间：
2007-06-01
影响因子：
0.7
通讯作者：
Kovarcik, K.
中科院分区：
文献类型：
--
作者：
Kosinova, E.;Psikal, I.;Kovarcik, K.
建立了定量真实的-时间RT- PCR（qRT- PCR）方法，用于检测和定量持续感染牛临床样品中的牛病毒性腹泻病毒（BVDV）。使用Light Cycler（R）检测系统和嵌入荧光染料SYBR绿色I对qRT-PCR进行优化，以定量BVD病毒拷贝数。从高度保守的5'非翻译区（5' UTR）中选择一组通用引物，以同时检测BVDV I型和II型。使用标准质粒DNA 10倍系列稀释液（1 - 10（8）拷贝/μ l）生成的校准曲线，完成BVDV cDNA的定量。通过分析290 bp扩增子，能够监测总共5种BVDV毒株（BVD- NADL、A03/ 3004、DB 03/ 2943、KA 04/ 3124、KV 05/ 3412）和16份散装牛奶样品中的病毒RNA/ BVDV水平，以及来自捷克农场的持久性携带者的牛血清和一批用于细胞培养的小牛血清中的病毒RNA/ BVDV水平。与NADL参考毒株相同的基因型组I的BVDV毒株的扩增子的解链温度（Tm）显示出热点的变异性，然而，在基因型组I和II的代表性之间观察到Tm值的显著差异。还通过常规RT-PCR定性鉴定了散装牛奶样品中的低浓度BVD病毒。批内和批间循环阈值的变异系数分别小于0.85%和2.76%，重复性好。结果充分证明了qRT-PCR法定量分析临床样本中BVDV的适用性。
Quantitative real- time RT- PCR ( qRT- PCR) assay was developed for the detection and quantification of bovine viral diarrhea virus ( BVDV) in clinical samples from persistently infected cattle. qRT- PCR was optimized to quantify the number of BVD virus copies using Light Cycler (R) detection system and intercalation fluorogenic dye SYBR Green I. A universal set of primers was selected from a highly conserved 5' untranslated region ( 5' UTR) to detect BVDV type I and II simultaneously. Quantification of BVDV cDNA was accomplished using a calibration curve generated from 10- fold serial dilutions of standard plasmid DNA in the range 1 - 10(8) copies/mu l. Analysis of 290 bp amplicons enabled monitoring of the viral RNA/ BVDV level in a total of five BVDV strains ( BVD- NADL, A03/ 3004, DB03/ 2943, KA04/ 3124, KV05/ 3412) and sixteen bulk milk samples, and in bovine sera of persistent carriers originating from Czech farms, as well as in a batch of calf serum for cell culture. Melting temperatures of amplicons ( Tm) of BVDV strains of the same genotype group I as the NADL reference strain showed variability of the thermal points, however significant differences were observed in Tm values between the representatives of genotype group I and II. Low concentrations of BVD virus in bulk milk samples were also qualitatively identified by conventional RT- PCR. Highly reproducible data were obtained as the coefficients of variation of threshold cycles values in intra- assay and inter- assay were less than 0.85% and 2.76%, respectively. The results give enough evidence of suitability of qRT- PCR assay for quantitative analysis of BVDV in clinical samples.