基于TaqMan探针的荧光定量PCR技术在PCV2检测中应用研究

A study using real-time fluorescence quantitative PCR based on TaqMan to detect PCV2

目的针对猪圆环病毒（PCV2）特点,利用TaqMan探针建立一种能够快速、准确并具有临床应用价值的PCV2检测方法。方法参照GenBank已收录PCV2ORF2,利用软件设计合成1对特异引物以及与之相匹配的特异性TapMan探针,采用PCR对PCV2衣壳蛋白部分基因进行克隆并鉴定,将纯化回收产物连接至pMT19-T载体,然后转化至大肠杆菌DH5a感受态细胞,涂布至Amp＋琼脂平板,筛选阳性克隆并扩大培养,进行2%琼脂糖凝胶电泳鉴定并测序。采用矩阵法确定引物和探针最佳浓度。对基于TapMan探针的实时荧光定量PCR循环进行优化,筛选最佳退火温度。以100-109倍稀释的标准品为模板,进行敏感性试验。提取CSFV、PRRSV、PRV和PCV1的DNA（CSFV和CSFV在提取RNA后反转录成cDNA）作为模板,进行TapMan荧光定量PCR,检测其特异性。采用105-108倍稀释的标准品为模板进行重复性实验,其中批内和批间均重复4次,根据所得值计算变异系数。结果 PCR产物经20g/L琼脂糖凝胶电泳鉴定后大小为114bp,与预期相符;测序分析PCR产物序列与参考序列同源性为100%。优化的PCR条件为：引物和探针浓度为10μmol/L,退火温度60℃。建立了PCV2TaqMan实时荧光定量PCR检测标准曲线,起始模板拷贝数为38.403,斜率为-3.69,相关系数R2为0.998,曲线呈线性。检测方法的敏感性为102拷贝/μl质粒标准品,是常规PCR检测敏感性的100倍。特异性检测显示,CSFV、PRRSV、PRV和PCV1扩增反应均阴性。重复性检测显示,PCR扩增循环阈值平均值基本一致,其变异系数均小于2%。采用TaqMan荧光定量PCR方法检测40份样品14份阳性,阳性率35.0%;普通PCR检测11份阳性,阳性率27.5%,差异无统计学意义（P〉0.05）。结论建立的PCV2TaqMan实时荧光定量PCR检测方法具有以下优点：1）敏感性高,能够用于精确计算PCV2病毒载量;2）特异性强,适用于检测PCV2与其他病原混合感染;3）用时短且可靠性强,可用于PCV2感染检测。

Objective In accordance with the characteristics of PCV2,a method of real-time fluorescence quantitative PCR based on TaqMan was established to accurately and quickly detect PCV2 in clinical settings. Methods In accordance with the sequence of ORF2 of PCV2in GenBank,apair of primers and a TaqMan probe were designed using software and then synthesized.PCR was used to clone and identify the capsid protein of PCV2.The PCR products were purified and collected and then ligated into a pMD19-T vector.This vector was then transformed into DH5 acompetent cells and cells were seeded onto Amp＋agar plates.Positive clones were selected,expanded,and cultured.Clones were identified and sequenced using 2%agarose gel electrophoresis.A pair of primers and a TaqMan probe were prepared at concentrations of 5μmol/L,10μmol/L,and 20μmol/L,and the optimal concentration of the primers and the TaqMan probe was determined using an optimization matrix with a 10-3 dilution as a template.The number of real-time fluorescence quantitative PCR cycles was optimized,and the optimal annealing temperature was selected.Sensitivity was tested with a sample diluted from 100 to 109 as a template and dddH2 Oas the negative control.Specificity was tested by extracting CSFV,PRRSV,PRV,and PCV1DNA（CSFV and PRRFV RNA was extracted and reverse-transcribed into cDNA）to serve as a template with a 102 dilution as the positive control and dddH2 Oas the negative control.Reproducibility was tested with a sample diluted from 105 to 108 as a template.Intra-and inter-assay reproducibility were tested 4times.Based on these results,the coefficient of variation was calculated. Results Electrophoresis on 20g/L agarose gels indicated that the PCR product produced a bright band at approximately 114 bp,and this agreed with the expected value.The sequence of the PCR product had 100%similarity to the sequence of the reference strain.Optimal results were achieved with an annealing temperature of 60 ℃ and a primer and TaqMan probe concentration of 10μmol/L.A method of real-time fluorescence quantitative PCR based on TaqMan was established.The initial number of template copies was 38.403,the slope of the standard curve was-3.69,and the correlation coefficient（R）was 0.998.The standard curve was linear.Testing indicated that sensitivity was 10-2plasmid copies/μl,which means that the current method has 100 times the sensitivity of conventional PCR.Testing of specificity indicated that the current method amplified PCV2 but not CSFV,PRRSV,PRV,or PCV1,indicating a good specificity.Reproducibility testing indicated that the cycle threshold（CT）values from the current method essentially coincided with average CT values.The coefficient of variation was smaller than 2%.In a test involving 40 clinical samples,conventional PCR detected PCV2 in 11samples for a detection rate of 27.5% while the current method detected PCV2 in 14samples for a detection rate of 35%. Conclusion The method of fluorescence PCR established here has the following advantages：a high level of sensitivity,allowing accurate calculation of the viral load;a high level of specificity,facilitating the detection of PCV2 in a mixed infection involving other pathogens;and rapid performance and a high level of reliability,which mean that the current method can be used to detect infection with PCV2.