猪Linda病毒实时荧光定量PCR检测方法的建立

Establishment of real-time fluorescent quantitative PCR assay for detection of Linda virus

为建立猪Linda病毒(LV)的实时荧光定量PCR检测方法,本研究基于LV的Erns基因序列,设计并合成了实时荧光定量PCR引物和探针,经过优化退火温度、引物和探针的比例,建立了一种灵敏度高、特异性好且操作简便的LV实时荧光定量PCR检测方法。该方法具有良好的线性、稳定性及准确性,最低检出限为1×10^(1)copies/μL,批内和批间变异系数均小于2%,且该方法检测非洲猪瘟病毒、猪瘟病毒、猪伪狂犬病病毒、高致病性猪繁殖与呼吸综合征病毒、猪圆环病毒2型时呈阴性。通过对不同模拟样品的检测结果进行比较分析,发现该方法能够检测到1×10^(1)copies/μL模拟DNA样品、1×10^(1)TU/mL模拟RNA样品。本研究建立的实时荧光定量PCR方法可用于LV的快速检测,为口岸检测、预防与控制由LV引起的进境仔猪先天性震颤提供了技术支持。

To establish a real-time fluorescence quantitative PCR assay for porcine Linda virus(LV),we designed and synthesized real-time fluorescent quantitative PCR primers and probe based on the Erns gene sequence of LV,and established a real-time fluorescent quantitative PCR assay for LV with high sensitivity,good specificity and easy operation by optimizing the annealing temperature and the ratio of primers to probes.The real-time fluorescence quantitative PCR standard curve showed good linearity,stability and accuracy with a minimum detection limit of 1×10^(1)copies/μL.The intra-and inter-batch coefficients of variation were less than 2%.And the assay was negative for African swine fever virus,swine fever virus,porcine pseudorabies virus,highly pathogenic porcine reproductive and respiratory syndrome virus and porcine circovirus type 2.The minimum detection limits of sensitivity for the simulated DNA sample assays were 1×10^(1)copies/μL and for the simulated RNA sample assays were 1×10^(1)TU/mL.The method in this study can be used for the rapid detection of LV and provides technical support for the detection,prevention and control of congenital tremor of piglets caused by Linda virus.