■猫冠状病毒(FCoV),猫泛白细胞减少症病毒(FPV),猫白血病病毒(FeLV)在中国普遍存在,并严重威胁猫的健康。这些病毒引起类似的表现和病理损害。快速准确的诊断取决于实验室的检测。本研究旨在建立一种可靠、快速、准确检测FCoV的方法,FPV,和FeLV,以便做出明确的诊断,并采取有效措施预防和控制病毒感染。
■我们设计了三对特异性引物和探针,用于检测FCoV5'非翻译区,FPV病毒蛋白2和FeLVpol基因。产生重组质粒构建体用作标准质粒构建体。最佳反应条件,包括引物和探针浓度,反应循环,和退火温度,在优化试验的基础上获得。成功建立了一步三螺旋实时逆转录-定量聚合酶链反应(RT-qPCR)同时检测FCoV,FPV,和FeLV。特异性,灵敏度,并分析了试验的可重复性,并通过检测1175份临床样本验证了其适用性。
■一步三联RT-qPCR仅对FCoV的检测具有高度特异性,FPV,和FeLV;它具有高灵敏度,p-FCoV的检出限为139.904、143.099和152.079拷贝/反应,p-FPV,和p-FeLV标准质粒构建体,分别,它具有可靠的可重复性,测定中的变异系数为0.06%-0.87%。总共对1175个临床样本进行了FCoV检查,FPV,和FeLV使用三重RT-qPCR,还有FCoV,FPV,FeLV阳性率为18.47%,19.91%,和47.57%,分别。一步法三组分RT-qPCR的临床敏感性和特异性分别为93.07%和97.99%,分别。
■我们开发了一种快速可靠的一步法三重RT-qPCR方法,用于检测FCoV,FPV,和FeLV,可以用作临床监测和诊断的诊断工具。
UNASSIGNED: Feline coronavirus (FCoV), feline panleukopenia virus (FPV), and feline leukemia virus (FeLV) are prevalent throughout China and significantly threaten cat health. These viruses cause similar manifestations and pathological damage. Rapid and accurate diagnosis depends on detection in the laboratory. This study aimed to establish a reliable and rapid method for accurate detection of FCoV, FPV, and FeLV so that a definite diagnosis can be made and effective measures can be taken to prevent and control viral infection.
UNASSIGNED: We designed three pairs of specific primers and probes for the detection of FCoV 5\' untranslated region, FPV viral protein 2, and FeLV pol genes. Recombinant plasmid constructs were generated for use as standard plasmid constructs. Optimal reaction conditions, including primer and probe concentrations, reaction cycles, and annealing temperatures, were obtained on the basis of optimization tests. One-step triplex real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was successfully established to simultaneously detect FCoV, FPV, and FeLV. The specificity, sensitivity, and repeatability of the assay were analyzed, and its applicability was validated by testing 1175 clinical samples.
UNASSIGNED: One-step triplex RT-qPCR had a high degree of specificity only for the detection of FCoV, FPV, and FeLV; it had high sensitivity with limits of detection of 139.904, 143.099, and 152.079 copies/reaction for p-FCoV, p-FPV, and p-FeLV standard plasmid constructs, respectively, and it had reliable repeatability with 0.06%-0.87% intra-assay coefficients of variations. A total of 1175 clinical samples were examined for FCoV, FPV, and FeLV using triplex RT-qPCR, and the FCoV, FPV, and FeLV positivity rates were 18.47%, 19.91%, and 47.57%, respectively. The clinical sensitivity and specificity of one-step triplex RT-qPCR were 93.07% and 97.99%, respectively.
UNASSIGNED: We developed a rapid and reliable one-step triplex RT-qPCR method for the detection of FCoV, FPV, and FeLV, which could be used as a diagnostic tool for clinical monitoring and diagnosis.