牛星状病毒、牛病毒性腹泻病毒1型、牛冠状病毒和牛轮状病毒四重实时荧光定量RT-PCR检测方法的建立

利用多重荧光定量RT-PCR （real-time RT-PCR）方法，提高对多病原检测的速度和灵敏度，促进对犊牛腹泻的快速诊断和及时治疗。分别在牛星状病毒（BAstV）ORF2基因，牛病毒性腹泻病毒1型（BVDV-1）5'端非编码区，牛冠状病毒（BCV）N pro基因和牛轮状病毒（BRV）VP6基因的保守基因序列设计、合成并试验筛选了四对有效的特异性引物和探针。进一步利用含4种病毒目的片段的重组质粒，对引物和探针的浓度以及反应条件进行了优化，建立了Real-time RT-PCR标准曲线，并对四重Real-time PCR方法的特异性、敏感性、重复性和各种临床样本的适用性进行了评价。结果显示：Real-time RT-PCR最适退火温度和时间分别为50.0℃和45 s，BAstV、BVDV-1、BCV和BRV的引物浓度分别为300、300、400和500 nmol·L^-1，探针浓度分别为250、150、100和300 nmol·L^-1。对BVDV-1、BCV和BRV的最低检测限均为10²copies·μL^-1，对BAstV的最低检测限为10³ copies·μL^-1，具有良好的特异性和重复性。该方法对临床采集的粪样的阳性检出率高于PCR方法。上述结果表明，建立的四重Real-time RT-PCR方法可以用于犊牛腹泻常见病原BAstV、BVDV-1、BCV和BRV的快速鉴别诊断。

The Development of the Quadruple Real-time RT-PCR for Bovine Astroviruses,Bovine Viral Diarrhea Virus 1, Bovine Coronavirus and Bovine Rotavirus

The multiple real-time RT-PCR method was used to improve the speed and sensitivity of multiple pathogen detection, which can promote rapid diagnosis and prompt treatment of calf diarrhea. Four pairs of effective primers and probes were designed,synthesized and experimentally screened targeting the conserved region of ORF2 Gene from bovine astroviruses (BAstV), 5'-UTR from bovine viral diarrhea virus 1 (BVDV-1), N pro gene from bovine coronavirus (BCV) and VP6 gene from bovine rotavirus (BRV), respectively. The concentrations of the primers, probes and the reaction parameters and conditions of the real-time RT-PCR reaction system were further optimized with four recombinant plasmids containing the target fragments of BAstV, BVDV-1, BCV and BRV respectively. The standard curves were drawn to assess the specificity, sensitivity and reproducibility of the real-time RT-PCR reaction system. The results showed that the optimum concentrations of the primers were 300, 300, 400 and 500 nmol·L^-1, and the optimum concentrations of the probes were 250, 150, 100 and 300 nmol·L^-1 for BAstV, BVDV-1, BCV and BRV respectively, and the annealing temperature and time were 50.0℃ and 45 s. The minimum detectable limits of BAstV, BVDV-1, BCV and BRV were 1 000, 100, 100 and 100 copies·μL^-1 respectively, and the quadruple real-time RT-PCR has good specificity and repeatability and the positive detection rate of clinical collection of fecal samples were higher than that of the PCR method. In conclusion, the results showed that the quadruple real-time RT-PCR method established in this paper can be used for the rapid differential diagnosis of BAstV, BVDV-1, BCV and BRV which are common causes of diarrhea in calves.