猪细小病毒荧光定量PCR检测方法的建立及应用

Development and application of real-time fluorescent quantitative PCR assay for detection of porcine parvovirus

为研究miRNA在猪细小病毒（PPV）复制中的作用机制,针对PPV的NS1基因设计引物,建立了实时荧光定量PCR;用RegRNA软件和RNAhybrid软件预测能靶向PPV基因组的miRNA,将筛选出的miR-34a与miR-181a、miR-16、miR-499-5p、miR-10b、miR-18a、miR-145-5p分别转染到PK-15细胞,然后用PPV感染细胞,每隔12h取细胞上清,用实时荧光定量PCR检测PPV的DNA拷贝数。结果显示,实时荧光定量PCR的最适退火温度为59℃,熔解温度在85℃左右,质粒浓度在1.59×10^9~1.59×10^4 copies/μL时可得到较为理想的标准曲线,相关系数为0.997,扩增效率为97.2%。用该实时荧光定量PCR对日本脑炎病毒、猪繁殖与呼吸综合征病毒、猪瘟病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪轮状病毒、伪狂犬病病毒、猪圆环病毒2型进行检测时均无荧光信号,表明具有良好的特异性。转染不同miRNA的细胞感染PPV后,用建立的实时荧光定量PCR检测发现miR-34a能抑制PPV的复制,其他miRNA对PPV增殖无明显影响。

To study the functional mechanism of miRNA in porcine parvovirus（PPV） duplication, a pair of specific primers was designed according to the NS1 gene of PPV,and real-time fluorescent quantita- tive PCR（FQ-PCR） was established to quantify the PPV. The specific miRNA of targeted to PPV was forecasted using the software of RegRNA and RNAhybrid,including miR-34a, miR-181a, miR-16, miR-499-Sp, miR-10b,miR-18a, and miR-145-Sp. Seven miRNAs were transfected into PK-15 cells, respectively, and then the transfected PK-15 cells were infected by PPV. The supernatant of infected cells was collected every 12 h,the DNA copies of PPV was detected by FQ-PCR. The results showed that the optimum annealing temperature of FQ-PCR is 59 ℃ ,the melting temperature is approximately 85 ℃. When the plasmids reach a concentration of 1.59 × 10^9 to 1.59 × 10^4 copies/μL, a relatively rational standard curve can be obtained, with a correlation coefficient of 0. 997 and an amplification efficiency of 97.2%. No fluorescent signal was detected in Japanese encephalitis virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus of swine, porcine rotavirus, pseudorabies virus, and porcine circovirus type 2, which is proved to be a good specificity of the method. The result showed that only miR-34a can suppress the duplication of PPV by detecting the DNA in the PK-15 cells transfected with different miRNAs after PPV infection, but the others have no influence.