牛病毒性腹泻病毒链特异性SYBR Green荧光定量PCR方法的建立及应用

doi:10.11843/j.issn.0366-6964.2019.10.014

摘要/Abstract

摘要： 为了对牛病毒性腹泻病毒（bovine viral diarrhea virus，BVDV）复制过程中正、负链RNA进行定量检测，通过生物学软件DNAStar和Primer express 3.0设计BVDV正、负链RNA特异性反转录引物并在其5'端添加非病毒核苷酸标签序列以区分BVDV正、负链RNA。同时设计荧光定量PCR引物与非病毒核苷酸标签序列组成引物对，建立了BVDV链特异性荧光定量RT-PCR（ssRT-qPCR）方法，对其敏感性、重复性、特异性进行评估并利用所建立BVDV ssRT-qPCR对不同接毒剂量下BVDV在细胞内复制过程中正、负链RNA进行定量检测。结果表明，以构建的pMD18T-BV+和pMD18T-BV-质粒为标准品建立的BVDV正链特异性荧光定量RT-PCR方法[ss（+）RT-qPCR]和负链特异性荧光定量RT-PCR方法[ss（-）RT-qPCR]在10²~10⁷拷贝范围内具有良好的线性关系，线性相关系数分别为0.998 1和0.995 3；敏感性试验结果显示，ssRT-qPCR敏感性较好，ss（+）RT-qPCR最低检测下限为10拷贝，ss（-）RT-qPCR最低检测下限为100拷贝。重复性试验结果显示，所建立的ssRT-qPCR批内和批间的变异系数为均小于1%，方法重复性良好。特异性试验显示，所建立的ssRT-qPCR方法与牛副流感病毒3型、牛传染性鼻气管炎病毒、牛呼吸道合胞体病毒等不存在交叉反应，特异性较好。利用建立的ssRT-qPCR对不同感染剂量下细胞内BVDV正、负链RNA进行定量分析，结果显示以10、0.1 MOI剂量接种BVDV于MDBK细胞后，BVDV正、负链RNA变化呈现先升后降再逐渐上升的趋势。以1 MOI剂量感染MDBK细胞，BVDV正、负链RNA的动态变化趋势略有差异，整体呈上升趋势。10、1 MOI接毒剂量接种细胞后，BVDV正、负链RNA在感染后36 h基本维持稳定水平，以0.1 MOI接种细胞后BVDV正、负链RNA在感染后24 h即达到稳定水平。BVDV链特异性检测进一步验证了所建立方法的适用性。该研究建立了BVDV链特异性荧光定量RT-PCR方法并利用该方法对BVDV在细胞内复制过程中正、负链RNA的动态变化过程进行描述，为研究宿主蛋白抗病毒机制、BVDV基因组RNA复制及调控机制等研究提供了研究手段。

Abstract: To quantitatively detect the positive and negative strand RNAs of bovine viral diarrhea virus (BVDV) during intracellular replication, the specific reverse transcription primers with non-viral nucleotide sequences for distinguishing the positive and negative strand RNAs of BVDV from each other and primers for real-time PCR were designed using DNAStar and Primer express 3.0 software. Primer pairs composed of real-time PCR primers and non-viral nucleotide sequences were used for the establishment of strand specific reverse transcription real-time quantitative PCR (ssRT-qPCR). The sensitivity, reproducibility and specificity of the ssRT-qPCR were evaluated. The dynamic changes of the positive and negative strand RNAs of BVDV during intracellular replication were described by the established ssRT-qPCR. Results were as follows:The standard curve of ss(+)RT-qPCR and ss(-)RT-qPCR had good linear relationships in the range of 10²-10⁷ copies of template with the linear correlation coefficients up to 0.998 1 and 0.995 3, respectively. Sensitivity test showed that the lowest detection limit of ss(+)RT-qPCR was 10 copies of template, while that of ss(-)RT-qPCR was 100 copies of template. The ssRT-qPCR was reproducibility with less than 1% of the coefficient of valuation. There were no cross-reactions with bovine parainfluenza virus type 3, infectious bovine rhinotracheitis virus, and bovine respiratory syncytial virus. The dynamic changes of the positive and negative strand RNAs of BVDV during replication in MDBK cells infected with different multiplicity of infection were analyzed using ssRT-qPCR. The results showed that the positive and negative strand RNAs of BVDV increased firstly, then decreased, and then gradually increased in MDBK cells inoculated with BVDV at 10 and 0.1 MOI. Meanwhile, the dynamic change of positive and negative strand RNAs of BVDV was different in MDBK cells infected with BVDV at 1 MOI which the overall upward trend. The positive and negative strand RNAs of BVDV eventually reached a plateau in MDBK cells inoculated with BVDV at 10 and 1 MOI after 36 hours. However, the positive and negative strand RNAs of BVDV eventually reached a plateau in MDBK cells inoculated with BVDV at 0.1 MOI after 24 hours. The applicability of ssRT-qPCR was further validated by BVDV chain specificity test. ssRT-qPCR of BVDV was established in this study and the dynamic changes of positive and negative strand RNAs of BVDV during intracellular replication were described. This study provided a research tool for the researches of the antiviral mechanisms of host proteins and replication and regulation mechanisms of BVDV.

中图分类号:

S852.659.6

刘存, 邓永, 梁琳, 李金祥, 张彦明, 崔尚金. 牛病毒性腹泻病毒链特异性SYBR Green荧光定量PCR方法的建立及应用[J]. 畜牧兽医学报, 2019, 50(10): 2079-2087.

LIU Cun, DENG Yong, LIANG Lin, LI Jinxiang, ZHANG Yanming, CUI Shangjin. Establishment and Application of Strand Specific SYBR Green Real-time PCR for Detection of Bovine Viral Diarrhea Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(10): 2079-2087.

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