摘要
为建立检测美洲型猪繁殖与呼吸综合征病毒(PRRSV)的荧光定量PCR方法,在本实验室前期试验筛选到扩增效果相对最好的3对引物(扩增基因区域对应ORF6)的基础上,本研究以PRRSV CH-1R株cDNA作为模板,利用这3对引物进行PCR扩增,将扩增产物克隆至pMD18-T载体,构建3个重组质粒标准品。采用方阵法对荧光定量PCR反应条件优化,最终建立了3对引物的SYBR Green Ⅰ荧光定量PCR检测方法。结果显示,标准曲线Ct值均与相应质粒标准品浓度存在良好的线性关系,相关系数(R~2)分别为0.992、0.999、0.999;该方法除对美洲型PRRSV有特异性扩增外,对猪圆环病毒2型(PCV2)、伪狂犬病毒(PRV)、猪瘟病毒(CSFV)、欧洲型PRRSV等病原的基因组DNA或cDNA均无扩增,特异性强;对3种质粒标准品的检测下限均为1.228×10^(1)拷贝/μL,敏感性高;组内与组间重复性试验变异系数均小于2%,重复性较好。利用该方法检测6份经预检的PRRS临床样品,3对引物的阳性检出率均为100%,与国标荧光定量PCR方法检出率相当,高于常规PCR(50%、50%和83.3%)的阳性检出率。尽管该方法的3对引物和国标方法均能检出,但部分样品不同引物检测的Ct值有差异。本研究通过利用多对引物并提高引物覆盖面,可减少因引物结合位点突变引起的检测不准确问题,3对引物中,若有1对引物检测样品的结果呈阳性,则该样品判为阳性样品,本研究为PRRSV的快速和定量检测提供了思路和方法。
In order to establish a detection and quantization method for type 2 porcine reproductive and respiratory syndrome virus(PRRSV), on the basis of the three pairs of primers with the best amplification effect(the amplified gene region corresponding to ORF6) that screened in the previous experiment, the cDNA of PRRSV CH-1R strain was used as template for PCR amplification with these three pairs of primers and the amplified products were cloned into pMD18-T vector to obtain three recombinant standard plasmids. Further, optimized SYBR Green Ⅰ quantitative PCR methods were established by using these three pairs of primers and the specificity test, sensitivity test and repeatability test were performed to verify the real-time PCR method.The results showed that strong correlations were obtained between the Ct values and the copy number of the corresponding plasmids standard, and the correlation coefficients(R^(2)) were 0.995, 0.999, 0.999, respectively. These assays showed high specificity,as there was no cross-reactions with the genomic DNA/cDNA of PCV2, PRV, CSFV and type 1 PRRSV. The sensitivity tests showed that the minimum detection limit of these methods was all 1.228 copies/μL and the coefficient of variation(CV) of intra and inter assay were less than 2%. Six pre-examined clinical PRRS samples were detected by these methods and the results showed positive detection rate was 100%, which were higher than the conventional PCR(50%, 50%, 83.3%, respectively). Although the established real-time PCR methods using the three pairs of primers could detect all six samples, the Ct values of some samples were different when using different primers. Using multiple primer pairs may mitigate the problem of inaccurate detection and quantitation that resulted from possible random mutations at primer sites. If any of the three primer pairs shows a positive result for the sample tested, it is considered as a positive sample. This study provides idea and technical support for the diagnosis of PRRSV.
作者
查帆
李倩文
孙广禄
余子豪
王晓洁
余旭平
ZHA Fan;LI Qian-wen;SUN Guang-lu;YU Zi-hao;WANG Xiao-jie;YU Xu-ping(College of Animal Science,Zhejiang University,Hangzhou 310058,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第7期731-736,749,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(31272582)
浙江省重点研发计划项目(2020C02032)。
作者简介
查帆(1995-),男,浙江诸暨人,硕士研究生,主要从事病原分子生物学研究;通信作者:余旭平,E-mail:xpyu@mail.zju.edu.cn。
