猪链球菌微滴数字PCR定量检测方法的建立

根据猪链球菌（Streptococcus suis，SS）gdh基因的保守序列设计特异性引物和TaqMan探针，通过反应条件优化和特异性、敏感性、重复性试验以及临床样品的检测，建立可对其准确定量的微滴数字PCR（droplet digital PCR，ddPCR）检测方法，并与实时荧光定量PCR（Quantitative real-time PCR，qPCR）检测方法进行比较分析。结果表明，该方法特异性良好，与猪传染性胸膜肺炎放线杆菌、副猪嗜血杆菌、大肠杆菌、沙门氏菌、金黄色葡萄球菌、猪圆环病毒2型和伪狂犬病毒无交叉反应；灵敏度优于qPCR，线性相关系数（R2）为0.991，呈良好线性关系，最低可检测到2.692 copies/μL的阳性质粒；稳定性好，变异系数为1.66%。临床样品定量检测结果表明，ddPCR具有敏感性高、特异性好等优点，能对qPCR检测的可疑样品进行精确定量。本研究建立的ddPCR能够准确定量检测SS，将为SS相关研究提供有益参考。
A droplet digital polymerase chain (ddPCR) detection method, based on gdh gene of Streptococcus suis was developed to improve the detection and quantification of S. suis. S. suis is an important pathogen, resulting in significant economic losses in pig farming. Prompt detection of S. suis in the field samples is important for effective control. ddPCR is a novel PCR technology, which offers good precision and direct quantification without using calibration curves. In this work, a ddPCR method for the sensitive and accurate quantification of S. suis was established. Under the optimized reaction conditions, results showed that ddPCR was specific to detect S. suis with detection limit of 2.692 copies/μL, and no cross-reactions with Actinobacillus pleuropneumoniae, Haemophilus parasuis, Bacillus subtilis, Escherichia coli, Salmonella, Staphylococcus aureu, Porcine circovirus type2 and Pseudorabies virus. The correlation coefficient (R2) obtained from linear regression analysis showed a good linearity of amplification for ddPCR (R2=0.991), and the repeatability test indicated that the coefficient of variation were 1.66%. The clinical application of ddPCR assay indicated that the sensitive and specific detection method could be useful to standardize quantitative detection of S. suis.