猪链球菌通用型和2型双重荧光定量PCR快速检测技术的建立和应用

doi:10.11843/j.issn.0366-6964.2018.02.016

摘要/Abstract

摘要：

为快速区分检测猪链球菌血清型2型和其他血清型，以猪链球菌的保守基因gdh和2型特异性基因cps2J为靶基因设计引物和TaqMan探针，建立猪链球菌通用型和2型特异性的双重荧光定量PCR检测方法，并与常规PCR方法一起对临床样品进行检测。结果显示：本方法在1.5 h内可完成猪链球菌和2型猪链球菌同时检测，与其他细菌无交叉反应；检测灵敏度可达5拷贝数，标准曲线相关系数大于0.999，批内和批间CV均小于1.25%。对67份临床样品检测显示猪链球菌和2型猪链球菌检出率分别为79.1%和35.8%，与常规PCR检测结果的符合率为92.5%和89.6%，kappa值为0.800和0.757，具有极好的一致性。成功建立了灵敏、特异和稳定的双重荧光定量PCR方法，实现了猪链球菌和2型猪链球菌同时及快速诊断。

Abstract:

In order to detect S. suis and S. suis serotype 2 rapidly, two sets of specific primers and TaqMan probes were designed according to the gdh and cps2J gene of S. suis serotype 2 to establish a duplex real-time PCR assay for detection of S. suis and S. suis serotype 2. Then the duplex real-time PCR was used to detect clinical samples and compare with conventional PCR. Results showed that S. suis and S. suis serotype 2 could be identified in 1.5 hours by the duplex real-time PCR assay and without any cross reaction with other bacteria. Besides, the detection limit was 5 copies/reaction. The correlation coefficient of standard curves was over 0.999, and intra-and inter-assay CV was less than 1.25%. Positive rate for sixty-seven clinical samples was 79.1% and 35.8%, respectively. Coincidence rates of duplex real-time PCR with conventional PCR for S. suis and S. suis serotype 2 were 92.5% and 89.6%, kappa values were 0.800 and 0.757, respectively. A sensitive, specific, reproducible assay is established successfully to detect S. suis and S. suis serotype 2 simultaneously and rapidly, and this assay is very suitable for detection of mass clinical samples.

中图分类号:

S852.611

吴静波, 南文金, 黄健强, 胡鸿惠, 彭国良, 彭凌, 董小英. 猪链球菌通用型和2型双重荧光定量PCR快速检测技术的建立和应用[J]. 畜牧兽医学报, 2018, 49(2): 368-377.

WU Jing-bo, NAN Wen-jin, HUANG Jian-qiang, HU Hong-hui, PENG Guo-liang, PENG Ling, DONG Xiao-ying. Development of A Rapid Duplex Real-Time PCR Assay for Streptococcus suis and S. suis Serotype 2[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 368-377.

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