Abstract

The aim of the research was to evaluate real-time PCR (qPCR) as an alternate method for quantitative detection of Brucella abortus strain 544 (S544) in the spleen of mice for potency testing of live B. abortus strain 19 (S19) vaccine. IS711 and eryC gene-based qPCR were optimized for calculating copy number. The copy number was further correlated with live Brucella count in the spleen by standard plate count (SPC) method. The mice were immunized with S19 and challenged with S544 on 30th Day post-immunization. The spleen of mice was collected at 15th, 21st, and 30th days post challenge (DPC) for estimation of S19 and S544 load via SPC as well as qPCR. The noteworthy difference was observed between immunized and unimmunized group by both methods at all time points. The maximum correlation between SPC and qPCR method was observed at 15th DPC in both immunized and unimmunized group. Repeated experiments at 15th DPC gave the parallel significant difference between immunized and unimmunized group by both methods. Thus novel, risk-free qPCR method can be used for the indirect culture-free potency evaluation of S19 vaccine in order to preclude the cultivation of zoonotic Brucella organisms from spleen samples.

摘要

该研究的目的是评估实时 PCR (qPCR) 作为定量检测小鼠脾脏中流产布鲁氏菌菌株 544 (S544)的替代方法，用于测试流产布鲁氏菌 19 (S19)活疫苗的效力。IS711和基于eryC基因的 qPCR 被优化用于计算拷贝数。拷贝数与活布鲁氏菌进一步相关通过标准平板计数 (SPC) 方法在脾脏中计数。小鼠用 S19 免疫并在免疫后第 30 天用 S544 攻击。在攻击后 (DPC) 的第 15、21 和 30 天收集小鼠的脾脏，用于通过 SPC 和 qPCR 估计 S19 和 S544 负载。两种方法在所有时间点均观察到免疫组和未免疫组之间的显着差异。在免疫组和未免疫组的第 15 次 DPC 观察到 SPC 和 qPCR 方法之间的最大相关性。在第 15 个 DPC 的重复实验给出了两种方法免疫组和未免疫组之间的平行显着差异。因此，新的、无风险的 qPCR 方法可用于 S19 疫苗的间接无培养效力评估，以排除人畜共患病布鲁氏菌的培养 来自脾脏样本的生物体。