外周血淋巴细胞布鲁氏菌核酸DNA检测的实验研究

目的基于目前布鲁氏菌病诊断现状，针对试管凝集滴度小于1∶100且布鲁氏菌病相应症状未持续1年患者，探讨其外周血淋巴细胞中布鲁氏菌DNA用于临床诊断的可能性。方法收集患者的血液，分离其外周血淋巴细胞和血清，分别提取其核酸，利用布鲁氏菌鉴定引物B4/B5进行普通PCR扩增，检测患者体内是否存在布鲁氏菌DNA。结果2017年1月1日至2018年2月28日期间共收集布鲁氏菌病患者血液样本278份，其中82份样品试管凝集检测滴度为小于1∶100，且患者病程未持续1年。 82份样品中分离自患者外周血淋巴细胞核酸中检测到布鲁氏菌核酸阳性样品为50份，而对应血清布鲁氏菌核酸阳性样品为15份，两者差异有统计学意义（P＜0.05）。 在血清抗体5个滴度段中SAT阴性、1∶25、1∶50、1∶100、1∶200，SAT阴性组患者全血淋巴细胞中布鲁氏菌核酸DNA检测率最高，1∶200组患者血清布鲁氏菌核酸DNA检出率最低。结论全血外周血淋巴细胞核酸布鲁氏菌DNA检测可用于血清抗体检测阴性患者的初步诊断，为布鲁氏菌病的诊断提供新的补充方法。

Experimental detection of DNA of Brucella in peripheral blood lymphocytes

ObjectiveTo investigate the feasibility of the diagnosis potential of Brucella nucleic acid detection in peripheral blood lymphocytes for patients with a tube agglutination titer (SAT) less than 1∶100 and disease duration less than one year.MethodsThe blood samples of the patients were collected for peripheral blood lymphocytes and serum isolations. Then, the nucleic acid extraction was conducted, and PCR amplification was performed by using the Brucella identification primer B4/B5 to detect the presence of Brucella DNA in the collected samples.ResultsA total of 278 blood samples from patients with brucellosis were collected from January 2017 to February 2018, of which 82 had a SAT of 1∶100 or less, and the disease duration did not last for one year. In 82 patient samples, 50 out of 82 peripheral blood lymphocyte samples were positive for nucleic acid of Brucella, while 15 of 82 serum samples were positive for nucleic acid of Brucella, the difference was significant (P＜0.05). Among the SAT (-), 1∶25, 1∶50, 1∶100, 1∶200 of serum antibody titers, the SAT(-) group had the highest detection rate of Brucella DNA in whole blood lymphocytes, while the 1∶200 group had the lowest detection rate.ConclusionThe detection of Brucella nucleic acid in peripheral blood lymphocyte can be used for the diagnosis of brucellosis in cases with negative serum antibody detection, providing a new method for the diagnosis of brucellosis.