Nested-PCR在人间布鲁氏菌病早期快速诊断中的探讨  

Evaluation of Nested-PCR for early and rapid diagnosis of brucellosis in humans

作　　者：刘志国 王妙[3] 塔娜 李晓燕 尉瑞平 米景川 李丹 崔步云 李振军[2] LIU Zhi-guo;WANG Miao;TA Na;LI Xiao-yan;YU Rui-ping;MI Jing-chuan;LI Dan;CUI Bu-yun;LI Zhen-jun(Inner Mongolia Autonomous Region Central for Comprehensive Disease Controled and Prevention,Huhhot 010031,China;National Institute of Infectious Diseases Control and Prevention,Chinese Center for Disease Control and Prevention/Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease,Beijing 102206,China;Ulanqab Center for Endemic Disease Control and Prevention,Ulanqab 012000,China)

机构地区：[1]内蒙古自治区综合疾病预防控制中心,呼和浩特010031 [2]中国疾病预防控制中心传染病预防控制所,传染病预防控制国家重点实验室,北京102206 [3]乌兰察布市地方病防治中心,集宁012000

出　　处：《中国人兽共患病学报》2021年第2期188-192,共5页Chinese Journal of Zoonoses

基　　金：内蒙古自治区自然科学基金项目(No.2018MS08004);国家重点研发计划(No.2019YFC1200705);传染病重大专项(No.2018ZX10734401和No.2018ZX10734404)联合资助。

摘　　要：目的探讨Nested-PCR在布鲁氏菌病(布病)患者早期快速诊断中的可行性。方法用Nested-PCR对布鲁氏菌标准菌株的19生物型、4个疫苗株(M5、A19、S2和104M)和有血清交叉反应的菌株(人苍白杆菌、大肠杆菌O∶157、小肠结肠炎耶尔森氏菌O∶9)进行扩增,评价该方法的特异性;用Nested-PCR对倍比稀释的16M菌株DNA(100~10^(-7))进行扩增,评价该方法的分析敏感性。用Nested-PCR对临床采集的200份布病样本DNA进行检测,评价该方法在临床样本检测中的可行性。结果Nested-PCR能特异性的检出所有布鲁氏菌种,而与布鲁氏菌有血清学交叉反应的菌株和阴性对照未见扩增。试验表明该方法的分析敏感性至少为10-6(相当于43 fg/μL羊种布鲁氏菌DNA),明显高于BCSP31-PCR。该方法首轮引物的扩增阳性率为4.5%(9/200),次轮引物扩增的阳性率为75.5%(151/200),与血清学检测结果有较高的一致性。结论Nested-PCR可尝试用于布病患者的早期筛查,但尚需优化改进。The purpose of this study was to explore the feasibility of using Nested-PCR for early and rapid detection of brucellosis in humans.In this study,19 biovars of the genus Brucella standard reference strain and four vaccine strains(M5,A19,S2 and 104 M)had serologic cross-reactivity with O.anthropic,E.coli O∶157 and Y.enterocolitica O∶9.We performed Nested-PCR amplification to evaluate the detection specificity of this method.Subsequently,a double dilution(100~10^(-7))of Brucella 16 M DNA was amplified by Nested-PCR to assess the analytical sensitivity of the assay.Finally,a total of 200 DNA samples from clinical human brucellosis samples were assayed to explore the feasibility of Nested-PCR in clinical diagnosis of human brucellosis.Our results confirmed the presence of a specific amplified band in all Brucella species but not in other strains.The analytical sensitivity of this method was at least 10-6(equal to 43 fg/μL of B.melitensis 16ΜDNA),which was significantly higher than that of BCSP31-PCR.The positive rates of amplification with the first-round and second-round primers were 4.5%(9/200)and 75.5%(151/200),respectively.High concordance was observed between serum agglutination tests and Nested-PCR.Consequently,we suggest that Nested-PCR may used for early screening of human brucellosis,although further improvement of the method is essential.

关 键 词：布病 NESTED-PCR 早期诊断 

分 类 号：R378.5[医药卫生—病原生物学]