新型分枝杆菌荧光报告噬菌体的构建及其在结核分枝杆菌药敏检测中的应用  

Development of a novel luminescence reporter mycobacteriophage for rapid drug susceptibility testing of Mycobacterium tuberculosis

作　　者：钱程成 马瑞青 郭明权 范小勇 Qian Chengcheng;Ma Ruiqing;Guo Mingquan;Fan Xiaoyong(School of Laboratory Medicine and Life Science,Wenzhou Medical University,Wenzhou 325035,China;Department of Scientific Research,Shanghai Public Health Clinical Center,Fudan University,Shanghai 201508,China)

机构地区：[1]温州医科大学检验医学院、生命科学学院,温州325035 [2]上海市(复旦大学附属)公共卫生临床中心科学研究部,上海201508

出　　处：《中华微生物学和免疫学杂志》2023年第10期749-755,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家重点研发项目(2022YFC2302900);上海市卫健委项目(20214Y0089,2022XD060);国家自然科学基金(82171815);上海市科委项目(20Y11903400)。

摘　　要：目的设计基于纳米荧光素酶(Nluc)的新型荧光报告系统,构建新型分枝杆菌荧光报告噬菌体ΦFN,分析该噬菌体检测结核分枝杆菌耐药性的能力。方法构建P_(furAma)启动子控制表达的Nluc荧光报告系统,将其整合至耻垢分枝杆菌基因组中,测试其在分枝杆菌的荧光报告能力;随后将P_(furAma)-nluc报告系统整合到TM4分枝杆菌噬菌体基因组中,构建新型分枝杆菌荧光报告噬菌体ΦFN;通过调整药物预处理时间、感染时间等条件,建立ΦFN检测分枝杆菌耐药性的快速检测流程,在96孔板中检测52株已知耐药性的结核分枝杆菌临床分离株对3种一线抗结核药物耐药的耐药性。结果整合型表达荧光报告系统P_(furAma)-nluc的耻垢分枝杆菌可报告下限达到100菌落形成单位(colony-forming unit,CFU),低于已报道的P_(left)启动子表达nluc系统。新型分枝杆菌荧光报告噬菌体ΦFN可通过孔板读值法在24 h内检测到分枝杆菌的存在,检测下限达到100 CFU。药物与待测菌预培养48 h后再加入噬菌体孵育24 h,敏感菌在105 CFU以下均无法检测到荧光信号,可有效区分敏感株并进行耐药性的快速检测。以罗氏药敏实验作为金标准,使用ΦFN检测52株结核分枝杆菌临床菌株对利福平、异烟肼和链霉素的耐药性,检测准确性分别是51/52、48/52、49/52。结论利用新型重组荧光报告噬菌体检测结核分枝杆菌对利福平、异烟肼、链霉素的耐药性具有极高的准确性,报告时间仅需3 d,有望成为一种快速简便的结核分枝杆菌耐药性检测新方法。ObjectiveTo construct a new reporter mycobacteriophage,ΦFN,based on a nanoluciferase(Nluc)reporter system,and to analyze its ability to detect drug resistance in Mycobacterium tuberculosis(Mtb).MethodsA Nluc reporter system controlled by P_(furAma) promoter was constructed and integrated into Mycobacterium smegmatis(Msm)genome to assess its reporting ability in mycobacteria.Then the P_(furAma)-nluc reporter system was integrated into TM4 mycobacteriophage genome to construct a new phage termedΦFN.A rapid procedure for detecting drug resistance in mycobacteria usingΦFN was established by adjusting conditions such as drug exposure time and time of infection.The susceptibility of 52 clinical isolates of Mtb with known drug resistance to three first-line anti-tuberculosis drugs were tested in 96-well plates.ResultsThe recombinant Msm mc2155 expressing P_(furAma)-nluc repoerter system could generate luminescence signal at a low limit of 100 colony-forming unit(CFU),which was lower than the previously reported nluc system controlled by P_(left) promoter.The detection limit ofΦFN for mycobacteria reached 100 CFU within 24 h by luminescent microplate reader.After 48 h of antibiotic exposure and 24 h of phage incubation,no luminescence signal could be detected when susceptible strains were below 105 CFU,which could effectively distinguish susceptible strains and rapidly detect drug resistance.The drug susceptibility of 52 clinical isolates of Mtb to rifampin,isoniazid and streptomycin was tested usingΦFN,and the accuracy was 51/52,48/52 and 49/52,respectively,by using the conventional drug susceptibility test,Lwenstein-Jensen culture medium assay,as reference.ConclusionsThe new recombinant luminescent reporter phage,ΦFN,showed high accuracy in detecting the drug susceptibility of Mtb to rifampicin,isoniazid and streptomycin and it only took three days.It is expected to be a new simple assay for the rapid detection of drug susceptibility of Mtb.

关 键 词：结核分枝杆菌 荧光报告噬菌体 药物敏感性测试 耐药结核 

分 类 号：R446.5[医药卫生—诊断学]