结核分枝杆菌复合群、副结核分枝杆菌和禽分枝杆菌禽亚种多重TaqMan荧光定量PCR方法的建立及初步应用

Establishment and preliminary application of multiplex 7aqMan fluoresce nee qua ntitative PCR method for detecti on of Mycobacterium tuberculosis complex,Mycobacterium avium subsp.paratuberculosis and Mycobacterium avium subsp.avium

结核分枝杆菌复合群(MTBC)、包括结核分枝杆菌(MTB)、牛分枝杆菌(MB)、非洲分枝杆菌、田鼠分枝杆菌等;非结核分枝杆菌(NTM)中禽分枝杆菌(MA)是自然界常见的动物致病菌,包含4个亚种,分别为禽分枝杆菌禽亚种(MAA)、禽分枝杆菌人/猪亚种(MAH)、禽分枝杆菌副结核亚种/副结核分枝杆菌(MAP)和禽分枝杆菌森林土壤亚种(MAS)。为建立MTBC、MAP和MAA的多重荧光定量PCR检测方法,本研究基于MTBC、MAP和MAA的特异性基因devR、F57和IS901分别设计引物及探针,通过优化各反应条件,建立了一种可同时检测3种分枝杆菌的多重Taq Man荧光定量PCR方法。特异性试验结果显示,该方法对MTB、MB、MAP、MAA和MAS检测为阳性,对其他畜禽病原菌,包括MAH、13种NTM和7种非分枝杆菌检测结果均为阴性,特异性较强。敏感性试验结果显示,该方法对MB、MAP和MAA重组质粒标准品的检测限均为1.0拷贝/μL,敏感性较高;重复性试验结果显示,批内与批间重复性试验变异系数均小于2.5%,重复性较好。利用该方法检测体外模拟污染(MB、MAP和MAA菌液浓度分别为1.0×10^(3)cfu/mL~1.0×10^(0)cfu/mL)的27份牛抗凝血和12份牛淋巴结组织样品,确定该方法对3种菌的检测限,结果显示该方法对3种菌的检测限均为1.0 cfu/mL~10.0 cfu/mL。将浓度为1.0×10^(6)cfu/mL的MB、MAP和MAA菌液分别单一、两两混合、三种菌混合感染BALB/c小鼠,5 d后,剖杀各组小鼠并取各组织及血液样品,同时采用该方法、常规单一PCR及细菌分离培养方法检测,比较三者的检测结果。结果显示,多重荧光定量PCR方法对不同感染组小鼠的各组织样品检出率为98.1%(212/216),常规单一PCR方法的检出率为81.5%(176/216),细菌分离培养的检出率为52.8%(114/216),多重荧光定量PCR与常规单一PCR的阳性符合率均为100.0%(176/176),与细菌分离培养鉴定结果的阳性符合率均为100.0%(114/114)。本研究首次建立了能够同时检测MTBC、MAP和MAA的Taq Man多重荧光定量PCR方法,其特异性强、敏感性高、重复性及稳定性好,检测性能强,可以用于临床各种样品的检测,为这3种病原的快速检测和流行病学调查提供了技术手段。

Mycobacterium tuberculosis complex(MTBC)is the main pathogen of human and animal tuberculosis,including Mycobacterium tuberculosis(MTB),Mycobacterium bovis(MB),Mycobacterium africans,and Mycobacterium vole,etc.In Nontuberculous mycobacteria(NTM),Mycobacterium avium(MA)is a common animal pathogenic bacterium in nature,which contains four subspecies including Mycobacterium avium subsp.avium(MAA),Mycobacterium avium subsp.hominissuis(MAH),Mycobacterium avium subsp.paratuberculosis(MAP)and Mycobacterium avium subsp.silvaticum(MAS).To establish a multiplex fluorescent quantitative PCR assay for detection of MTBC,MAP and MAA,three pairs of primers and TaqMan probes were designed according to the specific sequence of the devR gene,F57 gene and IS901 gene.After optimization of the reaction system and conditions,a multiplex Ta-Man fluorescent quantitative PCR method for the simultaneous detection of three mycobacteria was established.Specific test results showed that the method was positive for MTB,MB,MAP,MAA and MAS,and negative for other pathogens including MAH,13 NTMs(Mycobacterium vaccae,Mycobacterium intracellular Mycobacterium Kansasum,Mycobacterium accidentum,Mycobacterium microfidufo^Mycobacterium Gordonum,Mycobacterium gastritis,Mycobacterium Genevan,Mycobacterium abscess,Mycobacterium haliconisf Mycobacterium smegmatis9 Mycobacterium grasum)and 7 non-mycobacteria(Brucella,Mycoplasma bovis,Klebsiella pneumoniae,Staphylococcus aureus,Escherichia coli,Salmonella enteritidis,Streptococcus suis type II),indicating strong specificity.Sensitivity test results showed that the lower limit of detection for MB,MAP and MAA recombinant plasmid standards was 1.0 copies/gL,indicating high sensitivity.The results of repeatability test showed that the coefficient of variation in both intra-and inter-batch was less than 2.5%,suggesting good repeatability.A total of 27 samples of bovine anticoagulant blood and 12 samples of bovine lymph node tissue with simulated contamination in vitro(MB,MAP and MAA bacterial solution concentrations of 1.O×10^(3)cfu/mL-1.O×10^(0)cfu/mL,respectively)was used to determine the detection limits of the established method on the three kinds of bacteria.The results showed that the lower limit of detection for the three strains was 1.Ocfu/mL-10.0cfu/mL.The MB,MAP,and MAA bacterial culture were administered to BALB/c mice at doses of l.Ox 10^(6)cfii/niL for each single,double,and triple infections,respectively.The tissue and blood samples were collected 5 days post infection and tested by this method,conventional single PCR and bacterial isolation and culturing.The results showed that the detection rate of multiple fluorescent quantitative PCR was 98.1%(212/216),and it's 81.5%(176/216)for conventional single PCR and 52.8%(114/216)for bacterial isolation and culturing.The positive coincidence rate of multiplex fluorescence quantitative PCR with conventional single PCR was 100.0%(176/176),and the positive coincidence rate with the results of bacterial isolation and culturing was 100.0%(114/114).In this study,a multiplex ThgMan fluorescent quantitative PCR method,which can simultaneously detect MTBC,MAP and MAA,was established for the first time.It has strong specificity,high sensitivity and good stability;providing a technical means for rapid detection and epidemiological investigation of these three pathogens.