四种重要致病性分枝杆菌DHPLC检测鉴别方法的建立
陈茹;毕英佐;刘志玲;刘志辉;马静云;曾碧健;吴晓薇;周科;林志雄;
广东出入境检验检疫局;华南农业大学;广州市胸科医院;
Establishment of the denaturing high-performance liquid chromatography combined with multiplex nucleic acid amplification method for rapid identification of four important pathogenic mycobacteria
摘要 目的运用多重核酸扩增(PCR)联合变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)分析技术原理,针对结核分枝杆菌、牛分枝杆菌、禽分枝杆菌以及副结核分枝杆菌建立多重PCR-DHPLC快速检测方法,实现同时检测鉴别四种重要致病性分枝杆菌。方法根据四种分枝杆菌特异基因序列分别设计菌种特异核酸扩增引物,通过筛选优化试验建立四重核酸扩增体系,对核酸扩增产物采用DHPLC设备进行检测分析,每一菌种的核酸扩增产物分别形成DHPLC特征峰图。对结核分枝杆菌等51株分枝杆菌标准株和分离株样品以及沙门氏菌等22株常见微生物样品进行特异性检测试验;对四种分枝杆菌特异的核酸扩增产物分别制备克隆质粒,通过对梯度稀释的阳性质粒的检测试验,进行多重PCR-DHPLC灵敏度测试;对疑似病人痰液样品和疑似发病牛的组织样品进行检测,并与细菌分离培养法进行比较以验证临床检测效果。结果该方法能快速检测鉴别上述四种分枝杆菌,检测灵敏度达到102~103基因拷贝,从131份疑似病人临床样品中检出91份结核分枝杆菌阳性,从40份来自疑似发病牛群的临床样品中检出31份牛分枝杆菌阳性,检出率均高于细菌分离培养法。结论研究表明所建立的多重PCR-DHPLC方法能快速检测鉴别四种重要致病性分枝杆菌,为人畜结核、副结核病诊断提供一种新型分子生物学技术手段。
关键词 :
变性高效液相色谱 ,
结核分枝杆菌 ,
牛分枝杆菌 ,
禽分枝杆菌 ,
副结核分枝杆菌 ,
多重PCR
Abstract :A new molecular method for simultaneously rapid detection and differentiation of Mycobacterium tuberculosis,Mycobacterium bovis,Mycobacterium avium and Mycobacterium paratuberculosis was established by using denaturing high-performance liquid chromatography(DHPLC)combined with multiplex nucleic acid amplification.These 4 important pathogenic mycobacteria were identified by separation of 4 specific PCR-amplified target fragments by DHPLC analysis.A total of 51 Mycobacterium strains and 22 other bacterial species were tested to confirm the specificity of the multiplex PCR-DHPLC assay.The sensitivity of the assay was as low as 102-103 gene copies.This method rapidly identify the positive clinical samples from human and bovine with higher detection ratio than traditional culture method and was able to identify simultaneously four pathogenic Mycobacterium,which provided a new molecular tool for rapid detection of tuberculosis and paratuberculosis in human and animals.
Key words :
denaturing high-performance liquid chromatography
Mycobacterium tuberculosis
Mycobacterium bovis
Mycobacterium avium
Mycobacterium paratuberculosis
multiplex PCR
收稿日期: 2010-01-20
[1]刘志辉,罗春明,蔡杏姗.广州市旧城区1994-2003年非结核分枝杆菌流行状况分析[J].中华流行病学杂志,2005,26(6):424.
[2]Jacinto RC,Gomes BP,Desai M,et al.Bacterial examination of endodontic infections by clonal analysis in concert with denatu-ring high performance liquid chromatography[J].Oral Microbiol Immunol,2007,22(6):403-10.
[3]Hurtle W,Shoemaker D,Henchal E,et al.Denaturing HPLC for identifying bacteria[J].Biotechniques,2002,33(2):386.
[4]徐君怡,曹际娟,郑秋月,等.变性高效液相色谱检测食品中致泻大肠杆菌[J].微生物学报,2008,48(11):1526-1531.
[5]Nam YH,Lee SH,Ahn YC,et al.Detection of rifampin resist-ant mycobacteriumtuberculosis complex using denaturing HPLC[J].Korean J Lab Med,2008,28(2):95-102.
[6]Saramki OR,Waltering KK,Visakorpi T.Methods for identif-ying and studying genetic alterations in hormone-dependent canc-ers[J].Methods Mol Biol,2009;505:263-277.
[7]刘朝晖,王汉平,陈劲龙,等.运用变性高效液相色谱对肺炎克雷伯菌产ESBL进行基因分型[J].中华微生物学和免疫学杂志,2005,25(9):764-767.
[8]陈茹,刘中勇,杨国海,等.结核分枝杆菌和牛分枝杆菌Taq-Man-荧光PCR快速检测方法的建立[J].中国人兽共患病学报,2008,24(2):154-158.
[9]Hillemann D,Galle J,Voll mer E,et al.Real-time PCR assayfor improved detection ofMycobacteriumtuberculosiscomplexin paraffin-embedded tissues[J].Int J Tuberc Lung Dis,2006,10(3):340.
[10]Tasara T,Stephan R.Development of an F57sequence-based real-time PCRassay for detection ofMycobacteriumaviumsub-sp.paratuberculosisin milk[J].Appl Environ Microbiol,2005,71(10):5957-5968.
[11]Yip CW,Leung KL,Wong D,et al.Denaturing HPLC for high-throughput screening of rifampicin-resistantMycobacteri-umtuberculosisisolates[J].Int J Tuberc Lung Dis,2006,10(6):625.
[12]Shi R,Zhang J,Li C,et al.Detection of streptomycin resist-ance inMycobacteriumtuberculosisclinical isolates from China as determined by denaturing HPLC analysis and DNA sequen-cing[J].Microbes Infect,2007,9(14-15):1538-44.
[13]石瑞如,贾文斐,张国龙,等.DHPLC与SURVEYOR酶法在结核和牛分枝杆菌鉴别中的尝试[J].医学研究杂志,2007,36(3):68-70.
[14]Sreevatsan S,Pan X,Stockbauer KE,et al.Restricted struc-tural gene polymorphism in theMycobacterium tuberculosis complexindicates evolutionarily recent global dissemination[J].Proc Natl Acad Sci USA,1997,94:9869-9874.
[1]
鲁瑶, 赵丽丽, 李马超, 刘海灿, 赵秀芹, 刘志广, 万康林, 楼永良. RPA-LFD技术快速检测结核分枝杆菌利福平耐药性的应用 [J]. 中国人兽共患病学报, 2021, 37(5): 387-391.
[2]
王炜, 陈磊, 陈明心, 张岱, 蒋露, 任伟宏. 重组结核分枝杆菌精氨酰tRNA合成酶活性验证及晶体生长 [J]. 中国人兽共患病学报, 2021, 37(5): 430-434.
[3]
蓝如束, 叶婧, 罗丹, 覃慧芳, 黄莉雯, 苏华斌, 罗淋尹, 林玫. 基于PCR熔解曲线技术的结核分枝杆菌Spoligotyping基因分型的临床应用研究 [J]. 中国人兽共患病学报, 2021, 37(4): 285-291.
[4]
王炜, 任伟宏, 陈明心, 蒋露, 张岱, 陈磊. 结核分枝杆菌甲硫氨酰tRNA合成酶与底物及其类似物的结合比较 [J]. 中国人兽共患病学报, 2021, 37(4): 317-322.
[5]
全娟娟, 夏爱鸿, 孟闯, 李昕, 姚志鸿, 陈祥, 徐正中, 焦新安. 结核分枝杆菌靶向宿主细胞蛋白逃逸免疫杀伤的研究进展 [J]. 中国人兽共患病学报, 2021, 37(3): 259-263.
[6]
王悦, 孙颖, 孙炳奇. DNA微阵列芯片技术检测培阳肺结核患者痰样本中结核分枝杆菌耐药性的价值 [J]. 中国人兽共患病学报, 2021, 37(2): 109-114.
[7]
赵东阳, 王少华, 苏茹月, 郑丹薇, 朱岩坤, 马晓光, 石洁, 李辉, 孙定勇. 2013-2018年河南省耐药监测点非结核分枝杆菌流行特征分析 [J]. 中国人兽共患病学报, 2021, 37(2): 128-132.
[8]
周蕾, 安婷婷, 赵秀芹, 徐冬蕾, 蒋毅, 王瑞白, 万康林, 杨再昌. 独山瓜馥木总生物碱抗结核活性初探 [J]. 中国人兽共患病学报, 2020, 36(6): 487-490.
[9]
虞秀锋, 王启源, 姬文兰, 王亚梅. MiR-506-3p/SOCS-3促进BCG感染巨噬细胞引发的炎症应答反应 [J]. 中国人兽共患病学报, 2020, 36(4): 297-302.
[10]
王晓英,张汇征,罗明,曾婉婷. 结核分枝杆菌异烟肼和丙硫异烟胺耐药及其交叉耐药相关机制研究进展 [J]. 中国人兽共患病学报, 2020, 36(3): 234-238.
[11]
罗明, 张汇征, 严晓峰, 曹培明, 廖传玉, 何瑛, 李晓旭, 王静, 李同心, 陈耀凯. 重庆市耐多药结核分枝杆菌对贝达喹啉和德拉马尼的药物敏感性研究 [J]. 中国人兽共患病学报, 2020, 36(2): 100-104.
[12]
丁菁芸, 郭翰翔, 雒静, 许建华, 邵萌, 吴芳. 结核病疫苗的抗结核免疫机制的研究进展 [J]. 中国人兽共患病学报, 2020, 36(11): 940-948.
[13]
梁正敏, 宋银娟, 董玉慧, 屈孟锦, 周向梅. HspX的功能及其在结核疫苗研制中的应用进展 [J]. 中国人兽共患病学报, 2020, 36(11): 956-961.
[14]
王海宁, 金珊珊, 徐正中, 王蕾, 焦新安, 陈祥. 结核分枝杆菌基因敲除技术的研究进展 [J]. 中国人兽共患病学报, 2020, 36(11): 928-933.
[15]
鞠晓红, 王月华, 方芳. 结核分枝杆菌PE_PGRS33蛋白的研究进展 [J]. 中国人兽共患病学报, 2020, 36(11): 949-955.