CN101413031A - 鉴别结核分枝杆菌与非结核分枝杆菌的方法及其专用试剂盒 - Google Patents
鉴别结核分枝杆菌与非结核分枝杆菌的方法及其专用试剂盒 Download PDFInfo
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Abstract
本发明公开了一种鉴别结核分枝杆菌与非结核分枝杆菌的方法及其专用试剂盒。本发明的鉴别结核分枝杆菌与非结核分枝杆菌的试剂盒，包括引物和探针，其中所述引物包括4条引物，它们的核苷酸序列分别是序列表中序列1、序列2、序列4和序列5；所述探针包括2条，它们的核苷酸序列分别是序列表中序列3和序列6；所述探针的5’端带有荧光物质，3’端带有淬灭物质；所述2条探针的5’端的荧光物质不同。本发明的试剂盒能快速鉴别结核分枝杆菌与非结核分枝杆菌，且对仪器要求简单，费用低，操作简单，在结核病的早期诊断和鉴别诊断方面具有广泛的应用前景。
Description
技术领域
本发明涉及鉴别结核分枝杆菌与非结核分枝杆菌的方法及其专用试剂盒。
背景技术
结核病(Tuberculosis)是当今全球范围对人类最具威胁的感染性疾病之一，本世纪80年代中期以来在全球出现回升趋势，据世界卫生组织报道，我国是全球22个结核病高负担国家之一，活动性肺结核病人数居世界第二位。目前我国全年龄组结核菌感染率为44.5％，全国约5.5亿人受到了结核菌感染，结核菌感染率高于全球人口感染率为1/3的水平。同时，非结核分枝杆菌(NTM)病也呈逐年上升趋势，2000年卫生部组织的流调数据显示，NTM菌株分离率上升至11.1％，NTM病临床表现与结核病相似，在无菌种鉴定结果的情况下，经常被误诊为结核病。大量研究以及临床治疗病例表明多数传统抗痨药对NTM病很少或没有疗效，传统的菌种鉴定和药敏试验方法烦琐、费时，延误病人的治疗。因此，结核分枝杆菌与非结核分枝杆菌区分检测对结核病的早期诊断、鉴别诊断、个体化的有效化疗和控制传播都有极其重要的意义。
结核分枝杆菌与非结核分枝杆菌的区分目前仍然依靠传统的分离培养的方法，通常需要1-4周的时间才能得到确切的结果，这样大大的延误了病人的治疗，给病人造成额外的经济负担和精神负担。
发明内容
本发明的目的是提供一种鉴别结核分枝杆菌与非结核分枝杆菌的方法及其专用试剂盒。
本发明所提供的鉴别结核分枝杆菌与非结核分枝杆菌的试剂盒，包括进行PCR扩增的引物和探针，其中，所述引物包括4条引物，分别为引物1、2、3和4，所述引物1和引物2能与结核分枝杆菌的特异片段结合，所述引物3和引物4能与分枝杆菌特异片段结合；所述探针包括2条，分别为探针1和探针2，所述探针1能与结核分枝杆菌特异片段结合，并且其结合位置位于所述引物1和引物2与结核分枝杆菌特异片段结合位置之间；所述探针2能与分枝杆菌特异片段结合，并且其结合位置位于所述引物3和引物4与分枝杆菌特异片段结合位置之间；所述探针1和所述探针2的5’端带都有荧光物质，3’端都带有淬灭物质，所述探针1和所述探针2的5’端的荧光物质不同。
所述结核分枝杆菌的特异片段可选自如下：IS6110、16s、IS986、MTP40、38kDa、IS1081和ESAT-6。所述分枝杆菌特异片段可选自如下：16s、dnaj基因、编码65kDa蛋白的核苷酸序列、编码38kDa蛋白的核苷酸序列和编码32kDa蛋白的核苷酸序列。
所述引物1的核苷酸序列具体可为序列表中的序列1，所述引物2的核苷酸序列具体可为序列表中的序列2，所述引物3的核苷酸序列具体可为序列表中的序列4，所述引物4的核苷酸序列具体可为序列表中的序列5；所述探针1的核苷酸序列具体可为序列表中的序列3，所述探针2的核苷酸序列具体可为序列表中的序列6。
所述引物1的核苷酸序列还可为序列表中的序列7，所述引物2的核苷酸序列还可为序列表中的序列8，所述引物3的核苷酸序列还可为序列表中的序列10，所述引物4的核苷酸序列还可为序列表中的序列11；所述探针1的核苷酸序列还可为序列表中的序列9，所述探针2的核苷酸序列还可为序列表中的序列12。
所述荧光物质为FAM、HEX、TET、TAMRA、VIC、CY5、Texas Red或JOE。所述淬灭物质为TAMRA、Dabcyl或Eclipse。
所述探针合成时，荧光物质和淬灭物质被标记在探针的末端，所用荧光物质和淬灭物质可进行选择，能提供合成服务的公司有：宝生物工程有限公司、上海生工生物工程技术服务有限公司、上海基康生物技术有限公司、ABI公司等。
本发明的另一个目的是提供一种鉴别结核分枝杆菌与非结核分枝杆菌的方法。
本发明所提供的鉴别结核分枝杆菌与非结核分枝杆菌的方法，包括以下步骤：
用所述的试剂盒中的引物和探针对待测菌株的核酸进行荧光定量PCR扩增，检测所得到的PCR产物的荧光信号；如果能检测到探针1的荧光信号，则待测菌为结核分枝杆菌；如果不能检测到探针1的荧光信号，而能检测到探针2的荧光信号，则待测菌为非结核分枝杆菌。
所述非结核分枝杆菌为如下表1所示。
表1.非结核分枝杆菌
| Mycobacterium abortusMycobacterium abscessusMycobacterium acnesMycobacterium acnesMycobacterium acuminatum | 流产分枝杆菌脓肿分枝杆菌痤疮分枝杆菌粉刺分枝杆菌)尖形分枝杆菌 |
| Mycobacterium adamsoniMycobacterium adamsoniMycobacterium africanumMycobacterium agresteMycobacterium agriMycobacterium agriMycobacterium aichienseMycobacterium albumMycobacterium alveiMycobacterium ammethystinumMycobacterium anabantiMycobacterium anaerobiumMycobacterium aquaeMycobacterium asiaticumMycobacterium asiaticumMycobacterium asteroidsMycobacterium aurumMycobacterium austroafricanumMycobacterium aviumMycobacterium avium subsp.aviumMycobacterium avium subsp.paratuberculosisMycobacterium avium subsp.silvaticumMycobacterium avium subsp.suisMycobacterium balneiMycobacterium balneiMycobacterium berolinensisMycobacterium bifidumMycobacterium blattelaeMycobacterium brevicaleMycobacterium brumaeMycobacterium brunenseMycobacterium butanitrificansMycobacterium butyricumMycobacterium butyricumMycobacterium butyricumMycobacterium caesius | 亚当斯氏分枝杆菌亚氏分枝杆菌非洲分枝杆菌乡村分枝杆菌田野分枝杆菌土地分枝杆菌爱知分枝杆菌白色分枝杆菌蜂房分枝杆菌紫晶分枝杆菌攀登分枝杆菌厌氧分枝杆菌水分枝杆菌亚细亚分枝杆菌亚洲分枝杆菌星状分枝杆菌金色分枝杆菌南非分枝杆菌鸟分枝杆菌鸟分枝杆菌鸟亚种鸟分枝杆菌副结核亚种鸟分枝杆菌森林土壤亚种鸟分枝杆菌猪亚种巴尔内氏分枝杆菌巴氏分枝杆菌柏林分枝杆菌两形分枝杆菌蟑螂分枝杆菌短小分枝杆菌冬天分枝杆菌深褐色分枝杆菌生丁硝分枝杆菌丁酸分枝杆菌酪酸分枝杆菌乳酪分枝杆菌青灰分枝杆菌 |
| Mycobacterium carotenumMycobacterium catenaformisMycobacterium celatumMycobacterium chelonaeMycobacterium chelonae subsp.abscessusMycobacterium chelonae subsp.chelonaeMycobacterium chitaeMycobacterium chlorophenolicumMycobacterium cholesterolicumMycobacterium chubuenseMycobacterium citreochromogenumMycobacterium citreumMycobacterium coelicolorMycobacterium coerulatusMycobacterium coeruleumMycobacterium confluentisMycobacterium convolutumMycobacterium cookiiMycobacterium cookiiMycobacterium cornutumMycobacterium crystallophagumMycobacterium cuculiMycobacterium cuneatumMycobacterium cyaneumMycobacterium denitrificansMycobacterium diastaticumMycobacterium diernhoferiMycobacterium dimorphumMycobacterium duvaliiMycobacterium duvaliiMycobacterium engbaekiiMycobacterium enteritidisMycobacterium enzymicumMycobacterium eosMycobacterium equiMycobacterium erythrogenes | 胡萝卜素分枝杆菌链状分枝杆菌隐藏分枝杆菌龟分枝杆菌龟分枝杆菌脓肿亚种龟分枝杆菌龟亚种千田分枝杆菌氯酚红分枝杆菌胆固醇分枝杆菌楚布分枝杆菌柠檬产色分枝杆菌柠檬色分枝杆菌天蓝色分枝杆菌似天蓝分枝杆菌蓝色分枝杆菌汇合分枝杆菌旋卷分枝杆菌库克氏分枝杆菌库氏分枝杆菌角状分枝杆菌嗜晶体分枝杆菌布谷鸟分枝杆菌楔形分枝杆菌蓝色分枝杆菌反硝化分枝杆菌淀粉酶分枝杆菌迪氏分枝杆菌两形分枝杆菌杜氏分枝杆菌杜瓦氏分枝杆菌恩氏分枝杆菌肠炎分枝杆菌酶分枝杆菌曙光分枝杆菌马分枝杆菌产红分枝杆菌 |
| Mycobacterium fallaxMycobacterium farcinogenesMycobacterium farcinogenesMycobacterium farcinogenes subsp.senegalenMycobacterium farcinogenes subsp.tchadenseMycobacterium ferrosiliceumMycobacterium filamentosumMycobacterium filiformeMycobacterium fimiMycobacterium fimiMycobacterium flavescensMycobacterium flavovirideMycobacterium flavumMycobacterium flavum var.pseudoramnosumMycobacterium flavum var.pseudoramnosumMycobacterium flavum var.tortuosumMycobacterium fluorescensMycobacterium fortuitumMycobacterium fortuitumMycobacterium fortuitum subsp.acetamidolyticumMycobacterium fortuitum subsp.fortuitumMycobacterium fortuitum subsp.fortuitumMycobacterium fortuitum subsp.pelegrinumMycobacterium friedmaniiMycobacterium friedmaniiMycobacterium friedmaniiMycobacterium fuscumMycobacterium gadiumMycobacterium gallinarumMycobacterium gastriMycobacterium genavenseMycobacterium giaeMycobacterium gilvumMycobacterium gordonaeMycobacterium graminisMycobacterium graminis | 诡诈分枝杆菌鼻疽分枝杆菌产鼻疽分枝杆菌产鼻疽分枝杆菌塞内加尔亚种产鼻疽分枝杆菌乍得亚种铁硅分枝杆菌丝状分枝杆菌线形分枝杆菌肥料分枝杆菌粪肥分枝杆菌微黄分枝杆菌黄绿分枝杆菌黄色分枝杆菌假分枝杆菌假分枝状枝杆菌黄色分枝杆菌多曲变种荧光分枝杆菌偶发分枝杆菌偶然分枝杆菌偶发分枝杆菌解乙酰氨亚种偶发分枝杆菌偶发亚种偶然分枝杆菌偶然亚种偶然分枝杆菌外来亚种费氏分枝杆菌佛德曼氏分枝杆菌弗里德曼氏分枝杆菌褐色分枝杆菌加地斯分枝杆菌鸡分枝杆菌胃分枝杆菌日内瓦分枝杆菌盖氏分枝杆菌浅黄分枝杆菌戈登分枝杆菌草分枝杆菌禾草分枝杆菌 |
| Mycobacterium granulosumMycobacterium granulosumMycobacterium griseumMycobacterium habanaMycobacterium haemophilumMycobacterium hastilisMycobacterium hastilisMycobacterium hepatodystrophicansMycobacterium hiberniaeMycobacterium hodgkiniiMycobacterium hodgkiniiMycobacterium hominisMycobacterium humidusMycobacterium hyalinumMycobacterium insidiosumMycobacterium insidiosumMycobacterium intermediumMycobacterium intracellulareMycobacterium johneiMycobacterium kansasiiMycobacterium komossenseMycobacterium lacticolaMycobacterium lacticumMycobacterium lactisMycobacterium lepraeMycobacterium lichenifoemeMycobacterium limosusMycobacterium linensMycobacterium lipolyticumMycobacterium lipoptenaeMycobacterium liquefaciensMycobacterium luteumMycobacterium luteumMycobacterium madagascarienseMycobacterium malmoenseMycobacterium marianum | 颗粒分枝杆菌粒状分枝杆菌灰色分枝杆菌哈瓦那分枝杆菌嗜血分枝杆菌标枪分枝杆菌矛形分枝杆菌胀肝分枝杆菌爱尔兰分枝杆菌何杰金氏分枝杆菌霍金氏分枝杆菌人型分枝杆菌潮湿分枝杆菌透明分枝杆菌诡诈分枝杆菌谲诈分枝杆菌中间分枝杆菌胞内分枝杆菌约氏分枝杆菌堪萨斯分枝杆菌科莫斯分枝杆菌乳生分枝杆菌乳酸分枝杆菌乳分枝杆菌麻风分枝杆菌地衣分枝杆菌淤泥分枝杆菌扩展分枝杆菌解脂分枝杆菌扁蝇分枝杆菌液化分枝杆菌藤黄分枝杆菌无色分枝杆菌马达加斯加分枝杆菌玛尔摩分枝杆菌海分枝杆菌 |
| Mycobacterium marianumMycobacterium mesentericumMycobacterium methanicumMycobacterium michiganensisMycobacterium microtiMycobacterium moriokaenseMycobacterium mucogenicumMycobacterium mucosumMycobacterium murisMycobacterium murisepticumMycobacterium neoaurumMycobacterium nigrumMycobacterium nonchromogenicumMycobacterium nonchromogenicumMycobacterium nubi lumMycobacterium obuenseMycobacterium oligonitrophilumMycobacterium paraffinicumMycobacterium parafortuitumMycobacterium parasmegmatisMycobacterium parasmegmatisMycobacterium paratuberculosisMycobacterium pasteurianumMycobacterium pasteurianumMycobacterium paurometabolumMycobacterium peregrinumMycobacterium periplaneticumMycobacterium perrugosumMycobacterium petroleophilumMycobacterium phleiMycobacterium pisciumMycobacterium planumMycobacterium plumosusMycobacterium porcinumMycobacterium poriferaeMycobacterium pulveris | 海鱼分枝杆菌肠膜分枝杆菌甲烷分枝杆菌密执安分枝杆菌田鼠分枝杆菌莫里奥卡分枝杆菌产粘液分枝杆菌粘分枝杆菌鼠分枝杆菌鼠败血分枝杆菌新金色分枝杆菌黑色分枝杆菌不产色分枝杆菌无色分枝杆菌云雾状分枝杆菌奥布分枝杆菌嗜寡氮分枝杆菌石蜡分枝杆菌副偶然分枝杆菌副包皮垢分枝杆菌副耻垢分枝杆菌副结核分枝杆菌巴氏分枝杆菌巴斯德氏分枝杆菌稍变分枝杆菌外来分枝杆菌蟑螂分枝杆菌崎岖分枝杆菌嗜石油分枝杆菌草分枝杆菌鱼分枝杆菌平滑分枝杆菌羽状分枝杆菌猪分枝杆菌多孔分枝杆菌灰尘分枝杆菌 |
| Mycobacterium putricumMycobacterium pyogenesMycobacterium pyogenesMycobacterium pyogenes bovisMycobacterium pyogenes bovisMycobacterium radicicolaMycobacterium radicicolaMycobacterium rhodesiaeMycobacterium rhodochrousMycobacterium rhodochrousMycobacterium rubiacearumMycobacterium rubropertinctumMycobacterium rubrumMycobacterium rubrum nitrosumMycobacterium runyoniiMycobacterium salivariumMycobacterium salmoniphilumMycobacterium scrofulaceumMycobacterium segmentosumMycobacterium senegalenseMycobacterium sepedonicaMycobacterium sepedonicaMycobacterium shanghaienseMycobacterium shimoideiMycobacterium simiaeMycobacterium simiaeMycobacterium simplexMycobacterium smaragdinaMycobacterium smaragdinaMycobacterium smegmatisMycobacterium smegmatisMycobacterium sphagniMycobacterium stercorisMycobacterium szulgaiMycobacterium szulgaiMycobacterium terrae | 腐臭分枝杆菌化脓分枝杆菌酿脓分枝杆菌牛化脓性分枝杆菌牛酿脓分枝杆菌根瘤分枝杆菌生根内分枝杆菌罗德岛分枝杆菌胭脂红分枝杆菌紫红色分枝杆菌茜草分枝杆菌深红分枝杆菌红色分枝杆菌亚硝酸红色分枝杆菌润氏分枝杆菌唾液分枝杆菌喜鲑分枝杆菌瘰疬分枝杆菌横裂分枝杆菌塞内加尔分枝杆菌腐烂分枝杆菌坏腐分枝杆菌上海分枝杆菌石氏分枝杆菌猿分枝杆菌猿猴分枝杆菌单纯分枝杆菌翠绿分枝杆菌绿柱玉分枝杆菌包皮垢分枝杆菌耻垢分枝杆菌泥炭藓分枝杆菌粪堆分枝杆菌斯氏分枝杆菌苏加分枝杆菌土地分枝杆菌 |
| Mycobacterium terraeMycobacterium testudinisMycobacterium thamnopheosMycobacterium thermoresistibleMycobacterium tokaienseMycobacterium tomidaeMycobacterium tortuosumMycobacterium trivialeMycobacterium trivialeMycobacterium tropidonotiMycobacterium tropidonotiMycobacterium tumescensMycobacterium ulceransMycobacterium ulcerogenesMycobacterium vaccaeMycobacterium vadosumMycobacterium valentiaeMycobacterium viridisMycobacterium xenopiMycobacterium xenopi | 土分枝杆菌龟分枝杆菌蛇分枝杆菌抗热分枝杆菌东海分枝杆菌托氏分枝杆菌多曲分枝杆菌次要分枝杆菌通俗分枝杆菌赤炼蛇分枝杆菌水蛇分枝杆菌肿胀分枝杆菌溃疡分枝杆菌产溃疡分枝杆菌母牛分枝杆菌浅滩分枝杆菌瓦伦西亚分枝杆菌绿色分枝杆菌蟾蜍分枝杆菌蟾分枝杆菌 |
使用本发明的鉴别结核分枝杆菌与非结核分枝杆菌的试剂盒能快速鉴别结核分枝杆菌与非结核分枝杆菌，且对仪器要求简单，费用低，操作简单，在结核病的早期诊断和鉴别诊断方面具有广泛的应用前景。
附图说明
图1为实施例1中结核分枝杆菌FAM通道和HEX通道扩增曲线图。
图2为实施例1中堪萨斯分枝杆菌FAM通道和HEX通道扩增曲线图。
图3为实施例2中结核分枝杆菌FAM通道和HEX通道扩增曲线图。
图4为实施例2中堪萨斯分枝杆菌FAM通道和HEX通道扩增曲线图。
具体实施方式
实施例1、结核分枝杆菌与非结核分枝杆菌的鉴别
鉴别结核分枝杆菌与非结核分枝杆菌的试剂盒由如下部分组成：提取核酸的试剂、PCR反应试剂、引物、探针、阳性标本对照和阴性标本对照及说明书。其中的引物为引物P1和P2，引物P3和P4，探针1的5’端带有FAM荧光标记，3’端带有Eclipse荧光淬灭基团。探针2的5’端带有HEX荧光标记，3’端带有Eclipse荧光淬灭基团。
引物P1和P2是针对结核分枝杆菌IS6110序列设计，引物P1和P2的核苷酸序列如下：
引物P1：5’-AAGCCCGCAGGACCACGAT-3’(序列表中的序列1)；
引物P2：5’-CGTAGGCGTCGGTGACAAAGG-3’(序列表中的序列2)。
探针1的序列如下：
5’-CCACAGCCCGTCCCGCCGATCTCG-3’(序列表中的序列3)。
引物P3和P4是针对分枝杆菌65kDa蛋白的核苷酸序列设计的，引物P3和P4的核苷酸序列如下：
引物P3：5’-AGACCAAGGAGCAGATCGCT-3’(序列表中的序列4)；
引物P4：5’-CGCATACCCTCGGTGAGCT-3’(序列表中的序列5)。
探针2的序列如下：
5’-TGATGACACCCTCGTTGCCGACC-3’(序列表中的序列6)。
以结核分枝杆菌的核酸和堪萨斯分枝杆菌的核酸作为模板。结核分枝杆菌的核酸和堪萨斯分枝杆菌的核酸(结核PCR检测参考品)购自中国生物制品鉴定所。
反应体系如下：2×Real time taqman buffer(博奥公司)10μl，Taq酶0.3μl，引物P1和P2(10uM)各0.6μl，探针1(10uM)0.8μl；引物P3和P4(10uM)各1.2μl，探针2(10uM)0.8μl；模板2μl，H2O 2.5μl。
在ABI 7700进行反应检测，程序如下：先94℃ 3min，然后94℃ 15sec、60℃ 30sec进行40个循环，荧光信号采集点在60℃ 30sec。
PCR扩增结果如图1和2所示，表明以结核分枝杆菌核酸为模板进行扩增时，FAM通道出现扩增信号；以堪萨斯分枝杆菌(非结核分枝杆菌)核酸为模板进行扩增时，FAM通道没有扩增信号，仅有HEX通道有扩增信号。
实施例2、结核分枝杆菌与非结核分枝杆菌的鉴别
鉴别结核分枝杆菌与非结核分枝杆菌的试剂盒由如下部分组成：提取核酸的试剂、PCR反应试剂、引物、探针、阳性标本对照和阴性标本对照及说明书。其中的引物为引物P1和P2，引物P3和P4，探针1的5’端带有FAM荧光标记，3’端带有Eclipse荧光淬灭基团。探针2的5’端带有HEX荧光标记，3’端带有Eclipse荧光淬灭基团。
引物P1和P2是针对结核分枝杆菌MTP40序列设计，引物P1和P2的核苷酸序列如下：
引物P1：5’-CAAGTTGGGTCTGGTCGAATTC-3’(序列表中的序列7)；
引物P2：5’-CACACCTCGCAATTGAAGTTGA-3’(序列表中的序列8)。
探针3的序列如下：
5’-TGAGGTCATGTCGCCAACCACGCC-3’(序列表中的序列9)。
引物P3和P4是针对分支杆菌16s序列的核苷酸序列设计的，引物P3和P4的核苷酸序列如下：
引物P3：5’-GGATTGACGGTAGGTGGAGAAG-3’(序列表中的序列10)；
引物P4：5’-TTCACGAACAACGCGACAAAC-3’(序列表中的序列11)。
探针2的序列如下：
5’-CCAACTACGTGCCAGCAGCCGC-3’(序列表中的序列12)。
以结核分枝杆菌的核酸和堪萨斯分枝杆菌的核酸作为模板。结核分枝杆菌的核酸和堪萨斯分枝杆菌的核酸(结核PCR检测参考品)购自中国生物制品鉴定所。
反应体系如下：2×Real time taqman buffer(博奥公司)10μl，Taq酶0.3μl，引物P1和P2(10uM)各0.6μl，探针1(10uM)0.8μl；引物P3和P4(10uM)各1.2μl，探针2(10uM)0.8μl；模板2μl，H2O 2.5μl。
在ABI7700进行反应检测，程序如下：先94℃ 3min，然后94℃ 15sec、60℃ 30sec进行40个循环，荧光信号采集点在60℃ 30sec。
PCR扩增结果如图3和4所示，表明以结核分枝杆菌核酸为模板进行扩增时，FAM通道出现扩增信号；以对堪萨斯分枝杆菌(非结核分枝杆菌)核酸为模板进行扩增时，FAM通道没有扩增信号，仅有HEX通道有扩增信号。
序列表
<110>博奥生物有限公司 清华大学
<120>鉴别结核分枝杆菌与非结核分枝杆菌的方法及其专用试剂盒
<130>CGGNARW81966
<160>12
<210>1
<211>19
<212>DNA
<213>人工序列
<220>
<223>
<400>1
<210>2
<211>21
<212>DNA
<213>人工序列
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<212>DNA
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<210>6
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<212>DNA
<213>人工序列
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<213>人工序列
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<212>DNA
<213>人工序列
<220>
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Claims (8)
1、鉴别结核分枝杆菌与非结核分枝杆菌的试剂盒，包括进行PCR扩增的引物和探针，其特征在于：所述引物包括4条引物，分别为引物1、2、3和4，所述引物1和引物2能与结核分枝杆菌的特异片段结合，所述引物3和引物4能与分枝杆菌特异片段结合；所述探针包括2条，分别为探针1和探针2，所述探针1能与结核分枝杆菌特异片段结合，并且其结合位置位于所述引物1和引物2与结核分枝杆菌特异片段结合位置之间；所述探针2能与分枝杆菌特异片段结合，并且其结合位置位于所述引物3和引物4与分枝杆菌特异片段结合位置之间；所述探针1和所述探针2的5’端带都有荧光物质，3’端都带有淬灭物质，所述探针1和所述探针2的5’端的荧光物质不同。
2、根据权利要求1所述的试剂盒，其特征在于：所述结核分枝杆菌的特异片段选自如下：IS6110、16s、IS986、MTP40、38kDa、IS1081和ESAT-6。
3、根据权利要求1所述的试剂盒，其特征在于：所述分枝杆菌特异片段选自如下：16s、dnaj基因、编码65kDa蛋白的核苷酸序列、编码38kDa蛋白的核苷酸序列和编码32kDa蛋白的核苷酸序列。
4、根据权利要求1至3中任一所述的试剂盒，其特征在于：所述引物1的核苷酸序列为序列表中的序列1，所述引物2的核苷酸序列为序列表中的序列2，所述引物3的核苷酸序列为序列表中的序列4，所述引物4的核苷酸序列为序列表中的序列5；所述探针1的核苷酸序列为序列表中的序列3，所述探针2的核苷酸序列为序列表中的序列6。
5、根据权利要求1至3中任一所述的试剂盒，其特征在于：所述引物1的核苷酸序列为序列表中的序列7，所述引物2的核苷酸序列为序列表中的序列8，所述引物3的核苷酸序列为序列表中的序列10，所述引物4的核苷酸序列为序列表中的序列11；所述探针1的核苷酸序列为序列表中的序列9，所述探针2的核苷酸序列为序列表中的序列12。
6、根据权利要求4或5中任一所述的试剂盒，其特征在于：所述荧光物质为FAM、HEX、TET、TAMRA、CY3、VIC、CY5、Texas Red或JOE。
7、根据权利要求6所述的试剂盒，其特征在于：所述淬灭物质为TAMRA、Dabcyl或Eclipse。
8、一种鉴别结核分枝杆菌与非结核分枝杆菌的方法，包括以下步骤：
用权利要求1至7中任一项所述的试剂盒中的引物和探针对待测菌株的核酸进行荧光定量PCR扩增，检测所得到的PCR产物的荧光信号；如果能检测到探针1的荧光信号，则待测菌为结核分枝杆菌；如果不能检测到探针1的荧光信号，而能检测到探针2的荧光信号，则待测菌为非结核分枝杆菌。
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