微生物之克雷伯菌

肺炎克雷伯菌分子检测,陈颖曦江苏嘉语生物医药科技股份有限公司,目录,肺炎克雷伯种属鉴定常用靶点,肺炎克雷伯和鲍曼不动杆菌简介,鲍曼不动杆菌靶点的确定与序列比对,参考文献,肺炎克雷伯氏菌简介,本菌革兰氏阴性，革兰阴性杆菌，大小为(0 . 3 1. 5 )m X (0. 6 6. 0 )m 。单个、成双或短链排列。痰标本直接涂片染色，菌体呈卵圆形或球杆状，单个或成双排列。英膜染色外周可见透明、环状荚膜。 氧化酶试验阴性，发酵葡萄糖、乳糖、蔗糖等多种糖类， TSI 为A/ A ，IMViC- -+ ，硝酸盐还原、脲酶和赖氨酸脱竣酶试验阳性，动力、H2 S、鸟氨酸脱竣酶和精氨酸双水解酶试验均为阴性。 肺炎克雷伯菌广泛分布于自然界水和土壤中，是人类呼吸道的常居菌，在人和动物肠道内也常见，是-种条件致病菌。在临床分离到的克雷伯菌属中，肺炎克雷伯菌占80 % 以上，是本属中最为常见的病原菌，可引起肺炎、脑膜炎、腹膜炎、泌尿系统感染、败血症等疾病。 1。,肺炎克雷伯氏菌革兰氏染色（阴性）,克雷伯杆菌肺炎,分子检测常用靶点,KP肺炎克雷伯菌，K.ssp 克雷伯属,AB 鲍曼不动杆菌，A.ssp 不动杆菌属，A.genogroup 不动杆菌基因型分组,肺炎克雷伯菌16S序列比对,选取551株肺炎克雷伯菌16S序列进行比对，产生比对文件,由于仅有一篇文献描述了16S引物检测KP,且未将引物序列写明，故肺炎克雷伯16S没有“文献引物”,肺炎克雷伯菌16S进化树,肺炎克雷伯菌ITS序列比对,选取254株肺炎克雷伯菌ITS序列进行比对，生成比对文件肺炎克雷伯菌ITS.apr,肺炎克雷伯菌ITS进化树,文献中肺炎克雷伯菌ITS引物,肺炎克雷伯菌phoE序列比对,选取26株肺炎克雷伯菌phoE序列进行比对，生成比对文件phoE.apr,肺炎克雷伯phoE进化树,文献中肺炎克雷伯菌phoE引物,肺炎克雷伯菌耐药基因比对,肺炎克雷伯菌耐药基因,“?”表明该基因未找到对应序列进行比对，已比对的基因没进行任何标记。,比对文件注意事项,耐药基因多聚集成“簇”，有的单个比对文件中包含多个基因，在设计引物时，找准目标基因序列是关键。如果比对文件中包含多种目的基因，如-内酰胺酶“IMP”基因，有“IMP-1，IMP-2等等”，在FASTA文件中改成了相应的名字，以示区别，如果比对文件中只包含一种目的基因，FASTA文件中仍保留其原始名称和名称格式，未作修改。 虽然在FASTA文件中，绝大部分成簇基因名称都改成“X（靶基因名） and other genes”,以表示该条序列是靶基因和其他非靶标基因成的“簇”。但成簇基因名称在NTI序列比对软件中显示不完整，只显示把基因名，未显示“and other genes”字样，在查看比对结果时，可打开FASTA原始文件一一对应查看，不过也可以根据基因序列长度来判断，一般的说，碱基数3000bp的序列是成簇基因序列，请注意鉴别。有些铜绿耐药基因序列在NCBI和欧洲数据库中找不到，就用了其他种的对应耐药基因替代，FASTA文件中基因名包含了种名，但在NTI序列比对软件中未显示，只显示了基因名。有些耐药基因未找到文献引物，或是有限的文献中的引物不便于复制操作而未归纳总结，还需另行设计。一些基因在文献中未提及但在NCBI上有记载，也选下来进行比对。,TEM序列比对,以下是肺炎克雷伯TEM序列比对文件，其中有TEM-1（75株），TEM-1b(3株)，TEM-1a(1株)，TEM-10（2株），TEM-11（1株），TEM-12（1株），TEM-24（2株），TEM-26B（1株），TEM-47（1株），TEM-48（1株），TEM-52（1株），TEM-54(1株)，TEM-86（1株），TEM-130(1株)，TEM-132（1株）共93株TEM序列。,-内酰胺类耐药基因比对,文献中的TEM引物（通用引物）,KPC序列比对,选取肺炎克雷伯KPC-1(1株)，KPC-2（12株），KPC-3（2株），KPC-7（1株）进行比对。,文献中KPC引物（通用引物）,GES序列比对,选取肺炎克雷伯菌GES-1（1株），GES-3（1株），GES-4（2株），GES-7（2株）序列进行比对。,文献中GES引物（通用引物）,VEB序列比对,由于NCBI中缺少肺炎克雷伯VEB序列，仅1株，另加入3株铜绿假单胞菌（PA）VEB-1进行比对。,VIM序列比对,选取肺炎克雷伯VIM-1,2,3,5各1株，VIM-4（2株），VIM-12（1株），VIM-12（1株），VIM-27（2株），VIM-34（3株），VIM-like(2株)进行比对。,文献中VIM引物(通用引物),NDM（新德里金属蛋白酶）序列比对,选取23株NDM序列进行比对。,AmpC(CMY)序列比对,选取CMY-2（2株），CMY-4（5株），CMY-6（2株），CMY-12（1株），CMY-31（1株），CMY-56（3株），CMY-87（2株）进行比对。,文献中CMY引物,DHA序列比对,NCBI中选取17株的DHA-1和1株的DHA-2序列进行比对。,文献中DHA引物,Target 里面的“uni”全称是“universal”，表明该基因对应的通用引物。,AmpC(FOX)序列比对,选取肺炎克雷伯FOX-1,5,7,9各一条序列进行比对。,OKP序列比对,12株肺炎克雷伯OKP-A序列。,选取肺炎克雷伯OKP-B序列11条和OKP-B-13，14各一条进行比对。,文献中OKP-A和OKP-B引物,文献中的引物均不能与比对文件匹配！,OXA序列比对,选取肺炎克雷伯OXA-1（13株），OXA-4（1株），绿脓（PA）OXA-30（2株）进行比对。,2株肺炎克雷伯OXA-2,1株肺炎克雷伯OXA-10和1株肺炎克雷伯OXA-17,2株肺炎克雷伯OXA-48,3株肺炎克雷伯OXA-73,文献中OXA引物,AmpC(LEN)序列比对,选取LEN-1-like(2株)，LEN-2（1株），LEN-3（3株），LEN-4（2株），LEN-5（1株），LEN-25（1株），不知类型的LEN（2株）进行比对。,IMP序列比对,选取肺炎克雷伯菌IMP-4（3株），IMP-8（2株），IMP-13（1株），IMP-10（1株），IMP-38（1株），铜绿的IMP-1（7株）进行比对。,文献中IMP引物,SHV序列比对,选取肺炎克雷伯SHV-1（28株），SHV-2（11株），SHV-2a(2株)，SHV-5（27株），SHV-11（64株），SHV-12（22株），SHV-25（1株），SHV-26（2株），SHV-27(2株)， SHV-28（7株）， SHV-36（1株），SHV-40（2株），SHV-55（3株），SHV-60（2株），SHV-62（1株），SHV-103(1株)进行比对。,文献中SHV引物,CTX-M序列比对,选取肺炎克雷伯CTX-M-2（9株），CTX-M-3（12株），CTX-M-12（2株），CTX-M-15（55株），CTX-M-22（2株），CTX-M-24（1株），CTX-M-28（3株），CTX-M-32（1株）序列进行比对。,跟上面大文件未对其，单独拿出比对的9条CTX-M-15。,8条CTX-M-14（未能和大文件比对整齐而单独比对）和2条由国内毕业论文发现的两条新CTX-M（new）。,文献中的CTX-M引物,氨基糖苷类耐药基因,氨基糖苷修饰酶基因比对,选取肺炎克雷伯aac(6)-Ib-cr（1株），阴沟肠杆菌aac(6)-Ib-cr（4株）一起比对。,2株肺炎克雷伯AAC(3)-II(aacC2),ANT(3)-I(aadA1,2,8),ANT(3)-I(aadA5),3株 aac(6)-I(aacA1),13株 aac(6)-I(aacA4),8株肺炎克雷伯ant(2)-Ia(aadB),16S甲基化酶基因比对,3株armA,5株rmtB,文献中氨基糖苷耐药基因引物,喹诺酮耐药基因比对,质粒介导喹诺酮耐药基因比对,qnrA1,7,qnrA-like,qnrB4,qnrB1,10,42,53,qnrB6,文献中qnr引物,参考文献,1周庭银.临床微生物诊断与图谱M.上海：上海科学技术出版社， 2007.155-158,210-212.2 Prathiba Kurupati,Carol Chow, Gamini Kumarasinghe,et al. 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