应用滚环扩增技术对白念珠菌耐氟康唑相关基因点突变的研究
发布时间：2018-07-27 09:16
【摘要】： [目的]收集白念珠菌氟康唑耐药株和敏感株,应用滚环扩增技术检测菌株耐药相关基因ERG11和TAC1的点突变,并将所得结果与测序结果进行比较,以期建立一种准确、快速、特异的检测基因单点突变的分子生物学方法,同时进一步了解ERG11和TAC1突变与唑类药物耐药之间的关系。 [方法]从不同临床标本中收集白念珠菌并进行药物敏感性测定,得到白念珠菌氟康唑耐药株25株和敏感株21株；8株美国耐药株已知其ERG11突变位点。根据文献报道的与耐药有关的点突变和挂锁探针设计原则设计挂锁探针,其中ERG11设计24个挂锁探针,TAC1设计4个挂锁探针,每个探针可检测一种点突变。提取DNA、PCR扩增获得目的片段ERG11和TAC1的三个片段,纯化去除多余的缓冲液、引物和dNTP；然后通过挂锁探针的连接、核酸外切酶消化、超分支滚环扩增等过程,用滚环扩增技术分别检测耐药株和敏感株中ERG11的点突变和耐药株中TAC1的点突变。同时将目的片段纯化后送交测序,将滚环扩增所得结果与测序结果进行比较。 [结果]在8株己知突变位点的氟康唑耐药株中,滚环扩增技术准确地检测出与已知突变一致的点突变；应用滚环扩增技术最低可在含有5%目标模板的混合物中检测到滚环扩增信号。对全部临床菌株,应用滚环扩增技术检测到的点突变经测序验证全部正确,滚环扩增技术表现出和DNA测序很好的一致性。对于ERG11,25个氟康唑耐药株的24个中检测出了错义突变导致的20种氨基酸转换(突变率96%),分别是E266D(n=11),V488I(n=8),D116E(n=8),K128T(n=7),G464S(n=4),K143R(n=3),G448E(n=3),G307S(n=3),F145L(n=3),V437I(n=3),F449S(n=2),K108E(n=2),D153E(n=2),G465S(n=1),R467K(n=1),S405F(n=1),Y132H(n=1),F126L(n=1),D278E(n=1),G450V(n=1)。其中G450V以前未见报道,一株菌未检测到任何突变；23个氟康唑敏感株的18个中检测出5种氨基酸置换(突变率78%),分别是E266D(n=15),D116E(n=11),V488I(n=7),K128T(n=3),V437I(n=2)。两种菌株中共有的突变位点是D116E,E266D,K128T,V437I和V488I。对于TAC1,33株耐氟康唑白念珠菌(包括8株美耐药株)中,有5株菌株出现突变,分别为T225A(n=1)和A736V(n=4),其国中4株菌来自美国。 [结论]应用滚环扩增技术检测耐氟康唑白念珠菌耐药基因的点突变,挂锁探针准确地检测出受试菌株中ERG11基因和TAC1基因的突变,其中一些位置较接近的突变,滚环扩增技术均获得了准确的结果,表明滚环扩增技术检测基因单点突变具有良好的特异性和敏感性,是一种准确、快速的检测基因单点突变的分子生物学方法。ERG11和TAC1突变的数目或分布模式与唑类药物的MIC之间未见明显的关联,但是可能随着地理区域的不同而不同。ERG11点突变和耐药的发生密切相关,TAC1点突变与耐药的关系有待于进一步研究。白念珠菌对唑类药物耐药是多种分子机制同时作用的结果,因此今后应进一步探索和完善耐药产生的分子机制,为临床提供理论依据。
[Abstract]:[objective] to collect fluconazole-resistant and sensitive strains of Candida albicans, detect point mutations of drug-resistance-related genes ERG11 and TAC1 by rolling amplification technique, and compare the results with the sequencing results in order to establish an accurate and rapid method. A specific molecular biological method for the detection of single point mutations of genes, and further understanding of the relationship between ERG11 and TAC1 mutations and the resistance to azoles. [methods] Candida albicans were collected from different clinical specimens and their ERG11 mutation sites were identified in 25 fluconazole-resistant strains of Candida albicans and 21 susceptible strains in the United States. The padlock probe was designed according to the point mutation and padlock probe design principles reported in the literature, including 24 padlock probes designed by ERG11 and 4 padlock probes designed by TAC1, each probe could detect one point mutation. The three fragments of ERG11 and TAC1 were extracted and amplified by PCR, and the excess buffer, primer and dNTP were purified and removed, and then by padlock probe, nucleic acid exonuclease digestion, hyperbranching and rolling loop amplification, etc. The point mutation of ERG11 and the point mutation of TAC1 in resistant and sensitive strains were detected by rolling loop amplification technique. At the same time, the target fragment was purified and sent to sequencing, and the result of rolling loop amplification was compared with the result of sequencing. [results] among 8 fluconazole-resistant strains with known mutation sites, point mutations consistent with known mutations were detected by rolling loop amplification. The rolling amplification signal can be detected in the mixture containing 5% target template. For all clinical strains, the point mutations detected by rolling loop amplification technique were all correct by sequencing, and the rolling loop amplification technique showed good agreement with DNA sequencing. 瀵逛簬ERG11,25涓盁搴峰攽鑰愯嵂鏍殑24涓腑妫，
本文编号：2147332
[Abstract]:[objective] to collect fluconazole-resistant and sensitive strains of Candida albicans, detect point mutations of drug-resistance-related genes ERG11 and TAC1 by rolling amplification technique, and compare the results with the sequencing results in order to establish an accurate and rapid method. A specific molecular biological method for the detection of single point mutations of genes, and further understanding of the relationship between ERG11 and TAC1 mutations and the resistance to azoles. [methods] Candida albicans were collected from different clinical specimens and their ERG11 mutation sites were identified in 25 fluconazole-resistant strains of Candida albicans and 21 susceptible strains in the United States. The padlock probe was designed according to the point mutation and padlock probe design principles reported in the literature, including 24 padlock probes designed by ERG11 and 4 padlock probes designed by TAC1, each probe could detect one point mutation. The three fragments of ERG11 and TAC1 were extracted and amplified by PCR, and the excess buffer, primer and dNTP were purified and removed, and then by padlock probe, nucleic acid exonuclease digestion, hyperbranching and rolling loop amplification, etc. The point mutation of ERG11 and the point mutation of TAC1 in resistant and sensitive strains were detected by rolling loop amplification technique. At the same time, the target fragment was purified and sent to sequencing, and the result of rolling loop amplification was compared with the result of sequencing. [results] among 8 fluconazole-resistant strains with known mutation sites, point mutations consistent with known mutations were detected by rolling loop amplification. The rolling amplification signal can be detected in the mixture containing 5% target template. For all clinical strains, the point mutations detected by rolling loop amplification technique were all correct by sequencing, and the rolling loop amplification technique showed good agreement with DNA sequencing. 瀵逛簬ERG11,25涓盁搴峰攽鑰愯嵂鏍殑24涓腑妫，
本文编号：2147332
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