應(yīng)用滾環(huán)擴(kuò)增技術(shù)對(duì)白念珠菌耐氟康唑相關(guān)基因點(diǎn)突變的研究
發(fā)布時(shí)間：2018-07-27 09:16
【摘要】： [目的]收集白念珠菌氟康唑耐藥株和敏感株,應(yīng)用滾環(huán)擴(kuò)增技術(shù)檢測(cè)菌株耐藥相關(guān)基因ERG11和TAC1的點(diǎn)突變,并將所得結(jié)果與測(cè)序結(jié)果進(jìn)行比較,以期建立一種準(zhǔn)確、快速、特異的檢測(cè)基因單點(diǎn)突變的分子生物學(xué)方法,同時(shí)進(jìn)一步了解ERG11和TAC1突變與唑類藥物耐藥之間的關(guān)系。 [方法]從不同臨床標(biāo)本中收集白念珠菌并進(jìn)行藥物敏感性測(cè)定,得到白念珠菌氟康唑耐藥株25株和敏感株21株；8株美國耐藥株已知其ERG11突變位點(diǎn)。根據(jù)文獻(xiàn)報(bào)道的與耐藥有關(guān)的點(diǎn)突變和掛鎖探針設(shè)計(jì)原則設(shè)計(jì)掛鎖探針,其中ERG11設(shè)計(jì)24個(gè)掛鎖探針,TAC1設(shè)計(jì)4個(gè)掛鎖探針,每個(gè)探針可檢測(cè)一種點(diǎn)突變。提取DNA、PCR擴(kuò)增獲得目的片段ERG11和TAC1的三個(gè)片段,純化去除多余的緩沖液、引物和dNTP；然后通過掛鎖探針的連接、核酸外切酶消化、超分支滾環(huán)擴(kuò)增等過程,用滾環(huán)擴(kuò)增技術(shù)分別檢測(cè)耐藥株和敏感株中ERG11的點(diǎn)突變和耐藥株中TAC1的點(diǎn)突變。同時(shí)將目的片段純化后送交測(cè)序,將滾環(huán)擴(kuò)增所得結(jié)果與測(cè)序結(jié)果進(jìn)行比較。 [結(jié)果]在8株己知突變位點(diǎn)的氟康唑耐藥株中,滾環(huán)擴(kuò)增技術(shù)準(zhǔn)確地檢測(cè)出與已知突變一致的點(diǎn)突變；應(yīng)用滾環(huán)擴(kuò)增技術(shù)最低可在含有5%目標(biāo)模板的混合物中檢測(cè)到滾環(huán)擴(kuò)增信號(hào)。對(duì)全部臨床菌株,應(yīng)用滾環(huán)擴(kuò)增技術(shù)檢測(cè)到的點(diǎn)突變經(jīng)測(cè)序驗(yàn)證全部正確,滾環(huán)擴(kuò)增技術(shù)表現(xiàn)出和DNA測(cè)序很好的一致性。對(duì)于ERG11,25個(gè)氟康唑耐藥株的24個(gè)中檢測(cè)出了錯(cuò)義突變導(dǎo)致的20種氨基酸轉(zhuǎn)換(突變率96%),分別是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以前未見報(bào)道,一株菌未檢測(cè)到任何突變；23個(gè)氟康唑敏感株的18個(gè)中檢測(cè)出5種氨基酸置換(突變率78%),分別是E266D(n=15),D116E(n=11),V488I(n=7),K128T(n=3),V437I(n=2)。兩種菌株中共有的突變位點(diǎn)是D116E,E266D,K128T,V437I和V488I。對(duì)于TAC1,33株耐氟康唑白念珠菌(包括8株美耐藥株)中,有5株菌株出現(xiàn)突變,分別為T225A(n=1)和A736V(n=4),其國中4株菌來自美國。 [結(jié)論]應(yīng)用滾環(huán)擴(kuò)增技術(shù)檢測(cè)耐氟康唑白念珠菌耐藥基因的點(diǎn)突變,掛鎖探針準(zhǔn)確地檢測(cè)出受試菌株中ERG11基因和TAC1基因的突變,其中一些位置較接近的突變,滾環(huán)擴(kuò)增技術(shù)均獲得了準(zhǔn)確的結(jié)果,表明滾環(huán)擴(kuò)增技術(shù)檢測(cè)基因單點(diǎn)突變具有良好的特異性和敏感性,是一種準(zhǔn)確、快速的檢測(cè)基因單點(diǎn)突變的分子生物學(xué)方法。ERG11和TAC1突變的數(shù)目或分布模式與唑類藥物的MIC之間未見明顯的關(guān)聯(lián),但是可能隨著地理區(qū)域的不同而不同。ERG11點(diǎn)突變和耐藥的發(fā)生密切相關(guān),TAC1點(diǎn)突變與耐藥的關(guān)系有待于進(jìn)一步研究。白念珠菌對(duì)唑類藥物耐藥是多種分子機(jī)制同時(shí)作用的結(jié)果,因此今后應(yīng)進(jìn)一步探索和完善耐藥產(chǎn)生的分子機(jī)制,為臨床提供理論依據(jù)。
[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涓腑媯，
本文編號(hào)：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涓腑媯，
本文編號(hào)：2147332
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