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首页> 外文期刊>BMC Microbiology >Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing
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Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

机译：通过滚环扩增和DNA测序快速检测临床白色念珠菌分离株中对氟康唑敏感性降低的ERG11基因突变

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Background Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. Results The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate. Conclusion The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.
机译：背景唑类抗真菌药的目标酶Erg11p中的氨基酸取代有助于白色念珠菌的临床相关的唑耐药性。一种快速检测ERG11基因突变的简单分子方法作为鉴定潜在耐药菌株并追踪其运动的筛选工具将是一个优势。为了补充DNA测序，我们开发了一种基于挂锁探针和滚环扩增（RCA）的方法，以使用具有已知突变的“参考”抗唑类菌株来检测白色念珠菌ERG11基因中的一系列突变。然后，该方法用于估计25种对氟康唑敏感性降低的澳大利亚临床白色念珠菌分离株和23种氟康唑敏感性分离株中ERG11突变的频率及其类型。将RCA结果与DNA测序进行比较。结果RCA分析正确鉴定了八个“参考”白色念珠菌分离物中的所有ERG11突变。当应用于48个测试菌株时，RCA方法与使用ERG11突变特异性探针的DNA测序显示100％的一致性。在25个（96％）氟康唑敏感性降低的菌株中，有24个通过测序检测到20种不同的错义突变，其中RCA检测到16种。通过两种方法在23个氟康唑敏感性菌株中的18个（78％）中检测到5个错义突变。 DNA测序表明，非敏感分离株中的突变都是由于纯合核苷酸变化引起的。除了导致氨基酸取代E266D的突变外，对氟康唑敏感的菌株均为杂合子。两组分离物共有的氨基酸取代是D116E，E266D，K128T，V437I和V488I。氟康唑敏感性降低的菌株独特的替代品是G464 S（n = 4个菌株），G448E（n = 3），G307S（n = 3），K143R（n = 3）和Y123H，S405F和R467K（每个n = 1） 。 DNA测序揭示了一个分离株中的一种新型取代G450V。结论本文所述的灵敏RCA分析方法是一种简单，可靠且快速的（2 h）方法，用于检测ERG11多态性。它显示出与ERG11测序的极佳一致性，并且是追踪对唑类耐药的白色念珠菌的出现和传播以及研究ERG11突变的流行病学的潜在有价值的工具。 RCA方法适用于研究其他真菌对唑的抗性。

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