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付雷雯, 王玲, 乐婷婷, 郭辉杰, 杨钧, 陈清, 刘志华, 胡静. 耳念珠菌重组酶聚合酶扩增检测方法的建立与评价[J]. 疾病监测, 2023, 38(6): 729-734. DOI: 10.3784/jbjc.202302100031
引用本文: 付雷雯, 王玲, 乐婷婷, 郭辉杰, 杨钧, 陈清, 刘志华, 胡静. 耳念珠菌重组酶聚合酶扩增检测方法的建立与评价[J]. 疾病监测, 2023, 38(6): 729-734. DOI: 10.3784/jbjc.202302100031
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Fu Leiwen, Wang Ling, Le Tingting, Guo Huijie, Yang Jun, Chen Qing, Liu Zhihua, Hu Jing. Establishment and evaluation of recombinase polymerase amplification assay for identification of Candida auris[J]. Disease Surveillance, 2023, 38(6): 729-734. DOI: 10.3784/jbjc.202302100031
Citation: Fu Leiwen, Wang Ling, Le Tingting, Guo Huijie, Yang Jun, Chen Qing, Liu Zhihua, Hu Jing. Establishment and evaluation of recombinase polymerase amplification assay for identification of Candida auris[J]. Disease Surveillance, 2023, 38(6): 729-734. DOI: 10.3784/jbjc.202302100031
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耳念珠菌重组酶聚合酶扩增检测方法的建立与评价

Establishment and evaluation of recombinase polymerase amplification assay for identification of Candida auris

    摘要
  • 摘要:
      目的  耳念珠菌是一种新出现的多重耐药酵母菌，本研究拟建立基于重组酶聚合酶扩增（RPA）技术的耳念珠菌检测方法并评价。
      方法  针对念珠菌5.8S和ITS2基因区域设计耳念珠菌特异性引物和念珠菌通用引物，评价其最低检测限；选取65株菌对其特异度进行评价；同时测定其在非培养条件下对该病原的检出能力。 用5种常见念珠菌和耳念珠菌对念珠菌通用RPA检测方法进行评价。
      结果  耳念珠菌特异性RPA检测方法检测下限为103拷贝/反应，与聚合酶链式反应（PCR）检测下限相同，37 ℃下反应时间20 min，特异度为100.00%。 可以在2.5 h内检测出血液中的耳念珠菌，其检测下限为1.02×103 菌落形成单位（CFU）/mL，与PCR检测下限相同。 念珠菌通用引物可通过扩增产物大小不同，实现5种常见念珠菌与耳念珠菌的初步鉴别。 可在白色念珠菌与耳念珠菌菌量比（5∶1，1∶1，1∶5）的混合感染情况下产生两种易于区分的条带。种易于区分的条带。
      结论  本研究建立的耳念珠菌RPA 检测方法，具有简便、快速、成本低的特点，表现出了较高的灵敏度与特异度，可以实现5种临床常见念珠菌与耳念珠菌的初步鉴别诊断。

     

    Abstract:
      Objectives   Candida auris (C. auris) is an emerging multidrug-resistant yeast associated with high mortality. We developed a rapid assay for the specific identification of C. auris based on recombinase polymerase amplification (recombinase polymerase amplification, RPA).
      Methods  The fragment of 5.8S, all of ITS2 rRNA genes of Candida species were used as the target sequence to design C. auris specific primers and for Candida universal primer. The detection limit of RPS was tested and 61 yeast isolates and 4 bacterial strains were used to evaluate the specificity of RPA, and the detection power of RPA for C. auris under non-culture condition was evaluated. Five common Candida species and C. auris were used to evaluate the universal RPA assay.
      Results  The RPA assay could identify C. auris strains with a specificity of 100.00% at 37 ℃ in 20 minutes and the limit of detection was 103 copies/reaction to polymerase chain reaction(polymerase chain reaction, PCR) assay. The assay could detect C. auris in blood within 2.5 hours with the detection limit of 1.02×103 colony forming units (CFU)/mL same to PCR assay. The Candida universal RPA assay could realize the preliminary differential diagnose of infections of five common clinical Candida species and C. auris, by amplifying the products with different sizes. In the mixed pathogen experiment, two kinds of easily distinguishable bands can be produced in the case of the ratio of C. albicans to C. aurisat 5∶1, 1∶1 and 1∶5.
      Conclusion   A simple, rapid and cost effective RPA assay has been established, which can be used for the preliminary differential diagnose of infections of five common clinical Candida species and C. auris, with high specifity and sensitivity.

     

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