利用基于敏感通用引物的 PCR 和限制性内切酶消化与高分辨率熔解分析快速鉴定侵袭性真菌种属。

Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysis.

机构信息

Division of Neonatology and Pediatric Hematology/Oncology, Department of Pediatrics, Chang Gung Memorial Hospital, Yunlin, Taiwan; College of Medicine, Chang Gung University, Taiwan; Graduate Institute of Clinical Medical Science, College of Medicine, Chang Gung University, Taiwan.

Department of Laboratory Medicine, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan.

出版信息

J Microbiol Immunol Infect. 2019 Oct;52(5):728-735. doi: 10.1016/j.jmii.2019.06.001. Epub 2019 Jul 2.

DOI:10.1016/j.jmii.2019.06.001

PMID:31302087

Abstract

BACKGOUND

Conventional diagnosis of invasive fungal disease from blood cultures is often notoriously delayed and inadequately sensitive. We aimed to develop a universal primers-based polymerase chain reaction (PCR) assay and restriction fragment length polymorphisms (RFLP) for rapid identification of invasive fungal disease (IFD).

METHODS

We evaluated 16 clinical fungal species using a combination of PCR assays with 3 different restriction endonucleases targeting various internal transcribed spacer (ITS) regions and high resolution melting analysis (HRMA). Serial samples from 75 patients suspected to have IFD were analyzed for clinical verification.

RESULTS

We have designed a universal PCR capable of amplifying a portion of the 18S rRNA gene of 16 clinically important fungal species. The restriction patterns of most PCR products generated by EcoRI or double digested by ClaI and AvaI were different, except Aspergillus niger and Aspergillus flavus had a similar pattern, and Aspergillus fumigatus and Aspergillus terreus had a similar pattern. All these species had a unique melting curve shape using the HRMA. Both HRMA and universal PCR had adequate sensitivity, and all sixteen reference fungal species can be clearly distinguished by the universal PCR-RFLP-HRMA assay. With a reference library of 176 clinically relevant fungal strains, and 75 clinical samples from patients with suspicious IFD were tested, our assay identified 100% and 61.1% of isolates from the reference library and clinical samples, respectively.

CONCLUSIONS

Universal PCR and RFLP coupled with HRMA could be a highly discriminative and useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of invasive fungal disease.

摘要

背景

血液培养物常规诊断侵袭性真菌感染通常存在明显的延迟和不充分的敏感性。我们旨在开发一种基于通用引物的聚合酶链反应（PCR）检测和限制性片段长度多态性（RFLP），用于快速鉴定侵袭性真菌感染（IFD）。

方法

我们使用针对不同内部转录间隔区（ITS）区域的 3 种不同限制性内切酶的组合 PCR 检测评估了 16 种临床真菌。对 75 例疑似 IFD 的患者的连续样本进行分析以进行临床验证。

结果

我们设计了一种通用 PCR，能够扩增 16 种重要临床真菌的 18S rRNA 基因的一部分。除了黑曲霉和黄曲霉具有相似的模式外，大多数由 EcoRI 产生的 PCR 产物的限制图谱或由 ClaI 和 AvaI 双重消化的图谱都不同，烟曲霉和土曲霉具有相似的模式。所有这些物种在使用 HRMA 时都具有独特的熔解曲线形状。HRMA 和通用 PCR 都具有足够的灵敏度，并且通用 PCR-RFLP-HRMA 检测可以清楚地区分所有 16 种参考真菌。使用包含 176 种临床相关真菌株的参考文库，以及来自 75 例疑似 IFD 患者的 75 个临床样本进行测试，我们的检测方法分别鉴定了参考文库和临床样本中 100%和 61.1%的分离株。