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中华危重症医学杂志(电子版) ›› 2020, Vol. 13 ›› Issue (04) : 270 -276. doi: 10.3877/cma.j.issn.1674-6880.2020.04.006

所属专题： 总编推荐； 文献；

论著

多重荧光PCR-熔解曲线法同时检测常见病原真菌方法学的建立及诊断价值
朱芳1, 朱以军2,†(), 胡益飞2, 倪红英3, 单小云2   
  1. 1. 321000　浙江金华，金华市中心医院病理科
    2. 321000　浙江金华，金华市中心医院检验科
    3. 321000　浙江金华，金华市中心医院重症医学科
  • 收稿日期:2020-02-25 出版日期:2020-08-01
  • 通信作者: 朱以军
  • 基金资助:
    浙江省公益技术应用研究分析测试计划项目(2017C37050); 金华市中心医院中青年科研启动基金项目(JY2016-2-03)

Establishment and diagnostic value of multiplex fluorescent PCR-melting curve method for detection of common pathogenic fungi

Fang Zhu1, Yijun Zhu2,†(), Yifei Hu2, Hongying Ni3, Xiaoyun Shan2   

  1. 1. Department of Pathology, Jinhua Municipal Central Hospital, Jinhua 321000, China
    2. Department of Clinical Laboratory, Jinhua Municipal Central Hospital, Jinhua 321000, China
    3. Department of Critical Care Medicine, Jinhua Municipal Central Hospital, Jinhua 321000, China
  • Received:2020-02-25 Published:2020-08-01
  • Corresponding author: Yijun Zhu
  • About author:
    Corresponding author: Zhu Yijun, Email:
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朱芳, 朱以军, 胡益飞, 倪红英, 单小云. 多重荧光PCR-熔解曲线法同时检测常见病原真菌方法学的建立及诊断价值[J]. 中华危重症医学杂志(电子版), 2020, 13(04): 270-276.

Fang Zhu, Yijun Zhu, Yifei Hu, Hongying Ni, Xiaoyun Shan. Establishment and diagnostic value of multiplex fluorescent PCR-melting curve method for detection of common pathogenic fungi[J]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2020, 13(04): 270-276.

目的

建立多重荧光PCR-熔解曲线法，并评价其同时检测假丝酵母菌、曲霉菌及新型隐球菌的诊断价值。

方法

建立多重荧光PCR-熔解曲线法并对其检测体系进行优化。收集临床高度怀疑为侵袭性真菌感染（IFI）的各类临床样本179例，其中血液65例、深部痰液35例、尿液30例、脑脊液18例、胸腹水12例、肺泡灌洗液10例、新鲜肺组织9例。通过敏感性、特异性、重复性实验验证多重荧光PCR-熔解曲线法的检测性能，并应用受试者工作特征（ROC）曲线评价该方法的诊断效能。

结果

建立的多重荧光PCR-熔解曲线法可同时检测8种常见病原真菌，包括4种假丝酵母菌（白假丝酵母菌、热带假丝酵母菌、光滑假丝酵母菌、克柔假丝酵母菌）、3种曲霉菌（烟曲霉、黄曲霉、黑曲霉）和新型隐球菌。其最低检测限为1 × 104 cfu/mL，且常见细菌无扩增反应，重复性实验解链温度波动小于0.5 ℃。179例临床样本中，以培养法、镜检法、病理诊断等"金标准"方法诊断IFI阳性为112例，多重荧光PCR-熔解曲线法检测阳性为96例。多重荧光PCR-熔解曲线法同时检测假丝酵母菌、曲霉菌及新型隐球菌的敏感度为0.857，特异度为0.970，阳性预测值为0.980，阴性预测值为0.802，ROC曲线下面积为0.914，95%置信区间为0.868 ~ 0.959，P < 0.001。

结论

本研究建立的多重荧光PCR-熔解曲线法可快速、准确、高通量同时检测假丝酵母菌、曲霉菌及新型隐球菌，对于IFI的早期诊断具有重要意义。

关键词: 真菌, 多重荧光PCR, 熔解曲线
Objective

To establish a multiplex fluorescent PCR-melting curve method for simultaneous detection of Candida, Aspergillus and Cryptococcus, and to evaluate its diagnostic value.

Methods

A multiplex fluorescent PCR-melting curve method was established and its detection system was optimized. Totally 179 clinical specimens including 65 blood specimens, 35 deep sputum specimens, 30 urine specimens, 18 cerebrospinal fluid specimens, 12 hydrothorax and ascites specimens, 10 alveolar lavage fluid specimens and 9 fresh lung tissue specimens for highly suspected invasive fungal infection (IFI) were enrolled in this study. The sensitivity, specificity and repeatability experiments were used to verify the detection performance of this method, and the receiver operating characteristic (ROC) curve was used to evaluate its diagnostic efficiency.

Results

The multiplex fluorescent PCR-melting curve method could simultaneously detect 8 common pathogenic fungi, including 4 kinds of Candida (Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei), 3 kinds of Aspergillus (Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger) and Cryptococcus neoformans. The minimum detection limit was 1 × 104 cfu/mL. Common bacteria had no amplification reaction, and the melting temperature fluctuation of repetitive experiment was less than 0.5 ℃. Among 179 clinical specimens, 112 were positive by "gold standard" methods such as culture, microscopy and pathological diagnosis, and 96 were positive by the multiplex fluorescent PCR-melting curve method. The sensitivity, specificity, positive predictive value and negative predictive value for simultaneous detection of Candida, Aspergillus and Cryptococcus neoformans by this method were 0.857, 0.970, 0.980 and 0.802, respectively. The area under ROC curve was 0.914, and the 95% confidence interval was 0.868 ~ 0.959 (P < 0.001).

Conclusion

The multiplex fluorescent PCR-melting curve method can detect Candida, Aspergillus and Cryptococcus neoformans rapidly and accurately with high throughput, which is of great significance for the early diagnosis of IFI.

Key words: Fungi, Multiplex fluorescent-PCR, Melting curve
图1 多重荧光PCR Fam探针扩增体系8种真菌的熔解曲线及熔解峰
图2 多重荧光PCR Hex探针扩增体系8种真菌的熔解曲线及熔解峰
图3 多重荧光PCR Cy5探针扩增体系8种真菌的熔解曲线及熔解峰
表1 多重荧光PCR-熔解曲线法3组探针扩增体系中8种真菌标准菌株解链温度参考范围（℃）
菌种/菌属 Fam探针 Hex探针 Cy5探针
白假丝酵母菌 58.88 ~ 59.95 54.12 ~ 55.55 61.24 ~ 62.50
热带假丝酵母菌 58.45 ~ 60.02 60.87 ~ 61.81 61.40 ~ 62.25
光滑假丝酵母菌 52.00 ~ 60.01 59.17 ~ 60.09 65.20 ~ 65.80
克柔假丝酵母菌 52.00 ~ 55.64 58.36 ~ 59.76 67.12 ~ 67.90
黑曲霉 53.82 ~ 54.81 58.08 ~ 60.81 58.53 ~ 59.26
黄曲霉 53.96 ~ 54.82 54.04 ~ 54.96 62.60 ~ 63.18
烟曲霉 57.43 ~ 58.44 58.83 ~ 59.72 59.10 ~ 60.05
新型隐球菌 65.78 ~ 66.66 - 58.24 ~ 60.24
表2 多重荧光PCR-熔解曲线法检测结果与传统"金标准"诊断IFI结果汇总表（例）
多重荧光PCR-熔解曲线法检测结果 传统"金标准"诊断IFI结果 合计
假丝酵母菌阳性 曲霉菌阳性 隐球菌阳性 总阳性 阴性
阳性 66 23 7 96 2 98
阴性 10 5 1 16 65 81
合计 76 28 8 112 67 179
表3 多重荧光PCR-熔解曲线法检测各种真菌的ROC曲线分析结果
真菌菌种 AUC（95%CI） 标准误 P值 敏感度 特异度 阳性预测值 阴性预测值
假丝酵母菌 0.919（0.868～0.970） 0.026 <0.001 0.868 0.970 0.971 0.867
曲霉菌 0.896（0.808～0.983） 0.045 <0.001 0.821 0.970 0.920 0.929
隐球菌 0.923（0.786～1.000） 0.070 <0.001 0.875 0.970 0.778 0.985
假丝酵母菌+曲霉菌+隐球菌 0.914（0.868～0.959） 0.023 <0.001 0.857 0.970 0.980 0.802
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