MALDI-TOF MS直接涂靶法快速鑒定絲狀真菌的臨床應用

張桂 王麗赟 王玫 顧海彤 魯辛辛

引用本文: 張桂, 王麗赟, 王玫, 顧海彤, 魯辛辛. MALDI-TOF MS直接涂靶法快速鑒定絲狀真菌的臨床應用[J]. 疾病監測. DOI: 10.3784/j.issn.1003-9961.2019.11.008 shu
Citation:  Gui Zhang, Liyun Wang, Mei Wang, Haitong Gu and xinxin Lu. Evaluation of the clinical application of MALDI-TOF MS with directly smeared target in rapid identification of filamentous fungi[J]. Disease Surveillance. DOI: 10.3784/j.issn.1003-9961.2019.11.008 shu

MALDI-TOF MS直接涂靶法快速鑒定絲狀真菌的臨床應用

    作者簡介: 張桂,男,湖南省沅江市人,博士,主管檢驗師,主要從事微生物檢驗和分子生物學工作,Email:[email protected];
    通信作者: 魯辛辛, [email protected]
  • 基金項目: 首都醫科大學附屬北京同仁醫院科研骨干培育基金(No. 2014-YJJ-GGL-038);國家科技重大專項(No. 2014ZX10004005)

摘要: 目的評價基質輔助激光解吸電離飛行時間質譜直接涂靶法(質譜直涂法)快速鑒定絲狀真菌的臨床應用價值。方法對2010年1月至2017年12月首都醫科大學附屬北京同仁醫院臨床標本中分離的133株絲狀真菌分別進行形態學、基因序列分析及質譜直涂法鑒定,并將鑒定結果進行分析比較。 采用SPSS 16.0軟件進行統計分析,計數資料采用χ2檢驗。結果以基因序列分析結合形態學鑒定的結果為標準,將133株絲狀真菌鑒定到種、復合群和屬的鑒定率:質譜直涂法分別為64.66%、79.70%和96.24%,基因序列分析則分別為69.17%、83.46%和99.25%,而形態學鑒定分別為68.42%、84.21%和93.23%。 質譜直涂法與另外兩種方法的種、復合群和屬的鑒定率的差異無統計學意義(χ2值分別為0.611、0.625、2.728和0.516、0.915、1.206,P值均>0.05)。 質譜直涂法將77株曲霉屬中61.04%、88.31%和98.70%的菌株分別鑒定到種、復合群和屬,將14株青霉屬中64.29%和92.86%的菌株分別鑒定到種和屬,還將42株其他絲狀真菌中的71.43%和92.86%的菌株分別鑒定到種和屬;因數據庫中真菌種類不足,致3.76%(5/133)的真菌無效鑒定。結論MALDI-TOF MS直接涂靶法鑒定絲狀真菌的能力可與形態學鑒定和基因序列分析相媲美,因其快速且操作簡便,臨床應用值得推廣。

English

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  • 圖 1  絲狀真菌代表株的質譜圖、菌落形態圖及鏡下鑒定結果

    Figure 1.  Representative strains of filamentous fungi identified by mass spectrometry, colony morphology and microscopy

    表 1  本研究所用引物相關信息

    Table 1.  Information of primers used in the study

    引物編號引物名稱靶區域引物序列(5′~3′)參考文獻
    1ITS1-FITS1TCCGTAGGTGAACCTGCGG7, 1112
    2aITS1-RGCTGCGTTCTTCATCGATGC
    3ITS2-FITS2GCATCGATGAAGAACGCAGC1112
    4ITS2-RTCCTCCGCTTATTGATATGC1112
    1ITS1-FITS1-5.8S-ITS2CGTAGGTGAACCTGCGG7, 1112
    4ITS2-RTCCTCCGCTTATTGATATGC1112
    5D1/D2-FD1/D2GCATATCAATAAGCGGAGGAAAAG12
    6D1/D2-RGGTCCGTGTTTCAAGACGG12
    7MT-Fβ-tubulinAATTGGTGCCGCTTTCTGG7
    8MT-RAGTTGTCGGGACGGAATAG7
      注:a. 引物2是引物3的反向互補序列
    下載: 導出CSV

    表 2  形態學、基因序列分析及質譜直涂法鑒定絲狀真菌的能力比較

    Table 2.  Comparison of filamentous fungi identification powers of morphological observation,gene sequence analysis and mass spectrometry with directly smeared target

      菌株名稱形態學+基因序列分析鑒定(株)基因序列分析鑒定(株)形態學鑒定(株)質譜直涂法鑒定(株)
    復合群復合群復合群d復合群ef
    曲霉屬(Aspergillus 63 77 7757 76 7753 74 7747 68 76
     黑曲霉(A. niger 9 9 9 8 9a 9 9 9 9 9a 9 9
     黃曲霉(A. flavus 4 4 4 4 4 4 4 4 4 0 4 4
     黃曲霉/米曲霉(A. flavus/A. oryzae 0 10 10 0 10 10 0 10 10 0 8 10
     煙曲霉(A. fumigates 21 21 2121 21 2120 20 2121 21 21
     烏達加瓦曲霉(A. udagawae 1 1 1 1 1 1 0 1 1 0 0 0
     構巢曲霉(A. nidulans 7 7 7 7 7 7 5 5 7 7 7 7
     雜色曲霉(A.versicolor 6 6 6 4 5a 6b 6 6 6 6ab 6 6
     土曲霉(A. terreus 8 8 8 6 8a 8 8 8 8 4a 4 8
     阿姆斯特丹曲霉(A.amstelodami 1 1 1 0 1c 1 1 1 1 0 0 1c
     溜曲霉(A. tamarii 1 1 1 1 1 1 0 1 1 0 0 1
     雜色/聚多曲霉(A.versicolor/sydowii 0 4 4 0 4 4 0 4 4 0 4 4
     聚多曲霉(A. sydowii 5 5 5 5 5 5 0 5 5 0 4 5
    青霉屬(Penicillium 12 12 14 7 7 14 6 6 12 9 9 13
     產黃青霉(P. chrysogenum 4 4 4 0 0 4 4 4 4 3 3 4
     草酸青霉(P. oxalicum 3 3 3 3 3 3 0 0 3 0 0 2
     光孢青霉(P. glabrum 1 1 1 0 0 1 1 1 1 1 1 1
     桔青霉(P. citrinum 1 1 1 1 1 1 1 1 1 1 1 1
     普通青霉(P. commune 1 1 1 1 1 1 0 0 1 1 1 1
     繩狀青霉(P. funiculosum 0 0 1 0 0 1 0 0 1 1 1 1
     指狀青霉(P. digitatum 2 2 2 2 2 2 0 0 2 1 1 2
     青霉屬某種(Penicillium sp.) 0 0 1 0 0 1 0 0 1 1 1 1
    其他 36 36 4228 28 4132 32 3530 30 39
     紅色毛癬菌(Trichophyton rubum 2 2 2 2 2 2 2 2 2 2 2 2
     斷發毛癬菌(T. tonsurans 1 1 1 1 1 1 1 1 1 1 1 1
     犬小孢子菌(Microsporum canis 2 2 2 2 2 2 2 2 2 2 2 2
     石膏樣小孢子菌(M. gypseum 1 1 1 1 1 1 1 1 1 1 1 1
     絮狀表皮癬菌(Epidermorphyton floxxossum 1 1 1 1 1 1 1 1 1 1 1 1
     鐮刀菌屬(Fusarium 4 4 4 0 0 4 4 4 4 3 3 4
     鏈格孢霉(Alternaria alternata 7 7 7 7 7 7 7 7 7 7 7 7
     尖端賽多孢霉(Scedosporium apiospermum 1 1 1 0 0 0 1 1 1 1 1 1
     籃狀菌屬(Talaromyces 0 0 1 0 0 1 0 0 0 0 0 0
     宛氏擬青霉(Paecilomyces varioti 1 1 1 0 0 1 1 1 1 1 1 1
     葡枝根霉(Rhizopus stolonifer 3 3 3 3 3 3 3 3 3 1 1 3
     米根霉(R. oryzae 2 2 2 2 2 2 2 2 2 2 2 2
     出芽短梗霉(Aureobasidium pullulans 2 2 2 0 0 2 2 2 2 1 1 2
     帚霉屬(Scopulariopsis 0 0 1 0 0 1 0 0 1 0 0 1
     木霉屬(Trichoderma 0 0 2 0 0 2 0 0 2 1 1 2
     交枝頂孢(Sarocladium implicatum 1 1 1 1 1 1 0 0 0 0 0 0
     極細枝孢霉(Cladosporium tenuissimum 1 1 1 1 1 1 0 0 0 0 0 0
     枝孢霉屬(Cladosporium 0 0 2 0 0 2 0 0 0 0 0 2
     裂褶菌(Schizophyllum commune 7 7 7 7 7 7 7 7 7 6 6 7
       合計111125133921111329111212486106128
      注:a. 基因序列分析鑒定為復合群,質譜直涂法鑒定為相應的種(分值>2.00);b. 基因序列分析鑒定為曲霉屬,質譜直涂法鑒定為雜色曲霉(分值>2.00);c. 基因序列分析鑒定為阿姆斯特丹曲霉/冠突曲霉,質譜直涂法鑒定為阿姆斯特丹曲霉(1.70<分值<2.00);d. 種的鑒定率,質譜鑒定法與形態學+基因序列分析鑒定法、基因序列分析鑒定法、形態學鑒定法比較,其χ2=12.231,P<0.001;χ2=0.611,P =0.434;χ2=0.516,P=0.422;e. 復合群的鑒定率,質譜鑒定法與形態學+基因序列分析鑒定法、基因序列分析鑒定法、形態學鑒定法比較,其χ2=11.877,P<0.001;χ2=0.625,P=0.429;χ2 =0.915,P=0.339;f. 屬的鑒定率,質譜鑒定法與形態學+基因序列分析鑒定法、基因序列分析鑒定法、形態學鑒定法比較,其χ2=5.096,P=0.024;χ2=2.728,P=0.099;χ2 =1.206,P=0.272
    下載: 導出CSV

    表 3  以基因序列分析為參考的形態學、質譜直涂法的鑒定結果

    Table 3.  Identification results of morphological observation and mass spectrometry with directly smeared target based on gene sequence analysis

     基因序列分析鑒定菌株數(株)形態學鑒定菌株數(株)質譜直涂法鑒定菌株數(株)
    聚多曲霉(Aspergillus sydowii5雜色曲霉(A. versicolor5雜色曲霉(A.versicolor4+1a
    雜色曲霉/聚多曲霉(A.versicolor/A.sydowii4雜色曲霉(A. versicolor4雜色曲霉(A. versicolor4
    黃曲霉(A. flavus4NO4黃曲/米曲霉(A. flavus/oryzae4
    構巢曲霉(A. niduluns 2土曲霉(A. terreus2NO2
    煙曲霉(A. fumigates1局限曲霉(A. restrictus1NO1
    溜曲霉(A. tamari1黃曲霉(A. flavus1NO1
    烏達加瓦曲霉(A. udagawae1煙曲霉(A. fumigates1NID1
    草酸青霉(Penicillium oxalicum3青霉屬(Penicillium3青霉屬某種(Penicillium sp.)NID2
    1
    籃狀菌屬(Talaromyces1桔青霉(P. citrinum1NID1
    枝孢霉屬(Cladosporium2外瓶霉屬(Exophiala);
    棘狀外瓶霉(E. spinifera
    1
    1
    NO2
    極細枝孢霉(C. tenuissimum1棘狀外瓶霉(E. spinifera1NID1
    交枝頂孢霉(Sarocladium implicatum1外瓶霉屬(Exophiala1NID1
      注:a. 1株菌由質譜直涂法鑒定為雜色曲霉(1.70<分值<2.00);NO. 無鑒定錯誤;NID. 無效鑒定
    下載: 導出CSV
    广西体彩11选五开奖
  • 2019-0088 MALDI-TOF MS 直接涂靶法快速鑒定絲狀真菌的臨床應用.docx

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  • 通信作者:  魯辛辛, [email protected]
  • 收稿日期:  2019-03-11
  • 刊出日期:  2019-11-01
通信作者: 陳斌, [email protected]
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