應(yīng)用基因擴(kuò)增技術(shù)對三種臨床常用酵母樣真菌鑒定方法的評價
發(fā)布時間：2019-05-12 05:49
【摘要】：目的： 分子生物學(xué)技術(shù)的發(fā)展使真菌通過基因序列分析能夠準(zhǔn)確快速鑒定到種，但是臨床實驗室常使用商品化的生化鑒定方法來鑒定真菌。本實驗以分子生物學(xué)方法鑒定酵母樣真菌的結(jié)果為“金標(biāo)準(zhǔn)”，探討API20C AUX和BD PhoenixTM酵母菌鑒定板鑒定酵母樣真菌的臨床應(yīng)用價值。 方法： 收集吉林大學(xué)第一醫(yī)院二部微生物室2012~2013年無菌體液標(biāo)本分離出的酵母樣真菌121株，，已剔除同一患者同一部位或不同部位分離出的相同菌株。將121株菌株傳至沙氏培養(yǎng)基進(jìn)行充分復(fù)蘇、分純培養(yǎng)，并傳至科瑪嘉顯色培養(yǎng)基進(jìn)行初步顯色鑒定，同時對所有研究菌株平行進(jìn)行API20CAUX及BD PhoenixTM的鑒定。煮沸法提取DNA，以真菌通用引物ITS1和ITS4擴(kuò)增真菌rDNA，擴(kuò)增產(chǎn)物的測序結(jié)果提交GenBank，通過基因序列分析法將真菌準(zhǔn)確鑒定到種，不同鑒定方法的鑒定正確率行統(tǒng)計學(xué)分析，比較API20CAUX和BD PhoenixTM酵母菌鑒定板鑒定酵母樣真菌的臨床應(yīng)用價值。 結(jié)果： 1.121株酵母樣真菌經(jīng)分子生物學(xué)方法準(zhǔn)確鑒定到種，共包含8個菌種，均在API20C AUX的可鑒定菌種數(shù)據(jù)庫內(nèi)。除1株奧默畢赤酵母是BD PhoenixTM酵母菌鑒定板的庫外菌種外，其余120株菌株均在BD PhoenixTM酵母菌鑒定板的可鑒定菌種數(shù)據(jù)庫內(nèi)，占臨床分離菌株的99.2%（120/121）。 2.科瑪嘉顯色培養(yǎng)鑒定結(jié)果中有108株能夠鑒定至種，13株無法鑒定；108株鑒定至種的結(jié)果中有8株鑒定錯誤，總體鑒定正確率為82.65%（100/121）。API20CAUX鑒定結(jié)果中有5株菌株鑒定錯誤，4株菌株無鑒定結(jié)果，其余112株菌株均鑒定正確，鑒定正確率為92.56%（112/121）。BD PhoenixTM酵母菌鑒定板對庫內(nèi)菌種120株酵母樣真菌的鑒定結(jié)果中有1株葡萄牙念珠菌未能鑒定及1株季也蒙念珠菌鑒定錯誤，對庫外菌種1株奧默畢赤酵母鑒定錯誤，其余118株菌株均鑒定正確，庫內(nèi)鑒定正確率為98.33%（118/120），對全部121株菌株的總體鑒定正確率為97.52%（118/121）�？片敿物@色結(jié)合API20C AUX和科瑪嘉顯色結(jié)合BD PhoenixTM的總體鑒定正確率分別為90.08%（109/121）和91.74%（111/121）。BD PhoenixTM對庫內(nèi)菌種的鑒定正確率較API20C AUX的鑒定正確率高（2檢驗，P0.05）�？片敿物@色培養(yǎng)的總體鑒定正確率與其結(jié)合BD PhoenixTM的鑒定正確率之間有顯著差異（2檢驗，P0.05），與其結(jié)合API20CAUX的鑒定正確率之間無顯著差異（2檢驗，P0.05）。 結(jié)論： 1.科瑪嘉顯色培養(yǎng)、API20C AUX、BD PhoenixTM酵母菌鑒定板在鑒定酵母樣真菌中有較好的臨床應(yīng)用價值。 2. BD PhoenixTM酵母菌鑒定板較API20CAUX有更好的臨床應(yīng)用價值。 3. BD PhoenixTM酵母菌鑒定板和API20C AUX對少見菌種的鑒定效能有待進(jìn)一步驗證。
[Abstract]:Objective: with the development of molecular biology technology, fungi can be identified accurately and quickly by gene sequence analysis, but commercial biochemical identification methods are often used in clinical laboratories to identify fungi. In this experiment, the results of identification of yeast-like fungi by molecular biological method were "gold standard", and the clinical value of identification of yeast-like fungi by API20C AUX and BD PhoenixTM yeast identification plate was discussed. Methods: 121 strains of yeast-like fungi isolated from aseptic body fluid samples from the second microbiology department of the first Hospital of Jilin University in 2013 were collected and the same strains isolated from the same site or different parts of the same patient were eliminated. The strains were transferred to Shah's medium for full resuscitation, cultured in pure culture, and passed to Komaga color medium for preliminary color identification. At the same time, all the studied strains were identified by API20CAUX and BD PhoenixTM in parallel. DNA, was extracted by boiling method. The results of sequencing of fungal rDNA, products amplified by universal primers ITS1 and ITS4 were submitted to GenBank, for accurate identification of fungi to species by gene sequence analysis. The identification accuracy of different identification methods was statistically analyzed. The clinical value of API20CAUX and BD PhoenixTM yeast identification plate for identification of yeast-like fungi was compared. Results: 1.121 strains of yeast-like fungi were accurately identified by molecular biology, including 8 strains, all of which were in the identifiable strain database of API20C AUX. With the exception of one strain of Pichia omeptica which is outside the library of the BD PhoenixTM yeast identification board, all the other strains are in the identifiable strain database of the BD PhoenixTM yeast identification board, accounting for 99.2% (120 渭 121) of the clinical isolates. 2. 108 strains could be identified and 13 strains could not be identified. Of the 108 strains identified, 8 were wrong, the overall correct rate was 82.65% (100 脳 121). 5 of the 108 strains identified were wrong, 4 strains were not identified, and the other 112 strains were correctly identified, and the results of identification were as follows: (1) the total identification rate was 82.65% (100 脳 121), and that of the other 112 strains was correct. The correct rate of identification was 92.56% (112 / 121). BD PhoenixTM yeast identification board). One of the strains of Candida Portugal was not identified and one of the strains was wrong in the identification of Candida mongolicum in the library, and the results of the identification of yeast-like fungi in the library were as follows: (1) the identification rate was 92.56%. The identification error of one strain of Pichia pastoris outside the library was wrong, and the other 118 strains were correctly identified. The correct rate of identification in the library was 98.33% (118 鈮
本文編號：2475153
[Abstract]:Objective: with the development of molecular biology technology, fungi can be identified accurately and quickly by gene sequence analysis, but commercial biochemical identification methods are often used in clinical laboratories to identify fungi. In this experiment, the results of identification of yeast-like fungi by molecular biological method were "gold standard", and the clinical value of identification of yeast-like fungi by API20C AUX and BD PhoenixTM yeast identification plate was discussed. Methods: 121 strains of yeast-like fungi isolated from aseptic body fluid samples from the second microbiology department of the first Hospital of Jilin University in 2013 were collected and the same strains isolated from the same site or different parts of the same patient were eliminated. The strains were transferred to Shah's medium for full resuscitation, cultured in pure culture, and passed to Komaga color medium for preliminary color identification. At the same time, all the studied strains were identified by API20CAUX and BD PhoenixTM in parallel. DNA, was extracted by boiling method. The results of sequencing of fungal rDNA, products amplified by universal primers ITS1 and ITS4 were submitted to GenBank, for accurate identification of fungi to species by gene sequence analysis. The identification accuracy of different identification methods was statistically analyzed. The clinical value of API20CAUX and BD PhoenixTM yeast identification plate for identification of yeast-like fungi was compared. Results: 1.121 strains of yeast-like fungi were accurately identified by molecular biology, including 8 strains, all of which were in the identifiable strain database of API20C AUX. With the exception of one strain of Pichia omeptica which is outside the library of the BD PhoenixTM yeast identification board, all the other strains are in the identifiable strain database of the BD PhoenixTM yeast identification board, accounting for 99.2% (120 渭 121) of the clinical isolates. 2. 108 strains could be identified and 13 strains could not be identified. Of the 108 strains identified, 8 were wrong, the overall correct rate was 82.65% (100 脳 121). 5 of the 108 strains identified were wrong, 4 strains were not identified, and the other 112 strains were correctly identified, and the results of identification were as follows: (1) the total identification rate was 82.65% (100 脳 121), and that of the other 112 strains was correct. The correct rate of identification was 92.56% (112 / 121). BD PhoenixTM yeast identification board). One of the strains of Candida Portugal was not identified and one of the strains was wrong in the identification of Candida mongolicum in the library, and the results of the identification of yeast-like fungi in the library were as follows: (1) the identification rate was 92.56%. The identification error of one strain of Pichia pastoris outside the library was wrong, and the other 118 strains were correctly identified. The correct rate of identification in the library was 98.33% (118 鈮
本文編號：2475153
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