菌落PCR改良技术快速鉴定阿萨希毛孢子菌的实验研究

中国真菌学杂志　 2015, Vol. 10 　Issue (1): 6-10.

菌落PCR改良技术快速鉴定阿萨希毛孢子菌的实验研究

张德全^1,2, 刘涵¹, 廖勇¹, 夏志宽¹, 杨冬倩¹, 杨阳¹, 吕雪莲¹, 杨蓉娅¹

1. 北京军区总医院全军皮肤损伤修复研究所, 北京 100700;
2. 总政治部机关医院, 北京 100120

收稿日期:2014-11-04 出版日期:2015-02-28 发布日期:2015-02-28
通讯作者: 杨蓉娅,E-mail:yangrya@sina.com;吕雪莲,E-mail:mycoses@gmail.com E-mail:yangrya@sina.com;mycoses@gmail.com
作者简介:张德全,男(汉族),硕士研究生在读,主治医师.E-mail:mycologist@qq.com
基金资助:
国家自然科学基金(81471928,81271764,81301410)

Study of improved colony PCR technologies for the identification of Trichosporon asahii

ZHANG De-quan^1,2, LIU Han¹, LIAO Yong¹, XIA Zhi-kuan¹, YANG Dong-qian¹, YANG Yang¹, LV Xue-lian¹, YANG Rong-ya¹

1. The Military Institute of Injury and Reparation, General Hospital of Beijing Military Command of PLA, Beijing 100700, China;
2. Hospital of General Political Department of PLA, Beijing 100120, China

Received:2014-11-04 Online:2015-02-28 Published:2015-02-28

摘要/Abstract

摘要： 目的探索快速鉴定阿萨希毛孢子菌(Trichosporon asahii,T.asahii)的简便方法.方法分别应用直接法、直接离心法、酶解法、酶解改良法4种方法进行样本预处理后的菌落PCR技术检测16株T.asahii,评价上述4种方法的敏感性和微量样本阳性率,同时与DNA常规提取法PCR结果进行比较.结果酶解改良法、直接法、常规提取法敏感性均为10² CFU/mL,阳性率分别为93.75%、75%、50%;直接离心法敏感性为10⁴ CFU/mL,阳性率为43.75%;酶解法结果为阴性.结论酶解改良法是一种简便快速的PCR模板获取方法,适用于菌落PCR技术对T.asahii进行快速鉴定时的样本预处理.

关键词: 菌落PCR, 阿萨希毛孢子菌, DNA初提

Abstract: Objective To explore a direct colony PCR method for rapid identification of T.asahii.Methods Four methods including direct colony,direct colony with centrifugation,enzymolysis,and improved enzymolysis method were used to preliminarily sample treatment.The sensitivity and the positive rate of colony PCR based on four methods for isolates of T.asahii were evaluated and compared with the PCR results of the traditional DNA extraction.Results The sensitivity of the improved enzymolysis method,direct colony method and the traditional DNA extraction were both 10² CFU/mL,while the positive rates were 93.75%,75% and 50%,respectively.The direct colony with centrifugation presented the sensitivity of 10⁴ CFU/mL and the positive rate of 43.75%.The enzymolysis method presented total negative results in the detection of both sensitivity and positive rate.Conclusion The improved enzymolysis method which presented the advantages of high sensitivity and positive rate of trace samples,could be applied for the rapid identification of T.asahii.

Key words: Colony PCR, Trichosporon asahii, DNA preliminary extraction

中图分类号:

R379.9

张德全, 刘涵, 廖勇, 夏志宽, 杨冬倩, 杨阳, 吕雪莲, 杨蓉娅. 菌落PCR改良技术快速鉴定阿萨希毛孢子菌的实验研究[J]. 中国真菌学杂志, 2015, 10(1): 6-10.

ZHANG De-quan, LIU Han, LIAO Yong, XIA Zhi-kuan, YANG Dong-qian, YANG Yang, LV Xue-lian, YANG Rong-ya. Study of improved colony PCR technologies for the identification of Trichosporon asahii[J]. Chinese Journal of Mycology, 2015, 10(1): 6-10.

参考文献

[1] Baka S,Tsouma I,Kouskouni E.Fatal lower limb infection by Trichosporon asahii in an immunocompetent patient[J].Acta Dermatovenerol Croat,2013,21(4):241-244.
[2] Rubic Z,Novak A,Tomic Z,et al.Prompt diagnosis and effective treatment of Trichosporon asahii catheter-related infection in non-immunocompromised neurosurgical patient[J].Mycopathologia,2014,Sep 24.
[3] Colombo AL,Padovan AC,Chaves GM.Current knowledge of Trichosporon spp.and trichosporonosis[J].Clin Microbiol Rev,2011,24(4):682-700.
[4] Meshkat Z,Soleimanjahi H,Mahmoudi M,et al.Determination of human papillomavirus type 16 genotype and construction of cloning vector pTZ57R encoding HPV16 E7 gene[J].Saudi Med J,2006,28(10):1511-1515.
[5] 夏志宽,王文岭,杨蓉娅.国内临床分离阿萨希毛孢子菌3个新基因型分析[J].中华医院感染学杂志,2012,22(14):2988-2990.
[6] Makimura K,Murayama SY,Yamaguchi H.Detection of a wide range of medically important fungi by the polymerase chain reaction[J].J Med Microbiol,1994,40(5):358-364.
[7] 杨蓉娅,敖俊红,王文岭,等.阿萨希丝孢酵母引起播散性毛孢子菌病国内首例报告[J].中华皮肤科杂志,2001,34(5):329-332.
[8] 李新红,冯新国,欧阳淑兰.阿萨希毛孢子菌感染两例[J].中华医院感染学杂志,2012,22(19):2012.
[9] 郑晓丽,王志东,闫洪敏,等.异基因造血干细胞移植过程中并发播散性阿萨希毛孢子菌感染1例并文献复习[J].临床血液学杂志,2013,26(5):312-314.
[10] Gussow D,Clackson T.Direct clone characterization from plaques and colonies by the polymerase chain reaction[J].Nucleic Acids Res,1989,17(10):4000.
[11] Bergkessel M,Guthrie C.Colony PCR[J].Methods Enzymol,2013,529:299-309.
[12] 潘峰,周东风,肖晶,等.一步法PCR联合荧光原位杂交检测曲霉菌属感染标本[J].中华医院感染学杂志,2012,22(10):2223-2226.
[13] 陈吉良,黄小龙,吴安迪,等.一种快速高效提取病原真菌DNA作为PCR模板的方法[J].菌物学报,2011,30(1):147-149.
[14] Ezeronye OU,Okerentugba PO.Optimum conditions for yeast protoplast release and regeneration in Saccharomyces cerevisiae and Candida tropicalis using gut enzymes of the giant African snail Achatina achatina[J].Lett Appl Microbiol,2001,32(3):190-193.
[15] Alshahni MM,Makimura K,Yamada T,et al.Direct colony PCR of several medically important fungi using Ampdirect plus[J].Jpn J Infect Dis,2009,62(2):164-167.
[16] Petes TD,Botstein D.Simple Mendelian inheritance of the reiterated ribosomal DNA of yeast[J].Proc Natl Acad Sci USA,1977,74(11):5091-5095.