荧光定量PCR定量检测5种假丝酵母菌方法的建立

doi:10.3969/j.issn.1673-8640.2015.03.016

检验医学 ›› 2015, Vol. 30 ›› Issue (3): 265-268.DOI: 10.3969/j.issn.1673-8640.2015.03.016

• 技术研究与评价·论著 • 上一篇下一篇

荧光定量PCR定量检测5种假丝酵母菌方法的建立

夏乾峰¹, 傅琼瑶², 邬强², 覃西¹, 姚茂忠¹, 黄绵庆¹, 邢桂兰¹, 钱士匀¹

1.海南海医药物安全性评价研究有限责任公司,海南海口 570102
2.海南医学院热带医学与检验医学院,海南海口 570102

收稿日期:2014-10-20 出版日期:2015-03-30 发布日期:2015-04-16
作者简介:null
作者简介:夏乾峰,1979年生,博士,教授,从事临床微生物学检验教学和科研工作。
基金资助:
海南省自然科学基金项目(813187);海口市重点科技计划项目(2012-063)

Establishment of fluorescence quantitation PCR for the quantitative detection of five species of Candida

XIA Qianfeng¹, FU Qiongyao¹, WU Qiang¹, QIN Xi¹, YAO Maozhong¹, HUANG Mianqing¹, XING Guilan¹, QIAN Shiyun²

1.Research for Drug Safety Evaluation of Hainan Province, Hainan Haikou, China
2.The Faculty of Laboratory Medicine and Tropical Medicine, Hainan Medical College, Hainan Haikou 570102, China

Received:2014-10-20 Online:2015-03-30 Published:2015-04-16

摘要/Abstract

摘要： 目的

建立并评价从血液标本中检测5种假丝酵母菌的荧光定量聚合酶链反应(PCR)方法。

方法

以5种假丝酵母菌共有的高度保守序列为靶序列,设计特异引物和探针进行检测, 优化定量PCR体系,并进行方法学评价。

结果

建立的荧光定量PCR方法,其线性范围为10¹~10⁹ CFU/mL;灵敏度为5 CFU/mL;批内和批间CV值均<5%;对19份模拟血液标本进行检测,其中分别含有5种假丝酵母菌的标本均为阳性,而分别含有常见血液感染曲霉菌和细菌的检测结果均为阴性。

结论

建立的检测血中假丝酵母菌荧光定量PCR方法快速、准确,结果可靠,可对常见的临床血液感染假丝酵母菌进行定量检测。

关键词: 假丝酵母菌, 实时PCR, 定量

Abstract: Objective

To establish and evaluate fluorescence quantitation polymerase chain reaction (PCR) for the quantitative detection of 5 species of Candida.

Methods

The presence of DNA from pathogens in the Candida species was detected by the PCR targeting internal transcribed spacer region of fungal gene. The PCR was optimized and evaluated methodologically.

Results

The fluorescence quantitation PCR showed a wide range of linearity from 10¹-10⁹ CFU/mL, high sensitivity 5 CFU/mL, within-run and between-run coefficients of variation (CV) <5%. When the PCR was applied directly to identify 19 mocked double-blind blood samples, 5 of them were positive to 5 species of Candida, and the other 14 of them were negative to other pathogens.

Conclusions

The PCR may be appropriate for clinical laboratories as accurate, rapid and reliable determination for the most frequently encountered Candida species.

Key words: Candida, Fluorescence quantitation polymerase chain reaction, Quantitation

中图分类号:

Q503

夏乾峰, 傅琼瑶, 邬强, 覃西, 姚茂忠, 黄绵庆, 邢桂兰, 钱士匀. 荧光定量PCR定量检测5种假丝酵母菌方法的建立[J]. 检验医学, 2015, 30(3): 265-268.

XIA Qianfeng, FU Qiongyao, WU Qiang, QIN Xi, YAO Maozhong, HUANG Mianqing, XING Guilan, QIAN Shiyun. Establishment of fluorescence quantitation PCR for the quantitative detection of five species of Candida[J]. Laboratory Medicine, 2015, 30(3): 265-268.

图/表 1

参考文献 11

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荧光定量PCR定量检测5种假丝酵母菌方法的建立

Establishment of fluorescence quantitation PCR for the quantitative detection of five species of Candida

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