Quantitative TaqMan PCR for detection of Pneumocystis jiroveci

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• 作者：Brancart ; Fran??oise ; Rodriguez-Villalobos ; Hector ; Fonteyne ; Pierre-Alain ; Peres-Bota ; Daliana ; et. al.
• 刊名：Journal of Microbiological Methods
• 出版年：2005
• 出版时间：June, 2005
• 年：2005
• 卷：61
• 期：3
• 页码：381-387
• 全文大小：108 K

文摘

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46 % –4.27 % and 0.05–2.00 % over 5 log₁₀ values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45 % ) BAL found PCR positive, 8 were positive by microscopy (35 % ). The copy numbers of the target gene were between 4.4×10³ and 2.8×10⁶ per ml for the microscopically positive samples and between 8 and 9.2×10³ per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.