Identification of Bacteria by Polymerase Chain Reaction Followed by Liquid Chromatography-Mass Spectrometry

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• 作者：Bettina M. Mayr ; Uwe Kobold ; Martin Moczko ; Agnes Nyeki ; Thomas Koch ; and Christian G. Huber
• 刊名：Analytical Chemistry
• 出版年：2005
• 出版时间：July 15, 2005
• 年：2005
• 卷：77
• 期：14
• 页码：4563 - 4570
• 全文大小：150K
• 年卷期：v.77,no.14(July 15, 2005)
• ISSN：1520-6882

文摘

Bloodstream infections are an important cause of seriousmorbidity and mortality. Rapid detection and identification of specific pathogens from blood or other clinicalspecimens could improve the rational use of antimicrobialtherapy in clinical medicine and have a great impact onthe outcome of patients with systemic infections. Polymerase chain reaction using generic primers was used toamplify genomic DNA of different bacterial strains. Theidentification was accomplished by measuring the molecular masses of the PCR products using ion-pair reversed-phase high-performance liquid chromatography hyphenated to electrospray ionization mass spectrometry. DNAfrom 10 bacterial species was amplified by PCR, and theresulting amplification products were analyzed. In allcases, the measured molecular masses of the PCR products matched the theoretical value for the species-specificDNA sequence. However, three pairs of bacteria could notbe distinguished since the theoretical difference in amplicon molecular mass was <1.0 Da (different sequence,same base composition of amplicon). Determination ofintra- and interday mass reproducibility resulted in relative standard deviations of 0.0030 and 0.018%, respectively. The limit of detection of the presented method wasshown to be 0.5 genome equivalents/PCR. The specificityof the method in a human background was successfullytested by amplifying and analyzing 1000-10000 genomeequivalents of Staphylococcus aureus spiked into human plasma.