Multiplex Identification of Microbes

We have adapted molecular inversion probe technology to identify microbes in a highly multiplexed procedure. This procedure does not require growth of the microbes. Rather, the technology employs DNA homology twice: once for the molecular probe to hybridize to its homologous DNA and again for the 20-mer oligonucleotide barcode on the molecular probe to hybridize to a commercially available molecular barcode array. As proof of concept, we have designed, tested, and employed 192 molecular probes for 40 microbes. While these particular molecular probes are aimed at our interest in the microbes in the human vagina, this molecular probe method could be employed to identify the microbes in any ecological niche.The substantial majority of the earth''s bacteria have not been grown in culture and do not form colonies on agar plates (for examples, see references 19 and 21). These statements are particularly true of the bacteria living in or on human beings (for examples, see references 2, 6, and 7). The Human Microbiome Project (for examples, see references 2, 5, 24, and 27) is employing DNA sequencing and other genome-based technologies to reveal the plethora of microbes living in or on humans. Our goal was to develop a massively multiplex method employing currently commercially available reagents to identify those microbes at the species level.Several useful approaches to the identification of microbes that do not form colonies on agar plates have been published. Many scientists have employed dideoxy sequencing of the PCR-amplified 16S rRNA gene to identify microbes (for an example, see reference 22). Dideoxy sequencing is expensive, cumbersome, and slow. “Next-generation” sequencing reduces the cost of sequencing but produces much shorter read lengths. As examples, Tarnberg et al. (26), Jonasson et al. (11), and Sundquist et al. (25) employed pyrosequencing of small portions of the 16S rRNA gene to identify microbes. These scientists could not always identify the microbes down to the species level. “Checkerboard DNA-DNA hybridization” (CDH) is a technology that is more than a decade old (23). Nikolaitchouk et al. (14) have applied CDH to identify the microbes in the human female genital tract and achieved a 13-plex reaction. Dumonceaux et al. (4) coupled microbe-specific oligonucleotides to fluorescently labeled microspheres. Subsequently, the microbes were detected and counted by flow cytometry. Dumonceaux et al. (4) achieved a 9-plex reaction. DeSantis et al. (3) designed and employed a microarray containing 297,851 oligonucleotide probes derived from the 16S rRNA gene from 842 subfamilies of prokaryotes. DeSantis et al. (3) concluded that their microarray revealed greater prokaryotic diversity than dideoxy sequencing of a “typically sized clone library.”None of these ingenious methods meets the requirements for a robust, commercially available, highly multiplex technology. Therefore, we have adapted an independent method to identify microbes: molecular inversion probes (8) coupled with a commercial molecular barcode array. This method does not require growth of the microbes. Rather, molecular probe technology is a nucleic acid-based technology employing DNA homology twice: once for the molecular probe to hybridize to its homologous DNA and again for the 20-mer oligonucleotide barcode on the molecular probe to hybridize to a commercially available oligonucleotide barcode array. We present here data demonstrating proof of concept in which molecular probes were designed, tested, and employed to detect microbes in simulated clinical samples. Because of our ongoing interest in the bacteria that inhabit the adult human vagina (10), we focus on that ecological niche. However, this method is sufficiently general that it can be applied to detect the microbes in any ecological niche, e.g., soil and the ocean.