Targeted and Highly Multiplexed Detection of Microorganisms by Employing an Ensemble of Molecular Probes

Abstract

The vast majority of microscopic life on earth consists of microbes that do not grow in laboratory culture. To profile the microbial diversity in environmental and clinical samples, we have devised and employed molecular probe technology, which detects and identifies bacteria that do and do not grow in culture. The only requirement is a short sequence of contiguous bases (currently 60 bases) unique to the genome of the organism of interest. The procedure is relatively fast, inexpensive, customizable, robust, and culture independent and uses commercially available reagents and instruments. In this communication, we report improving the specificity of the molecular probes substantially and increasing the complexity of the molecular probe set by over an order of magnitude (>1,200 probes) and introduce a new final readout method based upon Illumina sequencing. In addition, we employed molecular probes to identify the bacteria from vaginal swabs and demonstrate how a deliberate selection of molecular probes can identify less abundant bacteria even in the presence of much more abundant species. It is now widely appreciated that only a small fraction of the earth's bacteria may be grown in laboratory culture (for example, see reference 1). That statement is particularly true for the bacteria found in and on human beings (2). The Human Microbiome Project employs a metagenomic approach, principally amplifying and sequencing a small portion of the 16S rRNA gene, to identify bacteria. Like any technology, sequencing rRNA genes has strengths and weaknesses. Its principal strengths are that it overcomes the serious limitations of bacterial unculturability and genomic diversity. Its principal weaknesses are that different bacteria have different copy numbers of rRNA genes (3)(4)(5), that the application of rRNA gene "universal" primers has specificity performance limitations for some bacterial species (6-8), that chimeras are formed at a relatively high frequency when rRNA genes are amplified and cloned (for example, see reference 9), and that the presence of a highly abundant bacterium can mask the detection of less abundant species. Microarrays containing probes that hybridize to rRNA genes sequences are also effective metagenomic tools for detecting unculturable bacteria (10), but, as mentioned above, they can be limited in their ability to discern between some species.To overcome these weaknesses, we developed a genome-based strategy that targets single-copy bacterial genome sequences with high specificity and sensitivity. Originally referred to as molecular inversion probes (11), the technology was simplified for highthroughput analysis, and thus, the name was revised simply to "molecular probes." This approach is rapid, scalable, robust, and culture independent (12, 13). This assay requires only a short stretch of contiguous DNA sequence unique to the bacterial genome of interest. That means that probe technology cannot detect novel microbes, i.e., microbes for which genome sequence information is not yet available. In ...

A method for high‐throughput production of sequence‐verified DNA libraries and strain collections

“…As a proof-of-concept, we applied REDI to a library of 7,051 DNA oligonucleotide probes for detection of 354 bacteria related to potable water quality (Table EV1). We designed multiple unique molecular probes (typically 20) for each bacterium, essentially as previously described (Xu et al, 2014). Each contained 60 nucleotides (nt) of homology to the target genome, an 8-nt "random" barcode, and 37 nt of internal priming sequence for amplification of probes that circularize upon successful hybridization to their targets (Fig 2A).…”

“…All of these probes could easily be omitted in future reactions, an advantage of the REDI method over employing arraysynthesized probes directly. Similarly, probes directed against bacteria found to be highly abundant in a sample can be removed from the library, enabling detection of organisms present at lower abundance (Xu et al, 2014).…”

Bacteria-Targeting Conjugates Based on Antimicrobial Peptide for Bacteria Diagnosis and Therapy

“…To investigate the dynamics of ICG02-UBI 29−41 (n = 5) in normal mice, 26 ICG02-UBI 29−41 (0.2 mL, 2 nmol) and ICG02 (as control) were injected respectively into the healthy ICR mice (n = 5) via tail vein and then the mice were monitored by NIR fluorescence imaging system over 48 h. The background images were taken for each mouse prior to the injection. The NIR fluorescent images were acquired at scheduled time points (1,4,6,12,24, and 48 h). To confirm the in vivo observations, the mice were sacrificed at predetermined time intervals (1, 2, 4, 6, 12, 24, and 48 h).…”

“…7,8 Hence, many researchers have switched to the direction of designing bacteria-targeting probes with tissue-selective ligands, e.g., antibodies, lectin, sugar, etc., 9,10 to improve the bacterial diagnosis. 11,12 A more effective approach to achieve broad bacteria-targeting has been utilized in nanoparticle surface modifications, ligand−acceptor interactions, and so on. 13−15 For example, cationic molecules on the surface of nanoparticles can be electrostatically attracted by the negatively charged bacteria, which are attributed to the high fraction of anionic phospholipids and amphiphile molecules.…”

“…Therefore, a rapid and accurate identification method for bacterial infections is critical for authentic anti-infective therapy. Traditionally, only infections that have become systemic or have caused significant anatomical tissue damages could be diagnosed using several inconvenient and time-consuming methods including laboratory culture tests, nuclear medicine imaging, and computed tomography (CT). , Hence, many researchers have switched to the direction of designing bacteria-targeting probes with tissue-selective ligands, e.g., antibodies, lectin, sugar, etc., , to improve the bacterial diagnosis. , A more effective approach to achieve broad bacteria-targeting has been utilized in nanoparticle surface modifications, ligand–acceptor interactions, and so on. − For example, cationic molecules on the surface of nanoparticles can be electrostatically attracted by the negatively charged bacteria, which are attributed to the high fraction of anionic phospholipids and amphiphile molecules. , …”