Nucleic acid detection technologies and marker molecules in bacterial diagnostics

Abstract

There is a growing need for quick and reliable methods for microorganism detection and identification worldwide. Although traditional culture-based technologies are trustworthy and accurate at a relatively low cost, they are also time- and labor-consuming and are limited to culturable bacteria. Those weaknesses have created a necessity for alternative technologies that are capable for faster and more precise bacterial identification from medical, food or environmental samples. The most common current approach is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to the specific nucleic acid profiles that are present. This review aims to give an up-to-date overview of different nucleic acid target sequences and respective analytical technologies.

Keywords::

• bacterial target molecules
• biosensors
• microarrays
• microbial diagnostics
• next-generation sequencing
• nucleic acid diagnostics

Financial & competing interests disclosure

O Scheler and A Kurg acknowledge support from SF0180027s10 grant from Estonian Ministry of Education and Research. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• Established methods (especially culture-based methods) have over a century of development and tradition behind them. Expecting an emerging technology to fully replace these methods in a short time frame is unrealistic whatever the demonstrated improvements in speed and precision.

• Nucleic acid diagnostics for microbial pathogens initially relied on PCR-based technologies. This established the gold standard in terms of sensitivity and specificity against which all subsequent technologies are measured.

• Alternative nucleic acid amplification technologies to PCR have made steady increases in their popularity as some of them have demonstrated sensitivity comparable to PCR. They have become popular in the development of point-of-care diagnostics systems due to simplified instrument requirements.

• Microarray-based methods are advantageous as they offer the possibility of highly parallel assays being included on a single array. Disadvantages include the requirement to include a pre-hybridization amplification step, a labeling step and typically a complex and expensive imaging system.

• Biosensor technologies have the primary advantage of detection of unlabeled nucleic acid targets in a multiplex assay format. Current systems compare poorly to amplification-based technologies in terms of sensitivity, but technology advances will overcome this difficulty.

• Combination of a brief isothermal amplification with a robust biosensor detection system may ultimately lead to the next wave of diagnostics platforms particularly for point-of-care and resource-limited scenarios.

• Close alignment with biomedical diagnostics and the electronics fabrication industry will see improvements in miniaturization of assay platforms as well as significant reductions in manufacture costs. The microring resonator biosensors described in this review are an initial example of this convergence.

• Specific nucleic acid target regions will remain central to nucleic acid diagnostics assays. Ribosomal RNA continues with great popularity, but on a smaller scale as other target regions are used more.

• The improvements in massively parallel next-generation sequencing technologies over the next 5–10 years will allow clinicians to routinely profile pathogenic organisms in extreme detail (including species, strain, pathogenicity and resistance profiles).