On the spot: Identifying common bacterial pathogens within hours
By: Jayanth Jawahar
Edited By: Madeline Nicol, Katherine Hill and Sienna Schaeffer
A new proof of concept device uses fluorescence to accurately detect genomic sequences unique to common pathogenic bacteria, and requires minimal training to use. If commercialized, this device could be used to quickly detect pathogenic bacteria in different settings.
Rapidly improving genome sequencing technologies are allowing us to understand human disease, aging, and evolution better than ever. However, these genetic technologies are currently only available to highly trained researchers, and are often expensive and time-consuming. In an attempt to apply modern genetic methods to patient-care settings (i.e. hospitals), Dr. Lars Renner and his colleagues at the University of Wisconsin-Madison recently developed a device nicknamed the “B-chip”.
The B-chip (short for bacterial chip) can be used to rapidly identify bacterial pathogens from patient samples, allowing doctors to modify antibiotic therapy accordingly. The chip is a hollow, transparent device that collects bacterial DNA and distributes it to 16 mini chambers (Fig. 1). Each of these 16 chambers contains a micro-sized version of a gene-amplifying reaction called a recombinase polymerase amplification (RPA) reaction. The RPA reaction uses short DNA sequences called ‘primers’ to guide an enzyme called recombinase to a specific target DNA sequence, and then amplify this sequence many times to make even the smallest amount detectable.
Different primers target different DNA DNA sequences, so each of the 16 chambers in the B-chip can be designed to amplify DNA from different bacteria. The different DNA samples allow clinicians to identify which type of bacteria is causing an infection. This amplification step allows the reaction to detect even the smallest amount of target DNA dispensed into the B-chip, which makes the B-chip a highly sensitive and accurate method to detect bacteria.
So how is this DNA amplification reaction visualized by users of the B-chip? The RPA reaction also contains “probes” which fluoresce when a DNA amplification product is detected. When observed on the B-chip, this fluorescence appears as a bright spot in each of the reaction chambers. The fluorescence can then be captured by a camera, and minute differences in fluorescence can be easily compared using software (Fig. 2).
The B-chip is an affordable, reliable, and an easy-to-use method to detect bacterial DNA for many reasons. First, it eliminates the need to manipulate several different chemical reagents and enzymes by pre-packaging them into its small chambers. Users of the B-chip would simply need to transfer a DNA extract to its reservoir, from where it is drawn into the chambers. Second, it can be read using a relatively simple setup consisting of a camera and computer software.
Finally, the RPA reaction used to amplify DNA is highly sensitive (requires ~10 target gene copies to produce detectable fluorescence) and works at 42 oC, allowing the reaction to be performed without expensive lab instruments. The DNA primers used to target bacterial DNA sequences can also be fine-tuned using freely available genetic libraries so that they only amplify target sequences unique to the bacteria of interest. This would help prevent false positive results arising from the primers binding to similar sequences in other organisms.
One problem not addressed by the B-chip is the extraction of DNA from patient samples. In this paper, the B-chip was loaded with DNA purified using DNA extraction kits, which are not cheap enough to be used on a day-to-day basis in healthcare settings. Thus, the team’s next goal is to develop a low-cost system for DNA extraction from a variety of samples such as blood and urine.
If commercialized, the B-chip could have a variety of applications in healthcare, agriculture, and sewage treatment, to name a few. The B-chip could be especially useful in helping doctors determine antibiotic regimens for patients, since different bacterial pathogens are susceptible to different kinds of treatment. It would also help doctors avoid prescribing antibiotics for viral infections, against which antibiotics are useless.
The current approach to antibiotic use is a trial-and-error method, which is risky and leads to the spread of antibiotic-resistant pathogens. Every time an antibiotic is used, it increases the chances of a resistant pathogen in the environment being selected over the others (which are killed). This resistant pathogen can then spread and, in some cases, transfer antibiotic resistance genes to other organisms, compounding the problem. The spread of antibiotic resistance renders lifesaving antibiotics useless. Since the development of new antibiotics can cost millions of dollars and take up to five years, it makes sense to use existing antibiotics wisely. Having a quick, easy-to-use device on hand can help us do just that.
References/ Further reading
Article: Renner, L. D. et al. Detection of ESKAPE bacterial pathogens at the point-of-care using isothermal DNA-based assays in a portable, de-gas microfluidic diagnostic assay platform. Appl. Environ. Microbiol. AEM.02449–16 (2016). doi:10.1128/AEM.02449–16
Recombinase polymerase amplification: Piepenburg, O., Williams, C. H., Stemple, D. L. & Armes, N. A. DNA Detection Using Recombination Proteins. PLOS Biology 4, e204 (2006).