Diagnostic Test Kit Offers Easy Identification of Virulent Pathogens

A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients.

A portable detection system has been developed that can rapidly identify some of the most virulent, often multi-drug resistant pathogens. This device was designed to be used in places where laboratory resources are lacking, such as isolated villages in developing countries.

An international team of scientists led by those from the University of Wisconsin-Madison developed a new battery powered detection system which is small and simple. Sixteen microchambers each contain a genetic sequence from one of the sixteen pathogens the device is designed to detect. When one of these sequences matches a sequence from a patient sample, a fluorescent light illuminates from within the relevant microchamber.

The investigators picked the sequences the device uses by screening the genomes of the relevant bacteria for unique genetic sequences. Pathogens can be identified using single drops of patient samples from blood, urine, or other bodily fluids. The system can identify Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and various species of the genus, Enterobacter.

To perform Recombinase Polymerase Amplification (RPA) assays in the B-chip, the team added the isothermal RPA mix including species specific primers, fluorescent exoprobe, and buffer to the TwistAmp exo kit lyophilized enzyme pellet using the TwistDX protocol. The investigators used microplate assays to assess the performance of RPA assays for detecting E. faecium, S. aureus, K. pneumoniae, A. baumannii, and P. aeruginosa. This very sensitive assay can provide positive identification with patient samples containing as few as 10 DNA sequences. They designed a portable B-chip detection system for measuring assay fluorescence containing an excitation source, camera, incubation heater, fluorescence filters and software to control the individual components.

The authors concluded that the combination of a simple and portable cartridge-based RPA assays and a versatile electronic reader that can be used to perform assays in the field. Using de-gas driven cartridges provides a unique platform for packaging RPA assays that can simplify their use and potentially reduce contamination by packaging and compartmentalization. The study was published on December 16, 2016, in the journal Applied and Environmental Microbiology.