Genetic Mimetics of Mycobacterium tuberculosis and Methicillin-Resistant Staphylococcus aureus as Verification Standards for Molecular Diagnostics.

Molecular diagnostics have revolutionized the management of health care through enhanced detection of disease or infection and effective enrollment into treatment. In recognition of this, the World Health Organization approved the rollout of nucleic acid amplification technologies for identification of using platforms such as GeneXpert MTB/RIF, the GenoType MTBDR line probe assay, and, more recently, GeneXpert MTB/RIF Ultra. These assays can simultaneously detect tuberculosis infection and assess rifampin resistance. However, their widespread use in health systems requires verification and quality assurance programs. To enable development of these, we report the construction of genetically modified strains of that mimic the profile of on both the GeneXpert MTB/RIF and the MTBDR line probe diagnostic tests. Using site-specific gene editing, we also created derivatives that faithfully mimic the diagnostic result of rifampin-resistant , with mutations at positions 513, 516, 526, 531, and 533 in the rifampin resistance-determining region of the gene. Next, we extended this approach to other diseases and demonstrated that a gene sequence can be introduced into to generate a positive response for the SCC probe in the GeneXpert SA Nasal Complete molecular diagnostic cartridge, designed for identification of methicillin-resistant These biomimetic strains are cost-effective, have low biohazard content, accurately mimic drug resistance, and can be produced with relative ease, thus illustrating their potential for widespread use as verification standards for diagnosis of a variety of diseases.