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Genetic mimetics of Mycobacterium tuberculosis and methicillin resistant Staphylococcus aureus as verification standards for molecular diagnostics.

Genetic mimetics of Mycobacterium tuberculosis and methicillin resistant Staphylococcus aureus as verification standards for molecular diagnostics.
Author Information (click to view)

Machowski EE, Kana BD,


Machowski EE, Kana BD, (click to view)

Machowski EE, Kana BD,

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Journal of clinical microbiology 2017 09 20() pii 10.1128/JCM.01111-17

Abstract

Molecular diagnostics have revolutionized the management of health care through enhanced detection of disease or infection and effective engagement into care. In recognition of this, the World Health Organization approved the rollout out of nucleic acid amplification technologies for identification of Mycobacterium tuberculosis using platforms such as GeneXpert MTB/RIF, the GenoType MTBDRplus Line Probe Assay and more recently, GeneXpert MTB/RIF Ultra. These assays can simultaneously detect tuberculosis infection and assess rifampicin 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 Mycobacterium smegmatis that mimic the profile of M. tuberculosis on both the GeneXpert MTB/RIF and the MTBDRplus Line Probe diagnostic tests. Using site-specific gene editing, we also created derivatives that faithfully mimic the diagnostic result of rifampicin resistant M. tuberculosis, with mutations at positions 513, 516, 526, 531, or 533 in the rifampicin resistance determining region of the rpoB gene. Next, we extended this approach to other diseases and demonstrated that a Staphylococcus aureus gene sequence can be introduced into M. smegmatis to generate a positive response for the SCCmec probe in the GeneXpert® SA Nasal Complete molecular diagnostic cartridge, designed for identification of methicillin resistant S. aureus 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.

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