title: Design and use of mouse control DNA for DNA biomarker extraction and PCR detection from urine: Application for transrenal Mycobacterium tuberculosis DNA detection.
abstract: Urine samples are increasingly used for diagnosing infections including Escherichia coli, Ebola virus, and Zika virus. However, extraction and concentration of nucleic acid biomarkers from urine is necessary for many molecular detection strategies such as polymerase chain reaction (PCR). Since urine samples typically have large volumes with dilute biomarker concentrations making them prone to false negatives, another impediment for urine-based diagnostics is the establishment of appropriate controls particularly to rule out false negatives. In this study, a mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA target was added to retrospectively collected urine samples from tuberculosis (TB)-infected and TB-uninfected patients to indicate extraction of intact DNA and removal of PCR inhibitors from urine samples. We tested this design on surrogate urine samples, retrospective 1milliliter (mL) urine samples from patients in Lima, Peru and retrospective 5mL urine samples from patients in Cape Town, South Africa. Extraction/PCR control DNA was detectable in 97% of clinical samples with no statistically significant differences among groups. Despite the inclusion of this control, there was no difference in the amount of TB IS6110 Tr-DNA detected between TB-infected and TB-uninfected groups except for samples from known HIV-infected patients. We found an increase in TB IS6110 Tr-DNA between TB/HIV co-infected patients compared to TB-uninfected/HIV-infected patients (N=18, p=0.037). The inclusion of an extraction/PCR control DNA to indicate successful DNA extraction and removal of PCR inhibitors should be easily adaptable as a sample preparation control for other acellular sample types.
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA.
authors.forename: Hali
authors.initials: H
authors.lastname: Bordelon
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN, USA.
authors.forename: Keersten M
authors.initials: KM
authors.lastname: Ricks
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA.
authors.forename: Megan E
authors.initials: ME
authors.lastname: Pask
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA.
authors.forename: Patricia K
authors.initials: PK
authors.lastname: Russ
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA.
authors.forename: Francesca
authors.initials: F
authors.lastname: Solinas
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA.
authors.forename: Mark L
authors.initials: ML
authors.lastname: Baglia
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Short
authors.affiliation: Institute of Infectious Disease and Molecular Medicine, Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa.
authors.forename: Andrew
authors.initials: A
authors.lastname: Nel
authors.affiliation: Institute of Infectious Disease and Molecular Medicine, Department of Integrative Biomedical Sciences, University of Cape Town, Cape Town, South Africa; Tuberculosis Center, Department of Medicine, Division of Infectious Diseases and Institute for Global Health, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Blackburn
authors.affiliation: Lung Infection and Immunity Unit, University of Cape Town, Cape Town, South Africa.
authors.forename: Keertan
authors.initials: K
authors.lastname: Dheda
authors.affiliation: de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Carlos
authors.initials: C
authors.lastname: Zamudio
authors.affiliation: de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Cáceres
authors.affiliation: Department of Chemistry, Vanderbilt University, Nashville, TN, USA; Tuberculosis Center, Department of Medicine, Division of Infectious Diseases and Institute for Global Health, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Wright
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA; Department of Chemistry, Vanderbilt University, Nashville, TN, USA; Tuberculosis Center, Department of Medicine, Division of Infectious Diseases and Institute for Global Health, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Haselton
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA; Tuberculosis Center, Department of Medicine, Division of Infectious Diseases and Institute for Global Health, Vanderbilt University School of Medicine, Nashville, TN, USA. Electronic address: april.pettit@vanderbilt.edu.
authors.forename: April C
authors.initials: AC
authors.lastname: Pettit
mesh_terms: D000818:Animals; D001483:Base Sequence; D060085:Coinfection; D004247:DNA / Q000302:isolation & purification*; D018390:Gene Targeting / Q000379:methods; D005819:Genetic Markers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D015658:HIV Infections / Q000150:complications; D006801:Humans; D051379:Mice / Q000235:genetics*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D013019:South Africa; D014376:Tuberculosis / Q000150:complications / Q000175:diagnosis / Q000382:microbiology / Q000652:urine*; D014556:Urine / Q000382:microbiology*
chemical_list: D005819:Genetic Markers; D010446:Peptide Fragments; C068142:glyceraldehyde 3-phosphate dehydrogenase (304-313); D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases