Design and use of mouse control DNA for DNA biomarker extraction and PCR detection from urine: Application for transrenal Mycobacterium tuberculosis DNA detection
| Autor: | April C. Pettit, Andrew J.M. Nel, David W. Wright, Carlos Zamudio, Frederick R. Haselton, Mark L. Baglia, Megan E. Pask, Keersten M. Ricks, Keertan Dheda, Hali Bordelon, Francesca Solinas, Patricia K. Russ, Tatiana Cáceres, Philip A. Short, Jonathan Michael Blackburn |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Urine/microbiology Polymerase Chain Reaction/methods HIV Infections Urine Polymerase Chain Reaction law.invention Mice chemistry.chemical_compound South Africa law Polymerase chain reaction Tuberculosis/complications/diagnosis/microbiology/urine Coinfection Glyceraldehyde-3-Phosphate Dehydrogenases Molecular Diagnostic Techniques Gene Targeting Microbiology (medical) Genetic Markers Tuberculosis 030106 microbiology HIV Infections/complications Gene Targeting/methods Mice/genetics Biology Mycobacterium tuberculosis/genetics/isolation & purification Microbiology Sensitivity and Specificity Article 03 medical and health sciences Tuberculosis diagnosis medicine Humans Animals Glyceraldehyde-3-Phosphate Dehydrogenases/genetics Peptide Fragments/genetics purl.org/pe-repo/ocde/ford#1.06.01 [https] Molecular Biology Retrospective Studies Base Sequence Molecular Diagnostic Techniques/methods DNA Mycobacterium tuberculosis DNA/isolation & purification medicine.disease DNA extraction Virology Molecular biology Peptide Fragments 030104 developmental biology chemistry Genetic marker Nucleic acid |
| Popis: | 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 1 milliliter (mL) urine samples from patients in Lima, Peru and retrospective 5 mL 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 a 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. |
| Databáze: | OpenAIRE |
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