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Article ; Online: Correction to "Low-Resource Nucleic Acid Extraction Method Enabled by High-Gradient Magnetic Separation".

Pearlman, Stephanie I / Leelawong, Mindy / Richardson, Kelly A / Adams, Nicholas M / Russ, Patricia K / Pask, Megan E / Wolfe, Anna E / Wessely, Cassandra / Haselton, Frederick R

ACS applied materials & interfaces

2023 Volume 16, Issue 1, Page(s) 1953

Language	English
Publishing date	2023-12-21
Publishing country	United States
Document type	Published Erratum
ISSN	1944-8252
ISSN (online)	1944-8252
DOI	10.1021/acsami.3c18419
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Low-Resource Nucleic Acid Extraction Method Enabled by High-Gradient Magnetic Separation

Pearlman, Stephanie I / Leelawong, Mindy / Richardson, Kelly A / Adams, Nicholas M / Russ, Patricia K / Pask, Megan E / Wolfe, Anna E / Wessely, Cassandra / Haselton, Frederick R

ACS applied materials & interfaces. 2020 Feb. 10, v. 12, no. 11

2020

Abstract: Nucleic acid-based diagnostic tests often require isolation and concentration of nucleic acids from biological samples. Commercial purification kits are difficult to use in low-resource settings because of their cost and insufficient laboratory ... ...

Abstract	Nucleic acid-based diagnostic tests often require isolation and concentration of nucleic acids from biological samples. Commercial purification kits are difficult to use in low-resource settings because of their cost and insufficient laboratory infrastructure. Several recent approaches based on the use of magnetic beads offer a potential solution but remain limited to small volume samples. We have developed a simple and low-cost nucleic acid extraction method suitable for isolation and concentration of nucleic acids from small or large sample volumes. The method uses magnetic beads, a transfer pipette, steel wool, and an external magnet to implement high-gradient magnetic separation (HGMS) to retain nucleic acid-magnetic bead complexes within the device’s steel wool matrix for subsequent processing steps. We demonstrate the method’s utility by extracting tuberculosis DNA from both sputum and urine, two typical large volume sample matrices (5–200 mL), using guanidine-based extraction chemistry. Our HGMS-enabled extraction method is statistically indistinguishable from commercial extraction kits when detecting a spiked 123-base DNA sequence. For our HGMS-enabled extraction method, we obtained extraction efficiencies for sputum and urine of approximately 10 and 90%, whereas commercial kits obtained 10–17 and 70–96%, respectively. We also used this method previously in a blinded sample preparation comparison study published by Beall et al., 2019. Our manual extraction method is insensitive to high flow rates and sample viscosity, with capture of ∼100% for flow rates up to 45 mL/min and viscosities up to 55 cP, possibly making it suitable for a wide variety of sample volumes and types and point-of-care users. This HGMS-enabled extraction method provides a robust instrument-free method for magnetic bead-based nucleic acid extraction, potentially suitable for field implementation of nucleic acid testing.
Keywords	DNA ; analytical kits ; chemistry ; diagnostic techniques ; infrastructure ; magnetic materials ; magnetic separation ; magnetism ; nucleotide sequences ; point-of-care systems ; steel wool ; tuberculosis ; urine ; viscosity
Language	English
Dates of publication	2020-0210
Size	p. 12457-12467.
Publishing place	American Chemical Society
Document type	Article
ISSN	1944-8252
DOI	10.1021/acsami.9b21564
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Low-Resource Nucleic Acid Extraction Method Enabled by High-Gradient Magnetic Separation.

Pearlman, Stephanie I / Leelawong, Mindy / Richardson, Kelly A / Adams, Nicholas M / Russ, Patricia K / Pask, Megan E / Wolfe, Anna E / Wessely, Cassandra / Haselton, Frederick R

ACS applied materials & interfaces

2020 Volume 12, Issue 11, Page(s) 12457–12467

Abstract	Nucleic acid-based diagnostic tests often require isolation and concentration of nucleic acids from biological samples. Commercial purification kits are difficult to use in low-resource settings because of their cost and insufficient laboratory infrastructure. Several recent approaches based on the use of magnetic beads offer a potential solution but remain limited to small volume samples. We have developed a simple and low-cost nucleic acid extraction method suitable for isolation and concentration of nucleic acids from small or large sample volumes. The method uses magnetic beads, a transfer pipette, steel wool, and an external magnet to implement high-gradient magnetic separation (HGMS) to retain nucleic acid-magnetic bead complexes within the device's steel wool matrix for subsequent processing steps. We demonstrate the method's utility by extracting tuberculosis DNA from both sputum and urine, two typical large volume sample matrices (5-200 mL), using guanidine-based extraction chemistry. Our HGMS-enabled extraction method is statistically indistinguishable from commercial extraction kits when detecting a spiked 123-base DNA sequence. For our HGMS-enabled extraction method, we obtained extraction efficiencies for sputum and urine of approximately 10 and 90%, whereas commercial kits obtained 10-17 and 70-96%, respectively. We also used this method previously in a blinded sample preparation comparison study published by Beall et al., 2019. Our manual extraction method is insensitive to high flow rates and sample viscosity, with capture of ∼100% for flow rates up to 45 mL/min and viscosities up to 55 cP, possibly making it suitable for a wide variety of sample volumes and types and point-of-care users. This HGMS-enabled extraction method provides a robust instrument-free method for magnetic bead-based nucleic acid extraction, potentially suitable for field implementation of nucleic acid testing.
MeSH term(s)	Bacteriological Techniques/methods ; DNA, Bacterial/analysis ; DNA, Bacterial/isolation & purification ; DNA, Bacterial/urine ; Humans ; Magnets/chemistry ; Mycobacterium tuberculosis/isolation & purification ; Nucleic Acids/analysis ; Nucleic Acids/isolation & purification ; Nucleic Acids/urine ; Real-Time Polymerase Chain Reaction ; Specimen Handling ; Sputum/chemistry ; Sputum/microbiology ; Tuberculosis/diagnosis
Chemical Substances	DNA, Bacterial ; Nucleic Acids
Language	English
Publishing date	2020-03-05
Publishing country	United States
Document type	Journal Article
ISSN	1944-8252
ISSN (online)	1944-8252
DOI	10.1021/acsami.9b21564
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: A magnetic bead-based method for concentrating DNA from human urine for downstream detection.

Bordelon, Hali / Russ, Patricia K / Wright, David W / Haselton, Frederick R

PloS one

2013 Volume 8, Issue 7, Page(s) e68369

Abstract: Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the ... ...

Abstract	Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×10(3) to 5×10(8) copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×10(6), 14×10(6), and 8×10(6) copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR.
MeSH term(s)	Adsorption ; Base Sequence ; Buffers ; DNA/urine ; Freeze Drying ; Humans ; Kinetics ; Limit of Detection ; Magnetics/methods ; Microspheres ; Molecular Weight ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Silicon Dioxide/chemistry ; Specimen Handling
Chemical Substances	Buffers ; Reagent Kits, Diagnostic ; Silicon Dioxide (7631-86-9) ; DNA (9007-49-2)
Language	English
Publishing date	2013-07-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0068369
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Tuberculosis Biomarker Extraction and Isothermal Amplification in an Integrated Diagnostic Device.

Creecy, Amy / Russ, Patricia K / Solinas, Francesca / Wright, David W / Haselton, Frederick R

PloS one

2015 Volume 10, Issue 7, Page(s) e0130260

Abstract: In this study, we integrated magnetic bead-based sample preparation and isothermal loop mediated amplification (LAMP) of TB in a single tube. Surrogate sputum samples produced by the Program for Appropriate Technology in Health containing inactivated TB ... ...

Abstract	In this study, we integrated magnetic bead-based sample preparation and isothermal loop mediated amplification (LAMP) of TB in a single tube. Surrogate sputum samples produced by the Program for Appropriate Technology in Health containing inactivated TB bacteria were used to test the diagnostic. In order to test the sample preparation method, samples were lysed, and DNA was manually extracted and eluted into water in the tube. In a thermal cycler, LAMP amplified TB DNA from 103 TB cells/mL of sputum at 53.5 ± 3.3 minutes, 104 cells/mL at 46.3 ± 2.2 minutes, and 105 cells/mL at 41.6 ± 1.9 minutes. Negative control samples did not amplify. Next, sample preparation was combined with in-tubing isothermal LAMP amplification by replacing the water elution chamber with a LAMP reaction chamber. In this intermediate configuration, LAMP amplified 103 cells/mL at 74 ± 10 minutes, 104 cells/mL at 60 ± 9 minutes, and 105 TB cells/mL of sputum at 54 ± 9 minutes. Two of three negative controls did not amplify; one amplified at 100 minutes. In the semi-automated system, DNA was eluted directly into an isothermal reaction solution containing the faster OptiGene DNA polymerase. The low surrogate sputum concentration, 103 TB cells/mL, amplified at 52.8 ± 3.3 minutes, 104 cells/mL at 45.4 ± 11.3 minutes, and 105 cells/mL at 31.8 ± 2.9 minutes. TB negative samples amplified at 66.4 ± 7.4 minutes. This study demonstrated the feasibility of a single tube design for integrating sample preparation and isothermal amplification, which with further development could be useful for point-of-care applications, particularly in a low-resource setting.
MeSH term(s)	DNA, Bacterial/chemistry ; Molecular Diagnostic Techniques/instrumentation ; Molecular Diagnostic Techniques/methods ; Mycobacterium tuberculosis/genetics ; Mycobacterium tuberculosis/isolation & purification ; Polymerase Chain Reaction/instrumentation ; Polymerase Chain Reaction/methods ; Sputum/microbiology
Chemical Substances	DNA, Bacterial
Language	English
Publishing date	2015-07-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0130260
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Article ; Online: A Prototype Biomarker Detector Combining Biomarker Extraction and Fixed Temperature PCR.

Russ, Patricia K / Karhade, Aditya V / Bitting, Anna L / Doyle, Andrew / Solinas, Francesca / Wright, David W / Haselton, Frederick R

Journal of laboratory automation

2016 Volume 21, Issue 4, Page(s) 590–598

Abstract: PCR is the most sensitive molecular diagnostic available for infectious diseases. The goal for low-resource settings is a simple, inexpensive instrument. Toward this goal, we previously published a self-contained sample preparation instrument that uses ... ...

Abstract	PCR is the most sensitive molecular diagnostic available for infectious diseases. The goal for low-resource settings is a simple, inexpensive instrument. Toward this goal, we previously published a self-contained sample preparation instrument that uses magnetics and prearrayed reagents in thin tubing to extract nucleic acids and perform isothermal amplification and detection of extracted biomarkers. To incorporate PCR thermal cycling, after biomarker is magnetically extracted from a patient sample, the section of tubing containing the extracted biomarker and PCR reagents is alternately positioned within two constant temperature blocks. This instrument was evaluated initially by extracting and amplifying a 140 bp fragment of the IS6110 sequence of tuberculosis from TE buffer. The mean cycle threshold for 5 × 10(6) copies of IS6110 was 25.5 ± 1.5 cycles (n = 4), which was significantly different from negative control samples (34.0 ± 2.6 cycles; n = 3). Using a more clinically relevant sample, we extracted and amplified Plasmodium falciparum DNA from malaria-infected human blood cultures. The average cycle threshold for 1% parasitemia samples was 24.7 ± 1.5 cycles (n = 3) and significantly different from negatives (31.5 ± 2.1 cycles; n = 3). This approach integrates biomarker extraction, PCR amplification, and detection in a simple, linear tubing design with potential for use as a low-resource instrument.
MeSH term(s)	Biomarkers/analysis ; Humans ; Molecular Diagnostic Techniques/instrumentation ; Molecular Diagnostic Techniques/methods ; Mycobacterium tuberculosis/genetics ; Mycobacterium tuberculosis/isolation & purification ; Plasmodium falciparum/genetics ; Plasmodium falciparum/isolation & purification ; Polymerase Chain Reaction/instrumentation ; Polymerase Chain Reaction/methods ; Specimen Handling/instrumentation ; Specimen Handling/methods ; Temperature
Chemical Substances	Biomarkers
Language	English
Publishing date	2016-08
Publishing country	United States
Document type	Journal Article
ISSN	2211-0690
ISSN (online)	2211-0690
DOI	10.1177/2211068216634072
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Surface engineering of quantum dots for in vivo vascular imaging.

Jayagopal, Ashwath / Russ, Patricia K / Haselton, Frederick R

Bioconjugate chemistry

2007 Volume 18, Issue 5, Page(s) 1424–1433

Abstract: Quantum dot-antibody bioconjugates (QD-mAb) were synthesized incorporating PEG cross-linkers and Fc-shielding mAb fragments to increase in vivo circulation times and targeting efficiency. Microscopy of endothelial cell cultures incubated with QD-mAb ... ...

Abstract	Quantum dot-antibody bioconjugates (QD-mAb) were synthesized incorporating PEG cross-linkers and Fc-shielding mAb fragments to increase in vivo circulation times and targeting efficiency. Microscopy of endothelial cell cultures incubated with QD-mAb directed against cell adhesion molecules (CAMs), when shielded to reduce Fc-mediated interactions, were more specific for their molecular targets. In vitro flow cytometry indicated that surface engineered QD-mAb labeled leukocyte subsets with minimal Fc-mediated binding. Nontargeted QD-mAb nanoparticles with Fc-blockade featured 64% (endothelial cells) and 53% (leukocytes) lower nonspecific binding than non-Fc-blocked nanoparticles. Spectrally distinct QD-mAb targeted to the cell adhesion molecules (CAMs) PECAM-1, ICAM-1, and VCAM-1 on the retinal endothelium in a rat model of diabetes were imaged in vivo using fluorescence angiography. Endogenously labeled circulating and adherent leukocyte subsets were imaged in rat models of diabetes and uveitis using QD-mAb targeted to RP-1 and CD45. Diabetic rats exhibited increased fluorescence in the retinal vasculature from QD bioconjugates to ICAM-1 and VCAM-1 but not PECAM-1. Both animal models exhibited leukocyte rolling and leukostasis in capillaries. Examination of retinal whole mounts prepared after in vivo imaging confirmed the fluorescence patterns seen in vivo. Comparison of the timecourse of retinal fluorescence from Fc-shielded and non-Fc-shielded bioconjugates indicated nonspecific uptake and increased clearance of the non-Fc-shielded QD-mAb. This combination of QD surface design elements offers a promising new in vivo approach to specifically label vascular cells and biomolecules of interest.
MeSH term(s)	Animals ; Antibodies, Monoclonal ; Cell Adhesion/physiology ; Cell Culture Techniques ; Diabetes Mellitus, Experimental/metabolism ; Diabetes Mellitus, Experimental/pathology ; Endothelium, Vascular/metabolism ; Endothelium, Vascular/pathology ; Flow Cytometry ; Fluorescent Antibody Technique/methods ; Microscopy, Fluorescence ; Quantum Dots ; Rats ; Receptors, Leukocyte-Adhesion/metabolism ; Retinal Vessels/metabolism ; Retinal Vessels/pathology ; Time Factors
Chemical Substances	Antibodies, Monoclonal ; Receptors, Leukocyte-Adhesion
Language	English
Publishing date	2007-09
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1024041-x
ISSN	1520-4812 ; 1043-1802
ISSN (online)	1520-4812
ISSN	1043-1802
DOI	10.1021/bc070020r
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Article ; Online: Detection of respiratory syncytial virus using nanoparticle amplified immuno-polymerase chain reaction.

Perez, Jonas W / Vargis, Elizabeth A / Russ, Patricia K / Haselton, Frederick R / Wright, David W

Analytical biochemistry

2010 Volume 410, Issue 1, Page(s) 141–148

Abstract: In traditional immuno-polymerase chain reaction (immuno-PCR), a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. Here we describe a nanoparticle-amplified immuno-PCR (NPA-IPCR) assay ... ...

Abstract	In traditional immuno-polymerase chain reaction (immuno-PCR), a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. Here we describe a nanoparticle-amplified immuno-PCR (NPA-IPCR) assay that combines antibody recognition of enzyme-linked immunosorbent assay (ELISA) with a 50-fold nanoparticle valence amplification step prior to tag amplification by PCR. The assay detects a respiratory syncytial virus (RSV) surface protein using an antibody bound to a 15-nm gold nanoparticle cofunctionalized with thiolated DNA complementary to a hybridized 76-base tag DNA with a tag DNA/antibody ratio of 50:1. The presence of virus particles triggers the formation of a "sandwich" complex composed of the gold nanoparticle construct, virus, and an antibody-functionalized magnetic particle used for extraction. After extraction, DNA tags are released by heating to 95°C and detected via real-time PCR. The limit of detection of the assay was compared with ELISA and reversion transcription (RT) PCR using RSV-infected HEp-2 cell extracts. NPA-IPCR showed an approximately 4000-fold improvement in the limit of detection compared with ELISA and a 4-fold improvement compared with viral RNA extraction followed by traditional RT-PCR. NPA-IPCR offers a viable platform for the development of early-stage diagnostics requiring an exceptionally low limit of detection.
MeSH term(s)	Animals ; Antibodies/immunology ; Antigens/chemistry ; Antigens/immunology ; Base Sequence ; Biosensing Techniques/methods ; Cell Extracts ; Cell Line ; DNA, Viral/analysis ; DNA, Viral/genetics ; DNA, Viral/immunology ; Gold/chemistry ; Immunoassay/methods ; Limit of Detection ; Metal Nanoparticles/chemistry ; Polymerase Chain Reaction/methods ; Quartz Crystal Microbalance Techniques ; Reproducibility of Results ; Respiratory Syncytial Viruses/genetics ; Respiratory Syncytial Viruses/immunology ; Respiratory Syncytial Viruses/isolation & purification
Chemical Substances	Antibodies ; Antigens ; Cell Extracts ; DNA, Viral ; Gold (7440-57-5)
Language	English
Publishing date	2010-11-25
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1110-1
ISSN	1096-0309 ; 0003-2697
ISSN (online)	1096-0309
ISSN	0003-2697
DOI	10.1016/j.ab.2010.11.033
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Article ; Online: Inhibition of RhoA signaling with increased Bves in trabecular meshwork cells.

Russ, Patricia K / Kupperman, Asher I / Presley, Sai-Han / Haselton, Frederick R / Chang, Min S

Investigative ophthalmology & visual science

2009 Volume 51, Issue 1, Page(s) 223–230

Abstract: Purpose: Blood vessel epicardial substance (Bves) is a novel adhesion molecule that regulates tight junction (TJ) formation. TJs also modulate RhoA signaling, which has been implicated in outflow regulation. Given that Bves has been reported in multiple ...

Abstract	Purpose: Blood vessel epicardial substance (Bves) is a novel adhesion molecule that regulates tight junction (TJ) formation. TJs also modulate RhoA signaling, which has been implicated in outflow regulation. Given that Bves has been reported in multiple ocular tissues, the authors hypothesize that Bves plays a role in the regulation of RhoA signaling in trabecular meshwork (TM) cells. Methods: Human TM cell lines NTM-5 and NTM-5 transfected to overexpress Bves (NTM-w) were evaluated for TJ formation, and levels of occludin, cingulin, and ZO-1 protein were compared. Assays of TJ function were carried out using diffusion of sodium fluorescein and transcellular electrical resistance (TER). Levels of activated RhoA were measured using FRET probes, and phosphorylation of myosin light chain (MLC-p), a downstream target of RhoA, was assessed by Western blot analysis. Results: Overexpression of Bves led to increased TJ formation in NTM-5 cells. Increased TJ formation was confirmed by increased occludin, cingulin, and ZO-1 protein. Functionally, NTM-w cells showed decreased permeability and increased TER compared with NTM-5 cells, consistent with increased TJ formation. NTM-w cells also exhibited decreased levels of active RhoA and lower levels of MLC-p than did NTM-5 cells. These findings support a TJ role in RhoA signaling. Conclusions: Increased Bves in TM cells leads to increased TJ formation with decreased RhoA activation and decreased MLC-p. This is the first report of a regulatory pathway upstream of RhoA in TM cells. In TM tissue, RhoA has been implicated in outflow regulation; thus, Bves may be a key regulatory molecule in aqueous outflow.
MeSH term(s)	Avian Proteins/physiology ; Blotting, Western ; Cell Adhesion Molecules/physiology ; Cell Line ; Electric Impedance ; Fluorescein/metabolism ; Fluorescent Antibody Technique, Indirect ; Humans ; Membrane Proteins/metabolism ; Microfilament Proteins/metabolism ; Muscle Proteins/physiology ; Myosin Light Chains/metabolism ; Occludin ; Permeability ; Phosphoproteins/metabolism ; Phosphorylation ; Signal Transduction ; Tight Junctions/metabolism ; Trabecular Meshwork/metabolism ; Transfection ; Zonula Occludens-1 Protein ; rhoA GTP-Binding Protein/metabolism
Chemical Substances	Avian Proteins ; Bves protein, Gallus gallus ; CGN protein, human ; Cell Adhesion Molecules ; Membrane Proteins ; Microfilament Proteins ; Muscle Proteins ; Myosin Light Chains ; OCLN protein, human ; Occludin ; Phosphoproteins ; TJP1 protein, human ; Zonula Occludens-1 Protein ; rhoA GTP-Binding Protein (EC 3.6.5.2) ; Fluorescein (TPY09G7XIR)
Language	English
Publishing date	2009-07-23
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	391794-0
ISSN	1552-5783 ; 0146-0404
ISSN (online)	1552-5783
ISSN	0146-0404
DOI	10.1167/iovs.09-3539
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Article ; Online: Design and use of mouse control DNA for DNA biomarker extraction and PCR detection from urine: Application for transrenal Mycobacterium tuberculosis DNA detection.

Bordelon, Hali / Ricks, Keersten M / Pask, Megan E / Russ, Patricia K / Solinas, Francesca / Baglia, Mark L / Short, Philip A / Nel, Andrew / Blackburn, Jonathan / Dheda, Keertan / Zamudio, Carlos / Cáceres, Tatiana / Wright, David W / Haselton, Frederick R / Pettit, April C

Journal of microbiological methods

2017 Volume 136, Page(s) 65–70

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 ... ...

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.
MeSH term(s)	Animals ; Base Sequence ; Coinfection ; DNA/isolation & purification ; Gene Targeting/methods ; Genetic Markers ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics ; HIV Infections/complications ; Humans ; Mice/genetics ; Molecular Diagnostic Techniques/methods ; Mycobacterium tuberculosis/genetics ; Mycobacterium tuberculosis/isolation & purification ; Peptide Fragments/genetics ; Polymerase Chain Reaction/methods ; Retrospective Studies ; Sensitivity and Specificity ; South Africa ; Tuberculosis/complications ; Tuberculosis/diagnosis ; Tuberculosis/microbiology ; Tuberculosis/urine ; Urine/microbiology
Chemical Substances	Genetic Markers ; Peptide Fragments ; glyceraldehyde 3-phosphate dehydrogenase (304-313) (130349-12-1) ; DNA (9007-49-2) ; Glyceraldehyde-3-Phosphate Dehydrogenases (EC 1.2.1.-)
Language	English
Publishing date	2017-03-09
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	604916-3
ISSN	1872-8359 ; 0167-7012
ISSN (online)	1872-8359
ISSN	0167-7012
DOI	10.1016/j.mimet.2017.02.010
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