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 J. M.; Blackburn, Jonathan M.; Dheda, Keertan; Zamudio, Carlos; Cáceres, Tatiana; Wright, David W.; Haselton, Frederick R.; Pettit, April C

doi:10.1016/j.mimet.2017.02.010

Cited by 6 publications

(8 citation statements)

References 36 publications

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“…The use of a larger sample can increase the sensitivity of low-resource detection methods. , This is simple to do with HGMS-enabled extraction. Pipettes with different sized bulbs or syringes could be easily interchanged for application-specific design, with larger, more dilute samples using larger volume actuators.…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, magnetic beads can be moved between different processing solutions while the liquid solutions remain stationary. The advantage of this approach is that it decreases the number of manual steps, eliminates the requirement of liquid handling, and allows for simple automation of bead manipulation. ,− The challenge with this approach, however, is maximizing the applied magnetic force on the beads, which is needed to efficiently transfer magnetic beads across fluid interfaces between processing solutions while minimizing bead loss. Despite this shortcoming, at least one sample-to-answer system based on this approach is commercially available, the Cobas Liat polymerase chain reaction (PCR) System (Roche Molecular Diagnostics, Pleasanton, CA).…”

Section: Introductionmentioning

confidence: 99%

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

Pearlman

Leelawong

Richardson

et al. 2020

ACS Appl. Mater. Interfaces

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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 acidmagnetic 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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Pearlman

Leelawong

Richardson

et al. 2020

ACS Appl. Mater. Interfaces

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“…The sensitivity and specificity of our assay for diagnosis of active pulmonary TB were 84% and 100%, respectively, the highest reported diagnostic accuracy of a TB cfDNA test. For comparison, previous TB urine cfDNA studies are summarized in Table 5 (8)(9)(10)(11)(12). For a comprehensive review of previous studies, refer to (14).…”

Section: Discussionmentioning

confidence: 99%

“…In patients with active TB disease, TB-specific cell-free DNA (cfDNA) fragments are released into the blood, a fraction of which are filtered through the kidneys and excreted in the urine as transrenal cfDNA (6)(7)(8). TB-specific cfDNA has been detected in urine from both HIV-negative and HIV-positive patients with pulmonary TB, but diagnostic sensitivities have been inconsistent (0 -79%) (8)(9)(10)(11)(12)(13). High variability in methodology and subsequent performance across studies have limited the understanding of TB urine cfDNA and hindered its clinical implementation (14,15).…”

Section: Introductionmentioning

confidence: 99%

Diagnosing pulmonary tuberculosis using sequence-specific purification of urine cell-free DNA

Oreskovic¹,

Panpradist²,

Marangu³

et al. 2021

Preprint

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Transrenal urine cell-free DNA (cfDNA) is a promising tuberculosis (TB) biomarker, but is challenging to detect because of the short length (<100 bp) and low concentration of TB-specific fragments. We aimed to improve the diagnostic sensitivity of TB urine cfDNA by increasing recovery of short fragments during sample preparation. We developed a highly sensitive sequence-specific purification method that uses hybridization probes immobilized on magnetic beads to capture short TB cfDNA (50 bp) with 91.8% average efficiency. Combined with short-target PCR, the assay limit of detection was ≤5 copies of cfDNA in 10 mL urine. In a clinical cohort study in South Africa, our urine cfDNA assay had 83.7% sensitivity (95% CI: 71.0– 91.5%) and 100% specificity (95% CI: 86.2–100%) for diagnosis of active pulmonary TB when using sputum Xpert MTB/RIF as the reference standard. The detected cfDNA concentration was 0.14–2804 copies/mL (median 14.6 copies/mL) and was inversely correlated with CD4 count and days to culture positivity. Sensitivity was non-significantly higher in HIV-positive (88.2%) compared to HIV-negative patients (73.3%), and was not dependent on CD4 count. Sensitivity remained high in sputum smear-negative (76.0%) and urine LAM-negative (76.5%) patients. With improved sample preparation, urine cfDNA is a viable biomarker for TB diagnosis. Our assay has the highest reported accuracy of any TB urine cfDNA test to date and has the potential to enable rapid non-sputum-based TB diagnosis across key underserved patient populations.

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“…Recent studies are demonstrating the presence of M . tuberculosis DNA in the urine of TB patients [ 23 , 24 ]. To evaluate the capacity of our biosensor, to detect M .…”

Section: Methodsmentioning

confidence: 99%

An electrochemical biosensor for the detection of Mycobacterium tuberculosis DNA from sputum and urine samples

et al. 2020

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Tuberculosis (TB) is a major global public health problem with high mortality and morbidity. In low-middle income countries (LMIC) a large number of respiratory symptomatic cases that require TB screening per year demands more accurate, fast and affordable testing for TB diagnostics. Sputum smear is the initial screening test in LMICs, however, its sensitivity is limited in patients with low sputum bacilli load. The same limitation is observed in the currently available molecular tests. We designed, standardized and evaluated an electrochemical biosensor that detects the highly specific DNA insertion element 6110 (IS6110). A PCR amplified DNA product is hybridized on the surface of the working electrode built on FTO-Glass with immobilized specific DNA probes, after which cyclic voltammetry is performed with an Ag/AgCl reference electrode and a platinum counter electrode. The response of the sensor was measured by the ratio (cathodic peak current of the hybridized sensor) / (cathodic peak current of the non-hybridized sensor). We tested the biosensor, using positive hybridization control sequences, genomic DNA extracted from M . tuberculosis strains and sputum of TB patients, and extracted DNA from the urine of healthy controls spiked with M . tuberculosis DNA. This biosensor was effective for the detection of M . tuberculosis DNA with a detection limit of 16 fM in sputum sample and 1 fM in spiked urine samples. The low cost and the relatively brief duration of the assay make this an important TB screening tool in the fight against tuberculosis.

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Design and use of mouse control DNA for DNA biomarker extraction and PCR detection from urine: Application for transrenal Mycobacterium tuberculosis DNA detection

Cited by 6 publications

References 36 publications

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

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

Diagnosing pulmonary tuberculosis using sequence-specific purification of urine cell-free DNA

An electrochemical biosensor for the detection of Mycobacterium tuberculosis DNA from sputum and urine samples

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