Culture‐free biphasic approach for sensitive detection of <i>Escherichia coli</i> O157:H7 from beef samples

Mostafa, Ariana; Ganguli, Anurup; Berger, Jacob; Rayabharam, Archith; Trujillo, Carlos Humberto Saavedra; Aluru, N. R.; Bashir, Rashid

doi:10.1002/bit.27920

Cited by 5 publications

(2 citation statements)

References 42 publications

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“…In our previous report, we developed a novel narrow-thermal-cycling accelerated strand exchange amplification (ASEA) method, which employed only one pair of primers and Bst DNA polymerase to rapidly accomplish exponential target amplification. 29 Moreover, due to the robustness of Bst DNA polymerase, which has been shown to perform amplification in tissue matrices, 30,31 we believe the ASEA method has great potential for pathogenic bacteria detection in complicated food matrices.…”

Section: Introductionmentioning

confidence: 99%

A biphasic accelerated strand exchange amplification strategy for culture-independent and rapid detection of Salmonella enterica in food samples

Zhang,

Wang,

Shi

et al. 2024

Anal. Methods

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Section: Introductionmentioning

confidence: 99%

A biphasic accelerated strand exchange amplification strategy for culture-independent and rapid detection of Salmonella enterica in food samples

Zhang,

Wang,

Shi

et al. 2024

Anal. Methods

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“…1 E and G ). We show that the generated porosity and the microfluidic and nanofluidic network allow for enzymes to access DNA in the liquid phase and initiate amplification with single-molecule sensitivity inside the dried blood matrix, thus bypassing the need for conventional DNA purification ( 46 ) ( Fig. 1 G ).…”

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confidence: 95%

A culture-free biphasic approach for sensitive and rapid detection of pathogens in dried whole-blood matrix

Ganguli

Lim

Mostafa

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Blood stream infections (BSIs) cause high mortality, and their rapid detection remains a significant diagnostic challenge. Timely and informed administration of antibiotics can significantly improve patient outcomes. However, blood culture, which takes up to 5 d for a negative result, followed by PCR remains the gold standard in diagnosing BSI. Here, we introduce a new approach to blood-based diagnostics where large blood volumes can be rapidly dried, resulting in inactivation of the inhibitory components in blood. Further thermal treatments then generate a physical microscale and nanoscale fluidic network inside the dried matrix to allow access to target nucleic acid. The amplification enzymes and primers initiate the reaction within the dried blood matrix through these networks, precluding any need for conventional nucleic acid purification. High heme background is confined to the solid phase, while amplicons are enriched in the clear supernatant (liquid phase), giving fluorescence change comparable to purified DNA reactions. We demonstrate single-molecule sensitivity using a loop-mediated isothermal amplification reaction in our platform and detect a broad spectrum of pathogens, including gram-positive methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria, gram-negative Escherichia coli bacteria, and Candida albicans (fungus) from whole blood with a limit of detection (LOD) of 1.2 colony-forming units (CFU)/mL from 0.8 to 1 mL of starting blood volume. We validated our assay using 63 clinical samples (100% sensitivity and specificity) and significantly reduced sample-to-result time from over 20 h to <2.5 h. The reduction in instrumentation complexity and costs compared to blood culture and alternate molecular diagnostic platforms can have broad applications in healthcare systems in developed world and resource-limited settings.

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A Blood Drying Process for DNA Amplification

Lim,

Zhou,

Baek

et al. 2023

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The presence of numerous inhibitors in blood makes their use in nucleic acid amplification techniques difficult. Current methods for extracting and purifying pathogenic DNA from blood involve removal of inhibitors, resulting in low and inconsistent DNA recovery rates. To address this issue, a biphasic method is developed that simultaneously achieves inhibitor inactivation and DNA amplification without the need for a purification step. Inhibitors are physically trapped in the solid‐phase dried blood matrix by blood drying, while amplification reagents can move into the solid nano‐porous dried blood and initiate the amplification. It is demonstrated that the biphasic method has significant improvement in detection limits for bacteria such as Escherichia coli, Methicillin‐resistant Staphylococcus aureus, Methicillin‐Sensitive Staphylococcus aureus using loop‐mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Several factors, such as drying time, sample volume, and material properties are characterized to increase sensitivity and expand the application of the biphasic assay to blood diagnostics. With further automation, this biphasic technique has the potential to be used as a diagnostic platform for the detection of pathogens eliminating lengthy culture steps.

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Culture‐free biphasic approach for sensitive detection of Escherichia coli O157:H7 from beef samples

Cited by 5 publications

References 42 publications

A biphasic accelerated strand exchange amplification strategy for culture-independent and rapid detection of Salmonella enterica in food samples

A biphasic accelerated strand exchange amplification strategy for culture-independent and rapid detection of Salmonella enterica in food samples

A culture-free biphasic approach for sensitive and rapid detection of pathogens in dried whole-blood matrix

A Blood Drying Process for DNA Amplification

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