Pathogenic Bacteria Detection Using RNA-Based Loop-Mediated Isothermal-Amplification-Assisted Nucleic Acid Amplification via Droplet Microfluidics

Azizi, Mohammad; Zaferani, Meisam; Cheong, Soon Hon; Abbaspourrad, Alireza

doi:10.1021/acssensors.8b01206

Cited by 97 publications

(57 citation statements)

References 41 publications

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“…The individual additives PEG-6K, BSA, and betaine were chosen as they were utilized in prior droplet PCR assays. 20, 35, 40, 41 By adding Tween-20 to each of these additives, ( D - D 0 )/ D 0 decreased below zero and were found to have lower coefficients of variation (CV) than with no additives and with each of the additives alone without Tween-20 ( Fig. 1B ).…”

Section: Resultsmentioning

confidence: 99%

Screening of additives for droplet qRT-PCR thermocycling enables single influenza A virus genome quantification

Loveday

Zath

Bikos

et al. 2020

Preprint

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18The miniaturization of real time quantitative polymerase chain reaction (qPCR) using 19 drop-based microfluidics, or droplet qPCR, allows for quantification of single nucleic acids. The 20 nucleic acids are compartmentalized into aqueous microdroplets, picoliters in volume, separated 21 by an immiscible oil, and stabilized by a surfactant. In droplet qPCR, accurate data can only be 22 obtained if the drops remain stable to coalescence upon thermocycling and drop contents do not 23 diffuse to neighboring drops. In this work, we present a droplet qRT-PCR assay for quantifying 24 influenza A virus (IAV) following systematic testing of different PCR additives, resulting in the 25 optimal combination of Tween-20 / BSA / betaine to maintain drop stability and limit dye 26 diffusion. We use a standard qPCR machine to generate real time amplification curves of 27 hundreds of thousands of drops and correlate this data with constructed amplification curves 28 obtained from hundreds of drops sampled at various cycle numbers and imaged using 29 epifluorescence microscopy. To demonstrate the utility of our method, we tested a range of in 30 vitro transcribed M gene and IAV viral supernatant from infected cells. We directly amplified 31 IAV genomes from infected supernatant without an RNA extraction step. Our droplet qPCR 32 assay enables detection of IAV down to 0.274 cpd, or a single viral genome per drop, 33 establishing the high sensitivity and precision of our method. 34 35 36 37 3 Introduction: 38

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Section: Resultsmentioning

confidence: 99%

Screening of additives for droplet qRT-PCR thermocycling enables single influenza A virus genome quantification

Loveday

Zath

Bikos

et al. 2020

Preprint

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“…The microfluidic device was made of polydimethylsiloxane using a standard soft lithography protocol (69, 70). The diameter of the curved quiescent zone was 200 µm and the height of the chamber was 25 µm.…”

Section: Methodsmentioning

confidence: 99%

Effect of flagellar beating pattern on sperm rheotaxis and boundary-dependent navigation

Zaferani

Javi

Mokhtare

et al. 2020

Preprint

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7The study of navigational mechanisms used by mammalian sperm inside a microenvironment 8 yields better understanding of sperm locomotion during the insemination process, which aids in 9 the design of tools for overcoming infertility. Near-and far-field hydrodynamic interactions with 10 nearby boundaries and rheotaxis are known to be some of the steering strategies that keep sperm 11 on the correct path toward the egg. However, it is not known how the beating patterns of sperm 12 may influence these navigational strategies. In this study, we investigate the effect of flagellar 13 beating pattern on navigation of sperm cells both theoretically and experimentally using a two-14 step approach. We first isolate bovine sperm based on their rheotactic behavior in a zone with 15 quiescent medium using a microfluidic system. This step ensures that the swimmers are able to 16 navigate upstream and have motilities higher than a selected value, even though they feature 17 various flagellar beating patterns. We then explore the flagellar beating pattern of these isolated 18 sperm and their subsequent influence on boundary-dependent navigation. Our findings indicate 19 that rheotaxis enables sperm to navigate upstream even in the presence of circular motion in their 20 motility, whereas boundary-dependent navigation is more sensitive to the circular motion and 21 selects for progressive motility. This finding may explain the clinical importance of progressive 23 the sperm cells throughout the process of insemination. 24 Keywords: Mammalian sperm | Navigation | Rheotaxis | Boundary-dependent navigation | 25 Progressive motility 26 Significance 27Finding the egg and moving toward it while traversing the complex structure of the female 28 reproductive tract is necessary for mammalian sperm. Previous studies have shown how sperm use 29 navigational steering mechanisms that are based on swimming upstream (i.e. rheotaxis) and along 30 the boundaries of the female reproductive tract. We demonstrate that the performance of theses 31 navigational mechanisms is associated with the primary characteristics of sperm motility. In fact, 32 sperm rheotaxis is more sensitive to the motility and thus average velocity of sperm while 33 navigation via rigid boundaries is more sensitive to the flagellar beating pattern and selects for 34 symmetric beating. Our results can be expanded to other autonomous microswimmers and their 35 subsequent navigation mechanisms. 36 37 38 39 40 41 42 43 48which vary among different species. For instance, marine plants and animals (10, 11) release their 49 gametes into the sea, where chemotactic behavior is observed (4,(11)(12)(13). Strikingly, the role of 50 this chemical communication observed in marine invertebrate sperm (which is reminiscent of 51 bacterial chemotaxis (14)) is uncertain in the navigation of mammalian sperm (13,(15)(16)(17)(18)(19). In 52 contrast, in vitro/vivo evidence suggests that the navigation of mammalian sperm within the female 53 reproductive tract depends more on fluid mechanical c...

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“…a1–a3) Typical devices to emulsify LAMP mixtures. (a1 is reproduced with permission, Copyright 2019, American Chemical Society; a2 is reproduced with permission, Copyright 2016, American Chemical Society; a3 reproduced with permission, Copyright 2016, Royal Society of Chemistry.) b1, b2) The multilayer approach for preventing evaporation during heating.…”

Section: Current Dlamp Techniquesmentioning

confidence: 99%

“…Nevertheless, LAMP can only detect pathogenic nucleic acid qualitatively . To quantify the target nucleic acid concentration, a calibration curve is always required . Typically, this calibration curve is obtained from tens or even hundreds of LAMP tests with various nucleic acid concentrations on a real‐time polymerase chain reaction (PCR) machine, which largely increases the instrumentation complexity and labor intensity.…”

Section: Introductionmentioning

confidence: 99%

Droplet and Microchamber‐Based Digital Loop‐Mediated Isothermal Amplification (dLAMP)

Yuan

Chao

Shum

2020

Small

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Digital loop‐mediated isothermal amplification (dLAMP) refers to compartmentalizing nucleic acids and LAMP reagents into a large number of individual partitions, such as microchambers and droplets. This compartmentalization enables dLAMP to be an excellent platform to quantify the absolute number of the target nucleic acids. Owing to its low requirement for instrumentation complexity, high specificity, and strong tolerance to inhibitors in the nucleic acid samples, dLAMP has been recognized as a simple and accurate technique to quantify pathogenic nucleic acid. Herein, the general process of dLAMP techniques is summarized, the current dLAMP techniques are categorized, and a comprehensive discussion on different types of dLAMP techniques is presented. Also, the challenges of the current dLAMP are illustrated together with the possible strategies to address these challenges. In the end, the future directions of the dLAMP developments, including multitarget detection, multisample detection, and processing nucleic acid extraction are outlined. With recently significant advances in dLAMP, this technology has the potential to see more widespread use beyond the laboratory in the future.

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Pathogenic Bacteria Detection Using RNA-Based Loop-Mediated Isothermal-Amplification-Assisted Nucleic Acid Amplification via Droplet Microfluidics

Cited by 97 publications

References 41 publications

Screening of additives for droplet qRT-PCR thermocycling enables single influenza A virus genome quantification

Screening of additives for droplet qRT-PCR thermocycling enables single influenza A virus genome quantification

Effect of flagellar beating pattern on sperm rheotaxis and boundary-dependent navigation

Droplet and Microchamber‐Based Digital Loop‐Mediated Isothermal Amplification (dLAMP)

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