Isolation and concentration of fungi in the blood improves sensitivity of the polymerase chain reaction (PCR) method to detect fungi in blood. This study demonstrates a sheathless, continuous ...separation and concentration method of candida cells using a viscoelastic fluid that enables rapid detection of rare candida cells by PCR analysis. To validate device performance using a viscoelastic fluid, flow characteristics of 2 μm particles were estimated at different flow rates. Additionally, a mixture of 2 μm and 13 μm particles was successfully separated based on size difference at 100 μl/min. Candida cells were successfully separated from the white blood cells (WBCs) with a separation efficiency of 99.1% and concentrated approximately 9.9-fold at the center outlet compared to the initial concentration (~2.5 × 10
cells/ml). Sequential 1st and 2nd concentration processes were used to increase the final number of candida cells to ~2.3 × 10
cells/ml, which was concentrated ~92-fold. Finally, despite the undetectable initial concentration of 10
CFU/ml, removal of WBCs and the additional buffer solution enabled the quantitative reverse transcription (RT)-PCR detection of candida cells after the 1st concentration (Ct = 31.43) and the 2nd concentration process (Ct = 29.30).
Pure separation and sorting of microparticles from complex fluids are essential for biochemical analyses and clinical diagnostics. However, conventional techniques require highly complex and ...expensive labeling processes for high purity separation. In this study, we present a simple and label-free method for separating microparticles with high purity using the elasto-inertial characteristic of a non-Newtonian fluid in microchannel flow. At the inlet, particle-containing sample flow was pushed toward the side walls by introducing sheath fluid from the center inlet. Particles of 1 μm and 5 μm in diameter, which were suspended in viscoelastic fluid, were successfully separated in the outlet channels: larger particles were notably focused on the centerline of the channel at the outlet, while smaller particles continued flowing along the side walls with minimal lateral migration towards the centerline. The same technique was further applied to separate platelets from diluted whole blood. Through cytometric analysis, we obtained a purity of collected platelets of close to 99.9%. Conclusively, our microparticle separation technique using elasto-inertial forces in non-Newtonian fluid is an effective method for separating and collecting microparticles on the basis of size differences with high purity.
A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six ...continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).
In this work, we investigated the lateral migration of microparticles suspended in two different viscoelastic fluids with or without the second normal stress difference. For the viscoelastic fluid ...without the second normal stress difference, competing forces existed between microfluidic inertia and the first normal stress difference (
N
1
), which resulted in a synergetic effect of particle focusing. For the fluid with the second normal stress difference (
N
2
), particles were greatly affected by a
N
2
-induced secondary flow, and the competition among the inertia,
N
1
, and
N
2
determined the lateral migration trajectories of the particles. The obtained results were delineated with the blockage ratio, which showed good agreement with the results of a recent numerical study (Villone et al. in J Non Newton Fluid Mech 195:1–8,
2013
). The present study also examined the possibility of particle separation in a size-dependent manner using the
N
2
-induced secondary flow in microchannel flow.
•Continuous leukocyte separation from high-hematocrit blood sample is proposed.•Effect of in vivo-margination and viscoelastic lateral migration is used.•From the blood sample with 25% hematocrit, ...leukocytes were successfully separated.•Our device could achieve 94% of separation efficiency and 10-fold enrichment.
The removal of erythrocytes from whole blood is an essential step during sample preparations intended for biomedical analyses and clinical diagnoses. To address the limitations of present methods, such as centrifugation and chemical lysis, we propose a novel microfluidic device for erythrocyte removal with high-efficiency and leukocyte separation from bulk flows of highly concentrated erythrocytes using a viscoelastic non-Newtonian fluid. The proposed device is designed based on the principle of viscoelasticity-induced particle migration toward the center of the microchannel. In addition, we based the functionality of our device on a bio-inspired phenomenon known as margination according to which erythrocytes migrate to the axial center of blood vessels. Fluorescent particles (10 μm) were added to blood suspensions of various concentrations (hematocrit) of erythrocytes in viscoelastic polymer solutions. Optimal hematocrit and flow rate conditions were determined for erythrocyte removal and for the separation of 10 μm particles. We also demonstrated the capability of our device to separate leukocytes with high efficiency (˜94%) and with a high-enrichment factor (10-fold).
Measuring red blood cell (RBC) deformability has become important for clinical disease diagnostics. Various methods for measuring RBC deformability have been developed; however, they require costly ...and large instruments, long measuring time, and skilled personnel. In this study, we present a three-dimensional-printed mini-disk (3D-PMD) for measuring RBC deformability to overcome the previous limitations. For a miniaturized and low-cost setup, the 3D-PMD was fabricated by a 3D printing technique, which had not yet been used for fabricating a lab-on-a-compact disk (LOCD). Using a 3D printing technique, a multi-layered fluidic channel on the mini CD could be fabricated easily. During rotation by a spinning motor, the difference of the length of compressed RBCs in the fluidic channel was measured and analysed as compressibility indices (CIs) of normal and glutaraldehyde-treated hardened RBCs. The rotation speed and time were decided as 3000 rpm and 30 min, respectively, at which the difference of CI values between normal and hardened RBCs was largest (CInormal-CIhardened = 0.195).
Dengue is an increasing public health concern worldwide and requires efficient laboratory diagnostics. We evaluated three commercially available dengue rapid diagnostic tests-the Humasis Dengue Combo ...NS1 & IgG/IgM (Humasis, Korea), SD Bioline Dengue Duo NS1 Ag & IgG/IgM (SD Bioline, Korea), and CareUS Dengue Combo NS1 and IgM/IgG kits (WellsBio, Korea)-and compared them to reference immunoglobulin M (IgM) or immunoglobulin G (IgG) ELISAs and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. In total, 109 dengue-positive samples from children with acute symptomatic dengue and 63 dengue-negative samples from febrile and asymptomatic individuals were collected. For the nonstructural 1 protein (NS1) Ag test, the sensitivity and specificity were in the following order: CareUS (79.82 and 100%), Humasis (63.30 and 100%), and SD Bioline (48.62 and 100%). For IgM and IgG, CareUS had the highest sensitivities and specificities (89.91 and 100%; 82.57 and 100%, respectively), followed by SD Bioline (60.55 and 100%, 77.98 and 100%, respectively), and Humasis (51.38 and 98.21%, 72.48 and 95.24%, respectively). The IgM kits were more sensitive than the NS1 Ag or IgG kits; however, combining NS1 Ag and IgM reduced the number of missed cases. Therefore, the NS1 Ag plus IgM dengue kits increase the accuracy of the results. In our study, the CareUS Dengue Combo NS1 and IgM/IgG kit showed higher accuracy in performance with reference to qRT-PCR and ELISA results.
The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time ...PCR-based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples.
For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method.
Of 123 NTM samples, 121 were identified as NTM and 72/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72/72) for MTB, and 98.4% (121/123) for NTM. And the specificity of assay was 100% (138/138) for all.
Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.
Severe fever with thrombocytopenia syndrome (SFTS) and scrub typhus are endemic zoonotic diseases that pose significant public health threats in East Asia. As these two diseases share common clinical ...features, as well as overlapping disease regions, it is difficult to differentiate between SFTS and scrub typhus. A multiplex reverse-transcription loop‑mediated isothermal amplification (RT-LAMP) assay was developed to detect large segments and GroES genes for SFTS virus (SFTSV) and Orientia tsutsugamushi (OT). The performance of the RT-LAMP assay was compared and evaluated with those of commercial PowerChek™ SFTSV real-time PCR and LiliF™ TSUTSU nested PCR for 23 SFTS and 12 scrub typhus clinical samples, respectively. The multiplex SFTSV/OT/Internal control (IC) RT-LAMP assay showed comparable sensitivity (91.3%) with that of commercial PowerChek™ SFTSV Real-time PCR (95.6%) and higher sensitivity (91.6%) than that of LiliF™ TSUTSU nested PCR (75%). In addition, the multiplex SFTSV/OT RT-LAMP assay showed 100% specificity and no cross-reactivity for blood from uninfected healthy patients and samples from patients infected with other fever viruses. Thus, the multiplex SFTSV/OT/IC RT-LAMP assay could serve as a useful point-of-care molecular diagnostic test for SFTS and scrub typhus.
Influenza, which is an acute respiratory disease caused by the influenza virus, represents a worldwide public health and economic problem owing to the significant morbidity and mortality caused by ...its seasonal epidemics and pandemics. Sensitive and convenient methodologies for the detection of influenza viruses are important for clinical care and infection control as well as epidemiological investigations. Here, we developed a multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) with quencher/fluorescence oligonucleotides connected by a 5' backward loop (LF or LB) primer for the detection of two subtypes of influenza viruses: Influenza A (A/H1 and A/H3) and influenza B. The detection limits of the multiplex RT-LAMP assay were 103 copies and 102 copies of RNA for influenza A and influenza B, respectively. The sensitivities of the multiplex influenza A/B/IC RT-LAMP assay were 94.62% and 97.50% for influenza A and influenza B clinical samples, respectively. The specificities of the multiplex influenza A/B/IC RT-LAMP assay were 100% for influenza A, influenza B, and healthy clinical samples. In addition, the multiplex influenza A/B/IC RT-LAMP assay had no cross-reactivity with other respiratory viruses.
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