RESUMEN
After three years of the SARS-CoV-2 pandemic, the demand for developing field-deployable point-of-care (PoC) molecular diagnostic tests has increased. Although RT-qPCR is the molecular diagnostic gold standard and is accurate, it is not readily applied to point-of-care testing (POCT). Meanwhile, rapid diagnostic kits have the disadvantage of low sensitivity. Recently, rapid isothermal nucleic acid amplification technology has emerged as an alternative for rapid diagnosis. Here, we developed a rapid SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP)-lateral flow assay (LFA) kit. This kit includes a Chelex-100/boiling nucleic acid extraction device and a one-step amplification detection apparatus capable of performing the entire process, from RNA extraction to detection, and diagnosing SARS-CoV-2 infection within 40 min without contamination. The detection limits of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 100 plaque-forming units (PFUs) mL-1 and 10-1 PFU mL-1 for RNA samples extracted using the Chelex-100/boiling nucleic acid extraction device and commercial AdvansureTM E3 system, respectively. The sensitivity and specificity of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 97.8% and 100%, respectively. Our SARS-CoV-2 RT-LAMP-LFA kit exhibited high sensitivity and specificity within 40 min without requiring laboratory instruments, suggesting that the kit could be used as a rapid POC molecular diagnostic test for SARS-CoV-2.
RESUMEN
Influenza and coronaviruses cause highly contagious respiratory diseases that cause millions of deaths worldwide. Public health measures implemented during the current coronavirus disease (COVID-19) pandemic have gradually reduced influenza circulation worldwide. As COVID-19 measures have relaxed, it is necessary to monitor and control seasonal influenza during this COVID-19 pandemic. In particular, the development of rapid and accurate diagnostic methods for influenza and COVID-19 is of paramount importance because both diseases have significant public health and economic impacts. To address this, we developed a multi-loop-mediated isothermal amplification (LAMP) kit capable of simultaneously detecting influenza A/B and SARS-CoV-2. The kit was optimized by testing various ratios of primer sets for influenza A/B (FluA/FluB) and SARS-CoV-2 and internal control (IC). The FluA/FluB/SARS-CoV-2 multiplex LAMP assay showed 100% specificity for uninfected clinical samples and sensitivities of 90.6%, 86.89%, and 98.96% for LAMP kits against influenza A, influenza B, and SARS-CoV-2 clinical samples, respectively. Finally, the attribute agreement analysis for clinical tests indicated substantial agreement between the multiplex FluA/FluB/SARS-CoV-2/IC LAMP and commercial AllplexTM SARS-CoV-2/FluA/FluB/RSV assays.
RESUMEN
Coronavirus disease (COVID-19) caused by SARS-CoV-2 infection has been a global pandemic for more than two years, and it is important to quickly and accurately diagnose and isolate patients with SARS-CoV-2 infection. The BZ COVID-19 NALF Assay could sensitively detect SARS-CoV-2 from a nasopharyngeal swab because it adopts both a loop-mediated isothermal amplification and lateral flow immunochromatography technology. In this study, a total of 389 nasopharyngeal swab samples, of which 182 were SARS-CoV-2 PCR positive and 207 were negative samples, were recruited. Compared to the Allplex™ SARS-CoV-2 Assay, the BZ COVID-19 NALF Assay showed 95.05% sensitivity and 99.03% specificity for detecting SARS-CoV-2. The concordance rate between the BZ COVID-19 NALF Assay and Allplex™ SARS-CoV-2 Assay was 97.69%. The turnaround time of the BZ COVID-19 NALF Assay is only about 40~55 min. The BZ COVID-19 NALF Assay is an accurate, easy, and quick molecular diagnostic test compared to the conventional PCR test for detection of SARS-CoV-2. In addition, the BZ COVID-19 NALF Assay is thought to be very useful in small size medical facilities or developing countries where it is difficult to operate a clinical laboratory.
RESUMEN
To reduce the morbidity and mortality of candidemia patients through rapid treatment, the development of a simple, rapid molecular diagnostic method that is based on nucleic acid extraction and is superior to conventional methods for detecting Candida in the blood is necessary. We developed a multiplex Candida Pan/internal control (IC) loop-mediated isothermal amplification (LAMP) assay and a simple DNA extraction boiling protocol using Chelex-100 that could extract yeast DNA in blood within 20 min. The Chelex-100/boiling method for DNA extraction showed comparable efficiency to that of the commercial QIAamp UCP Pathogen Mini Kit using Candida albicans qPCR. In addition, the Candida Pan/IC LAMP assay showed superior sensitivity to that of general Candida Pan and species qPCRs against clinical DNA samples extracted with the QIAamp UCP Pathogen Mini Kit and Chelex-100/boiling method. The Candida Pan/IC LAMP assay followed by Chelex-100/boiling-mediated DNA extraction showed high sensitivity (100%) and specificity (100%) against clinical samples infected with Candida. These results suggest that the Candida Pan/IC LAMP assay could be used as a rapid molecular diagnostic test for candidemia.
RESUMEN
Malaria, caused by the parasite Plasmodium and transmitted by mosquitoes, is an epidemic that mainly occurs in tropical and subtropical regions. As treatments differ across species of malarial parasites, there is a need to develop rapid diagnostic methods to differentiate malarial species. Herein, we developed a multiplex malaria Pan/Pf/Pv/actin beta loop-mediated isothermal amplification (LAMP) to diagnose Plasmodium spp., P. falciparum, and P. vivax, as well as the internal control (IC), within 40 min. The detection limits of the multiplex malaria Pan/Pf/Pv/IC LAMP were 1 × 102, 1 × 102, 1 × 102, and 1 × 103 copies/µL for four vectors, including the 18S rRNA gene (Plasmodium spp.), lactate dehydrogenase gene (P. falciparum), 16S rRNA gene (P. vivax), and human actin beta gene (IC), respectively. The performance of the LAMP assay was compared and evaluated by evaluating 208 clinical samples (118 positive and 90 negative samples) with the commercial RealStar® Malaria S&T PCR Kit 1.0. The developed multiplex malaria Pan/Pf/Pv/IC LAMP assay showed comparable sensitivity (100%) and specificity (100%) with the commercial RealStar® Malaria S&T PCR Kit 1.0 (100%). These results suggest that the multiplex malaria Pan/Pf/Pv/IC LAMP could be used as a point-of-care molecular diagnostic test for malaria.
RESUMEN
Cell concentration is a critical process in biological assays and clinical diagnostics for the pre-treatment of extremely rare disease-related cells. The conventional technique for sample preconcentration and centrifugation has the limitations of a batch process requiring expensive and large equipment. Therefore, a high-throughput continuous cell concentration technique needs to be developed. However, in single-pass operation, the required concentration ratio is hard to achieve. In this study, we propose a closed-loop continuous cell concentration system using a viscoelastic non-Newtonian fluid. For miniaturized and integrated systems, two piezoelectric pumps were adopted. The pumping capability generated by a piezoelectric pump in a microfluidic channel was evaluated depending on the applied voltage, frequency, sample viscosity, and channel length. The concentration performance of the device was evaluated using 13 µm particles and white blood cells (WBCs) with different channel lengths and voltages. In the closed-loop system, the focused cells collected at the center outlet were sent back to the inlet, while the buffer solution was removed to the side outlets. Finally, to expand the clinical applicability of our closed-loop system, WBCs in lysed blood samples with 70% hematocrit and prostate cancer cells in urine samples were used. Using the closed-loop system, WBCs were concentrated by ~63.4 ± 0.8-fold within 20 min to a final volume of 160 µL using 10 mL of lysed blood sample with 70% hematocrit (~3 cP). In addition, prostate cancer cells in 10 mL urine samples were concentrated by ~64.1-fold within ~11 min due to low viscosity (~1 cP).
RESUMEN
Rapid and accurate identification of Candida albicans from among other candida species is critical for cost-effective treatment and antifungal drug assays. Shape is a critical biomarker indicating cell type, cell cycle, and environmental conditions; however, most microfluidic techniques have been focused only on size-based particle/cell manipulation. This study demonstrates a sheathless shape-based separation of particles/cells using a viscoelastic non-Newtonian fluid. The size of C. albicans was measured at 37 °C depending on the incubation time (0 h, 1 h, and 2 h). The effects of flow rates on the flow patterns of candida cells with different shapes were examined. Finally, 2-h-incubated candida cells with germ tube formations (≥26 µm) were separated from spherical candida cells and shorter candida cells with a separation efficiency of 80.9% and a purity of 91.2% at 50 µL/min.