3-step Protocol Research Articles

Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices

Poly(methyl methacrylate) (PMMA) microfluidic devices have become promising platforms for a wide range of applications. Here we report a simple method for immobilising histidine-tagged enzymes suitable for PMMA microfluidic devices. The 1-step-immobilisation described is based on the affinity of the His-tag/Ni-NTA interaction and does not require prior amination of the PMMA surface, unlike many existing protocols. We compared it with a 3-step immobilisation protocol involving amination of PMMA and linking NTA via a glutaraldehyde cross-linker. These methods were applied to immobilise transketolase (TK) in PMMA microfluidic devices. Binding efficiency studies showed that about 15% of the supplied TK was bound using the 1-step method and about 26% of the enzyme was bound by the 3-step method. However, the TK-catalysed reaction producing l-erythrulose performed in microfluidic devices showed that specific activity of TK in the device utilising the 1-step immobilisation method was approximately 30% higher than that of its counterpart. Reusability of the microfluidic device produced via the 1-step method was tested for three cycles of enzymatic reaction and at least 85% of the initial productivity was maintained. The device could be operated for up to 40 h in a continuous flow and on average 70% of the initial productivity was maintained. The simplified immobilisation method required fewer chemicals and less time for preparation of the immobilised microfluidic device compared to the 3-step method while achieving higher specific enzyme activity. The method represents a promising approach for the development of immobilised enzymatic microfluidic devices and could potentially be applied to combine protein purification with immobilisation.

A comprehensive in vitro and in vivo metabolism study of hydroxysafflor yellow A.

As the most important marker component in Carthamus tinctorius L., hydroxysafflor yellow A (HSYA) was widely used in the prevention and treatment of cardiovascular diseases, due to its effect of improving blood supply, suppressing oxidative stress, and protecting against ischemia/reperfusion. In this paper, both an in vitro microsomal incubation and an in vivo animal experiment were conducted, along with an LC-Q-TOF/MS instrument and a 3-step protocol, to further explore the metabolism of HSYA. As a result, a total of 10 metabolites were searched and tentatively identified in plasma, urine, and feces after intravenous administration of HSYA to male rats, although no obvious biotransformation was found in the simulated rat liver microsomal system. The metabolites detected involving both phase I and phase II metabolism including dehydration, deglycosylation, methylation, and glucuronic acid conjugation. A few of the metabolites underwent more than one-step metabolic reactions, and some have not been reported before. The study would contribute to a further understanding of the metabolism of HSYA and provide scientific evidence for its pharmacodynamic mechanism research and clinical use.

Transcriptome Analysis of Induced Pluripotent Stem Cell (iPSC)-derived Pancreatic β-like Cell Differentiation

Diabetes affects millions of people worldwide, and β-cell replacement is one of the promising new strategies for treatment. Induced pluripotent stem cells (iPSCs) can differentiate into any cell type, including pancreatic β cells, providing a potential treatment for diabetes. However, the molecular mechanisms underlying the differentiation of iPSC-derived β cells have not yet been fully elucidated. Here, we generated pancreatic β-like cells from mouse iPSCs using a 3-step protocol and performed deep RNA sequencing to get a transcriptional landscape of iPSC-derived pancreatic β-like cells during the selective differentiation period. We then focused on the differentially expressed genes (DEGs) during the time course of the differentiation period, and these genes underwent Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis. In addition, gene-act networks were constructed for these DEGs, and the expression of pivotal genes detected by quantitative real-time polymerase chain reaction was well correlated with RNA sequence (RNA-seq). Overall, our study provides valuable information regarding the transcriptome changes in β cells derived from iPSCs during differentiation, elucidates the biological process and pathways underlying β-cell differentiation, and promotes the identification and functional analysis of potential genes that could be used for improving functional β-cell generation from iPSCs.

Lipidomic profiling of patient-specific induced pluripotent stem cell-derived hepatocyte-like cells

ABSTRACTHepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem cells (iPSCs) offer an alternative model to primary human hepatocytes to study lipid aberrations. However, the detailed lipid profile of HLCs is yet unknown. In the current study, functional HLCs were differentiated from iPSCs generated from dermal fibroblasts of three individuals by a three-step protocol through the definitive endoderm (DE) stage. In parallel, detailed lipidomic analyses as well as gene expression profiling of a set of lipid-metabolism-related genes were performed during the entire differentiation process from iPSCs to HLCs. Additionally, fatty acid (FA) composition of the cell culture media at different stages was determined. Our results show that major alterations in the molecular species of lipids occurring during DE and early hepatic differentiation stages mainly mirror the quality and quantity of the FAs supplied in culture medium at each stage. Polyunsaturated phospholipids and sphingolipids with a very long FA were produced in the cells at a later stage of differentiation. This work uncovers the previously unknown lipid composition of iPSC-HLCs and its alterations during the differentiation in conjunction with the expression of key lipid-associated genes. Together with biochemical, functional and gene expression measurements, the lipidomic analyses allowed us to improve our understanding of the concerted influence of the exogenous metabolite supply and cellular biosynthesis essential for iPSC-HLC differentiation and function. Importantly, the study describes in detail a cell model that can be applied in exploring, for example, the lipid metabolism involved in the development of fatty liver disease or atherosclerosis.

In Vitro Efficacy of XP-endo Finisher with 2 Different Protocols on Biofilm Removal from Apical Root Canals

IntroductionThe purpose of this study was to evaluate the effectiveness of the XP-endo Finisher (XPF; FKG Dentaire SA, La Chaux-de-Fonds, Switzerland) in biofilm removal in comparison with conventional needle irrigation (CNI) and passive ultrasonic irrigation (PUI) using an infected tooth model with an artificial apical groove. MethodsFifty-four extracted human single-rooted premolars were selected. Each tooth was split longitudinally into 2 halves, with a groove made in the apical segment of the canal wall. After growing mixed bacteria biofilm for 4 weeks, the split halves were reassembled and instrumented using Vortex Blue files (Dentsply Tulsa Dental, Tulsa, OK) to size 40/.06. The instrumented teeth were randomly assigned to 6 groups (n = 8) according to the final irrigation protocol. Three different techniques (CNI, PUI, and XPF) were performed each with either continuous irrigation or 3-step irrigation. Scanning electron microscopic images were taken to evaluate the amount of residual biofilm inside and outside the groove. ResultsRobust growth of biofilm was observed in each canal of the controls after 4 weeks. XPF showed the best biofilm removal efficacy inside and outside the groove followed by PUI and CNI (P < .05). The XPF 2 group using the 3-step protocol showed better antibiofilm efficiency than the XPF 1 group with continuous irrigation inside the groove (P < .05). ConclusionsThe XP-endo Finisher, as an irrigation agitation technique, may help to remove biofilm from hard-to-reach areas in the root canal system. The 3-step irrigation protocol was more effective than continuous irrigation when XPF was used.

Identifying Allergic Drug Reactions Through Placebo-Controlled Graded Challenges

Graded challenges are performed to exclude hypersensitivity reactions in patients with a low likelihood of drug allergy. Literature regarding optimal protocols with a defined number of steps and use of placebo is lacking. To identify allergic drug reactions (ADRs) through a 3-step protocol composed of placebo followed by a 2-step graded drug challenge. We performed a 5-year retrospective chart review of all patients with historical ADRs who underwent single-blind, placebo-controlled graded drug challenges between October 2010 and November 2015 at an outpatient drug allergy clinic. Patients' demographic characteristics and description of historical reaction were obtained. Outcomes of challenges to drug versus placebo were compared by drug class. Two hundred twenty-nine patients underwent at least 1 single-blind placebo-controlled graded challenge. The most commonly challenged drug class was beta-lactams (70.8%) followed by nonsteroidal anti-inflammatory drugs (17.5%). The reaction rate to drug and placebo was similar during beta-lactam challenges (9.4% vs 8.2%; P= .9) and during nonsteroidal anti-inflammatory drug challenges (14% vs 7%, P= .5), respectively. Only 10 patients (4.4%) had objective findings during drug challenges. Patients who reacted to placebo before beta-lactam challenges had an increased number of drug allergies (4.3 ± 1.0) compared with nonreactors (2.4 ± 0.1) and to beta-lactam reactors (3.3 ± 0.7) (P= .002). All placebo reactors were female (20 of 183 vs 0 of 46 males; P= .02). Two-step graded challenges are safe in appropriately selected patients with a low risk of reaction. Placebo should be considered to reduce false-positive results, especially in females and in patients with multiple drug allergies.

Managing weeds during wildflower meadow establishment in the arid Intermountain West: efficacy of a grass-first strategy for sites with heavy annual weed pressure

The creation of attractive, sustainable wildflower meadows in private and public green spaces is becoming increasingly popular. Establishment failure rate for new meadow plantings can be very high, primarily because of the initial annual weed pressure. We proposed a 3-step grass-first establishment protocol based on plant augmentative succession principles: 1) spring planting of grass component species; 2) application of standard turf-appropriate weed-control methods; and 3) early fall overseeding or outplanting of forbs into the established grasses. This grass-first meadow establishment protocol was successful, regardless of standard weed-control method employed (mowing; 2,4-D application; or application of a three-way pre-mix containing mecoprop, 2,-4-D, and dicamba [Ortho Weed B Gon]). Among the weed-control treatments, weed density during the establishment year did not affect ultimate meadow establishment success, as long as weeds were controlled sufficiently to allow grass and forb survival. Seeding resulted in a greater density of forbs and, ultimately, an overall more aesthetically pleasing mix of flowering plants and grasses as compared to outplanting. Among the grasses in the species mix, slender wheatgrass ( Elymus trachycaulus (Link) Gould ex Shinners [Poaceae]) was dominant. Among the forb species, 5 Asteraceae species persisted over the period of the study and contributed good color to the meadow plantings. An augmented succession protocol based on grass-first establishment will be valuable where native meadow plantings are desired for urban habitat development and beautification.

The All-Chemical Approach: A Solution for Converting Fibroblasts Into Myocytes.

Conversion of Human Fibroblasts Into Functional Cardiomyocytes by Small Molecules Cao et al Science . 2016;352:1216–1220. Converting cardiac fibroblasts to cardiomyocytes has been considered as a regenerative strategy for myocardial infarction and other disorders. Recently, Cao et al1 defined a cocktail of 9 chemical compounds with this capability, increasing the likelihood of clinical success. It has taken a while, but the concept that the terminally differentiated state is immutably stable no longer dominates modern biology. In 1938, Hans Spemann contemplated a fantastical experiment in which transfer of an egg nucleus could redirect a recipient somatic cell to become pluripotent, and Gurdon2 made this a reality in 1958 by converting gut epithelial cells into whole frogs. Inspired by Gurdon et al,2 Takahashi and Yamanaka3 developed the induced pluripotent stem cell (iPSC) technology where 4 transcription factors sufficed in generating PSCs. The last decade has witnessed an explosion in cell fate manipulations. Many somatic cell types were interconverted by the means of transcription factors and epigenetic and cellular pathway modulators. As one of the most difficult cells to regenerate, cardiomyocyte was successfully generated from fibroblasts by introducing cocktails of transcriptions factors.4–7 In a recent article in Science , Cao et al1 reported the successful reprograming of functional cardiomyocytes by small molecules and growth factors. Cao et al1 devised a 3-step protocol. Human foreskin fibroblasts or human …

The cytoprotective capacity of processed human cardiac extracellular matrix.

Freshly isolated human cardiac extracellular matrix sheets (cECM) have been shown to support stem cell proliferation and tissue-specific lineage commitment. We now developed a protocol for standardized production of durable, bio-functional hcECM microparticles and corresponding hydrogel, and tested its cytoprotective effects on contractile cells subjected to ischemia-like conditions. Human ventricular myocardium was decellularized by a 3-step protocol, including Tris/EDTA, SDS and serum incubation (cECM). Following snap-freezing and lyophilization, microparticles were created and characterized by laser diffraction, dynamic image analysis (DIA), and mass spectrometry. Moreover, cECM hydrogel was produced by pepsin digestion. Baseline cell-support characteristics were determined using murine HL-1 cardiomyocytes, and the cytoprotective effects of ECM products were tested under hypoxia and glucose/serum deprivation. In cECM, glycoproteins (thrombospondin 1, fibronectin, collagens and nidogen-1) and proteoglycans (dermatopontin, lumican and mimecan) were preserved, but residual intracellular and blood-borne proteins were also detected. The median particle feret diameter was 66μm (15-157μm) by laser diffraction, and 57μm (20-182μm) by DIA with crystal violet staining. HL-1 cells displayed enhanced metabolic activity (39±12%, P<0.05) and proliferation (16±3%, P<0.05) when grown on cECM microparticles in normoxia. During simulated ischemia, cECM microparticles exerted distinct cytoprotective effects (MTS conversion, 240±32%; BrdU uptake, 45±14%; LDH release, -72±7%; P<0.01, each). When cECM microparticles were solubilized to form a hydrogel, the cytoprotective effect was initially abolished. However, modifying the preparation process (pepsin digestion at pH 2 and 25°C, 1mg/ml final cECM concentration) restored the cytoprotective cECM activity. Extracellular matrix from human myocardium can be processed to yield standardized durable microparticles that exert specific cytoprotective effects on cardiomyocyte-like cells. The use of processed cECM may help to optimize future clinical-grade myocardial tissue engineering approaches.

Semimembranosus Release for Medial Soft Tissue Balancing Does Not Weaken Knee Flexion Strength in Patients Undergoing Varus Total Knee Arthroplasty

BackgroundThe sequential medial release technique including semimembranosus (semiM) release is effective and safe during varus total knee arthroplasty (TKA). However, there are concerns about weakening of knee flexion strength after semiM release. We determined whether semiM release to balance the medial soft tissue decreased knee flexion strength after TKA. MethodsFifty-nine consecutive varus knees undergoing TKA were prospectively enrolled. A 3-step sequential release protocol which consisted of release of (1) the deep medial collateral ligament (dMCL), (2) the semiM, and (3) the superficial medial collateral ligament based on medial tightness. Gap balancing was obtained after dMCL release in 31 knees. However, 28 knees required semiM release or more after dMCL release. Isometric muscle strength of the knee was compared 6 months postoperatively between the semiM release and semiM nonrelease groups. Knee stability and clinical outcomes were also compared. ResultsNo differences in knee flexor or extensor peak torque were observed between the groups 6 months postoperatively (P = .322 and P = .383, respectively). No group difference was observed in medial joint opening angle on valgus stress radiographs (P = .327). No differences in the Knee Society or Western Ontario and McMaster Universities Osteoarthritis Index scores were detected between the groups (P = .840 and P = .682, respectively). ConclusionThese results demonstrate that semiM release as a sequential step to balance medial soft tissue in varus knees did not affect knee flexion strength after TKA.

Fast and efficient three-step target-specific curing of a virulence plasmid in Salmonella enterica.

Virulence plasmids borne by serovars of Salmonella enterica carry genes involved in its pathogenicity, as well as other functions. Characterization of phenotypes associated with virulence plasmids requires a system for efficiently curing strains of their virulence plasmids. Here, we developed a 3-step protocol for targeted curing of virulence plasmids. The protocol involves insertion of an I-SecI restriction site linked to an antibiotic resistance gene into the target plasmid using λ-Red mutagenesis, followed by the transformation with a temperature-sensitive auxiliary plasmid which carries I-SecI nuclease expressed from a tetracycline-inducible promoter. Finally, the auxiliary plasmid is removed by incubation at 42 °C and the plasmid-less strains are verified on antibiotic-containing media. This method is fast and very efficient: over 90 % of recovered colonies lacked their virulence plasmid.

Pressure Relief, Visco-Elastic Foam with Inflated Air? A Pilot Study in a Dutch Nursing Home.

Objective: There is still little evidence regarding the type of mattress that is the best for preventing pressure ulcers (PUs). In a Dutch nursing home, a new type of overlay mattress (air inflated visco-elastic foam) was tested to analyze the opportunity for replacement of the normally used static air overlay mattress in its three-step PU prevention protocol In this small pilot the outcome measures were: healing of a category one pressure ulcer, new development or deterioration of a category one PU and need for repositioning. Methods: We included 20 nursing home residents with a new category one pressure ulcer, existing for no longer than 48 h following a consecutive sampling technic. All residents were staying for more than 30 days in the nursing home and were lying on a visco-elastic foam mattress without repositioning (step one of the 3-step protocol) at the start of the pilot study. They had not suffered from a PU in the month before. The intervention involved use of an air inflated foam overlay instead of a static air overlay (normally step 2 of the 3-step protocol). At the start; the following data were registered: age; gender; main diagnosis and presence of incontinence. Thereafter; all participating residents were checked weekly for PU healing tendency; deterioration of PUs; new PUs and need of repositioning. Only when residents showed still a category one PU after 48 h or deterioration of an existing pressure ulcer or if there was development of a new pressure ulcer, repositioning was put into practice (step 3 of the PU protocol). All residents participated during 8 weeks. Results: Seven residents developed a new pressure ulcer category one and still had a category one pressure ulcer at the end of the study period. One resident developed a pressure ulcer category 2. Fifteen residents needed repositioning from one week after start of the study until the end of the study. Conclusions: Overall 40% of the residents developed a pressure ulcer. Seventy five percent of the residents started with repositioning because there was no healing tendency of their category one PU diagnosed at the start of the pilot. Because this new type of overlay mattress resulted in an increased PU incidence, and almost standard need of repositioning with accompanied high costs, this type of overlay mattress gives no benefit above the traditional visco-elastic foam mattresses in combination with the originally used static air overlay.

Isolation and characterization of recombinant murine Wnt3a

Wnt proteins are a family of morphogens that possess potent biological activity. Structure–function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu2+-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30mM methyl-ß-cyclodextrin (MßCD). Wnt3a recovered in MßCD-containing buffer was soluble and biologically active. Insofar as MßCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt–cyclodextrin complexes may facilitate structure–activity studies in the absence of adverse detergent effects.

Late-life cynical distrust, risk of incident dementia, and mortality in a population-based cohort.

We investigated the association between late-life cynical distrust and incident dementia and mortality (mean follow-up times of 8.4 and 10.4 years, respectively) in the Cardiovascular Risk Factors, Aging and Dementia Study. Cynical distrust was measured based on the Cook-Medley Scale and categorized into tertiles. Cognitive status was evaluated with a 3-step protocol including screening, clinical phase, and differential diagnostic phase. Dementia was diagnosed according to DSM-IV criteria. Complete data on exposure, outcome, and confounders were available from 622 persons (46 dementia cases) for the dementia analyses and from 1,146 persons (361 deaths) for the mortality analyses. Age, sex, systolic blood pressure, total cholesterol, fasting glucose, body mass index, socioeconomic background, smoking, alcohol use, self-reported health, and APOE genotype were considered as confounders. Cynical distrust was not associated with dementia in the crude analyses, but those with the highest level of cynical distrust had higher risk of dementia after adjusting for confounders (relative risk 3.13; 95% confidence interval [CI] 1.15-8.55). Higher cynical distrust was associated with higher mortality in the crude analyses (hazard ratio 1.40; 95% CI 1.05-1.87) but the association was explained by confounders (adjusted hazard ratio 1.19; 95% CI 0.86-1.61). Higher cynical distrust in late life was associated with higher mortality, but this association was explained by socioeconomic position, lifestyle, and health status. Association between cynical distrust and incident dementia became evident when confounders were considered. This novel finding suggests that both psychosocial and lifestyle-related risk factors may be modifiable targets for interventions. We acknowledge the need for larger replication studies.

A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers

• We present a detailed 3-step protocol for live imaging of polarized primary RPE . • Step 1: a protocol for primary porcine retinal pigment epithelial culture. • This method results in highly polarized monolayers within 7 days. • Step 2: two gene delivery protocols that yield efficient transgene expression. • Step 3: High speed live imaging to visualize intracellular traffic in the RPE.

Drainage of pleural effusion in mechanically ventilated patients: Time to measure chest wall compliance?

PurposePleural effusion (PE) is commonly encountered in mechanically ventilated, critically ill patients and is generally addressed with evacuation or by fluid displacement using increased airway pressure (PAW). However, except when massive or infected, clear evidence is lacking to guide its management. The aim of this study was to investigate the effect of recruitment maneuvers and drainage of unilateral PE on respiratory mechanics, gas exchange, and lung volume. Materials and methodsFifteen critically ill and mechanically ventilated patients with unilateral PE were enrolled. A 3-step protocol (baseline, recruitment, and effusion drainage) was applied to patients with more than 400 mL of PE, as estimated by chest ultrasound. Predefined subgroup analysis compared patients with normal vs reduced chest wall compliance (CCW). Esophageal and PAWs, respiratory system, lung and CCWs, arterial blood gases, and end-expiratory lung volumes were recorded. ResultsIn the whole case mix, neither recruitment nor drainage improved gas exchange, lung volume, or tidal mechanics. When CCW was normal, recruitment improved lung compliance (81.9 [64.8-104.1] vs 103.7 [91.5-111.7] mL/cm H2O, P < .05), whereas drainage had no significant effect on total respiratory system mechanics or gas exchange, although it measurably increased lung volume (1717 vs 2150 mL, P < .05). In the setting of reduced CCW, however, recruitment had no significant effect on total respiratory system mechanics or gas exchange, whereas pleural drainage improved respiratory system and CCWs as well as lung volume (42.7 [38.9-50.0] vs 47.0 [43.8-63.3], P < .05 and 97.4 [89.3-97.9] vs 126.7 [92.3-153.8] mL/cm H2O, P < .05 and 1580 vs 1750 mL, P < .05, respectively). ConclusionsDrainage of a moderate-sized effusion should not be routinely performed in unselected population of critically ill patients. We suggest that measurement of CCW may help in the decision-making process.

Dimethyl homophthalates to naphthopyrans: the total synthesis of arnottin I and the formal synthesis of (−)-arnottin II

A simple and efficient 3-step synthetic protocol has been reported for dimethyl homophthalates to naphthopyrans. Starting from dimethyl 2,3-dimethoxyhomophthalate, a practical synthesis of arnottin I has been described via a base catalyzed mono-alkylation, the selective hydrolysis of an aliphatic ester moiety, two consecutive intramolecular cyclizations and an oxidative aromatization pathway with a very good overall yield. The involved intramolecular acylation followed by an associated enolative lactonization was the decisive step. The synthesis of dihydroarnottin I also completes the formal synthesis of (−)-arnottin II.

Five treatments nurses can better direct

Specimen contamination generates unreliable results, leading to potential misdiagnosis and improper treatments. The purpose of this multidisciplinary continuous quality improvement project was to implement an evidence-based diagnostic stewardship program to reduce urine specimen contamination rates. We conducted educational in-service sessions introducing a 3-step preanalytical protocol within the emergency department. Pre- and postintervention chart review was used to evaluate the impact on urine contamination. Urine culture contamination rates were significantly reduced between the pre- and postintervention phases (χ2 = 3.78, P = .05). An evidence-based preanalytical protocol supplemented with an educational intervention reduced the contamination of urine specimens.

3-Step vs 2-step protocol for difficult biliary access with frequent pancreatic cannulation

3-Step vs 2-step protocol for difficult biliary access with frequent pancreatic cannulation

Detection of viruses directly from the fresh leaves of a Phalaenopsis orchid using a microfluidic system

Early detection of pathogens is crucial for the effective surveillance of diseases. Many efforts have been made to explore methods which can detect these pathogens within a short period of time without requiring a tedious protocol. However, these developed methods have disadvantages such as they are relatively time-consuming or require specialized laboratory facilities. In this work, we have developed an integrated microfluidic system for rapid and automatic detection of viruses by direct analysis from fresh Phalaenopsis orchid leaves. The entire protocol, including ribonucleic acid (RNA) purification, reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) and optical detection by measuring changes in turbidity was performed on a single chip. This is the first time that an integrated microfluidic system for the detection of viruses infecting the Phalaenopsis orchid has been demonstrated. The sensitivity of the developed system was also explored in this study to validate its performance. From the Clinical EditorIn this study, the authors report the development of an integrated microfluidic system for rapid and automatic detection of viruses by direct analysis of fresh Phalaenopsis orchid leaves, performing the 3-step protocol using a single chip. Similar methods may find clinical application for fast and accurate detection of viral infections.