scholarly journals Detection of Microorganisms in Body Fluids via MTT-PMS Assay

Diagnostics ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 46
Author(s):  
Cheng-Han Chen ◽  
Yu-Ting Tsao ◽  
Po-Ting Yeh ◽  
Yu-Hsiang Liao ◽  
Yi-Tzu Lee ◽  
...  
Keyword(s):  
Low Cost ◽  
Traditional Culture ◽  
Live Cells ◽  
Clinical Samples ◽  
Culture Methods ◽  
Bacterial Detection ◽  
Clinical Evaluations ◽  
Diphenyltetrazolium Bromide ◽  
Potential Tool ◽  
Rule Out

Early detection of microorganisms is essential for the management of infectious diseases. However, this is challenging, as traditional culture methods are labor-intensive and time-consuming. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-phenazine methosulfate (MTT-PMS) assay has been used to evaluate the metabolic activity in live cells and can thus be used for detecting living microorganisms. With the addition of NaOH and Tris-EDTA, the same approach can be accelerated (within 15 min) and used for the quick detection of common bacterial pathogens. The assay results can be evaluated colorimetrically or semi-quantitatively. Here, the quick detection by MTT-PMS assay was further investigated. The assay had a detection limit of approximately 104 CFU/mL. In clinical evaluations, we used the MTT-PMS assay to detect clinical samples and bacteriuria (>105 CFU/mL). The negative predictive value of the MTT-PMS assay for determining bacteriuria was 79.59% but was 100% when the interference of abnormal blood was excluded. Thus, the MTT-PMS assay might be a potential “rule-out” tool for bacterial detection in clinical samples, at a cost of approximately USD 1 per test. Owing to its low cost, rapid results, and easy-to-use characteristics, the MTT-PMS assay may be a potential tool for microorganism detection.

scholarly journals Comparison of culture methodology for the detection of methicillin-resistant Staphylococcus pseudintermedius in clinical specimens collected from dogs

2017 ◽  
Vol 30 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Matthew E. Saab ◽  
C. Anne Muckle ◽  
Henrik Stryhn ◽  
J. Trenton McClure
Keyword(s):  
High Risk ◽  
Traditional Culture ◽  
Culture Method ◽  
Confirmatory Test ◽  
Clinical Samples ◽  
Culture Methods ◽  
Staphylococcus Pseudintermedius ◽  
Mannitol Salt Agar ◽  
Methicillin Resistant ◽  
Oxacillin Resistance

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged as a major pathogen in dogs and has been implicated as a hospital-acquired pathogen in veterinary hospitals. We attempted to determine if selective culture methods will detect more MRSP when compared to the traditional culture methods in clinical samples from dogs in Atlantic Canada with a high risk for MRSP infection. Each sample was tested using 4 culture methods: traditional culture; mannitol salt agar with 2 μg/mL of oxacillin (MSAox); enrichment broth (EB) with MSAox; and EB with traditional culture. Detection of penicillin-binding protein 2’, via latex agglutination, was used as a confirmatory test for oxacillin resistance. We analyzed 741 samples from 556 dogs between February 2013 and April 2014. The prevalence of MRSP in samples detected by any method was estimated at 13.4% (95% CI: 11.1–16.0%). When the prevalence of MRSP was determined according to culture method, EB with MSAox detected the highest prevalence (11.2% [9.1–13.7%]), followed by EB with traditional (10.8% [8.8–13.2%]), traditional (10.1% [8.1–12.5%]), and MSAox (8.9% [7.1–11.2%]). The prevalence using the traditional culture method did not differ significantly from any of the 3 selective culture methods. Culture with MSAox detected significantly fewer MRSP than either of the EB methods. The addition of EB to current methodology is recommended, particularly for patients considered at high risk for MRSP infection.

scholarly journals Molecular detection and identification of Enterococcus faecium isolated from dental root canals

2021 ◽  
pp. 1447-1451
Author(s):  
Eman A. Mustafa ◽  
Suhad M. Hamdoon ◽  
Enas Y. Shehab
Keyword(s):  
Root Canal ◽  
Enterococcus Faecium ◽  
Brain Heart Infusion ◽  
Traditional Culture ◽  
Clinical Samples ◽  
Biochemical Tests ◽  
Culture Methods ◽  
Oral Infections ◽  
Root Canals ◽  
Infected Root

Enterococci are usually encountered and predominate in oral infections, especially those associated with dental root canal infections of necrotic pulp and periodontitis. This study aimed to detect and identify Enterococcus faecium isolated from infected root canals, using polymerase chain reaction ( PCR). Thirty samples were collected from patients with  necrotic pulp, infected root canals, and endodontic treatment failure, attending the Conservative Treatment Department, College of Dentistry, Mosul University, Dental Teaching Hospital. The samples were obtained by inserting sterile paper points into the root canals and transferred in brain heart infusion broth vials to be  inoculated in a selective M-Enterococcus Agar Base . Twenty five isolates that belong to the genus Enterococcus were   recognized by traditional culture methods and biochemical tests. Then, DNA extractions of these isolates were carried out for  identification  with PCR by the amplification of ddI (D-Ala-D-Ala Ligase) chromosomal genes of Enterococcus faecium. Among the 25  isolates, twenty (80%) were identified to the level of Enterococcus faecium by traditional culture methods and biochemical tests, in comparison to 17 (68%) identified by  molecular identification. The PCR products for the specific primer produced bands on agarose gel at the position of 658bp. The study showed that the use of PCR with primers for the E. faecium ddI gene may be the most accurate method for rapid identification of Enterococci. Molecular identification of Enterococcus spp. revealed a significant role of E. feacium in root canal infections. Also, the detection of ddI gene  using PCR provides a definitive target that could be used for the detection of E. faecium  from clinical samples.

scholarly journals RT-qPCR Assays for Rapid Detection of the N501Y, 69-70del, K417N, and E484K SARS-CoV-2 Mutations: A Screening Strategy to Identify Variants With Clinical Impact

2021 ◽  
Vol 11 ◽  
Author(s):  
Natali Vega-Magaña ◽  
Rocío Sánchez-Sánchez ◽  
Jorge Hernández-Bello ◽  
Alberto Antony Venancio-Landeros ◽  
Marcela Peña-Rodríguez ◽  
...  
Keyword(s):  
Biological Effects ◽  
Low Cost ◽  
Gene Mutations ◽  
High Specificity ◽  
Screening Strategy ◽  
Clinical Impact ◽  
Mexican Population ◽  
Rapid Screening ◽  
Clinical Samples ◽  
Reverse Transcription Pcr

BackgroundSeveral variants of the SARS-CoV-2 have been documented globally during the current COVID-19 pandemic. The N501Y, 69-70del, K417N, and E484K SARS-CoV-2 mutations have been documented among the most relevant due to their potential pathogenic biological effects. This study aimed to design, validate, and propose a fast real-time RT-qPCR assay to detect SARS-CoV-2 mutations with possible clinical and epidemiological relevance in the Mexican population.MethodsTargeting spike (S) gene mutations of SARS-CoV-2 (N501Y, 69-70del, K417N, and E484K), specific primers, and probes for three specific quantitative reverse transcription PCR (RT-qPCR) assays were designed, and validated using Sanger sequencing. These assays were applied in clinical samples of 1060 COVID-19 patients from Jalisco Mexico.ResultsIn silico analyzes showed high specificity of the three assays. Amplicons of samples were confirmed through sequencing. The screening of samples of COVID-19 patients allowed the identification of the E484K mutation in nine individuals and the identification of P.2 Brazilian variant in Mexico.ConclusionThis work provides low-cost RT-qPCR assays for rapid screening and molecular surveillance of mutations with potential clinical impact. This strategy allowed the detection of E484K mutation and P.2 variant for the first time in samples from the Mexican population.

scholarly journals LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus

2021 ◽  
Author(s):  
Bo YANG ◽  
zhengwang shi ◽  
Yuan Ma ◽  
Lijuan Wang ◽  
Liyan Cao ◽  
...  
Keyword(s):  
Real Time ◽  
Detection System ◽  
African Swine Fever Virus ◽  
Low Cost ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Portable Detection ◽  
Real Time Qpcr

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a system was established in one tube for the detection of the ASFV p72 gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporters and CRISPR-derived RNA (crRNAs) were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples, a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. In the current study, a LAMP coupled with the CRISPR detection method was developed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

Clinical applications of spectroscopic techniques in conjunction with multivariate analysis in virus diagnosis

2021 ◽  
pp. 1-27
Author(s):  
Marfran C. D. Santos ◽  
João V. M. Mariz ◽  
Raissa V. O. Silva ◽  
Camilo L. M. Morais ◽  
Kássio M. G. Lima
Keyword(s):  
Multivariate Analysis ◽  
Near Infrared ◽  
Low Cost ◽  
Diagnostic Techniques ◽  
Clinical Samples ◽  
Spectroscopic Techniques ◽  
Global Pandemic ◽  
Molecular Fluorescence ◽  
Viral Diagnosis ◽  
Near Future

In view of the global pandemic that started in 2020, caused by COVID-19, the importance of the existence of fast, reliable, cheap diagnostic techniques capable of detecting the virus even in the first days of infection became evident. This review discusses studies involving the use of spectroscopic techniques in the detection of viruses in clinical samples. Techniques based on mid-infrared, near-infrared, Raman, and molecular fluorescence are explained and it was demonstrated how they can be used in conjunction with computational tools of multivariate analysis to build models capable of detecting viruses. Studies that used real clinical samples from 2011 to 2021 were analyzed. The results demonstrate the potential of the techniques in detecting viruses. Spectroscopic techniques, as well as chemometric techniques, were also explained. Viral diagnosis based on spectroscopy has interesting advantages compared to standard techniques such as: fast results, no need for reagents, non-destructiveness for the sample, no need for sample preparation, relatively low cost, among others. Several studies have corroborated the real possibility that, in the near future, we may have spectroscopic tools being successfully applied in viral diagnosis.

scholarly journals A Simple Off-Grid Incubator for Microbiological Water Quality Analysis

Water ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 240 ◽  
Author(s):  
Carolina Bernardes ◽  
Ricardo Bernardes ◽  
Camille Zimmer ◽  
Caetano C. Dorea
Keyword(s):  
Water Quality ◽  
Low Cost ◽  
Expanded Polystyrene ◽  
Hot Water ◽  
Quality Analysis ◽  
Health Concern ◽  
Culture Methods ◽  
Conventional Culture ◽  
Quality Testing ◽  
Microbiological Water Quality

There is a need for accessible and low-cost microbiological water quality testing in contexts where diarrheal illness is a major public health concern. In most cases, the quantification of Escherichia coli and other microbial indicators by conventional culture methods requires an incubation step for processed samples at specific temperatures for bacterial growth over a prescribed time. However, incubators can be the most expensive equipment required for such microbial analyses, limiting the number and scope of water quality testing available in low-resource contexts. In this study, a low-cost incubator was developed using a locally available expanded polystyrene (EPS) foam cooler, with two water bottles filled with hot water to heat incubator to a target of 35 °C. The EPS incubator performance was validated by processing 150 water samples in duplicates using the Colilert Quanti-tray/2000 system, incubated in either the EPS incubator or a standard laboratory incubator set at 35 °C. Statistically significant correlations of results indicated that the quantification of E. coli was comparable between both methods. Risk categorizations from standard and EPS incubation results agreed for 141 of 150 (94%) samples, with zero false negatives. In addition to being reasonably mobile the EPS incubator would reduce the cost of such water quality testing, thus potentially increasing the scope of water quality testing coverage.

scholarly journals Crocetin Isolated from the Natural Food Colorant Saffron Reduces Intracellular Fat in 3T3-L1 Adipocytes

Foods ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1648
Author(s):  
Elena Jiménez-Ortega ◽  
Aitana Braza-Boïls ◽  
Miguel Burgos ◽  
Natalia Moratalla-López ◽  
Manuel Vicente ◽  
...  
Keyword(s):  
Lipid Accumulation ◽  
Low Cost ◽  
Natural Food ◽  
Peroxisome Proliferator ◽  
Synthetic Dyes ◽  
Potential Candidate ◽  
Peroxisome Proliferator Activated Receptor ◽  
Food Colorant ◽  
Enhancer Binding Protein ◽  
Diphenyltetrazolium Bromide

Saffron, as a food colorant, has been displaced by low-cost synthetic dyes. These have unhealthy properties; thus, their replacement with natural food colorants is an emerging trend. Obesity is a worldwide health problem due to its associated comorbidities. Crocetin esters (crocins) are responsible for the red saffron color. Crocetin (CCT) exhibits healthful properties. We aimed to broaden the existing knowledge on the health properties of CCT isolated from saffron, to facilitate its consideration as a healthy natural food colorant in the future. We evaluated the ability of CCT (1 and 5 μM) to reduce lipid accumulation during the differentiation of 3T3-L1 preadipocytes. Intracellular fat was quantified by Oil Red O staining. CTT cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The number and size of lipid droplets were analyzed using WimLipid software. The expression of adipogenic genes (CCAAT/enhancer-binding protein (C/EBPβ, C/EBPδ, C/EBPα), and peroxisome proliferator-activated receptor γ (PPARγ)) was analyzed using quantitative real-time PCR (qRT-PCR). CCT 5 μM decreased intracellular fat by 22.6%, without affecting viability or lipid droplet generation, via a decrease in C/EBPα expression, implicated in lipid accumulation. Thus, CCT is a potential candidate to be included in dietary therapies aimed at reversing adipose tissue accumulation in obesity.

A Low-Cost Electrochemical Biosensor for Rapid Bacterial Detection

2011 ◽  
Vol 11 (1) ◽  
pp. 210-216 ◽  
Author(s):  
Vivek Nandakumar ◽  
Daniel Bishop ◽  
Eric Alonas ◽  
Jeffrey LaBelle ◽  
Lokesh Joshi ◽  
...  
Keyword(s):  
Electrochemical Biosensor ◽  
Low Cost ◽  
Bacterial Detection

scholarly journals Fringe optimization for structured illumination super-resolution microscope with digital micromirror device

2019 ◽  
Vol 12 (03) ◽  
pp. 1950014 ◽  
Author(s):  
Xibin Yang ◽  
Qian Zhu ◽  
Zhenglong Sun ◽  
Gang Wen ◽  
Xin Jin ◽  
...  
Keyword(s):  
Modulation Depth ◽  
Low Cost ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Live Cells ◽  
Digital Micromirror Device ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Wide Range ◽  
Fringe Patterns

Structured illumination microscopy (SIM) is a promising super-resolution technique for imaging subcellular structures and dynamics due to its compatibility with most commonly used fluorescent labeling methods. Structured illumination can be obtained by either laser interference or projection of fringe patterns. Here, we proposed a fringe projector composed of a compact multi-wavelength LEDs module and a digital micromirror device (DMD) which can be directly attached to most commercial inverted fluorescent microscopes and update it into a SIM system. The effects of the period and duty cycle of fringe patterns on the modulation depth of the structured light field were studied. With the optimized fringe pattern, [Formula: see text] resolution improvement could be obtained with high-end oil objectives. Multicolor imaging and dynamics of subcellular organelles in live cells were also demonstrated. Our method provides a low-cost solution for SIM setup to expand its wide range of applications to most research labs in the field of life science and medicine.

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