scholarly journals Ratiometric Electrochemical Biosensor Based on Internally Controlled Duplex PCR for Detection of Mycobacterium Tuberculosis

2022 ◽  
Author(s):  
SASINEE BUNYARATAPHAN ◽  
Therdsak Prammananan ◽  
Deanpen Japrung
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Internal Control ◽  
Pathogenic Bacteria ◽  
False Negative ◽  
Duplex Pcr ◽  
Self Assembled Monolayer ◽  
Extrinsic Factors ◽  
Detection Range ◽  
Wave Voltammetry ◽  
C Terminus

Abstract The pathogenic bacteria Mycobacterium tuberculosis (MTB) is responsible for tuberculosis, which is well known as the globally leading cause of death. The likelihood of false negative interpretation as well as potential influence from intrinsic and extrinsic factors are considerably minimized by the incorporation of internal control (IC) detection in the developed assay platform. Ratiometric electrochemical (REC) biosensor for detection of MTB was developed based on the IC integration via duplex PCR (dPCR) and a dual-signal electrochemical readout. The MTB- or IC-specific PNA probe was labeled with methylene blue (MB) or ferrocene (FC), respectively at the C terminus, producing a strong square wave voltammetry signal. Interaction of the ICdPCR product could induce changes in the dynamics of these two redox-labeled PNA probes (MTB-MB and IC-FC) that were attached to the screen-printed gold electrode via formation of a self-assembled monolayer. Using this MB as a reporter and FC as an IC, the REC ICdPCR biosensor achieved a broad detection range from 10 fM to 10 nM and a detection limit of 1.26 fM, corresponding to approximately 2.5 bacteria cells. The REC ICdPCR biosensor was applied to MTB measurement in practical samples, exhibiting high accuracy and more importantly high practicability.

scholarly journals Internal control in PCR for Mycobacterium tuberculosis: usefulness and improvement of the diagnosis

2008 ◽  
Vol 51 (4) ◽  
pp. 485-491 ◽  
Author(s):  
Elizabeth Cortez-Herrera ◽  
Rosa Dea Sperhacke ◽  
Daniela Becker ◽  
Afrânio Kritski ◽  
Arnaldo Zaha ◽  
...  
Keyword(s):  
Public Health ◽  
Mycobacterium Tuberculosis ◽  
Internal Control ◽  
False Negative ◽  
Clinical Results ◽  
Public Health Laboratory ◽  
Negative Results ◽  
False Negative Results ◽  
Diagnostic Fragment ◽  
Pcr Test

The aim of this work was to construct and test a plasmidial Internal Control (IC) to detect the inhibition in the PCR test for M. tuberculosis and also its contribution for a Public Health Laboratory routine. The IC was a 600-bp of DNA linked to a plasmid with the same primer sites, allowing the amplification with the 245-bp diagnostic fragment. The amplification of the positive samples rendered the IC and the diagnostic fragment; instead negative samples only showed the IC. A total of 149 tuberculosis samples were studied and introduced the IC to monitor. Results showed 3.3% of the samples without amplification of the IC, suggesting the inhibition. These samples showed results in accordance with the clinical results. The objective of the IC was to identify the false negative results.

DETECTING MYCOBACTERIUM TUBERCULOSIS RAPIDLY AND MYCOBACTERIUM TUBERCULOSIS STRAINS WITHOUT IS6110 SEQUENCE BY PCR ASSAY

2012 ◽  
pp. 15-19
Author(s):  
Thi Chau Anh Nguyen ◽  
Hoang Bach Nguyen ◽  
Hai Duong Huynh ◽  
Nu Xuan Thanh Le ◽  
Xuan Cuong Le ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
16S Rdna ◽  
False Negative ◽  
Clinical Samples ◽  
Tuberculosis Complex ◽  
Pcr Assay ◽  
Complex Objects ◽  
Method Performance ◽  
Gene Coding ◽  
16S Rdna Pcr

Background: The Nested IS6110 PCR is used for detecting tuberculosis, however IS6110 sequence is not present in the genome of all strains of M.tuberculosis, the result may be false negative. The gene coding 16S ribosome always contains a short sequence specific to M. tuberculosis complex. Objects: Performance of the 16S Real-time PCR to detect M. tuberculosis and combining to the nested IS6110 PCR to determine the rate of Mtb strains without IS6110 from clinical samples. Materials and method: Performance of 16S rDNA PCR by commercial kit of Viet A Inc. for all 480 samples, the samples which were positive with the 16S rDNA PCR were retested in IS6110 PCR assay by in-house kit. Results: The Realtime 16S rDNA PCR detected 258 cases (53.8%) of tuberculosis. There were 3 (1.2 %) M. tuberculosis strains which do not harbor IS6110 sequence in genome. Conclusion: The IS6110 nested PCR can be applied more widely than the 16S rDNA realtime PCR. In case of using IS6110 PCR assay, results may show a low proportion of false negative. Combining 16S rDNA PCR with the IS6110 based PCR allowed detection of deletion of IS6110 sequence in M. tuberculosis isolates.

scholarly journals Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Bruna de Oliveira Coelho ◽  
Heloisa Bruna Soligo Sanchuki ◽  
Dalila Luciola Zanette ◽  
Jeanine Marie Nardin ◽  
Hugo Manuel Paz Morales ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Reverse Transcription ◽  
Color Change ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
False Negative ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

scholarly journals Label-Free Electrochemical Biosensor Based on Au@MoS₂–PANI for Escherichia coli Detection

Chemosensors ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 49
Author(s):  
Pushap Raj ◽  
Man Hwan Oh ◽  
Kyudong Han ◽  
Tae Yoon Lee
Keyword(s):  
Escherichia Coli ◽  
Bacterial Infections ◽  
Limit Of Detection ◽  
Bacterial Pathogens ◽  
Cost Effective ◽  
Covalent Immobilization ◽  
Label Free ◽  
Self Assembled Monolayer ◽  
Detection Range ◽  
Linear Detection

Bacterial infections have become a significant challenge in terms of public health, the food industry, and the environment. Therefore, it is necessary to address these challenges by developing a rapid, cost-effective, and easy-to-use biosensor for early diagnosis of bacterial pathogens. Herein, we developed a simple, label-free, and highly sensitive immunosensor based on electrochemical detection using the Au@MoS₂–PANI nanocomposite. The conductivity of the glassy carbon electrode is greatly enhanced using the Au@MoS₂–PANI nanocomposite and a self-assembled monolayer of mercaptopropionic acid on the gold nanoparticle surface was employed for the covalent immobilization of antibodies to minimize the nonspecific adsorption of bacterial pathogens on the electrode surface. The biosensor established a high selectivity and sensitivity with a low limit of detection of 10 CFU/mL, and detected Escherichia coli within 30 min. Moreover, the developed biosensor demonstrated a good linear detection range, practical utility in urine samples, and electrode regenerative studies.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

scholarly journals Percutaneous Discectomy—Continuous Irrigation and Drainage for Tuberculous Lumbar Spondylitis: A Report of Two Cases

2009 ◽  
Vol 2009 ◽  
pp. 1-4
Author(s):  
Sei Shibuya ◽  
Satoshi Komatsubara ◽  
Tetsuji Yamamoto ◽  
Nobuo Arima ◽  
Yoshiaki Kanda ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pathogenic Bacteria ◽  
Definitive Diagnosis ◽  
Continuous Irrigation ◽  
Percutaneous Discectomy ◽  
Caseous Necrosis ◽  
Postoperative Mri ◽  
Tuberculous Lesion ◽  
Vertebral Bodies ◽  
Over Time

Percutaneous curettage and continuous irrigation were performed for definitive diagnosis and treatment of tuberculous (TB) lumbar spondylitis. Under local anaesthesia, affected lumbar discs were curetted using a procedure of percutaneous nucleotomy, and in-tube and the out-tube were placed for continuous irrigation. The period of continuous irrigation was 12–16 days.Mycobacterium tuberculosiswas demonstrated in case 1 by culture and PCR, whereas histology showed tuberculous lesion with caseous necrosis in both cases. Postoperative MRI showed markedly reduced abscesses after 3 months in both cases. The signal intensity in vertebral bodies was improved. In Case 2, CT observations showed remodeling over time in the vertebral body cavities. This method is advantageous in that although minimally invasive, it achieves identification of pathogenic bacteria and treatment simultaneously. This surgical procedure is expected to prove effective for both TB and pyogenic spondylitis.

Crystal structure of hexanoyl-CoA bound to β-ketoacyl reductase FabG4 of Mycobacterium tuberculosis

2013 ◽  
Vol 450 (1) ◽  
pp. 127-139 ◽  
Author(s):  
Debajyoti Dutta ◽  
Sudipta Bhattacharyya ◽  
Amlan Roychowdhury ◽  
Rupam Biswas ◽  
Amit Kumar Das
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Ternary Complex ◽  
Catalytic Mechanism ◽  
Acyl Carrier Protein ◽  
Structural Data ◽  
Acid Synthesis ◽  
Binary Complex ◽  
C Terminus ◽  
Covalently Linked

FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β-oxoacyl-ACP (acyl-carrier protein) into β-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal ‘flavodoxintype’ domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-oxoacyl-CoA to β-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4–NADH binary complex and the FabG4–NAD+–hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg146 and Arg445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

scholarly journals Factors affecting removal of bacterial pathogens from healthcare surfaces during dynamic wiping

2018 ◽  
Vol 89 (4) ◽  
pp. 580-589 ◽  
Author(s):  
NWM Edwards ◽  
EL Best ◽  
P Goswami ◽  
MH Wilcox ◽  
SJ Russell
Keyword(s):  
Removal Efficiency ◽  
Surface Density ◽  
Pathogenic Bacteria ◽  
Best Practice ◽  
Regenerated Cellulose ◽  
Extrinsic Factors ◽  
Factors Affecting ◽  
E Coli ◽  
Liquid Removal ◽  
Fabric Surface

Wiping of surfaces contaminated with pathogenic bacteria is a key strategy for combating the transmission of healthcare associated infections. It is essential to understand the extent to which removal of bacteria is modulated by fiber properties, biocidal liquid impregnation and applied hand pressure. The influence of intrinsic and extrinsic factors on the removal efficiencies of pathogenic bacteria was studied. Nonwoven wipes made of either hydrophobic (polypropylene) or hygroscopic (lyocell) fibers were manufactured and dynamic removal efficiency of bacteria studied. The single most important parameter affecting bacterial removal efficiency was impregnation with biocidal liquid ( p < 0.05). For inherently hygroscopic 100% regenerated cellulose (lyocell) wipes impregnated with biocidal liquid, removal of E. coli, S. aureus and E. faecalis improved by increasing the fabric surface density and wiping pressure to their maximal values – 150 g.m–2 and 13.80 kN.m–2, respectively. For inherently hydrophobic 100% polypropylene nonwoven wipes, the same conditions maximized the removal efficiency of S. aureus, but for E. coli and E. faecalis a reduction in the wiping pressure to 4.68 kN.m–2 was required. Best practice involves the use of higher surface density wipes (150 g.m–2) containing regenerated cellulose fibers loaded with liquid biocide, and applied with the greatest possible wiping pressure.

Antimicrobial effects of plant extracts against Clostridium perfringens with respect to food-relevant influencing factors

2021 ◽  
Author(s):  
Ulrike Friedlein ◽  
Samart Dorn-In ◽  
Karin Schwaiger
Keyword(s):  
Influencing Factors ◽  
Plant Extract ◽  
Plant Extracts ◽  
Pathogenic Bacteria ◽  
Screening Method ◽  
Extrinsic Factors ◽  
Antimicrobial Effects ◽  
Extract Concentration ◽  
Microdilution Method

The application of plant extracts (PEs) could be a promising option to satisfy consumers’ demand for natural additives to inhibit growth of variable pathogenic bacteria. Thus, the aim of this study was to develop a standardized microdilution method to examine the antimicrobial effects of ten hydrophilic plant extracts against two strains of C. perfringens facing various food-relevant influencing factors. Due to the high opacity of PEs, resazurin was used as an indicator for bacterial growth instead of pellet formation. The highest value of the minimum inhibitory concentration (MIC) of the replications of each PE was defined as effective plant extract concentration (EPC), whereas the next concentration beneath the lowest MIC value was defined as the ineffective plant extract concentration (IEPC). The EPC of seven PEs: allspice, cardamom, cinnamon, clove, coriander, ginger and mace were between 0.625 - 10 g/kg, whereas extracts of caravey, nutmeg and thyme showed no antimicrobial activity up to the maximum concentration tested (10 g/kg) against C. perfringens in vitro. Two intrinsic factors, sodium chloride and sodium nitrite, displayed either synergistic/additive effects or no interaction with most PEs. By combination with PEs at its ineffective plant concentration (IEPC, 0.08 – 1.25 g/kg), MIC of NaCl and NaNO2 decreased from 25 – 50 g/kg to 6 – 25 g/kg and &gt; 200 mg/kg to 0.2 – 100 mg/kg respectively. On the contrary, lipid (sun flower oil) at a low concentration inhibited the antimicrobial effects of all tested PEs. For extrinsic factors, only allspice, ginger and coriander could maintain their antimicrobial effects after being heated to 78 °C for 30 min. The synergistic effect between PEs and pH values (5.0 and 5.5) was also found for all PEs. The established screening method with resazurin and defining EPC and IEPC values allows the verification of antimicrobial effects of PEs under various food-relevant influencing factors in a fast and reproducible way.

Sign in / Sign up

Export Citation Format

Share Document