Academic literature on the topic 'Isothermal pcr'
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Journal articles on the topic "Isothermal pcr":
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Park, Jee-Woong. "Principles and Applications of Loop-Mediated Isothermal Amplification to Point-of-Care Tests." Biosensors 12, no. 10 (October 10, 2022): 857. http://dx.doi.org/10.3390/bios12100857.
Abstract:
For the identification of nucleic acids, which are important biomarkers of pathogen-mediated diseases and viruses, the gold standard for NA-based diagnostic applications is polymerase chain reaction (PCR). However, the requirements of PCR limit its application as a rapid point-of-care diagnostic technique. To address the challenges associated with regular PCR, many isothermal amplification methods have been developed to accurately detect NAs. Isothermal amplification methods enable NA amplification without changes in temperature with simple devices, as well as faster amplification times compared with regular PCR. Of the isothermal amplifications, loop-mediated isothermal amplification (LAMP) is the most studied because it amplifies NAs rapidly and specifically. This review describes the principles of LAMP, the methods used to monitor the process of LAMP, and examples of biosensors that detect the amplicons of LAMP. In addition, current trends in the application of LAMP to smartphones and self-diagnosis systems for point-of-care tests are also discussed.
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Yunita, Lisa, Dian Rachma Wijayanti, and Apriani Riyanti. "Sensitivitas Pemeriksaan Covid-19: Insulated Isothermal PCR (iiPCR) Dan Reverse Transcription PCR (RT-PCR)." JOURNAL OF MUHAMMADIYAH MEDICAL LABORATORY TECHNOLOGIST 6, no. 1 (May 19, 2023): 37. http://dx.doi.org/10.30651/jmlt.v6i1.12441.
Abstract:
Covid-19 (Corona Virus disease 2019) is a new type of disease caused by a virus from the Corona Virus group, namely SARS-CoV-2. Covid-19 may cause respiratory system disorders, ranging from mild symptoms such as flu to lung infections, such as pneumonia. Laboratory diagnoses for Covid-19 disease generally include hematological (complete blood examinations), and molecular or a combination of serology and molecular. PCR examination that can be carried out using the Insulated Isothermal PCR and Reverse Transcription PCR methods. This research is descriptive observational research. Data were collected at Grha Kedoya Hospital, West Jakarta, North Kedoya. The result showed a sensitivity of iiPCR score 82,6%, which is relatively lower than gold-standard RT-PCR. Our research suggested that RT-PCR is still an effective and sensitive method for Covid-19 examination.
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Bouam, Amar, Jean-Jacques Vincent, Elisabeth Le Glass, Lionel Almeras, Pierre-Yves Levy, Hervé Tissot-Dupont, Jean-Christophe Lagier, Pierre-Edward Fournier, Didier Raoult, and Michel Drancourt. "Rapid Isothermal Amplification for the Buccal Detection SARS-CoV-2 in the Context of Out-Patient COVID-19 Screening." Journal of Clinical Medicine 10, no. 12 (June 16, 2021): 2643. http://dx.doi.org/10.3390/jcm10122643.
Abstract:
A commercially available isothermal amplification of SARS-CoV-2 RNA was applied to self-collected saliva samples using dry dental cotton rolls, which were held in the mouth for two minutes. Of 212 tests, isothermal amplification yielded three (0.14%) invalid results, 120 (56.6%) positive results and 89 (42%) negative results. Compared to reference RT-PCR assays routinely performed simultaneously on nasopharyngeal swabs, excluding the three invalid isothermal amplification assays and one RT-PCR invalid assay, these figures indicated that 119/123 (96.7%) samples were positive in both methods and 85/85 samples were negative in both methods. Four positive buccal swabs which were missed by the isothermal amplification, exhibited Ct values of 26–34 in reference RT-PCR assays. Positive isothermal amplification detection was achieved in less than 10 min. Supervision of the self-sampling procedure was key to achieve these performances. These data support the proposal to use the protocol reported in this paper, including supervised buccal self-sampling, to screen people suspected of having COVID-19 at the point of care.
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Shahrajabian, Mohamad H., Wenli Sun, and Qi Cheng. "Different Methods for Molecular and Rapid Detection of Human Novel Coronavirus." Current Pharmaceutical Design 27, no. 25 (September 15, 2021): 2893–903. http://dx.doi.org/10.2174/1381612827666210604114411.
Abstract:
Introduction: While PCR has been recognized as one of the appropriate ways to diagnose infectious diseases, Loop-mediated isothermal amplification (LAMP) which is a nucleic acid amplification method, can be considered as an alternative to PCR, and it is faster, cost-effective, and easier to perform than nested PCR. Patients and Methods: Keywords were searched in PubMed/MEDLINE, Scopus and Institute for Scientific Information Web of Science, as well as the search engine of Google Scholar. Keywords included PCR, LAMP, RAA, RPA, Virus and COVID-19. Results: LAMP technology has been extensively applied for the detection of human pathogenic bacteria, crop pests, pathogenic organisms and components in meat products. A new isotheral method, Recombinase polymerase amplification (RPA), can amplify the DNA as well as RPA. RPA involves benefits of isothermal PCR as well as simplicity and rapid amplification. Recombinase aided amplification (RAA) assay has been favorably used in the detection of bacterial and viral pathogens and solved the technical difficulties posed by DNA amplification methods because it does not need thermal denaturation of the template and involves a low and constant temperature. Conclusions: Reverse transcription polymerase chain reaction, digital PCR, LAMP, nicking endonuclease amplification reaction, recombinase polymerase amplification, and clustered regularly interspaced short palindromic repeats are different nucleic acid amplification tests of COVID-19. LAMP methods can be more specific than qPCR and immunoassays. The LAMP assay can be applied for rapid detection of SARS-CoV, MERS-CoV, SARS-CoV-2, and influenza, because LAMP is a highly sensitive and specific DNA/RNA amplification technique.
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Ahrberg, Christian D., Andreas Manz, and Bong Geun Chung. "Polymerase chain reaction in microfluidic devices." Lab on a Chip 16, no. 20 (2016): 3866–84. http://dx.doi.org/10.1039/c6lc00984k.
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Maltzeva, Yulia I., Daria A. Gorbenko, Ekaterina V. Nikitina, Maria S. Rubel, and Dmitry M. Kolpashchikov. "Visual Detection of Stem-Loop Primer Amplification (SPA) Products without Denaturation Using Peroxidase-like DNA Machines (PxDM)." International Journal of Molecular Sciences 24, no. 9 (April 25, 2023): 7812. http://dx.doi.org/10.3390/ijms24097812.
Abstract:
Rapid, inexpensive, and accurate determination of nucleic acids is a decisive factor in evaluating population’s health and monitoring treatment at point-of-care (POC) settings. Testing systems with visual outputs can provide instrument-free signal detection. Isothermal amplification technologies can substitute conventional polymerase chain reaction (PCR) testing due to compatibility with the POC diagnostic. Here, we have visually detected DNA fragments obtained by stem-loop-primer-assisted isothermal amplification (SPA), but not those obtained by PCR or LAMP amplification using DNA nanomachines with peroxidase-like activity (PxDM) with sensitivity to a single nucleotide substitution. Compared to the diagnostics with conventional loop-mediated isothermal amplification (LAMP), the PxDM method produces no false positive signals with the non-specific amplification products. The study suggests that PxDM, in conjunction with SPA isothermal amplification, can become a valid platform for POC testing systems.
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Rostamkhani, N., A. Haghnazari, M. Tohidfar, and A. Moradi. "Rapid identification of transgenic cotton (Gossypium hirsutum L.) plants by loop-mediated isothermal amplification." Czech Journal of Genetics and Plant Breeding 47, No. 4 (December 15, 2011): 140–48. http://dx.doi.org/10.17221/7/2011-cjgpb.
Abstract:
In an attempt to speed up the process of screening of transgenic cotton (G. hirsutum L.) plants, a visual and rapid loop-mediated isothermal amplification (LAMP) assay was adopted. Genomic DNA was extracted from fresh leaf tissues of T<sub>2</sub> transgenic cotton containing chitinase (chi) and cry1A(b) genes. Detection of genes of interest was performed by polymerase chain reaction (PCR), LAMP and real-time PCR methods. In LAMP assay the amplification was performed after 30 min at 65°C when loop primers were involved in the reaction. The involvement of loop primers decreased the time needed for amplification. By testing serial tenfold dilutions (10<sup>–1</sup> to 10<sup>–8</sup>) of the genes of interest, the detection sensitivity of LAMP was found to be 100-fold higher than that of PCR. The rapid DNA extraction method and LAMP assay can be performed within 30 min and the derived LAMP products can be directly observed as visually detectable based on turbidity in the reaction tube. The accuracy of LAMP method in the screening of transgenes was confirmed by PCR and real-time PCR. The developed method was efficient, rapid and sensitive in the screening of cotton transgenic plants. This method can be applied to any other crops.
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TSEN, HAU-YANG, CHIA-MING SHIH, PING-HUA TENG, HSIN-YEN CHEN, CHIA-WEI LIN, CHIEN-SHUN CHIOU, HWA-TANG THOMAS WANG, et al. "Detection of Salmonella in Chicken Meat by Insulated Isothermal PCR." Journal of Food Protection 76, no. 8 (August 1, 2013): 1322–29. http://dx.doi.org/10.4315/0362-028x.jfp-12-553.
Abstract:
Consumption of Salmonella-contaminated foods, such as poultry and fresh eggs, is known to be one of the main causes of salmonellosis. Conventional PCR methods, including real-time PCR for rapid detection of Salmonella, in general require skilled technicians and costly instruments. A recently developed novel convective PCR, insulated isothermal PCR (iiPCR), is carried out in polycarbonate capillary tubes. In this study, we designed TaqMan probes and PCR primers based on the yrfH gene encoding a heat shock protein for the iiPCR detection of Salmonella in chicken meat samples. The TaqMan probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine at the 5′ and 3′ ends, respectively. The PCR amplicon was 133 bp. A typical run of this iiPCR assay was completed within 1 h. Specific PCR products were obtained for 148 strains representing 49 serotypes of Salmonella tested. Under the same conditions, false-positive results were not obtained for 98 non-Salmonella strains tested, including strains of Enterobacteriaceae closely related to Salmonella. For chicken meat samples, with a 5-h enrichment step Salmonella at as low as 100 CFU/g of poultry meat could be detected. Because the amplification signals from the probes are detectable at 520 nm, identification of the PCR products by gel electrophoresis is not required. Compared with conventional PCR, the iiPCR system requires less expertise and provides an economical, reliable, and rapid tool for result interpretation. Detection results can be obtained within 8 h, including the enrichment and DNA extraction steps.
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Du, Tian, Ji-hong Lin, Jun-hua Zhao, Hai-bo Wang, and Qiu-hua Mo. "Development and Evaluation of an iiPCR Assay for Salmonella and Shigella Detection on a Field-Deployable PCR System." Canadian Journal of Infectious Diseases and Medical Microbiology 2020 (September 7, 2020): 1–5. http://dx.doi.org/10.1155/2020/9373984.
Abstract:
Background. Salmonella and Shigella are often associated with fecal-oral transmission and cause large-scale outbreaks in centralized catering units and, therefore, should be frequently and strictly monitored, especially among food handlers. However, no specific and sensitive on-site detection method is available until now. Methods. In this study, an insulated isothermal PCR assay for the detection of Salmonella and Shigella on a field-deployable PCR system was developed. Specificity, sensitivity, reproducibility, and clinical accuracy of the assay were characterized and evaluated. Results. The insulated isothermal PCR assay could be completed within 58 minutes with minimal pretreatment needed. The assay was specific and with good reproducibility. The limit of detection was 103 CFU/mL and 101 CFU/mL for Salmonella and Shigella, respectively, which was comparable to multiplex real-time PCR. Mock on-site clinical evaluation results showed that the analytical sensitivity and specificity of the insulated isothermal PCR assay were 100% and 96.6%, while the positive predictive value and negative predictive value were 94.1% and 100%, respectively. Conclusion. Based on our results, we believe that the assay developed herein could serve as an alternative method for preliminary screening and provide a valuable platform for the on-site detection of Salmonella and Shigella, especially in resource-limited and developing countries.
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Daher, Rana K., Gale Stewart, Maurice Boissinot, and Michel G. Bergeron. "Isothermal Recombinase Polymerase Amplification Assay Applied to the Detection of Group B Streptococci in Vaginal/Anal Samples." Clinical Chemistry 60, no. 4 (April 1, 2014): 660–66. http://dx.doi.org/10.1373/clinchem.2013.213504.
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Abstract BACKGROUND Group B streptococcal infections are the leading cause of sepsis and meningitis in newborns. A rapid and reliable method for the detection of this pathogen at the time of delivery is needed for the early treatment of neonates. Isothermal amplification techniques such as recombinase polymerase amplification have advantages relative to PCR in terms of the speed of reaction and simplicity. METHODS We studied the clinical performance of recombinase polymerase amplification for the screening of group B streptococci in vaginal/anal samples from 50 pregnant women. We also compared the limit of detection and the analytical specificity of this isothermal assay to real-time PCR (RT-PCR). RESULTS Compared to RT-PCR, the recombinase polymerase amplification assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The limit of detection was 98 genome copies and the analytical specificity was 100% for a panel of 15 bacterial and/or fungal strains naturally found in the vaginal/anal flora. Time-to-result for the recombinase polymerase amplification assay was <20 min compared to 45 min for the RT-PCR assay; a positive sample could be detected as early as 8 min. CONCLUSIONS We demonstrate the potential of isothermal recombinase polymerase amplification assay as a clinically useful molecular diagnostic tool that is simple and faster than PCR/RT-PCR. Recombinase polymerase amplification offers great potential for nucleic acid–based diagnostics at the point of care.
Dissertations / Theses on the topic "Isothermal pcr":
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McClean, Jennifer Natalie. "Novel isothermal PCR methodologies for the selective detection and analysis of microorganisms in environmental samples." Thesis, Queen's University Belfast, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.557957.
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SUMMARY (To be printed on this form) Phosphoramidite derivatives of a nucleoside analogue bearing photoswitchable ortho, meta and para-azobenzene moieties were synthesized and used to incorporate the photoswitchable azobenzene group proximal to the 5' and 3' ends of deoxyoligonucIeotide primers (12mers) of the glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene. The photochemical E~Z isomerisation of azobenzene appended primers was investigated by UV/vis spectroscopy and RP-HPLC. Melting studies were also performed to determine the Tm of the primers. Isothermal primer extension reactions were performed with Klenow fragment at the dark- adapted and irradiated photo stationary states. This revealed that only the para-azobenzene perturbed the extension by the enzyme. Further investigation with time course experiments to determine a yield using fluorescent fluorescein label and explore the possibility of photoswitchable DNA amplification using the para-azobenzene primers. OligonucIeotides bearing a novel photoswitchable moiety, phenylazopyridine, have been prepared. Directed Michaelis-Arbuzov reactions of support-bound internucleotide phosphite triesters with alkane-diamines were effected in the presence of iodine. The primary amine functionalised oligonucleotides were subsequently purified trityl on. This enabled resolution offast- and slow-isomers of oligomers.
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Pommies, Lilas. "Intégration de la préparation des échantillons dans une analyse par spectrométrie de masse et PCR isotherme." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASQ001.
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La bio-analyse correspond à l'identification et la quantification des molécules biologiques ou activement biologiques dans un échantillon. Elle se divise en plusieurs étapes : la collecte de l'échantillon et l'envoi au laboratoire ; la préparation des échantillons qui permet de le rendre compatible à la méthode d'analyse ; l'analyse et enfin la mesure des événements ayant eu lieu pendant l'analyse.Au cours de cette thèse, deux types d'analyse ont été étudiées : la spectrométrie de masse, plus particulièrement le MALDI-TOF, et une PCR isotherme nommée amplification isotherme médiée par les boucles (LAMP).L'objectif principale de la thèse était de rendre compatible ces méthodes d'analyse à une utilisation terrain. Pour cela, le SPID, un dispositif breveté par le CEA, a été adapté à ces deux technologies. Ce dispositif permet la filtration, la concentration, l'extraction d'une suspension bactérienne et la détection par immunochromatographie à flux latéral. Le SPID est un outil qui permet de s'affranchir des étapes de centrifugations ou lavages, méthodes classiquement utilisées pour préparer les échantillons pour le MALDI et la LAMP.Pour extraire les protéines ribosomales, pour une analyse au MALDI-TOF, il a fallu développer un tampon d'extraction compatible avec une telle analyse. En effet, pour faciliter la lyse bactérienne, l'utilisation de détergents est souvent recommandée ; or, les détergents peuvent empêcher l'identification des bactéries en spectrométrie de masse. Les résultats ont été comparés avec un protocole de référence proposé par Bruker. L'utilisation du SPID a permis d'identifier trois espèces bactériennes à une concentration de 107 UFC/mL dans un milieu simple et en urine. Le protocole de référence, quant à lui, a permis ces mêmes identifications à une concentration de 106 UFC/mL.Pour extraire l'ADN des bactéries avec le SPID suivi d'une amplification LAMP, de nombreuses mises au point ont été faites. Après avoir implanté la technique au laboratoire, différentes méthodes d'extraction avec le SPID ont été développées. Celle qui permet d'obtenir les meilleurs résultats est la création d'un tampon d'extraction combiné aux réactifs nécessaires à la LAMP. L'autre méthode étudiée est l'utilisation de billes de silice pour purifier l'ADN avant la réaction d'amplification.Pour rendre le chauffage compatible à une utilisation terrain, une station de chauffage a été conçue au cours de la thèse. Cette station permet de chauffer le réservoir du SPID pendant 40 minutes à 65 °C. Elle pourrait être alimentée par batterie.La détection des produits de l'amplification, appelés amplicons, est faite par immunochromatographie à flux latéral. Les amplicons sont marqués à leurs extrémités grâce à deux amorces. Une des amorces est marquée à la digoxigénine, l'autre à la biotine. L'amplicon est capturé par un anti-anticorps anti-digoxigénine, immobilisé sur une membrane de nitrocellulose, et est révélé à l'aide de la streptavidine couplé à des nanoparticules d'or.Avec les différents éléments développés au cours de cette thèse, le protocole pour réaliser une LAMP sur le terrain dure moins d'une heure : de la préparation des échantillons à la détectionBioanalysis is the identification and the quantification of biological molecules or active biological agent in a sample. It is divided into several stages: sample collection and sending to the laboratory; sample preparation to make it compatible with the analysis method; analysis and, finally, measurement of the events occurring during the analysis.During this thesis, two analysis were studied: mass spectrometry, more specifically MALDI-TOF and an isothermal PCR called Loop-mediated isothermal amplification (LAMP).The main aim was to make these methods compatible with field use. To achieve this, the SPID, a device patented by CEA, was adapted to both technologies. This device enables a bacterial suspension to be filtered, concentrated, extracted and detected by lateral flow immunoassay. The SPID is a tool that eliminate the need for centrifugation or washing, methods traditionally used to prepare sample for MALDI and LAMP.To analyze ribosomal proteins by MALDI-TOF, an extraction buffer compatible with this technic had to be developed. Indeed, to facilitate bacterial lysis, the use of detergents is often recommended but detergents can prevent the identification of bacteria by mass spectrometry. The results were compared with a reference protocol proposed by Bruker. Using SPID, three bacterial species were identified at a concentration of 107 CFU/mL in a simple medium and in urine. In contrast, the reference protocol identified the same species at a concentration of 106 CFU/mL.To make heating step compatible with filed use, an autonomous heating station was designed during the thesis. This station heats the SPID tank for 40 minutes at 65°C. It could be battery-powered.The amplification products, called amplicons, are detected by lateral flow immunoassay. Amplicons are end-labeled using two pirmers. One primer is labeled with digoxygenin, the other with biotin. The amplicon is captured by an anti-digoxigenin antibody, immobilized on a nitrocellulose membrane; and revealed using streptavidin coupled to gold nanoparticles.With the optimizations made during the thesis, the complete LAMP field protocol takes less than an hour : from de sample preparation to the detection
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Ghosh, Satyaki. "Molecular Detection and Quantification of the Fish Pathogen Saprolegnia spp. Using qPCR and Loop Mediated Isothermal Amplification." Bowling Green State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1573814311236554.
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RAVI, RANJANI. "A Novel RNA Virus Detection System Based on Duplex Specific Nuclease." University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1413820090.
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Stiedl, Cathrin [Verfasser], and Karin [Akademischer Betreuer] Weber. "Bestimmung des MDR1 (Multidrug resistance 1)-Genstatus beim Hund mittels allel-spezifischer PCR und allel-spezifischer Loop-mediated Isothermal Amplification (LAMP) / Cathrin Stiedl ; Betreuer: Karin Weber." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1141053756/34.
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Santos, Marcos André Ferreira. "Desenvolvimento de métodos moleculares para detecção de Theileria annulata." Master's thesis, Faculdade de Ciências e Tecnologia, 2011. http://hdl.handle.net/10362/6271.
Abstract:
Dissertação para a obtenção do Grau de Mestre em Genética Molecular e BiomedicinaA theileriose tropical (ou mediterrânica) é uma doença transmitida por carraças e que afecta bovinos, resultando em elevadas perdas económicas na produção e comércio internacional destes animais. O agente da doença é o hemoparasita Theileria annulata e a sua detecção em animais infectados é habitualmente realizada através de exame microscópico de esfregaços de sangue, que apresenta baixa sensibilidade na identificação de animais portadores, onde apenas um número diminuto de eritrócitos se mantém infectado. O objectivo deste estudo foi desenvolver métodos melhorados de detecção de T. annulata em amostras de sangue de bovino, baseados nas tecnologias de PCR em tempo real e de amplificação isotérmica de ácidos nucleicos (LAMP). Foram desenhados oligonucleótidos iniciadores com alvos complementares no gene Tams1, específico de T. annulata,para o método de LAMP. O DNA foi extraído a partir de amostras de sangue de bovino, usando métodos comerciais robotizados e kits de extracção, e foi usado como molde nas reacções de PCR em tempo real (com o corante intercalante EvaGreen®) e LAMP. Estas amostras de DNA encontravam-se testadas para a presença de parasitas dos géneros Theileria e Babesia, usando um método de hibridação reversa em membrana (Reverse Line Blotting – RLB). A detecção de T. annulata por PCR em tempo real nestas amostras revelou uma especificidade e sensibilidade de 100% e 80%,respectivamente, usando o teste de RLB como referência. A técnica de LAMP, por sua vez, conseguiu detectar uma amostra com baixa parasitémia correspondente a 0,03%. Ambas as técnicas usadas neste trabalho demonstraram ser eficientes na detecção de T. annulata, constituindo a técnica de PCR em tempo real um bom método de detecção em laboratórios bem equipados e a técnica de LAMP uma promissora ferramenta em laboratórios de diagnóstico com menores recursos.
Apoiado pelo Deputado Europeu Rui Tavares através do financiamento de uma bolsa de estudo
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Batinga, Maria Cryskely Agra. "Diagnóstico molecular comparativo da brucelose canina pela aplicação das técnicas de reação em cadeia pela polimerase (PCR) e amplificação isotérmica do DNA mediada por loop (LAMP)." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-25052017-105756/.
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A Brucella canis é a bactéria responsável pela brucelose nos cães, pode ser transmitida para os seres humanos, ocasionalmente resultando em doença grave, com impacto na saúde pública. A brucelose canina desencadeia inúmeras perdas econômicas em canis comerciais, com a ocorrência de abortamentos, morte embrionária, natimortos e nascimento de filhotes debilitados. O diagnóstico sorológico é rotineiramente realizado, contudo a hemocultura é o teste \"padrão-ouro\". A técnica de reação em cadeia pela polimerase (PCR) pode ser aplicada no diagnóstico direto como alternativa à hemocultura, pela rapidez, alta especificidade e sensibilidade do teste, mas apresenta alto custo com infraestrutura e equipamentos. A amplificação isotérmica mediada por loop (LAMP) constitui outra alternativa para amplificação do DNA em um curto período de tempo, com simplicidade e menor custo. O projeto avaliou comparativamente o desempenho dos testes moleculares de PCR e LAMP com primers direcionados à sequência de inserção IS711 de Brucella spp. em 98 amostras de sangue total, obtidas de 57 cães. Os 57 cães foram divididos em três grupos: infectados por B. canis (cães positivos na hemocultura) não infectados por B. canis (negativos na hemocultura e sem evidências clínicas e epidemiológicas de brucelose) e suspeitos de brucelose (cães negativos na hemocultura, mas com suspeita clínica e/ou epidemiológica da infecção). A sensibilidade e especificidade diagnóstica das reações de LAMP e PCR foram calculadas, utilizando-se os grupos de cães infectados e não infectados, respectivamente. O desempenho dos testes foi analisado, utilizando-se as 98 amostras, comparadas duas a duas, pelos testes estatísticos de Coeficiente Kappa e McNemar. A proporção de amostras positivas foi de 43,87% (43/98) na hemocultura, 46,93% (46/98) na PCR e 16,33% (16/98) na LAMP. A concordância entre a hemocultura e a PCR foi ótima, enquanto que a concordância entre a LAMP e a hemocultura e entre a LAMP e a PCR foi sofrível. A sensibilidade diagnóstica foi de 100% (18/18) na PCR e 44,44% (8/18) na LAMP, enquanto que a especificidade diagnóstica foi de 96% (20/21) na PCR e 100% (21/21) na LAMP. O desempenho da reação de LAMP foi insatisfatório para o diagnóstico da brucelose nos cães, em razão dos baixos valores de sensibilidade do teste. A PCR, por outro lado, apresentou desempenho similar à hemocultura, o que a torna uma alternativa para uso no diagnóstico da brucelose canina.Brucella canis is the etiological agent responsible for brucellosis in dogs and can be transmitted to human beings, occasionally resulting in severe disease, and leading to impacts on public health. Canine brucellosis triggers numerous economic losses in commercial kennels, causing abortions, embryonic death, stillbirths and birth of debilitated puppies. Serological diagnosis is routinely performed, but blood culture is the gold standard test. Polymerase chain reaction (PCR) can be used to the direct diagnosis of infection in view of its speed and high specificity and sensitivity values, however it has high cost because of the laboratory infrastructure and equipments needed. The loop-mediated isothermal amplification (LAMP) may be an alternative to DNA amplification in a shorter period of time, with simplicity and low cost. This project evaluated the potential of the molecular tests of PCR and LAMP using primers targeting the insertion sequence IS711 of Brucella, using 98 whole blood samples of 57 dogs. The 57 dogs were divided into three groups: infected by B. canis (dogs with positive results in blood culture), non-infected by B. canis (dogs with negative results by blood culture and showing no clinical or epidemiological evidences of brucellosis) and dogs suspected of brucellosis (those with negative blood culture but with clinical and/or epidemiological evidences of infection). The diagnostic sensitivity and specificity of PCR and LAMP were calculated using the infected and non-infected groups, respectively. The performance of the three diagnostic tests was pair compared using the 98 samples using McNemar test and Kappa coefficient. The proportion of positive samples detected by blood culture, PCR and LAMP was respectively 43.87% (43/98), 46.93% (46/98), and 16.33% (16/98). The concordance between blood culture and PCR was almost perfect, while the concordance between LAMP and blood culture and between LAMP and PCR was fair. The diagnostic sensitivity of PCR and LAMP was, respectively, 100% (18/18) and 44.44% (8/18), while the diagnostic specificity of the tests was 96% (20/21) and 100% (21/21), respectively. LAMP performance was not satisfactory for canine brucellosis diagnosis because of the low sensitivity of the test. PCR showed similar performance when compared to blood culture, which makes it a good alternative for use for the diagnosis of canine brucellosis.
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Oliveira, Maythsulene Inácio de Sousa. "Desenvolvimento de métodos moleculares para detecção simultânea de fusarium oxysporum f. sp. phaseoli, fusarium solani e curtobacterium flaccumfaciens pv. flaccumfaciens." Universidade Federal de Goiás, 2015. http://repositorio.bc.ufg.br/tede/handle/tede/8762.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Common bean (Phaseolus vulgaris L.) is grown in Brazil in three different cropping seasons, and in diverse agroecosystems. In such different environments, the crop is exposed to several constraints responsible for yield losses, such as pathogenic organisms. Among common bean relevant diseases, fusarium wilt (Fusarium oxysporum f. sp. phaseoli), dry root-rot (Fusarium solani) and Curtobacterium wilt (Curtobacterium flaccumfaciens pv. flaccumfaciens) have similar symptoms, hindering diagnosis in the field, and whose identification in seed health testing is also limited. In both cases, identification at species level is an important step to manage this root pathogen complex, whose detection can be improved by molecular biology tools. Therefore, this study aimed to: 1) to develop and validate a multiplex PCR (m-PCR) method for simultaneous identification of three common bean pathogens, F. oxysporum f. sp. phaseoli, F. solani and C. flaccumfaciens pv. flaccumfaciens; and 2) develop an isothermal amplification of DNA (LAMP) method to detect of F. oxysporum f. sp. phaseoli on seeds. M-PCR method was developed for identification of isolated colonies, as well as infected seeds. In seeds, total DNA was obtained by alkaline lysis method, which inactivates nucleases during the extraction process. M-PCR allowed the identification of all pathogens, with detection of C. flaccumfaciens pv. flaccumfaciens, F. oxysporum f. sp. phaseoli and F. solani amplicons in agarose gel with respectively 306, 609 and 143 base pairs. Furthermore, m-PCR also reduced costs and time to detect Fusarium oxysporum f. sp. phaseoli from 10 days to three hours. It was not possible to develop an optimized protocol for detection of F. oxysporum f. sp. phaseoli by the LAMP method, using only the tf1 gene for design of primers, since such primers were functional only for amplifying large amounts of target DNA. Based on the negative results with LAMP, it is suggested that further studies should be performed using other DNA sequences available in GenBank database.
O feijoeiro-comum (Phaseolus vulgaris L.) é cultivado durante todo o ano no território brasileiro, em três épocas distintas e em vários agroecosistemas. Nestes ambientes distintos, a cultura está exposta a diversos fatores que causam perdas de rendimento, como o ataque de patógenos. Dentre as doenças do feijoeiro-comum encontram-se a murcha-de-fusarium (Fusarium oxysporum f. sp. phaseoli), a podridão-radicular-seca (Fusarium solani) e a murcha-de-curtobacterium (Curtobacterium flaccumfaciens pv. flaccumfaciens) que apresentam sintomas semelhantes, dificultando seu diagnóstico no campo, e cuja identificação em testes de sanidade de sementes também é limitada. Em ambos os casos, a identificação em nível de espécie é uma importante etapa do manejo deste complexo de patógenos, cuja detecção pode ser aperfeiçoada com a adoção de ferramentas de biologia molecular. Portanto, este estudo teve como objetivos: 1) Desenvolver e validar um método de multiplex PCR (m-PCR) para identificação simultânea de três espécies de patógenos do feijoeiro-comum, F. oxysporum f. sp. phaseoli, F. solani e C. flaccumfaciens pv. flaccumfaciens; e 2) desenvolver a técnica de amplificação isotérmica de DNA (LAMP) para detecção de F. oxysporum f. sp. phaseoli em sementes. O método de m-PCR foi desenvolvido para identificação de colônias isoladas bem como sementes infectadas. Nas sementes, o DNA total foi obtido pela lise alcalina, método que inativa nucleases durante o processo de extração. A m-PCR possibilitou a identificação de todos os patógenos, com detecção de C. flaccumfaciens pv. flaccumfaciens, F. oxysporum f. sp. phaseoli e F. solani em bandas formadas em gel de agarose respectivamente com 306, 609 e 143 pares de base. Além disso, a extração do DNA total das sementes pela lise alcalina em combinação com a m-PCR também possibilitou redução de custos e tempo de realização do diagnóstico de Fusarium oxysporum f. sp. phaseoli, de 10 dias para três horas. Não foi possível estabelecer um protocolo otimizado para detecção de F. oxysporum f. sp. phaseoli pelo método LAMP, utilizando somente o gene tf1 para desenho dos iniciadores, uma vez que, os iniciadores revelaram-se funcionais apenas para a amplificação com grandes quantidades de DNA alvo. Diante dos resultados obtidos com LAMP, sugere-se que estudos posteriores sejam realizados empregando outras sequências de DNA disponíveis no banco de dados GenBank.
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Tafoukt, Boulous Djida. "Suivi de réactions biochimiques par calorimétrie en vue de la production de biocarburants de 2ème génération." Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM4332/document.
Abstract:
C'est dans un contexte marqué par une demande croissante en énergie primaire, une diminution des ressources et dans un souci de protection de l'environnement que le biocarburant de 2ème génération est développé. Cependant, ce biocarburant est non viable économiquement. L’optimisation, le contrôle et la connaissance des cinétiques régissant les procédés de fabrication de ce bioéthanol sont donc des éléments capitaux. Dans cette étude, le potentiel de la calorimétrie isotherme pour surveiller les réactions d'hydrolyse et de fermentation est testé.Les résultats montrent que cette méthode est efficace. En effet, celle-ci a permis de mettre en évidence l'importance du ratio enzyme/substrat pour maximiser le rendement et de déterminer un meilleur cocktail composé de cellulases + cellobiose déshydrogénase (CDH) qui permet la production d'une certaine quantité d'acide gluconique, qui pourrait améliorer l'attractivité de ce biocarburant. Ces mêmes essais ont également permis de déterminer la chaleur de l'hydrolyse de la paille de blé, qui est 32,18 ± 3,18 J.g-1 (gramme de sucres produits).Les mesures obtenues ont été utilisées pour déterminer les constantes cinétiques des cellulases + CDH sur la paille de blé et les résultats montrent que ce cocktail enzymatique est plus rapide à 45 °C dans la gamme de températures testée (40-55°C) avec une vitesse de 7,36 ± 0,62 mmol/L.min.Par ailleurs, les essais avec un calorimètre à échelle laboratoire ont montré que même si celui-ci ne mesure pas avec précision les chaleurs engendrées par les réactions d'hydrolyse et de fermentation, celui-ci donne de bonnes indications sur le déroulement et l'avancement de ces réactionsSecond generation biofuel is developed in a context marked by an increasing demand for primary energy, a decrease in resources and in environmental protection concernsHowever, this biofuel is not economically viable. Optimization, control and knowledge of the kinetics governing this bioethanol production processes are crucial elements.In this study the potential of isothermal calorimetry to monitor hydrolysis and fermentation reactions is tested.The results show that the isothermal calorimetry is an effective method. Indeed this method allowed determining that the substrate/enzyme ratio is an important parameter of the hydrolysis yield.Furthermore it has determined a better enzyme cocktail consisting of Cellulases + Cellobiose Dehydrogenase (CDH) which allows the production of a certain amount of gluconic acid, which could improve the attractiveness of these second-generation biofuels. These same tests also determined the hydrolysis heat of wheat straw which is 32.18 ± 3.18 J.g-1 (gram reducing sugars product).The measurements obtained were used to determine kinetic constants cellulases + CDH on wheat straw and the results show that this enzyme cocktail is faster at 45 ° C in the range of temperatures tested (40 - 55°C) with a speed of 7.36 ± 0.62 mmol/L.min.In addition, testing with a laboratory-scale calorimeter showed that even if this tool does not accurately measure the heat generated by the hydrolysis reaction and fermentation, it gives a good indication of the development and advancement of these reactions
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Yu, Hao. "Matériaux hydrures pour le stockage irréversible ou réversible de l’hydrogène." Thesis, Lyon 1, 2012. http://www.theses.fr/2012LYO10247.
Abstract:
L’utilisation des combustibles fossiles (énergies non renouvelables) est responsable de l’augmentation de la concentration en gaz à effet de serre dans l’atmosphère. Lors de l'examen des solutions de rechange, l’hydrogène comme vecteur énergétique est le plus séduisant. Le stockage de l’hydrogène en phase solide sous forme d’hydrures, est l’une des solutions non polluantes futures pour le stockage et le transport de l’énergie. Parmi les matériaux candidats, le borohydrure de sodium (NaBH4) et l’hydrure de magnésium (MgH2) ont été sélectionnés au vu de leur capacité gravimétrique élevée en hydrogène. La réaction d'hydrolyse de NaBH4 a été étudiée dans un calorimètre en phase liquide couplée à un compteur à gaz, afin de suivre en même temps, la cinétique de production d’hydrogène et l’évolution de la chaleur de réaction. Nous avons préparé des catalyseurs à base de cobalt supporté sur différents supports (hydrotalcites, KF/Al2O3, hétéropolyanions) ayant des propriétés acido-basiques différentes. Les supports et les catalyseurs à base de cobalt ont été caractérisés par DRX, MEB+EDX, ICP et BET. Co/hétéropolyanions a montré une cinétique très élevée pour la production d'hydrogène accompagnée d'une conversion totale dans la réaction d'hydrolyse. L’absorption et la désorption de l’hydrogène ont été étudiées sur l’hydrure de magnésium. Afin d’améliorer la cinétique de sorption de MgH2, nous avons préparé des mélanges MgH2-MT (MT = métal de transition Co, Ni, Fe, Cr, Mn), MgH2-MTmélangé (MT = métal de transition Co, Ni, Fe,), MgH2-MTnano (MT = métal de transition Conano, Ninano, Fenano, Cunano, Znnano) et MgH2-nLiBH4-MTnano (MT = métal de transition Conano, Ninano, Fenano) par broyage à billes de haute énergie. Leurs propriétés physico-chimiques ont été étudiées par DRX et MEB+EDX. La température de désorption de l’hydrogène et la quantité d’hydrogène dégagée ont été étudiées par TPD. La cinétique d’absorption de l’hydrogène et la réversibilité du stockage de l’hydrogène ont été étudiées par isotherme PCT pour le système MgH2-MTnano. MgH2-10-Ninano présente la meilleure propriété de stockage réversible de l’hydrogène, MgH2-10-Conano et MgH2- 10-Fenano sont aussi de bons candidats potentielsThe use of fossil fuels (non-renewable) is the main raison of increasing the green house in the atmosphere. Among the considered alternatives, hydrogen is seen as the most attractive energy carrier. The storage of the hydrogen in the solid phase in the form of hydrides is one of the clean future solutions for storage and transport of energy. Among potential materials, sodium borohydride (NaBH4) and magnesium hydride (MgH2) were selected regarding their high hydrogen gravimetric capacity. The hydrolysis reaction of NaBH4 was studied in a liquid phase calorimetry coupled to a gas-meter, in order to monitor simultaneously the kinetics of the hydrogen production and the evolution of the reaction heat. We prepared cobalt supported catalysts using various supports (hydrotalcites, KF/Al2O3, heteropolyanions) with different acid-base properties. The supports and the catalysts were characterized by XRD, SEM+EDX, ICP and BET. Co/heteropolyanions showed a very high kinetics for the production of hydrogen accompanied by a total conversion in the hydrolysis reaction. The absorption and desorption of hydrogen were studied using magnesium hydride. In order to improve the sorption kinetics of MgH2, we have prepared the MgH2-MT (MT= transition metal Co, Ni, Fe, Cr, Mn), MgH2-MTmixture (MT= transition metal Co, Ni, Fe), MgH2-MTnano (MT = transition metal Conano, Ninano, Fenano, Cunano, Znnano) and MgH2-nLiBH4-MTnano (MT = transition metal Conano, Ninano, Fenano) mixtures by high energy ball milling. Their physicochemical properties were studied by XRD and SEM+EDX. The temperature of hydrogen desorption and the amount of hydrogen generated were investigated by TPD. The kinetics of hydrogen absorption and the reversibility of hydrogen storage were investigated with PCT isotherm for the system of MgH2-MTnano. The sample MgH2-10-Ninano presents the best property for reversible hydrogen storage; MgH2- 10-Conano and MgH2-10-Fenano are also good potential candidates
Books on the topic "Isothermal pcr":
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Scialpi, Angela, and Alessio Mengoni, eds. La PCR e le sue varianti. Florence: Firenze University Press, 2008. http://dx.doi.org/10.36253/978-88-6453-159-5.
Abstract:
The book "La PCR e le sue varianti" is designed as a reference tool for those whose laboratory activities deal with methods based on nucleic acid amplification. The text provides the theoretical bases of the polymerase chain reaction (PCR) and its variants (e.g. RT-PCR, quantitative PCR, isothermic PCR) in a rapid and concise manner and describes the principal applications used for genetic identification and the study of genetic polymorphism, in the form of a protocol that can be easily consulted by the users.
Book chapters on the topic "Isothermal pcr":
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Bartholomew, Rachel A., Janine R. Hutchison, Timothy M. Straub, and Douglas R. Call. "PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification." In Manual of Environmental Microbiology, 2.3.2–1–2.3.2–13. Washington, DC, USA: ASM Press, 2015. http://dx.doi.org/10.1128/9781555818821.ch2.3.2.
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Reif, John H., and Urmi Majumder. "Isothermal Reactivating Whiplash PCR for Locally Programmable Molecular Computation." In DNA Computing, 41–56. Berlin, Heidelberg: Springer Berlin Heidelberg, 2009. http://dx.doi.org/10.1007/978-3-642-03076-5_5.
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Yamazaki, Wataru. "Sensitive and Rapid Detection of Campylobacter jejuni and Campylobacter coli Using Loop-Mediated Isothermal Amplification." In PCR Detection of Microbial Pathogens, 267–77. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-60327-353-4_18.
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Balasuriya, Udeni B. R. "Type A Influenza Virus Detection from Horses by Real-Time RT-PCR and Insulated Isothermal RT-PCR." In Methods in Molecular Biology, 393–402. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-0758-8_34.
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Tortajada-Genaro, Luis Antonio. "Design of Oligonucleotides for Allele-Specific Amplification Based on PCR and Isothermal Techniques." In Methods in Molecular Biology, 35–51. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1799-1_3.
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V., Santhy, Nagamani Sandra, Kundapura V. Ravishankar, and Bhavya Chidambara. "Molecular Techniques for Testing Genetic Purity and Seed Health." In Seed Science and Technology, 365–89. Singapore: Springer Nature Singapore, 2023. http://dx.doi.org/10.1007/978-981-19-5888-5_15.
Abstract:
AbstractWith the globalization of seed trade and transgenic variety development, the application of molecular technologies for seed quality gained more significance in both the internal and international markets. Besides germination, genetic purity and seed health are the two most important seed quality parameters that determine the planting value of a seed lot. Compared to the conventional methods of testing, molecular marker technologies are more efficient for quality analysis as these are more accurate, robust, abundant, and faster. Among the various markers, simple sequence repeats (SSRs), due to their genome-wide presence, reproducibility, multi-allelic nature, and co-dominant inheritance, have emerged as the best markers, for establishing varietal distinctness, identity, and variety/hybrid seed purity testing. With the advent of the next-generation sequencing (NGS) technology, single nucleotide polymorphic (SNP) markers also became widely popular, and the closest to being an ideal marker besides SSRs, in seed genetic purity testing. With large-scale GM crop cultivation, testing for the adventitious presence and trait purity are two added components of seed quality testing. The methods of GM seed quality testing include DNA-based (conventional and real-time PCR), protein-based (lateral flow test and ELISA), and bioassay-based technologies. DNA-based methods including PCR/real-time PCR assays have been successfully employed to detect the adventitious presence of transgenic seeds in seed trade especially at international level, as well as in the national gene banks for germplasm conservation. ISTA plays a prominent role in international harmonization and providing universal guidelines on use of different methods to detect GM seeds. The BMT group of UPOV and the Working Group on DNA Methods of the Variety Committee of ISTA, work in tandem to standardize suitable molecular techniques for establishing variety identity and purity testing, respectively. In the area of seed health testing also, molecular detection assays such as, PCR (nested PCR, multiplex PCR, real-time PCR), loop-mediated isothermal amplification (LAMP), and DNA microarray with many advantages over the conventional assays have been proven highly useful. However, there is a need to validate the usefulness of molecular markers through stringent multi-laboratory tests for their reproducibility before recommending them in routine seed purity and health testing.
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Balasuriya, Udeni B. R. "Type A Influenza Virus Detection from Horses by Real-Time RT-qPCR and Insulated Isothermal RT-PCR." In Methods in Molecular Biology, 383–92. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0346-8_29.
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Jain, Nityanand, Tungki Pratama Umar, Reem Sayad, Muhammed Edib Mokresh, Kevin Tandarto, Reynold Siburian, Phey Liana, Sniedze Laivacuma, and Aigars Reinis. "Monkeypox Diagnosis in Clinical Settings: A Comprehensive Review of Best Laboratory Practices." In Advances in Experimental Medicine and Biology, 253–71. Cham: Springer Nature Switzerland, 2024. http://dx.doi.org/10.1007/978-3-031-57165-7_16.
Abstract:
AbstractAn outbreak of monkeypox (Mpox) was reported in more than 40 countries in early 2022. Accurate diagnosis of Mpox can be challenging, but history, clinical findings, and laboratory diagnosis can establish the diagnosis. The pre-analytic phase of testing includes collecting, storing, and transporting specimens. It is advised to swab the lesion site with virus transport medium (VTM) containing Dacron or polyester flock swabs from two different sites. Blood, urine, and semen samples may also be used. Timely sampling is necessary to obtain a sufficient amount of virus or antibodies. The analytical phase of infectious disease control involves diagnostic tools to determine the presence of the virus. While polymerase chain reaction (PCR) is the gold standard for detecting Mpox, genome sequencing is for identifying new or modified viruses. As a complement to these methods, isothermal amplification methods have been designed. ELISAassays are also available for the determination of antibodies. Electron microscopy is another effective diagnostic method for tissue identification of the virus. Wastewater fingerprinting provides some of the most effective diagnostic methods for virus identification at the community level. The advantages and disadvantages of these methods are further discussed. Post-analytic phase requires proper interpretation of test results and the preparation of accurate patient reports that include relevant medical history, clinical guidelines, and recommendations for follow-up testing or treatment.
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Razizy, F. Muhamad, N. Zhen Zhang, M. S. Hashim, O. Saliza Azlina, and O. Shahrul Azmir. "The Effect of Isothermal Ageing Treatment on Different PCB Surface Finishes: Simulation and Experimental." In Recent Progress in Lead-Free Solder Technology, 171–94. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-030-93441-5_8.
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Nixon, Gavin, and Claire Bushell. "A Review of Isothermal Nucleic Acid Amplification Technologies." In PCR Technology, 363–92. CRC Press, 2013. http://dx.doi.org/10.1201/b14930-33.
Conference papers on the topic "Isothermal pcr":
1
Mesforush, Shaghayegh, Amir Jahanshahi, and Mohesen Khajeh Zadeh. "Finite element simulation of isothermal regions in serpentine shaped PCB electrodes of a micro-PCR device." In 2019 27th Iranian Conference on Electrical Engineering (ICEE). IEEE, 2019. http://dx.doi.org/10.1109/iraniancee.2019.8786721.
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Persat, Alexandre, Tomoyuki Morita, and Juan G. Santiago. "On-Chip Isothermal Polymerase Chain Reaction." In ASME 2007 International Mechanical Engineering Congress and Exposition. ASMEDC, 2007. http://dx.doi.org/10.1115/imece2007-43070.
Abstract:
We present a novel technique for on-chip PCR where temperature is held constant and uniform in the reactor. Specific chemicals, known as denaturants, have the ability to melt DNA. A flow control scheme establishes spatio-temporal fluctuations in the concentration of denaturants along a microchannel, while electromigration drives DNA through this spatially varying denaturant concentration field. Preliminary results show denaturation and extension of a 200 base pairs (bp) DNA template.
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Yu, Eun-Sil, Byoung-Hoon Kang, Hamin Na, and Ki-Hun Jeong. "Nanoplasmonic isothermal PCR assay with CRISPR/Cas for real-time SARS-CoV-2 detection." In MOEMS and Miniaturized Systems XXII, edited by Wibool Piyawattanametha, Yong-Hwa Park, and Hans Zappe. SPIE, 2023. http://dx.doi.org/10.1117/12.2650879.
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Chen, Pin-Chuan, Masahiko Hashimoto, Michael W. Mitchell, Dimitris E. Nikitopoulos, Steven A. Soper, and Michael C. Murphy. "Limiting Performance of High Throughput Continuous Flow Micro-PCR." In ASME 2004 International Mechanical Engineering Congress and Exposition. ASMEDC, 2004. http://dx.doi.org/10.1115/imece2004-62091.
Abstract:
Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, potentially affecting amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95°C) and renaturation (55°C-68°C) zones; above 6 mm/s the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from λ-DNA. Amplifications at velocities ranging from 1 mm/s to 20 mm/s were investigated. Alternative design of PCR was investigated. Shuttle PCR has a single straight channel and a DNA plug, driven by electrokinetic flow, will move forward and backward in the microchannel to achieve the repetitive thermal cycles. Thermal performance, independent insulated temperature blocks, and molecular and thermal diffusion were evaluated.
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Ide, Tatsuya. "Molecular identification of invasive drywood wood boring species using frass and fecal pellets by loop-mediated isothermal amplification and nested PCR assays." In 2016 International Congress of Entomology. Entomological Society of America, 2016. http://dx.doi.org/10.1603/ice.2016.93886.
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Sussman, Michael. "ISO TC 34/SC 16 Horizontal methods for molecular biomarker analysis—international standards for molecular biomarker analysis/isothermal nucleic acid amplification methods." In 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/fnwh5573.
Abstract:
Harmonized, easy to handle methods of analysis with defined patterns and known nomenclatures bring more customers to the market. The International Organization for Standardization (ISO.org) was formed in 1946. It is an independent, non-governmental voluntary consensus standard body based in Geneva, Switzerland with a membership of 165 national standards bodies. The US ISO member is the American National Standards Institute (ANSI.org), a consortium of US standardization organizations. There are 45 participating countries. The US delegation responsible for developing the US position for standards development in agricultural molecular biomarker analysis was delegated to the American Oil Chemist’s Society (AOCS.org) by ANSI. The AOCS US TAG also hosts the TC 34/SC 16 international secretariat. TC 34/SC 16 has published 31 standards with another 6 under development. The six under development are: ISO/AWI 5354 Molecular biomarkers of agricultural fibers. Screening of genetically modified organisms (GMOs) in cotton and textiles; ISO/DIS 16577 Molecular biomarker analysis. Vocabulary for molecular biomarker analytical methods in agriculture and food production; ISO/CD 16578 Molecular biomarker analysis. Requirements for microarray detection of specific nucleic acid sequences; ISO/DTS 20224-8 Molecular biomarker analysis. Detection of animal-derived materials in foodstuffs and feedstuffs by real-time PCR Part 8: Turkey DNA detection method; ISO/DTS 20224-9; Molecular biomarker analysis. Detection of animal-derived materials in foodstuffs and feedstuffs by real-time PCR. Part 9: Goose DNA detection method and ISO/FDIS 22942-1 Molecular biomarker analysis. Isothermal polymerase chain reaction (isoPCR) methods. Part 1: General requirements. We will discuss details and publication of these new standards.
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Daly, John, and Mark Davies. "A Quantitative Free Convection DNA Amplifier." In ASME/JSME 2007 Thermal Engineering Heat Transfer Summer Conference collocated with the ASME 2007 InterPACK Conference. ASMEDC, 2007. http://dx.doi.org/10.1115/ht2007-32381.
Abstract:
The Polymerase Chain Reaction (PCR) has been used extensively to amplify targeted nucleic acids for many applications in molecular biology and, increasingly, in medical diagnostics. Outlined in this paper is a PCR device which takes account of the advantages offered by free convection. The design is, in it fundamental format a time-wise isothermal well-based thermocycler. A temperature gradient induced across the well causes convection forces to circulate the sample through the required temperatures necessary for amplification. Quantitative amplification is demonstrated with real time measurements of SYBR Green I fluorescence within the free convective DNA amplifier. Amplification of an 86-bp fragment of the pGEM®-T vector (Promega) is performed in a 25μl volume in eight minutes. A 10-fold dilution series and methods for calculating effective cycle times are presented. Also detailed within this paper are PIV and thermal imaging results of the free convection cavity. This device presents an opportunity for the development of a practical and inexpensive gene-expression measurement system.
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Chen, Jyh Jian, Yao Tsung Yang, and Tse Yu Hsieh. "Modeling and Experiment of Shuttling Speed Effects on the Oscillatory Thermal Cycler Chamber (OSTRYCH)." In ASME 2010 International Mechanical Engineering Congress and Exposition. ASMEDC, 2010. http://dx.doi.org/10.1115/imece2010-37011.
Abstract:
Polymerase chain reaction (PCR) has emerged as a powerful tool in genetic analysis. The PCR products are closely linked with thermal cycles. Therefore, to reduce the reaction time and make temperature distribution uniform in the reaction chamber, a novel oscillatory thermal cycler (OSTRYCH) is designed. The sample is placed in a fixed chamber, and three constant isothermal zones are established and lined in the system. The sample is oscillated and contacted with three different isothermal zones to complete thermal cycles. This study presents the analyses of the operational parameters of the chamber. The commercial software CFD-ACE+™ is utilized to investigate the influences of various chamber materials, boundary conditions and moving speed of the chamber on the temperature distributions inside the chamber. The chamber moves at a specific speed and the boundary conditions with time variations are related to the moving speed. Whereas the chamber moves, the boundary is specified at the conditions of the convection or the uniform temperature. The user subroutines compiled by the FORTRAN language are used to make the numerical results realistically. The effects of various chamber materials, boundary conditions, moving speeds of the rectangular chamber on the temperature distributions are examined. Results show that regarding to the temperature profiles and the standard deviation of the temperature at the Y-cut cross section; the effects of various moving speeds of the chamber on the temperature distributions are negligible at the assigned time duration. The central temperatures of the chamber with various moving speeds are measured. The repeatability and stability of the OSTRYCH are examined. Finally, the experimental results and numerical simulations are compared.
9
Ahortor, E., S. Mahazu, T. Manful, A. Erber, and A. Ablordey. "Evaluation of an IS2404 LAMP protocol, a simple and rapid test for diagnosis of Buruli ulcer in low-resource settings." In MSF Scientific Days International 2024. NYC: MSF-USA, 2024. http://dx.doi.org/10.57740/mat4h7.
Abstract:
INTRODUCTION Buruli ulcer caused by Mycobacterium ulcerans is a devastating necrotic skin disease. PCR, recommended for confirmation of Buruli ulcer by WHO, requires an adequately equipped laboratory, often delaying diagnosis and treatment of patients in remote or humanitarian settings. We aimed to assess loop- mediated isothermal amplification (LAMP), which is a molecular assay for isothermal amplification of DNA suggested for timely diagnosis of Buruli ulcer in low-resource settings. METHODS This study combines quantitative and qualitative methods. First, we evaluated a simple rapid syringe DNA extraction method (SM) in comparison with a conventional extraction method (CM), followed by a LAMP assay targeting IS2404 for the detection of M ulcerans, either using a pocket warmer (pw) or a heat block (hb) for incubation of the reaction. 83 clinical specimens (swabs and fine-needle aspirates from different centres in Ghana) were tested. We assessed sensitivity, specificity, and positive and negative predictive value (PPV and NPV). Second, we explored the diagnostic workflow for Buruli ulcer at a community-based health centre in rural Ghana, a potential target setting. We used observations and interviews with researchers and healthcare workers (HCWs) and community-based surveillance volunteers. We discuss evaluation results in relation to the target setting and requirements of a target product profile for Buruli ulcer diagnosis. RESULTS DNA extraction using SM followed by IS2404 PCR (IS2404 PCRSM) identified M ulcerans DNA in 73 of 83 clinical specimens. The sensitivity, specificity, PPV, and NPV of IS2404 PCRSM were 90.12%, 100%, 100%, and 65.21%, respectively, compared with the reference standard IS2404 PCR with the CM protocol. Evaluation of the LAMP assay on 64 SM DNA extracts showed a sensitivity, specificity, PPV, and NPV of 83.6%, 100%, 100%, and 50%, respectively, using either pw (pwLAMPSM) or hb (hbLAMPSM) for incubation, compared with the same reference standard. The limit of detection of both pwLAMPSM and hbLAMPSM was 30 target copies. Interviews confirmed that, despite great engagement from HCWs and volunteers, patients met challenges regarding transport and costs for initial diagnosis and follow- up and often sought alternative treatments first. Diagnostic confirmation via PCR in a reference laboratory led to a delay in the initiation of treatment. A diagnosis at the point of care, following clinical screening, was considered advantageous to prevent delays and loss to follow-up, therefore ensuring timely patient treatment. CONCLUSION Our findings support the potential use of pwLAMP for rapid diagnosis of Buruli Ulcer in patients with a suspected infection at the community or primary health-care level, with limited equipment and without reliable electricity supply such as found in humanitarian settings.
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Lusiastuti, Angela M., Bambang S. Sihananto, and Christina Wianty. "Etiologi, Deteksi dan Pengendalian Penyakit Tumor Mutiara pada Ikan Gabus, Channa striata." In Seminar Nasional Ikan XI. Masyarakat Iktiologi Indonesia, 2022. http://dx.doi.org/10.32491/semnasikan-mii-2022-p.1-7.
Abstract:
Penyakit tumor mutiara (pearl-like nodul disease) yang disebabkan oleh virus dari genus Lymphocyctis, Family Iridoviridae merupakan salah satu kendala pada budidaya ikan gabus. Tujuan penulisan ini adalah mendiskripsikan penyakit tumor mutiara (LCDV) pada ikan gabus dari identifikasi virus penyebabnya, cara deteksi dan pengendalian LCDV di akuakultur. Kondisi stress merupakan pemicu berkembangnya penyakit LCDV atau Lymphocystis Disease Virus ini pada tubuh ikan. Virus yang bersifat dermotropic ini menyebabkan penyakit yang bersifat self-limiting disease, atau dapat sembuh dengan sendirinya jika kondisi tubuh dan lingkungannya membaik. Deteksi level 1 seperti gejala klinis, level 2 melalui histopatologis dan level 3 yang bersifat konfirmasi dengan PCR, in situ hybridisasi maupun loop mediated isothermal amplification untuk skrining infeksi yang bersifat subklinis telah banyak digunakan. Gejala klinis yang khas adalah timbulnya tumor menyerupai buah anggur atau mutiara. Histopatologi yang pathognomonis adalah sel limfosit hipertrofi dikelilingi kapsul hyaline dan ditemukan basophilic intracytoplasmic inclusion. PCR konvensional dan qPCR digunakan untuk deteksi kualitatif dan kuantitatif. Sinyal hibridisasi pada deteksi ISH menunjukkan adanya perkembangan virus di dalam tubuh ikan. Ikan dapat sembuh dengan sendirinya dan mempunyai kekebalan perolehan. Kondisi ikan yang sehat dan lingkungan yang baik dapat mencegah berkembangnya virus.
Reports on the topic "Isothermal pcr":
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Baral, Aniruddha, Jeffery Roesler, and Junryu Fu. Early-age Properties of High-volume Fly Ash Concrete Mixes for Pavement: Volume 2. Illinois Center for Transportation, September 2021. http://dx.doi.org/10.36501/0197-9191/21-031.
Abstract:
High-volume fly ash concrete (HVFAC) is more cost-efficient, sustainable, and durable than conventional concrete. This report presents a state-of-the-art review of HVFAC properties and different fly ash characterization methods. The main challenges identified for HVFAC for pavements are its early-age properties such as air entrainment, setting time, and strength gain, which are the focus of this research. Five fly ash sources in Illinois have been repeatedly characterized through x-ray diffraction, x-ray fluorescence, and laser diffraction over time. The fly ash oxide compositions from the same source but different quarterly samples were overall consistent with most variations observed in SO3 and MgO content. The minerals present in various fly ash sources were similar over multiple quarters, with the mineral content varying. The types of carbon present in the fly ash were also characterized through x-ray photoelectron spectroscopy, loss on ignition, and foam index tests. A new computer vision–based digital foam index test was developed to automatically capture and quantify a video of the foam layer for better operator and laboratory reliability. The heat of hydration and setting times of HVFAC mixes for different cement and fly ash sources as well as chemical admixtures were investigated using an isothermal calorimeter. Class C HVFAC mixes had a higher sulfate imbalance than Class F mixes. The addition of chemical admixtures (both PCE- and lignosulfonate-based) delayed the hydration, with the delay higher for the PCE-based admixture. Both micro- and nano-limestone replacement were successful in accelerating the setting times, with nano-limestone being more effective than micro-limestone. A field test section constructed of HVFAC showed the feasibility and importance of using the noncontact ultrasound device to measure the final setting time as well as determine the saw-cutting time. Moreover, field implementation of the maturity method based on wireless thermal sensors demonstrated its viability for early opening strength, and only a few sensors with pavement depth are needed to estimate the field maturity.