Relevant bibliographies by topics / PCR-based diagnostic

Academic literature on the topic 'PCR-based diagnostic'

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Published: 10 March 2023

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Journal articles on the topic "PCR-based diagnostic"

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Mirmajlessi, Seyed Mahyar, Maria Jennifer Sjölund, Marika Mänd, Marianne Loiseau, Vincenza Ilardi, Geert Haesaert, Reet Karise, Richard Alexander Gottsberger, Jason Sumner-Kalkun, and Assunta Bertaccini. "PCR-based diagnostic methods for ‘Candidatus Liberibacter solanacearum’ – Review." Plant Protection Science 55, No. 4 (September 13, 2019): 228–41. http://dx.doi.org/10.17221/145/2018-pps.

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‘Candidatus Liberibacter solanacearum’ is an economically important pathogen in the Americas, New Zealand and Europe. The primary objective of this review is to systematically investigate the polymerase chain reaction (PCR)-based methods used for its detection in plant samples. Several databases were searched from the inception of the relevant literature up to August 2018. This review identified 53 studies that met all the inclusion criteria. The performance of the different methods was also compared, however due to data heterogeneity and insufficient evidence on the sensitivity of all assays used, a meta-analysis of the data was not possible. Nonetheless, the review indicates that the rtPCR designed to the 16S ribosomal RNA gene can be routinely employed as a fast, cost-effective, and reliable detection technique in diagnostic laboratories.
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Mangayarkarasi, V., Sneka P, Sujith R, and Jayaprakash Jayaprakash. "Ergonomic Diagnostic Tool based on Chip Mini RT-PCR for Diagnosis of Pulmonary and Extra Pulmonary Tuberculosis." Journal of Pure and Applied Microbiology 13, no. 2 (June 30, 2019): 1185–90. http://dx.doi.org/10.22207/jpam.13.2.58.

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Volkov, A. N., L. V. Nacheva, and Yu V. Zakharova. "Molecular genetic techniques in current biomedical research. Part II: PCR applications in diagnostics of human infectious diseases." Fundamental and Clinical Medicine 6, no. 1 (March 29, 2021): 77–85. http://dx.doi.org/10.23946/2500-0764-2021-6-1-77-85.

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Polymerase chain reaction (PCR)-based diagnostics is currently established as a gold standard for the detection of microorganisms. The features of PCR include rapid amplification of DNA and RNA as well as high sensitivity and specificity. In contrast to diagnostic microbiology, PCR diagnostics does not require preliminary culture of the microorganisms for their identification, reducing both time and costs of the diagnostic procedure. The lecture discusses the molecular basis behind the modern technical solutions for the PCR diagnostics of human infectious diseases including multiplex and reverse transcription PCR. We describe the principles of qualitative and quantitative PCR-based detection of pathogens in biological samples and provide the examples of PCR application for solving specific diagnostic scenarios. The lecture is primarily designed for students of biomedical specialties and healthcare professionals using molecular genetic techniques in their practice.
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Reinitz, David M., Timothy P. Yoshino, and Rebecca A. Cole. "A Ribeiroia Spp. (Class: Trematoda)–-Specific PCR-Based Diagnostic." Journal of Parasitology 93, no. 5 (October 2007): 1234–38. http://dx.doi.org/10.1645/ge-3584rn.1.

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Kumar, Vinay, Nitish Bansal, Trilok Nanda, Aman Kumar, Rajni Kumari, and Sushila Maan. "PCR Based Molecular Diagnostic Assays for Brucellosis: A Review." International Journal of Current Microbiology and Applied Sciences 8, no. 02 (February 10, 2019): 2666–81. http://dx.doi.org/10.20546/ijcmas.2019.802.312.

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Kugeler, Kiersten J., Nikos Gurfield, Jean G. Creek, Kerry S. Mahoney, Jessica L. Versage, and Jeannine M. Petersen. "Discrimination between Francisella tularensis and Francisella-Like Endosymbionts when Screening Ticks by PCR." Applied and Environmental Microbiology 71, no. 11 (November 2005): 7594–97. http://dx.doi.org/10.1128/aem.71.11.7594-7597.2005.

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ABSTRACT The presence of Francisella-like endosymbionts in tick species known to transmit tularemia poses a potential diagnostic problem for laboratories that screen tick samples by PCR for Francisella tularensis. Tick samples initially considered positive for F. tularensis based on standard 16S rRNA gene PCR were found to be positive only for Francisella-like endosymbionts using a multitarget F. tularensis TaqMan assay (ISFtu2, tul4, and iglC) and 16S rRNA gene sequencing. Specificity of PCR-based diagnostics for F. tularensis should be carefully evaluated with appropriate specimen types prior to diagnostic use.
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Agrawal, Megha. "On-Chip PCR Based Plasmonic Microfluidic Platform: Ultrafast Point-of-Care Diagnostics of SARS-CoV-2." Biotechnology Kiosk 3, no. 1 (January 7, 2021): 5–11. http://dx.doi.org/10.37756/bk.21.3.1.1.

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It is critically important to have rapid screening and identification of contagious viral diseases such as the current COVID-19 pandemic that is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Rapid and accurate diagnostic is essential for preventing worldwide spread of virus and ensuring in-time care for patients during the fast spread of pandemic diseases. Nanobiotechnology enabled tools have allowed to develop advanced polymerase chain reaction (PCR) based diagnostics of contagious viral diseases. To this end, microfluidic on-chip PCR platforms have shown huge promise for highly efficient, rapid and small-volume bioassay for point-of-care (POC) diagnostic applications in mitigating the challenges of SARS-CoV-2. Here, we discuss latest advances in ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at POC level.
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Li, Xiaolei, Weiping Zeng, Jing Liao, Zhenbiao Liang, Shuhua Huang, and Zhi Chao. "DNA Barcode-Based PCR-RFLP and Diagnostic PCR for Authentication of Jinqian Baihua She (Bungarus Parvus)." Evidence-Based Complementary and Alternative Medicine 2015 (2015): 1–7. http://dx.doi.org/10.1155/2015/402820.

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We established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and diagnostic PCR based on cytochrome C oxidase subunit I (COI) barcodes ofBungarus multicinctus, genuine Jinqian Baihua She (JBS), and adulterant snake species. The PCR-RFLP system utilizes the specific restriction sites ofSpeI andBstEII in the COI sequence ofB. multicinctusto allow its cleavage into 3 fragments (120 bp, 230 bp, and 340 bp); the COI sequences of the adulterants do not contain these restriction sites and therefore remained intact after digestion withSpeI andBstEII (except for that ofZaocys dhumnades, which could be cleaved into a 120 bp and a 570 bp fragment). For diagnostic PCR, a pair of species-specific primers (COI37 and COI337) was designed to amplify a specific 300 bp amplicon from the genomic DNA ofB. multicinctus; no such amplicons were found in other allied species. We tested the two methods using 11 commercial JBS samples, and the results demonstrated that barcode-based PCR-RFLP and diagnostic PCR both allowed effective and accurate authentication of JBS.
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Malorny, B. "Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method." International Journal of Food Microbiology 89, no. 2-3 (December 31, 2003): 241–49. http://dx.doi.org/10.1016/s0168-1605(03)00154-5.

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Clancy, Cornelius J., and M. Hong Nguyen. "Polymerase Chain Reaction (PCR)–Based Diagnostic Assays for Invasive Candidiasis." Current Fungal Infection Reports 5, no. 3 (August 4, 2011): 135–40. http://dx.doi.org/10.1007/s12281-011-0062-x.

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Dissertations / Theses on the topic "PCR-based diagnostic"

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Smith, Shelle Ann. "A comparison of diagnostic techniques for detecting salmonella spp in equine fecal samples using culture methods, gel-based pcr, and real-time pcr assays." Thesis, Texas A&M University, 2003. http://hdl.handle.net/1969.1/5735.

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Salmonellae are enteric bacteria infecting animals and humans. Large animal clinics and Veterinary Teaching Hospitals are greatly affected by Salmonella outbreaks and nosocomial infection. The risk of environmental contamination and spread of infection is increased when animals are confined in close contact with each other and subjected to increased stress factors. This study was designed to compare double-enrichment culture techniques with Gel-based and Real-time PCR assays in the quest for improved diagnostic methods for detecting Salmonella in equine fecal samples. 120 fecal samples submitted to the Clinical Microbiology Laboratory of the Veterinary Medical Teaching Hospital at Texas A&M University (CML, VMTH, TAMU) were tested for Salmonella using all three techniques. Double-enrichment bacterial culture detected 29 positive results (24%), Real-time PCR detected 33 positive results (27.5%), and Gel-based PCR detected 73 positives results (60.8%). While culture and real-time PCR methods had similar results, the gel-based PCR method detected many more positive results, indicating probable amplicon contamination. Real-time PCR can be completed as soon as the day after submission while culture techniques may take 2 to 5 days to complete. However, viable bacterial cells are needed for antimicrobial susceptibility testing and serotyping: both important for epidemiological studies. Therefore, double-enrichment bacterial culture performed concurrently with real-time PCR methods could be efficient in clinical settings where both accurate and expedient results are required.
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Gunson, Rory Niall. "The development, implementation and evaluation of a real-time PCR-based diagnostic service for viral causes of infectious intestinal disease." Connect to e-thesis, 2007. http://theses.gla.ac.uk/108/.

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Thesis (Ph.D.) - University of Glasgow, 2007.
Ph.D. thesis submitted to the Division of Community Based Sciences, Faculty of Medicine, University of Glasgow, 2007. Includes bibliographical references. Print version also available.
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Gunson, Rory N. "The development, implementation and evaluation of a real-time PCR-based diagnostic service for viral causes of infectious intestinal disease." Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/108/.

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Outbreaks and sporadic cases of viral Infectious Intestinal Disease (IID) are a major public health issue resulting in significant morbidity and sometimes mortality each year. The economic costs associated are substantial. Laboratory diagnosis of viral IID is important as the many infectious and non-infectious causes cannot be reliably differentiated using clinical or epidemiological characteristics alone. An accurate diagnosis can aid patient management, infection control procedures and reduce health care costs by preventing unnecessary treatments, testing for alternative causes and hospital stay. It also aids public health surveillance. At the start of the research described in this thesis the West of Scotland Specialist Virology Centre (WOSSVC) used Electron Microscopy (EM) as the frontline test for outbreaks and sporadic cases of IID. However, although rapid on a small number of samples, this technique has been shown to be insensitive, laborious and is not suited to testing large numbers of samples. The research presented in this thesis sought to examine whether molecular diagnostic techniques such as conventional gel-based or real-time Polymerase Chain Reaction (PCR) assays could be a viable replacement for EM as the frontline test(s) for viral IID in a routine laboratory service of this type, and whether their implementation could bring benefits to the laboratory service in terms of improved rapidity, sensitivity and throughput. The aim was to adapt published PCR methods for use in routine diagnostic work rather than for research purposes, an approach that distinguishes this research from previous work in this area. In order to achieve this aim, the appropriate PCR techniques were first selected from the literature, based on a combination of clinical and laboratory requirements, and were adapted for use in the laboratory service. A series of laboratory experiments was then carried out in order to compare the sensitivity of the adapted methods to existing techniques such as EM and antigen detection assays (EIAs) and to other methods that emerged during the period of study including alternative PCR assays. Where found to be suitable, the selected PCR tests were implemented in the routine diagnostic service for viral IID. The effects of these changes on the laboratory service were then examined. The results show that since the introduction of molecular tests at WOSSVC for the detection of viral pathogens in cases of gastroenteritis the number of samples tested has risen steadily, as have the detection rates for each of the main viral causes of IID. Furthermore, this has been achieved at the same time as a substantial reduction in sample turn-around-times. Such improvements will have a positive impact in several areas of public health relating to viral IID and are discussed fully, including patient management, infection control and national surveillance.
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Wayland, Jennifer. "Development and optimization of a diagnostic system based on Illumina sequencing of genus wide PCR amplicons for the detection of viruses of grapevines." Diss., University of Pretoria, 2017. http://hdl.handle.net/2263/63362.

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In this study we present a poly-specific PCR-based, high-throughput sequencing (HTS) diagnostic system together with an appropriate data analysis pipeline for the diagnosis of grapevine viruses. Poly-specific and virus-specific primers were established to be capable of detecting and identifying 37 grapevine infecting viruses from 11 genera. An analysis pipeline using CLC Genomics workbench was developed by utilising various defined artificial samples which were assembled and sequenced on the Illumina MiSeq platform. A threshold for percentage mapped reads of 0.4% during reference mapping was established to discriminate between presence or absence of viruses associated with reads. Various criteria for the evaluation of de novo assembled contigs and BLAST results were identified based on virus hits, E-value, percentage query overlap and percentage amplicon overlap. Various RT-PCR systems were used to screen 62 grapevine samples (field collected and candidate nuclear vines) for their virus populations. Seven samples were selected for Illumina MiSeq sequencing, and the data was analysed as per the optimized pipeline. The threshold established for reference mapping and the criteria for BLAST analysis was successfully implemented, proving the applicability of this PCR and HTS-based system in grapevine diagnostics. This system was compared to the standard ELISA system routinely utilised during certification. In our study, when samples evaluated by RT-PCR were tested using ELISA for the presence of GLRaV-1, -2 and -3, a false-negative rate in ELISA of 14.3% was observed, confirming that RT-PCR is the more sensitive test of the two. The capability of RTPCR to readily detect viruses present in low concentrations in woody plants, the availability of primers for virus identification, the ease and rapidity of the technique, together with constant improvement of HTS platforms especially in the area of cost makes this an extremely useful method for grapevine virus diagnostics.
Dissertation (MSc)--University of Pretoria, 2017.
Winetech
Microbiology and Plant Pathology
MSc
Unrestricted
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Jethani, Kajal. "The development of a real-time diagnostic RT-PCR based on the molecular analysis of Aspergillus fumigatus genes regulated during the early stages of lung invasion." Thesis, King's College London (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441363.

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Trouchet, Amandine. "PCR digitale pour la détection et la caractérisation de micro-organismes pathogènes au niveau de la cellule unique." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC170/document.

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Nous avons pour but de développer un système microfluidique en gouttes, capable, à l’échelle de la cellule/bactérie unique, de détecter et de co-localiser plusieurs marqueurs génétiques, en utilisant une version digitale et multiplexée de la réaction de polymérisation en chaîne (PCR). Les systèmes de PCR digitale actuellement commercialisés ne le permettent toujours pas. Un tel prototype garantira la présence de multiples marqueurs à l’intérieur d’un même génome, ce qui permettra l’identification du pathogène avec précision et un taux de faux-positifs proche de zéro. Comme première application, nous démontrerons la possibilité de co-localiser quatre gènes de virulence de la souche O157:H7 d’Escherichia coli, un pathogène majeur, qui est détecté dans des échantillons alimentaires ou provenant de fèces cliniques pouvant aussi contenir des E. coli non pathogènes porteuses d’une partie des gènes de virulence. Avant de procéder à des tests TaqMan multicolores en point final, E. coli sera d’abord encapsulée dans des gouttes micrométriques et lysée par la chaleur in situ. Notre objectif est de démontrer que ce test peut être appliqué avec succès à un petit ensemble d’échantillons cliniques ou alimentaires
We aim to develop a prototype of droplet-based microfluidic system capable of detecting and colocalizing multiple genetic markers at the single cell/bacteria level, using the Polymerase Chain Reaction (PCR) in a digital multiplexed version. This cannot be achieved using current commercial digital PCR systems, and should increase the sensitivity and reliability of the detection of pathogens. Importantly, the system will guarantee the presence of multiple markers within the same genome and enable accurate identification, and bring the false positive rate close to zero. As a first application, we will demonstrate the possibility to co-localize 3 virulence genes in the E.coli strain O157:H7, a major foodborne pathogen, which has to be detected in clinical feces samples or food samples, which may also contain non pathogenic E. coli carrying only a subset of these virulence genes. E. Coli will be encapsulated in micrometric droplets, lysed by heating in situ prior performing a multicolour end-point Taqman assay. Our objective is to demonstrate that this test can be successfully applied to real clinical or food samples
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Mohamed, Nahla. "Molecular Diagnosis of Common Viral Infectious Diseases Based on Real-Time PCR." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7118.

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Xu, Jiru. "Application of PCR and DNA sequencing based molecular diagnosis in infectious diseases." Thesis, University of Ulster, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399727.

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Cheng, Wing-suen, and 鄭穎璿. "Rapid diagnosis of isoniazid resistant mycobacterium tuberculosis by hybridization probe based real time PCR." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B4833361X.

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Background Tuberculosis (TB) infection is a contagious disease due to infection by Mycobacterium tuberculosis(MTB) causing global health burden. There is increasing effort to develop early case detection methods and also to address the issue of multidrug resistance TB (MDR-TB). Molecular methods have been applied to provide rapid and accurate diagnosis. In addition to commercial kits being available for the detection of MTB from clinical specimens, In-house PCR assays have also been developed for the detection of MTB, and can be adjusted according to the laboratories’ own demand. Several molecular techniques like TaqMan probes and Hybridization probes may be applied to target for markers of MTB, e.g. 16s rRNA and IS6110.Detection of the mutation genes, for example, katGfor isoniazid (INH), enables determination of susceptibility of the antibiotic more rapidly than traditional culture methods, and is especially useful due to the increasing emergence of MDR-TB. A wide range of genes have been reported to be related to the resistance of INH, katG315 mutation is the most common gene among them. Therefore, genotyping katG315 allows determination of the susceptibility of INH. Objective The first objective is to evaluate the diagnostic performance of IS6110 One-tube Nested Real-Time PCR for the detection of MTB. Clinical pulmonary specimens collected from Tuen Mun Hospital were retrieved for investigation. All the specimens have already been tested for COBAS TaqMan MTB test and culture results have been obtained for all the samples. During the first stage of the study, all the specimens were tested with IS6110 One-tube Nested Real-Time PCR. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic odds ratio were obtained from the comparison with the gold standard of MTB detection, i.e., culture. During the second stage of the study, samples were selected to undergo katG315 HybProbe Real-Time PCR assay to determine the genotype of katG. The performance of the assay was evaluated statistically. Result In the first stage of the study, 200 samples were tested with IS6110 One-tube Nested Real-Time PCR. The assay was found to have a sensitivity of 76.92%, specificity of 98.52%, positive predictive value of 96.15%, negative predictive value of 89.86% and the diagnostic odds ratio of 221.667. In the second stage of the study, 66 samples were selected and tested for katG315 HybProbe Real-Time PCR assay, 36 samples were successfully genotyped while 30 samples failed to be genotyped. The only culture proven INH resistance specimen was not amplified at first, and culture isolate was extracted for genotyping again. The repeated test confirmed the genotype of the resistance strain to be a mutant. Conclusion katG315 HybProbe Real-Time PCR assay is a valid approach for genotyping katG. However, the sensitivity and efficiency has to be improved before application for clinical use. From the statistics obtained, COBAS TaqMan PCR assay, which is routinely used in Tuen Mun Hospital, is statistically proven to have comparatively better performance than the IS6110 One-tube Nested Real-Time PCR. Improvement on the assay is required for IS6110 One-tube Nested Real-Time PCR. However, there is great potential of applying both IS6110 One-tube Nested Real-Time PCR and katG315 HybProbe Real-Time PCR assay in clinical use with the same platform available.
published_or_final_version
Microbiology
Master
Master of Medical Sciences
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Abusedra, Amina Saleh. "The molecular characterisation of variable segment usage of the immunoglobulin genes and its application to the diagnosis and monitoring of lymphoproliferative disorders." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321931.

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Books on the topic "PCR-based diagnostic"

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D, Ehrlich Garth, and Greenberg Steven J, eds. PCR-based diagnostics in infectious disease. Boston: Blackwell Scientific Publications, 1994.

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Xu, Jiru. Application of PCR and DNA sequencing based molecular diagnosis in infectious diseases. [S.l: The Author], 2003.

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Book chapters on the topic "PCR-based diagnostic"

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da Silva, Alexandre J., and Norman J. Pieniazek. "Latest Advances and Trends in PCR-Based Diagnostic Methods." In Textbook-Atlas of Intestinal Infections in AIDS, 397–412. Milano: Springer Milan, 2003. http://dx.doi.org/10.1007/978-88-470-2091-7_25.

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Wang, Zhuang, and Joanne Spadoro. "Determination of Target Copy Number of Quantitative Standards Used in PCR-Based Diagnostic Assays." In Gene Quantification, 31–43. Boston, MA: Birkhäuser Boston, 1998. http://dx.doi.org/10.1007/978-1-4612-4164-5_3.

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Opota, Onya, René Brouillet, Gilbert Greub, and Katia Jaton. "Methods for Real-Time PCR-Based Diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus Infections in an Opened Molecular Diagnostic Platform." In Methods in Molecular Biology, 171–81. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7037-7_11.

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Rohlfs, Elizabeth M., and W. Edward Highsmith. "PCR-Based Methods for Mutation Detection." In Molecular Diagnostics, 123–62. Totowa, NJ: Humana Press, 1997. http://dx.doi.org/10.1007/978-1-4757-2588-9_7.

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Frayling, Ian M., Emma Monk, and Rachel Butler. "PCR-Based Methods for Mutation Detection." In Molecular Diagnostics, 65–74. Totowa, NJ: Humana Press, 2006. http://dx.doi.org/10.1385/1-59259-928-1:065.

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Subbotin, Sergei A. "Molecular identification of nematodes using polymerase chain reaction (PCR)." In Techniques for work with plant and soil nematodes, 218–39. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781786391759.0218.

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Abstract Molecular diagnostics are a vital component of the management of economically important pests, including plant-parasitic nematodes. Various molecular techniques for diagnostics have been introduced to nematology during last decades, but the most popular is Polymerase Chain Reaction (PCR) based. This chapter presents procedures for DNA extraction, PCR techniques, cloning and DNA sequencing of nematodes.
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Subbotin, Sergei A. "Molecular identification of nematodes using polymerase chain reaction (PCR)." In Techniques for work with plant and soil nematodes, 218–39. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781786391759.0012a.

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Abstract Molecular diagnostics are a vital component of the management of economically important pests, including plant-parasitic nematodes. Various molecular techniques for diagnostics have been introduced to nematology during last decades, but the most popular is Polymerase Chain Reaction (PCR) based. This chapter presents procedures for DNA extraction, PCR techniques, cloning and DNA sequencing of nematodes.
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Fegan, M., G. Holoway, A. C. Hayward, and J. Timmis. "Development of a Diagnostic Test Based on the Polymerase Chain Reaction (PCR) to Identify Strains of R. solanacearum Exhibiting the Biovar 2 Genotype." In Bacterial Wilt Disease, 34–43. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-662-03592-4_5.

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White, P. Lewis, and Rosemary A. Barnes. "Polymerase Chain Reaction (PCR)-Based Tests." In Aspergillosis: From Diagnosis to Prevention, 135–57. Dordrecht: Springer Netherlands, 2009. http://dx.doi.org/10.1007/978-90-481-2408-4_9.

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Dykas, Daniel J., and Allen E. Bale. "PCR Based Diagnosis of Fragile X Syndrome." In Modern Clinical Molecular Techniques, 363–72. New York, NY: Springer New York, 2012. http://dx.doi.org/10.1007/978-1-4614-2170-2_24.

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Conference papers on the topic "PCR-based diagnostic"

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Ramalingam, Naveen, Long-Qing Chen, Xin-Hao Yang, Liqun Deng, Qing-Hui Wang, Eric Yap Peng Huat, Chiew Hoon Neo, and Hai-Qing Gong. "A Surface-Directed Microfluidic Scheme for Parallel Nanoliter PCR Array Suitable for Point-of-Care Testing." In ASME 2009 7th International Conference on Nanochannels, Microchannels, and Minichannels. ASMEDC, 2009. http://dx.doi.org/10.1115/icnmm2009-82052.

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In resource-limited settings, it is impractical to get access to a diagnostic laboratory having sophisticated instruments, and it is desirable to use disposable point-of-care diagnostic chips that do not require liquid handling or pumping instruments for sample distribution among an array of reactors. In addition to the pump-less sample loading method, the challenge to seal an array of reactors without the use of microvalves or mechanical parts still persists. Implementation of microvalve array adds complexity to the chip fabrication and operation processes, and also reduces the space on the microchip. In this paper, we report the development of a high-throughput quantitative PCR chip platform for parallel analyses of multiple gene targets. The PCR mixture distribution among an array of 80 microreactors and subsequent isolation of the reactors were solely realized by a two-step surface tension-based microfluidic scheme, which eliminates the use of pumps, valves and liquid handling instruments. Confinement of the PCR mixture inside the micro reactors was achieved by implementing hybrid flow-restriction passive valves. The microreactors were isolated from each other by the flow of a curable liquid sealant delivered through microchannels by capillary action. We also investigated the effect of detergents that are present in most commercial PCR buffers. Presence of detergents makes the PCR buffer much more wetting on the passive capillary valve surface and this imposes another challenge to the design of the conventional hydrophobic patch valves which has been successfully used for deionized water. We demonstrated a successful capillary valve array with a common air venting channel having a hydrophobic surface for restricting the flow of PCR buffer containing surfactant. The interconnected microreactor array was fabricated on a glass chip substrate with approximate volume of 250 nl microreactor volume for PCR. A different set of PCR primers were preloaded into different microreactor on the PCR array chip for simultaneous amplification of multiple genes. Fluorescent signals from all the microreactors were simultaneously detected at every PCR thermal cycle using EvaGreen fluorescent dye on an in-house real-time PCR instrument. The capability of the scalable PCR array chip was demonstrated by amplifying a fragment of uidA gene for beta-glucuronidase of E. coli genome. Key technical issues related to chip operation such as PCR inhibition on the acid-washed glass substrate, and PCR compatibility of the sealant in both the uncured and cured states have been addressed.
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"Allelic drop-out is a common phenomenon reducing the diagnostic yield of PCR-based target sequencing." In Bioinformatics of Genome Regulation and Structure/ Systems Biology. institute of cytology and genetics siberian branch of the russian academy of science, Novosibirsk State University, 2020. http://dx.doi.org/10.18699/bgrs/sb-2020-070.

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Daly, John, and Mark Davies. "A Natural Convection DNA Amplifier." In ASME 4th International Conference on Nanochannels, Microchannels, and Minichannels. ASMEDC, 2006. http://dx.doi.org/10.1115/icnmm2006-96244.

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Natural convection is the driver of innumerable natural world phenomena. Within the laboratory, it offers simplified geometries and flow structures without the need for auxiliary flow inducement, thereby greatly reducing the risk of external contamination within biomedical applications. Outlined in this paper is a polymerase chain reaction (PCR) device which takes advantage of these distinct qualities. PCR has become synonymous with DNA amplification in molecular biology laboratories throughout the world, and at the heart of PCR is thermal cycling. Commonly PCR is accomplished utilising a three stage thermal cycle, however, the device presented employs an alternative two stage cycle which facilitates a simplified natural convection flow structure. The device is, in its fundamental design format, a well-based thermocycler with fast reaction times of 15 minutes. Through the use of Particle Image Velocimetry (PIV) and flow visualisation techniques, a better understanding of the flow structures and their effect on PCR is attained within a device of dimensions of 1 mm depth by 10mm width and 10mm height. This device may present an opportunity for the development of a practical and inexpensive single gene diagnostic tool. Presented here are the findings of the amplification of an 86-bp fragment of the pGEM®-T vector (Promega) within the convective flow loop.
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Allen, AJ, A. Gonzalez-Ciscar, C. Lendrem, AJ Simpson, P. Kumar, K. Eastham, and M. Brodlie. "G499(P) Clinical diagnostic evaluation of a point of care pcr-based rsv test – a prospective, observational multi-centre cohort study." In Royal College of Paediatrics and Child Health, Abstracts of the RCPCH Conference and exhibition, 13–15 May 2019, ICC, Birmingham, Paediatrics: pathways to a brighter future. BMJ Publishing Group Ltd and Royal College of Paediatrics and Child Health, 2019. http://dx.doi.org/10.1136/archdischild-2019-rcpch.483.

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Hirama, Takashi, Shohei Minezaki, Takefumi Yamaguchi, Taro Nakayama, Koichi Hagiwara, and Minoru Kanazawa. "HIRA-TAN, A Real-Time PCR-Based Diagnostic Test In The Respiratory Tract Secretions, Identifies The Pathogens Therapeutically Targeted In Pneumonia." In American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California. American Thoracic Society, 2012. http://dx.doi.org/10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a5239.

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Hirama, T., S. Minezaki, T. Yamaguchi, M. Kanazawa, and K. Hagiwara. "Multicenter Prospective Study for the Validation of the Novel amd Multiplex Real-Time PCR-Based Diagnostic Test for 20 Pneumonic Pathogens in the Respiratory Tract Secretions." In American Thoracic Society 2009 International Conference, May 15-20, 2009 • San Diego, California. American Thoracic Society, 2009. http://dx.doi.org/10.1164/ajrccm-conference.2009.179.1_meetingabstracts.a2583.

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Mainar-Jaime, R. C., N. Atashparvar, and M. Chirino-Trejo. "Assessment of the diagnostic accuracy of bacteriological culture and the invA-gen-based PCR for the detection of Salmonella organisms from caecal content from slaughtered pigs through Bayesian approaches." In Seventh International Symposium on the Epidemiology and Control of Foodborne Pathogens in Pork. Iowa State University, Digital Press, 2007. http://dx.doi.org/10.31274/safepork-180809-104.

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Halait, Harkanwal, Kelli DeMartin, Sweta Shah, Stephen Soviero, Rachel Langland, Suzanne Cheng, Grantland Hillman, Lin Wu, and Jeffrey Lawrence. "Abstract 2212: The analytic performance of a real-time PCR-based assay for the BRAF V600E mutation used as the companion diagnostic test for the novel BRAF inhibitor RG7204 (PLX4032) in metastatic melanoma." In Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL. American Association for Cancer Research, 2011. http://dx.doi.org/10.1158/1538-7445.am2011-2212.

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Ritzi-Lehnert, Marion, Jan Claussen, Eva Schaeffer, Ole Wiborg, Isabell Wick, Klaus S. Drese, Ralf Himmelreich, et al. "New Lab-on-a-Chip System for Infectious Disease Analysis." In ASME 2010 8th International Conference on Nanochannels, Microchannels, and Minichannels collocated with 3rd Joint US-European Fluids Engineering Summer Meeting. ASMEDC, 2010. http://dx.doi.org/10.1115/fedsm-icnmm2010-31048.

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Early diagnosis followed by personalised efficient therapy of infectious diseases (e.g. respiratory diseases, meningitis, sepsis) can lead to considerable reduction of costs in health care. Point-of-care testing (POCT) can provide early detection since this kind of decentralised analysis can be done by unskilled personnel at any time. Other advantages of automated miniaturised Lab-on-a-Chip systems (LoC) are reduction of time and reagents, elimination of cross-contamination and enhanced reproducibility due to enhanced process control. Such Lab-on-a-Chip systems will establish themselves on market only when sensitivity and specificity meet clinical requirements. An integrated cost-efficient lab-on-a-chip system is presented which allows performing all diagnostic process steps for pathogen analysis of respiratory viruses from nasopharyngeal samples. The microfluidic disposable chip comprises structures for lysis of nasopharyngeal swab samples, preparation of total nucleic acids using magnetic silica beads, reverse transcription followed by QIAplex PCR technology and labelling of the nucleic acids by hybridisation with LiquiChip Beads and streptavidin-R-phycoerythrin. Labelled target sequences are transferred for analysis into a QIAGEN LiquiChip 200 workstation. The core of the instrument is a construction based on rotating heating bars allowing for fast cycling. All chemicals needed for performing of 24 analyses are either stored freeze-dried on the single-use disposable microfluidic chip (processing cartridge) or as liquids in a separate reagent cartridge. After introducing the sample into the lysis chamber of the microfluidic chip and inserting the chip into the device all steps are done automatically. To realise these steps, fluidic control in terms of light barriers and turning valves are integrated into the injection moulded disposable chip. This includes metering structures as well as magnetic stir bars for mixing. The functionality was proven by direct comparison of samples processed manually vs. automatically using the “ResPlex Panel II” for detection of respiratory viruses from nasopharyngeal samples. The efficiency of the automated LoC system yields at about 30–60% as compared to the manually performed reference experiments. Comparing the performance of the instrument with commercially available kits and nucleic acid preparation devices showed slightly weaker but clearly positive final signal intensities obtained from the prototype device even without protocol optimization.
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Nielsen, Søren J., Nana Jacobsen, Jacob U. Fog, Hanni Willenbrock, Adam Baker, Thorarinn Blondal, Martin Vincent, et al. "Early detection of colorectal cancer from patient blood plasma using microRNA-based q-rt-PCR." In AACR International Conference: Molecular Diagnostics in Cancer Therapeutic Development– Sep 27-30, 2010; Denver, CO. American Association for Cancer Research, 2010. http://dx.doi.org/10.1158/diag-10-b2.

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Reports on the topic "PCR-based diagnostic"

1

Levisohn, Sharon, Mark Jackwood, and Stanley Kleven. New Approaches for Detection of Mycoplasma iowae Infection in Turkeys. United States Department of Agriculture, February 1995. http://dx.doi.org/10.32747/1995.7612834.bard.

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Mycoplasma iowae (Mi) is a pathogenic avian mycoplasma which causes mortality in turkey embryos and as such has clinical and economic significance for the turkey breeder industry. Control of Mi infection is severely hampered by lack of adequate diagnostic tests, together with resistance to most antibiotics and resilience to environment. A markedly high degree of intra-species antigenic variation also contributes to difficulties in detection and control of infection. In this project we have designed an innovative gene-based diagnostic test based on specific amplification of the 16S rRNA gene of Mi. This reaction, designed Multi-species PCR-RFLP test, also amplifies the DNA of the pathogenic avian mycoplasmas M. gallisepticum (Mg) and M. synoviae (Ms). This test detects DNA equivalent to about 300 cfu Mi or either of the other two target mycoplasmas, individually or in mixed infection. It is a quick test, applicable to a wide variety of clinical samples, such as allantoic fluid or tracheal or cloacal swab suspensions. Differential diagnosis is carried out by gel electro-phoresis of the PCR amplicon digested with selected restriction enzymes (Restriction Fragment Length Polymorphism). This can also be readily accomplished by using a simple Dot-Blot hybridization assay with digoxigenin-labeled oligonucleotide probes reacting specifically with unique Mi, Mg or Ms sequences in the PCR amplicon. The PCR/OLIGO test increased sensitivity by at least 10-fold with a capacity for rapid testing of large numbers of samples. Experimental infection trials were carried out to evaluate the diagnostic tools and to study pathogenesis of Mi infection. Field studies and experimental infection of embryonated eggs indicated both synergistic and competitive interaction of mycoplasma pathogens in mixed infection. The value of the PCR diagnostic tests for following the time course of egg transmission was shown. A workable serological test (Dot Immunobinding Assay) was also developed but there was no clear-cut evidence that infected turkeys develop an immune response. Typing of a wide spectrum of Mi field isolates by a variety of gene-based molecular techniques indicated a higher degree of genetic homogeneity than predicted on the basis of the phenotypic variability. All known strains of Mi were detected by the method developed. Together with an M. meleagridis-PCR test based on the same gene, the Multi-species PCR test is a highly valuable tool for diagnosis of pathogenic mycoplasmas in single or mixed infection. The further application of this rapid and specific test as a part of Mi and overall mycoplasma control programs will be dependent on developments in the turkey industry.
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Hedrick, Ronald, and Herve Bercovier. Characterization and Control of KHV, A New Herpes Viral Pathogen of Koi and Common Carp. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7695871.bard.

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In this project we proposed to characterize the virus genome and the structural virion polypeptides to allow development of improved diagnostic approaches and potential vaccination strategies. These goals have been mostly achieved and the corresponding data were published in three papers (see below) and three more manuscripts are in preparation. The virion polypeptides of KHV strains isolated from USA (KHV-U) and Israel (KHV-I) were found to be identical. Purified viral DNA analyzed with a total of 5 restriction enzymes demonstrated no fragment length polymorphism between KHV-I and KHV-U but both KHV isolates differed significantly from the cyprinid herpesvirus (CHV) and the ictalurid herpesvirus (channel catfish virus or CCV). Using newly obtained viral DNA sequences two different PCR assays were developed that need to be now further tested in the field. We determined by pulse field analysis that the size of KHV genome is around 280 kbp (1-1. Bercovier, unpublished results). Sequencing of the viral genome of KHV has reached the stage where 180 kbp are sequenced (twice and both strands). Four hypothetical genes were detected when DNA sequences were translated into amino acid sequences. The finding of a gene of real importance, the thymidine kinase (TK) led us to extend the study of this specific gene. Four other genes related to DNA synthesis were found. PCR assays based on defined sequences were developed. The PCR assay based on TK gene sequence has shown improved sensitivity in the detection of KHV DNA compared to regular PCR assays. </P> <P><SPAN>With the ability to induce experimental infections in koi with KHV under controlled laboratory conditions we have studied the progress and distribution of virus in host tissues, the development of immunity and the establishment of latent infections. Also, we have investigated the important role of water temperature on severity of infections and mortality of koi following infections with KHV. These initial studies need to be followed by an increased focus on long-term fate of the virus in survivors. This is essential in light of the current &quot;controlled exposure program&quot; used by farmers to produce KHV &quot;naturally resistant fish&quot; that may result in virus or DNA carriers. </SPAN></P> <P><SPAN>The information gained from the research of this project was designed to allow implementation of control measures to prevent the spread of the virus both by improved diagnostic approaches and preventive measures. We have accomplished most of these goals but further studies are needed to establish even more reliable methods of prevention with increased emphases on improved diagnosis and a better understanding of the ecology of KHV. </SPAN>
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Levisohn, Sharon, Maricarmen Garcia, David Yogev, and Stanley Kleven. Targeted Molecular Typing of Pathogenic Avian Mycoplasmas. United States Department of Agriculture, January 2006. http://dx.doi.org/10.32747/2006.7695853.bard.

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Intraspecies identification (DNA "fingerprinting") of pathogenic avian mycoplasmas is a powerful tool for epidemiological studies and monitoring strain identity. However the only widely method available for Mycoplasma gallisepticum (MG) and M. synoviae (MS)wasrandom amplified polymorphic DNA (RAPD). This project aimed to develop alternative and supplementary typing methods that will overcome the major constraints of RAPD, such as the need for isolation of the organism in pure culture and the lack of reproducibility intrinsic in the method. Our strategy focussed on recognition of molecular markers enabling identification of MG and MS vaccine strains and, by extension, pathogenic potential of field isolates. Our first aim was to develop PCR-based systems which will allow amplification of specific targeted genes directly from clinical material. For this purpose we evaluated the degree of intraspecies heterogeneity in genes encoding variable surface antigens uniquely found in MG all of which are putative pathogenicity factors. Phylogenic analysis of targeted sequences of selected genes (pvpA, gapA, mgc2, and lp) was employed to determine the relationship among MG strains.. This method, designated gene targeted sequencing (GTS), was successfully employed to identify strains and to establish epidemiologically-linked strain clusters. Diagnostic PCR tests were designed and validated for each of the target genes, allowing amplification of specific nucleotide sequences from clinical samples. An mgc2-PCR-RFLP test was designed for rapid differential diagnosis of MG vaccine strains in Israel. Addressing other project goals, we used transposon mutagenesis and in vivo and in vitro models for pathogenicity to correlated specific changes in target genes with biological properties that may impact the course of infection. An innovative method for specific detection and typing of MS strains was based on the hemagglutinin-encoding gene vlhA, uniquely found in this species. In parallel, we evaluated the application of amplified fragment length polymorphism (AFLP) in avian mycoplasmas. AFLP is a highly discriminatory method that scans the entire genome using infrequent restriction site PCR. As a first step the method was found to be highly correlated with other DNA typing methods for MG species and strain differentiation. The method is highly reproducible and relatively rapid, although it is necessary to isolate the strain to be tested. Both AFLP and GTS are readily to amenable to computer-assisted analysis of similarity and construction of a data-base resource. The availability of improved and diverse tools will help realize the full potential of molecular typing of avian mycoplasmas as an integral and essential part of mycoplasma control programs.
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Bercovier, Herve, and Paul Frelier. Pathogenic Streptococcus in Tilapia: Rapid Diagnosis, Epidemiology and Pathophysiology. United States Department of Agriculture, October 1994. http://dx.doi.org/10.32747/1994.7568776.bard.

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Within the project "Pathogenic Streptococcus in Tilapia", gram positive cocci pathogens of fish in Israel and in the United States were characterized. We showed that Streptococcus shiloi, the name for an agent causing septicemic infection in fish, is a junior synonym of Streptococcus iniae and that Enterococcus seriolicida is a junior synonym of Lactococcus garvieae, a causative agent of septicemia and meningo-encephalitis in fish. Molecular epidemiology studies on these two pathogens, based on 16S rDNA sequences and ribotyping showed that although each country had specific clones, S. iniae originated probably from the U.S. and L. garvieae from Japan. PCR assays were developed for both pathogens and applied to clinical samples. S. agalactiael S. difficile was also recognized for the first time in the U.S. in tilapia. Our histopathological studies explained the noted paradox (abundant in vitro growth often accompanied by scant to small numbers of organisms within the meninges in histologic sections of brain) in diagnostic of fish streptococcus. The greatest concentration of cocci were consistently observed within macrophages infiltrating the extrameningeal fibroadipose tissue surrounding the brain within the calvarium. These results also suggests that the primary route of meningeal infection may be extension from the extrameningeal connective tissue rather than meningeal vascular emigration of cocci-containing macrophages. Our work has resulted in a cognizance of streptococcus as fish pathogen which goes beyond the pathology observed in tilapia and is already extended to many aquaculture fish species in Israel and in the United States. Finally, our data suggest that vaccines (bivalent or trivalent) could be developed to prevent most of the damages caused by streptococcus in aquaculture.
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Mackey, Katherine, Irina Arkhipova-Jenkins, Charlotte Armstrong, Emily Gean, Johanna Anderson, Robin A. Paynter, and Mark Helfand. Antibody Response Following SARS-CoV-2 Infection and Implications for Immunity: A Rapid Living Review. Agency for Healthcare Research and Quality (AHRQ), March 2021. http://dx.doi.org/10.23970/ahrqepccovidimmunity.

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 Evidence suggests that the majority of adults develop detectable levels of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies following infection with SARS-CoV-2 (moderate strength of evidence* [SoE]).  IgM levels peak approximately 20 days after symptom onset or RT-PCR diagnosis and subsequently decline. IgG levels peak approximately 25 days after symptom onset or RT-PCR diagnosis and may remain detectable for at least 120 days (moderate SoE*).  Almost all adults develop neutralizing antibodies in response to SARS-CoV-2 infection, and these antibodies may remain detectable for at least 152 days (low SoE*).  A small percentage of people do not develop antibodies in response to SARS-CoV-2 infection for reasons that are largely unclear but may be related to less severe disease or absence of symptoms.  Antibody prevalence does not appear to vary by age or sex, but older age may be associated with higher antibody levels (low SoE*). Non-White race may be associated with higher antibody prevalence and levels (low SoE*). COVID-19 severity and presence of symptoms may also be associated with higher antibody prevalence or levels (low SoE*). More evidence is needed to draw stronger conclusions regarding how the antibody response varies by patient characteristics and disease factors.  Studies to date have not established the relationship between the development of antibodies after RT-PCR-diagnosed SARS-CoV-2 infection and the risk of reinfection. Studies based on index serologic testing suggest that the presence of antibodies is associated with a lower risk of a subsequent positive SARS-CoV-2 RT-PCR test.
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Ficht, Thomas, Gary Splitter, Menachem Banai, and Menachem Davidson. Characterization of B. Melinensis REV 1 Attenuated Mutants. United States Department of Agriculture, December 2000. http://dx.doi.org/10.32747/2000.7580667.bard.

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Brucella Mutagenesis (TAMU) The working hypothesis for this study was that survival of Brucella vaccines was directly related to their persistence in the host. This premise is based on previously published work detailing the survival of the currently employed vaccine strains S19 and Rev 1. The approach employed signature-tagged mutagenesis to construct mutants interrupted in individual genes, and the mouse model to identify mutants with attenuated virulence/survival. Intracellular survival in macrophages is the key to both reproductive disease in ruminants and reticuloendothelial disease observed in most other species. Therefore, the mouse model permitted selection of mutants of reduced intracellular survival that would limit their ability to cause reproductive disease in ruminants. Several classes of mutants were expected. Colonization/invasion requires gene products that enhance host-agent interaction or increase resistance to antibacterial activity in macrophages. The establishment of chronic infection requires gene products necessary for intracellular bacterial growth. Maintenance of chronic infection requires gene products that sustain a low-level metabolism during periods characterized little or no growth (1, 2). Of these mutants, the latter group was of greatest interest with regard to our originally stated premise. However, the results obtained do not necessarily support a simplistic model of vaccine efficacy, i.e., long-survival of vaccine strains provides better immunity. Our conclusion can only be that optimal vaccines will only be developed with a thorough understanding of host agent interaction, and will be preferable to the use of fortuitous isolates of unknown genetic background. Each mutant could be distinguished from among a group of mutants by PCR amplification of the signature tag (5). This approach permitted infection of mice with pools of different mutants (including the parental wild-type as a control) and identified 40 mutants with apparently defective survival characteristics that were tentatively assigned to three distinct classes or groups. Group I (n=13) contained organisms that exhibited reduced survival at two weeks post-infection. Organisms in this group were recovered at normal levels by eight weeks and were not studied further, since they may persist in the host. Group II (n=11) contained organisms that were reduced by 2 weeks post infection and remained at reduced levels at eight weeks post-infection. Group III (n=16) contained mutants that were normal at two weeks, but recovered at reduced levels at eight weeks. A subset of these mutants (n= 15) was confirmed to be attenuated in mixed infections (1:1) with the parental wild-type. One of these mutants was eliminated from consideration due to a reduced growth rate in vitro that may account for its apparent growth defect in the mouse model. Although the original plan involved construction of the mutant bank in B. melitensis Rev 1 the low transformability of this strain, prevented accumulation of the necessary number of mutants. In addition, the probability that Rev 1 already carries one genetic defect increases the likelihood that a second defect will severely compromise the survival of this organism. Once key genes have been identified, it is relatively easy to prepare the appropriate genetic constructs (knockouts) lacking these genes in B. melitensis Rev 1 or any other genetic background. The construction of "designer" vaccines is expected to improve immune protection resulting from minor sequence variation corresponding to geographically distinct isolates or to design vaccines for use in specific hosts. A.2 Mouse Model of Brucella Infection (UWISC) Interferon regulatory factor-1-deficient (IRF-1-/- mice have diverse immunodeficient phenotypes that are necessary for conferring proper immune protection to intracellular bacterial infection, such as a 90% reduction of CD8+ T cells, functionally impaired NK cells, as well as a deficiency in iNOS and IL-12p40 induction. Interestingly, IRF-1-/- mice infected with diverse Brucella abortus strains reacted differently in a death and survival manner depending on the dose of injection and the level of virulence. Notably, 50% of IRF-1-/- mice intraperitoneally infected with a sublethal dose in C57BL/6 mice, i.e., 5 x 105 CFU of virulent S2308 or the attenuated vaccine S19, died at 10 and 20 days post-infection, respectively. Interestingly, the same dose of RB51, an attenuated new vaccine strain, did not induce the death of IRF-1-/- mice for the 4 weeks of infection. IRF-1-/- mice infected with four more other genetically manipulated S2308 mutants at 5 x 105 CFU also reacted in a death or survival manner depending on the level of virulence. Splenic CFU from C57BL/6 mice infected with 5 x 105 CFU of S2308, S19, or RB51, as well as four different S2308 mutants supports the finding that reduced virulence correlates with survival Of IRF-1-/- mice. Therefore, these results suggest that IRF-1 regulation of multi-gene transcription plays a crucial role in controlling B. abortus infection, and IRF-1 mice could be used as an animal model to determine the degree of B. abortus virulence by examining death or survival. A3 Diagnostic Tests for Detection of B. melitensis Rev 1 (Kimron) In this project we developed an effective PCR tool that can distinguish between Rev1 field isolates and B. melitensis virulent field strains. This has allowed, for the first time, to monitor epidemiological outbreaks of Rev1 infection in vaccinated flocks and to clearly demonstrate horizontal transfer of the strain from vaccinated ewes to unvaccinated ones. Moreover, two human isolates were characterized as Rev1 isolates implying the risk of use of improperly controlled lots of the vaccine in the national campaign. Since atypical B. melitensis biotype 1 strains have been characterized in Israel, the PCR technique has unequivocally demonstrated that strain Rev1 has not diverted into a virulent mutant. In addition, we could demonstrate that very likely a new prototype biotype 1 strain has evolved in the Middle East compared to the classical strain 16M. All the Israeli field strains have been shown to differ from strain 16M in the PstI digestion profile of the omp2a gene sequence suggesting that the local strains were possibly developed as a separate branch of B. melitensis. Should this be confirmed these data suggest that the Rev1 vaccine may not be an optimal vaccine strain for the Israeli flocks as it shares the same omp2 PstI digestion profile as strain 16M.
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7

Jordan, Ramon L., Abed Gera, Hei-Ti Hsu, Andre Franck, and Gad Loebenstein. Detection and Diagnosis of Virus Diseases of Pelargonium. United States Department of Agriculture, July 1994. http://dx.doi.org/10.32747/1994.7568793.bard.

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Pelargonium (Geranium) is the number one pot plant in many areas of the United States and Europe. Israel and the U.S. send to Europe rooted cuttings, foundation stocks and finished plants to supply a certain share of the market. Geraniums are propagated mainly vegetatively from cuttings. Consequently, viral diseases have been and remain a major threat to the production and quality of the crop. Among the viruses isolated from naturally infected geraniums, 11 are not specific to Pelargonium and occur in other crops while 6 other viruses seem to be limited to geranium. However, several of these viruses are not sufficiently characterized to conclude that they are distinct agents and their nomenclature and taxonomy are confusing. The ability to separate, distinguish and detect the different viruses in geranium will overcome obstacles te developing effective detection and certification schemes. Our focus was to further characterize some of these viruses and develop better methods for their detection and control. These viruses include: isolates of pelargonium line pattern virus (PLPV), pelargonium ringspot virus (PelRSV), pelargonium flower break virus (PFBV), pelargonium leaf curl (PLCV), and tomato ringspot virus (TomRSV). Twelve hybridoma cell lines secreting monoclonal antibodies specific to a geranium isolate of TomRSV were produced. These antibodies are currently being characterized and will be tested for the ability to detect TomRSV in infected geraniums. The biological, biochemical and serological properties of four isometric viruses - PLPV, PelRSV, and PFBV (and a PelRSV-like isolate from Italy called GR57) isolated from geraniums exhibiting line and ring pattern or flower break symptoms - and an isolate ol elderbeny latent virus (ELV; which the literature indicates is the same as PelRSV) have been determined Cloned cDNA copies of the genomic RNAs of these viruses were sequenced and the sizes and locations of predicted viral proteins deduced. A portion of the putative replicase genes was also sequenced from cloned RT-PCR fragments. We have shown that, when compared to the published biochemical and serological properties, and sequences and genome organizations of other small isometric plant viruses, all of these viruses should each be considered new, distinct members of the Carmovirus group of the family Tombusviridae. Hybridization assays using recombinant DNA probes also demonstrated that PLPV, PelRSV, and ELV produce only one subgenomic RNA in infected plants. This unusual property of the gene expression of these three viruses suggests that they are unique among the Carmoviruses. The development of new technologies for the detection of these viruses in geranium was also demonstrated. Hybridization probes developed to PFBV (radioactively-labeled cRNA riboprobes) and to PLPV (non-radioactive digoxigenin-labeled cDNAs) were generally shown to be no more sensitive for the detection of virus in infected plants than the standard ELISA serology-based assays. However, a reverse transcriptase-polymerase chain reaction assay was shown to be over 1000 times more sensitive in detecting PFBV in leaf extracts of infected geranium than was ELISA. This research has lead to a better understanding of the identity of the viruses infecting pelargonium and to the development of new tools that can be used in an improved scheme of providing virus-indexed pelargonium plants. The sequence information, and the serological and cloned DNA probes generated from this work, will allow the application of these new tools for virus detection, which will be useful in domestic and international indexing programs which are essential for the production of virus-free germplasm both for domestic markets and the international exchange of plant material.
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