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Artykuły w czasopismach na temat "PCR-RFLP"
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Karaca, Mehmet, Ayse Gul Ince, Saadet Tugrul Ay, Kenan Turgut i Ahmet Naci Onus. "PCR-RFLP and DAMD-PCR genotyping forSalviaspecies". Journal of the Science of Food and Agriculture 88, nr 14 (listopad 2008): 2508–16. http://dx.doi.org/10.1002/jsfa.3372.
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Trejaut, Jean, i Heather Dunckley. "HLA-DRB5 genotyping by PCR-RFLP". Tissue Antigens 43, nr 1 (styczeń 1994): 60–63. http://dx.doi.org/10.1111/j.1399-0039.1994.tb02300.x.
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MacAllister, Thomas W. "{BLR 2083} DNA Fingerprinting - PCR - RFLP". Biotechnology Law Report 14, nr 4 (lipiec 1995): 641–48. http://dx.doi.org/10.1089/blr.1995.14.641a.
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Pourzand, Charareh, i Peter Cerutti. "Genotypic mutation analysis by RFLP/PCR". Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 288, nr 1 (lipiec 1993): 113–21. http://dx.doi.org/10.1016/0027-5107(93)90213-y.
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VICTOIR, K., A. L. BAÑULS, J. AREVALO, A. LLANOS-CUENTAS, R. HAMERS, S. NOËL, S. DE DONCKER, D. LE RAY, M. TIBAYRENC i J. C. DUJARDIN. "The gp63 gene locus, a target for genetic characterization of Leishmania belonging to subgenus Viannia". Parasitology 117, nr 1 (lipiec 1998): 1–13. http://dx.doi.org/10.1017/s0031182098002789.
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In the present study the gp63 gene locus was used as a target for genetic characterization of Leishmania parasites by 2 methods: (i) RFLP analysis with several restriction enzymes (gp63–RFLP), and (ii) intra-genic PCR amplification coupled with restriction analysis (PCR–RFLP). Both methods were applied to a large number of natural isolates belonging to 4 species of the subgenus Viannia, namely L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis and L. (V.) lainsoni: reference stocks of subgenus Leishmania were included as outgroups. Multilocus isoenzyme typing (MLEE) was used as a reference. On the one hand gp63–RFLP evidenced an extensive polymorphism and revealed specific markers for subgenus, species and geographical populations: congruence with MLEE was demonstrated statistically. The particular interest of gp63–RFLP was illustrated by infra-specific polymorphism, because of the possible relationship with phenotype diversity. On the other hand intra-genic amplification was less resolutive than gp63–RFLP, but also allowed discrimination of the 2 subgenera (PCR alone) and all the species tested in the subgenus Viannia (PCR–RFLP). PCR–RFLP presents an important operational advantage as it allows genetic characterization of minute amounts of parasites, using Leishmania specific primers. The polymorphism revealed by gp63–RFLP and PCR–RFLP illustrates the very high genomic and genetic plasticity of gp63 genes.
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Ivona Djurkin, Kušec, Samac Danijela, Margeta Vladimir, Radišić Žarko, Vincek Dragutin i Kušec Goran. "Efficiency of PCR-RFLP and species-specific PCR for the identification of meat origin in dry sausages". Czech Journal of Food Sciences 35, No. 5 (20.10.2017): 386–91. http://dx.doi.org/10.17221/243/2016-cjfs.
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The purpose of this investigation was the identification of chicken, beef and sheep meat in pork sausages using PCR-RFLP and PCR with pecies-specific primers. Six dry fermented pork sausages were produced by adding beef, sheep and chicken meat to each in the amount of 1 and 5%. DNA was extracted from five regions of each sausage and PCR-RFLP together with PCR using species-specific primers was performed. PCR-RFLP analysis was successful only for chicken meat, while species-specific PCR was effective for identification of chicken, eef and sheep meat in all ratios and from all regions of the sausages. The results of our study show that discovering adulteration using PCR-RFLP is suitable only for chicken meat in the investigated products, while for detection of beef and sheep meat use of species-specific oligonucleotides is more effective.
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Hazlianda, Cut, Kamaliah Muis i Isma Lubis. "A Comparative Study of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism and Fungal Culture for the Evaluation of Fungal Species in Patients with Tinea Cruris". Open Access Macedonian Journal of Medical Sciences 5, nr 7 (21.11.2017): 844–47. http://dx.doi.org/10.3889/oamjms.2017.197.
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BACKGROUND: Tinea cruris is the second most common dermatophytosis in the world and the most common in Indonesia. The conventional laboratory tests for dermatophyte infection are slow and less specific. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) is a PCR method with the addition of enzyme after amplification, therefore enabling for more specific results.AIM: This study aimed to find whether the PCR-RFLP test could yield the same fungal species result as a fungal culture.METHODS: The specimens were skin scrapings from thirty-one patients suspected tinea cruris. The tools and materials that were used were Sabaroud’s dextrose agar media, primer ITS 1 and ITS 4 and MvaI.RESULTS: The equation percentage of the test result species between PCR-RFLP and fungal culture was 50% of 12 subjects whose the test results were both positive from the fungal culture and PCR-RFLP. The percentage of the test result with fungal culture the fungal species were found, but in the PCR-RFLP test which the fungal species was not found, the percentage was 50% of 12 subjects which the test results were both positive as fungi from the culture and PCR-RFLP test.CONCLUSIONS: The species from PCR-RFLP examination was the same with the fungal culture.
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Khayyira, Amalia Sitti, Viktoria Mardhika Estepane i Amarila Malik. "RAPID PCR–BASED DETECTION OPTIMIZATION OF PORCINE DNA IN GELATIN CAPSULE SHELL". International Journal of Applied Pharmaceutics 10, nr 6 (22.11.2018): 217. http://dx.doi.org/10.22159/ijap.2018v10i6.29346.
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Objective: This study was conducted to optimize the genomic deoxyribonucleic acid (DNA) based molecular detection of gelatin derived from porcine by performing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and duplex PCR method employing cyt B gene.Methods: Optimization was carried out for DNA extraction, PCR conditions, and the sensitivity of the PCR-RFLP method. Due to the very low DNA trace in gelatin after the various manufacturing process, the extraction was optimized to obtain sufficient DNA which was visible on the agarose gel. PCR-RFLP was carried out using universal primers and BsaJI restriction enzyme, and duplex PCR was carried out using two sets of porcine-specific primers. Porcine and bovine DNA were mixed in various concentration to confirm sensitivity of both methods, i.e. 100%, 50%, 10%, 1%, 0.5%, 0.1%, 0.05%, and 0.01%Results: Both methods, PCR-RFLP, and Duplex PCR, were able to detect as low as 0.01% porcine DNA, indicated by the presence of porcine DNA amplicon bands (131 bp and 228 bp for PCR-RFLP, 212 bp and 398 bp for duplex PCR). Although DNA bands presented in low intensity, identification of porcine and bovine species and estimation of DNA quantities were possible.Conclusion: Both conventional PCR methods, i.e. PCR-RFLP and Duplex PCR, were sensitive, specific, and suitable as a rapid initial detection method for molecular detection of porcine in gelatin capsule shells.
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Bouzouita, Imen, Henda Draoui, Samia Mahdhi, Leila Essalah i Leila Slim Saidi. "Evaluation of PCR pncA-restriction fragment length polymorphism and PCR amplification of genomic regions of difference for the identification of M. bovis strains in lymph nodes cultures". African Health Sciences 21, nr 3 (27.09.2021): 985–89. http://dx.doi.org/10.4314/ahs.v21i3.4.
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Background: A rapid accurate identification of Mycobacterium bovis is essential for surveillance purposes.
Objectives: A PCR pncA-Restriction Fragment Length Polymorphism (RFLP) and a multiplex PCR based on the detection of 3 regions of difference (RD-PCR): RD9, RD4 and RD1 were evaluated for the identification of M. bovis in lymph nodes cultures, in Tunisia, during 2013-2015.
Methods: Eighty-two M. tuberculosis complex strains were identified using the biochemical tests, GenoType MTBC assay, PCR pncA-RFLP and RD-PCR.
Results: The PCR pncA-RFLP showed that 54 M. bovis strains, identified by GenoType MTBC, had a mutation at position 169 of pncA gene. Twenty-eight strains did not show any mutation at this position 27 M. tuberculosis isolates and one M. caprae. The PCR pncA-RFLP had a sensitivity of 100.0% (95%CI: 93.3 -100.0) and a specificity of 100.0% (95%CI: 87.9- 100.0) for identifying M. bovis. The RD-PCR showed that all M. bovis strains had the RD9 and RD4 deleted but presented RD1. RD-PCR also presented high sensitivity and specificity in detecting M. bovis strains (100.0%).
Conclusions: PCR pncA-RFLP and RD-PCR represent very accurate and rapid tools to identify M. bovis. They can be easily implemented in each laboratory due to their low cost and easy use.
Keywords: GenoType MTBC; lymph nodes; Mycobacterium bovis; PCR pncA-RFLP; RD-PCR.
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Andrini, Fauzia, Imam Supardi, Sunarjati Sudigdoadi i Sadeli Masria. "Detection of Staphylococcus aureus’s Strain Similarity on Surgical Ward Nurses’s Hand and Nose and Post Operative Wound Infection Using Coa Gene Through PCR-RFLP Method". Jurnal Ilmu Kedokteran 4, nr 2 (23.11.2017): 116. http://dx.doi.org/10.26891/jik.v4i2.2010.116-122.
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Staphylococcus aureus (S.aureus) remains to be the most important cause of post operative wound infection. Nursescould become reservoirs to transmit S.aureus through contaminated hands transiently, or through colonized nose.Strain polymorphism could be determined by Restriction Fragment Length Polymorphism (RFLP), using coa gene andrestriction endonuclease enzyme Alu1. There were 30 isolates of S.aureus’s infection, and 20 isolates taken from handsand nose of the nurses in charge. From 50 isolate positive S.aureus, PCR results showed single and multiple bandswithin 300 to 900 base pairs (bp) in length, and multiple bands within 200 to 600 bp. Five out of 30 patients (17%)showed no PCR-RFLP similarity with any of the nurses. Ten out of 15 nurses which hands were positive for S.aureus,has PCR-RFLP similarity with some patients. There was only 1 out of 5 nurses which nose was positive for S.aureus,showed PCR-RFLP similarity with some patients. Statistically, the proportion of the similar PCR-RFLP between thosesamples in this study is 0.12 (12%). Conclusion: Nurses had 12 % PCR-RFLP similarity for S.aureus with post operativewound infection.
Rozprawy doktorskie na temat "PCR-RFLP"
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Šuranská, Hana. "Identifikace vinných kvasinek metodou PCR-RFLP". Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2009. http://www.nusl.cz/ntk/nusl-216494.
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This thesis deals with identification of the wine yeasts by applying the PCR-RFLP method. The identification and characteristic of the yeasts has gone through substantial changes in recent years. There have been introduced new methods of taxonomic classifying based on the molecular methods, which are oriented to easy and fast identification. One of these methods is the PCR-RFLP method. The amplification of the 5•8S-ITS rDNA sequence by the polymerase chain reaction with use of the primers ITS1 and ITS4 leads to the amplification of the specific sequence of DNA. Such multiplied DNA is after repurifying by the ethanol and drying submitted to the restriction analysis. With use of the restriction endonuklases DNA is chopped into the specific segments typical for the particular genus. The chopped fragments can be separated in the electric field in the agarose gel and subsequently evaluated. In this thesis together 63 type yeasts were used. These yeasts were analysed by applying of the seven restriction endonuklases – HaeIII, HhaI, HinfI, HpaII, TaqI, AluI a MseI. The final image of type yeasts splitting was compared to the results of splitting of already identified wine yeasts and these yeasts were subsequently taxonomically classified. Evaluation of genetic similarity was conducted by program BioNumerics and as the results the dendrograms that were created with use of Jaccard‘s coefficients are obtained.
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Ziebell, Kim. "Evaluation of PCR and PCR-RFLP protocols to identify the Shiga toxins". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0026/MQ51107.pdf.
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Olivová, Radana. "Optimalizace metody PCR-RFLP pro taxonomické zařazení kvasinek". Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2009. http://www.nusl.cz/ntk/nusl-216560.
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This thesis deal with optimalization method PCR – RFLP for taxonomy enlistment of yeasts. Conventional identification methods for yeasts are time-consuming. Molecular biological method based on PCR are instrumental towards fast and precise identification as compared to conventional phenotypic methods. In this thesis molecular biological method PCR – RFLP was used for identification and enlistment of yeasts. This metod follow repeating spacers of ribozomal DNA of yeast, characteristic for each species and strain. By the help of PCR were amplified specific partitions of DNA. These fragments of DNA were split by restriction endonucleases and identified by horizontal electroforesis. In background of this thesis there are information about yeasts, their taxonomy and molecular biological methods.
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Arraes, Ana Carolina Palmeira. "Detecção da diversidade molecular de Candida spp. isoladas de UTI neonatal". reponame:Repositório Institucional da UFBA, 2013. http://www.repositorio.ufba.br/ri/handle/ri/11817.
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A incidência de candidemia tem aumentado nos últimos anos e o espectro das espécies de Candida tem mudado com a emergência das espécies não-albicans e com o aumento da resistência antifúngica. A técnica de PCR associada à análise dos polimorfismos de fragmentos de restrição (PCR-RFLP) e AP-PCR são exemplos de técnicas moleculares utilizadas para a detecção da variabilidade genética desses micro-organismos. O objetivo deste trabalho foi caracterizar molecularmente Candida spp. clinicamente importantes. Foram estudadas 82 leveduras do gênero Candida, 73 isolados clínicos e nove cepas padrão de referência da “American Type Culture Collection” (ATCC). O DNA genômico foi extraído através de lise mecânica/química e fenol-clorofórmio. Após amplificação com iniciadores ITS1 e ITS4, os produtos da PCR foram digeridos com as enzimas DdeI, HaeIII, BfaI e MspI. Outra amplificação foi realizada com o iniciador arbitrário AP-4. A identificação fenotípica revelou a frequência de 38% de C. parapsilosis, 33% de C. albicans, 27,5% de C. tropicalis e 1,5% de C. guilliermondii. Após amplificação, foi possível identificar e diferenciar as espécies C. lusitaniae, C. guilliermondii, C. famata e C. glabrata. A diferenciação entre C. albicans, C. dubliniensis C. tropicalis, C. krusei e C. parapsilosis não foi bem evidenciada com as enzimas DdeI e HaeIII. Porém, MspI conseguiu diferenciar C. tropicalis, C. krusei, C. parapsilosis, C. glabrata, C. guilliermondii, C. lusitaniae e C. famata, juntamente com BfaI que separou C. albicans de C. dubliniensis. Já a técnica de AP-PCR com o iniciador AP-4, possibilitou a distinção das nove espécies cepas padrão ATCC de Candida, pois todas apresentaram perfis de amplificação com número, intensidade e tamanho de banda específico, onde cada espécie foi alocada em grupo distinto e a relação de similaridade variou de 38-69%, ratificando o poder de diferenciação das espécies. As técnicas moleculares foram reprodutíveis e relativamente rápidas, de fácil interpretação e podem ser aplicadas na identificação de espécies clinicamente importantes de Candida para auxílio no diagnóstico mais rápido e tratamento mais eficiente.
Salvador
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Malvárez, Macedo Gabriela. "Aplicação das metodologias PCR/RFLP para caracterização de fungos ectomicorrízicos em eucalipto /". reponame:Repositório Institucional da UFSC, 1999. https://repositorio.ufsc.br/handle/123456789/112290.
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Björkbom, Tommy. "PCR-RFLP analys av mt-DNA hos Öring (Salmo trutta) i Gävleborgs län". Thesis, University of Gävle, Department of Electronics, Mathematics and Natural Sciences, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:hig:diva-7624.
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Sultan, Amal Hassan. "The development of molecular tools for identification of Leishmania species by PCR-RFLP". Thesis, University of Liverpool, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479067.
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MEDEIROS, Rafael Acioli. "Caracterização da Leishmania infantum e Leishmania (Viannia) braziliensis em cães provenientes da Região Metropolitana do Recife, Pernambuco". Universidade Federal de Pernambuco, 2013. https://repositorio.ufpe.br/handle/123456789/12218.
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As Leishmanioses são uma antropozoonose com ampla distribuição geográfica e ocorrência em torno de 88 países. Na Leishmaniose visceral americana (LVA), os cães domésticos são considerados os principais reservatórios da L. infantum no ambiente domiciliar. Diferentes perfis epidemiológicos da Leishmaniose Tegumentar Americana (LTA) têm sido caracterizados pela presença de casos humanos em áreas de colonizações antigas, sugerindo uma antropozoonose entre os animais domésticos por demonstrarem lesões tegumentares causadas pela Leishmania (Vianna) braziliensis. O diagnóstico das leishmanioses pode ser realizado através de métodos clínicos, epidemiológicos, parasitológicos, imunológicos e moleculares. Em áreas endêmicas o diagnóstico clínico e epidemiológico dessas zoonoses é dificultado pela similaridade clínica com outras doenças, tornando a utilização de exames laboratoriais de suma importância para confirmação diagnóstica. Em virtude da necessidade de diagnóstico especifico das leishmanioses na espécie canina, este trabalho tem como objetivo a utilização da PCR-RFLP, tendo como alvo o espaçador interno transcrito (ITS-1), para diferenciação das espécies de Leishmania em cães provenientes da Região Metropolitana do Recife. Foram coletadas 40 amostras de soro e medula de cães, 30 machos e 10 fêmeas, com idades entre 2 e 7 anos , que apresentaram suspeita clinica de leishmaniose quando atendidos no Hospital Veterinário da UFRPE. Foram realizados os testes diagnósticos de exame direto em medula e Reação indireta de imunoflorescência tanto para LTA quanto LVA. Além disso foi realizado a PCR-RFLP nas amostras de soro e urina para a pesquisa de L. infantum e L. (viannia) braziliensis. Dos 40 cães pesquisados 23 foram positivos para L.infantum e 17 foram negativos para as duas espécies pesquisadas. Quando comparado com os testes diagnósticos convencionais , 3 dos 23 cães foram positivos apenas na técnica molecular. Portanto, a ITS1-PCR RFLP pode ser uma importante ferramenta para o diagnostico e caracterização da leishmaniose canina em uma determinada região.
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Abreu, Daniel Paiva Barros de. "Caracteriza??o fenot?pica, genot?pica e perfil de sensibilidade a antif?ngicos de isolados cl?nicos de c?es e gatos pertencentes ao Complexo Sporothrix schenckii oriundos do estado do Rio de Janeiro". Universidade Federal Rural do Rio de Janeiro, 2017. https://tede.ufrrj.br/jspui/handle/jspui/2031.
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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq
Dimorphic fungi belonging to Sporothrix schenckii complex are responsible for sporotrichosis, important fungal infection with worldwide distribution. The anthropozoonotic characteristic is of high relevance in the state of Rio de Janeiro, where an increasing in the number of cases in human patients was observed in the last decades, highlighting the role of domestic cat as a transmitter agent. The description of new species compounding de Sporothrix genus, based on phenotypic and genotypic evaluations, showed the involvement of other members of this group in the epidemic status installed in Rio de Janeiro. The verification of strains resistant to itraconazole, a widely used antifungal in human and animal medicine for the treatment of this mycosis, is an important factor that possibly results in relapse and therapeutic failure of this disease. The present study aimed to identify, by phenotypic and molecular approaches, 168 isolates obtained from the routine of Veterinary Clinical Microbiology Laboratory ? UFRRJ, and the determination of minimal inhibitory concentration (MIC) for amphotericin B (AMB), ketoconazole (KTC), itraconazole (ITC), terbinafine (TRB) and voriconazole (VRC). Based on morphophysiological characteristics it was possible to identify 159 (94.64%) isolates as S. brasiliensis and 9 (5.36%) as S. luriei. However, applying PCR-RFLP of calmodulin 168 (100%) samples were identified as S. brasilensis. The susceptibility test, based on M38-A2 document (CLSI), showed that TRB was the most effective antifungal tested, followed by ITC, KTC, AMB, and VRC, respectively. No ITC resistant isolates were detected in the present study. These results demonstrate that the identification reached only by phenotypic evaluation is not recommended for the characterization of Sporothrix schenckii complex components. It also proves the predominance of S. brasiliensis in other regions of RJ state. The better efficacy of TRB added to the absence of isolates resistant to ITC support the necessity of pharmacodynamics and pharmacokinetics studies for the optimization of the therapeutic protocols. More information about isolates from dogs and cats correlated with the species from the S. schenckii complex, as well as in vitro antifungal efficacy evaluation provide knowledge about therapeutic alternatives. In this way, the present study also provides relevant information about the endemic status in Rio de Janeiro and important data for the treatment of human and animal sporotrichosis.
Fungos dim?rficos pertencentes ao complexo Sporothrix schenckii s?o respons?veis pela esporotricose, importante infec??o f?ngica que apresenta distribui??o mundial. Sua conhecida caracter?stica antropozoon?tica apresenta grande relev?ncia no estado do Rio de Janeiro, onde se verifica aumento significativo no n?mero de pacientes humanos e animais acometidos pela doen?a nas ?ltimas d?cadas, destacando-se em tais casos o papel do felino como agente transmissor. A descri??o de novas esp?cies pertencentes ao g?nero Sporothrix, baseada em caracter?sticas fenot?picas e genot?picas, demonstrou o envolvimento de outros componentes deste g?nero na epidemia instalada no estado. A verifica??o de isolados resistentes a itraconazol, antif?ngico amplamente utilizado na medicina humana e veterin?ria para o tratamento da doen?a, ? fato preocupante e tem poss?vel associa??o a recidivas e falhas terap?uticas. O presente estudo objetiva a identifica??o fenot?pica e genot?pica de 168 exemplares oriundos de pacientes felinos e caninos, obtidos na rotina do Diagn?stico Microbiol?gico Veterin?rio - UFRRJ, com determina??o da Concentra??o Inibit?ria M?nima (CIM) frente ? anfotericina B (AMB), cetoconazol (KTC), itraconazol (ITC), terbinafina (TRB) e voriconazol (VRC). A partir de caracter?sticas morfofisiol?gicas foi poss?vel identificar 159 (94,64%) isolados como S. brasiliensis e 9 (5,36%) como S. luriei. Contudo, metodologias moleculares identificaram 168 (100%) S. brasiliensis, a partir de PCR-RFLP em gene respons?vel pela s?ntese de calmodulina. O teste de sensibilidade, realizado a partir do documento M38-A2 (CLSI) determinou maior efic?cia in vitro para TRB, seguido por ITC, KTC, AMB e VRC, respectivamente. Cepas resistentes a ITC n?o foram detectadas no presente estudo. Tais resultados demonstram que a identifica??o alcan?ada exclusivamente por m?todos fenot?picos n?o ? recomendada para caracteriza??o de componentes do complexo Sporothrix schenckii. Comprova-se ainda a predomin?ncia de S. brasiliensis em outras regi?es do estado do RJ. A maior efic?cia de TRB, somada a aus?ncia de exemplares resistentes a ITC, refor?a a necessidade de estudos farmacodin?micos e farmacocin?ticos para otimiza??o dos protocolos terap?uticos atualmente utilizados. Obten??o de maiores informa??es acerca dos isolados provenientes de amostras provenientes de c?es e gatos correlacionados a esp?cies dentro do complexo S. schenckii, bem como a avalia??o da efic?cia in vitro de antif?ngicos proporcionam conhecimento sobre alternativas terap?uticas. Tais informa??es auxiliam no entendimento do quadro instalado no estado do Rio de Janeiro e fornece dados de grande utilidade para o tratamento humano e veterin?rio
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Spengler, C. J. "PCR-RFLP typification of microbes used in the production of a fermented fish product". Thesis, Stellenbosch : Stellenbosch University, 2001. http://hdl.handle.net/10019.1/52397.
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Thesis (MScFoodSc)--Stellenbosch University, 2001.ENGLISH ABSTRACT: The preservation of various fresh fish products is achieved by either smoking, salting, canning, freezing or fermenting a highly perishable raw product. Since many of these facilities are not readily available, the use of fermentation as a means of preserving the product has been extensively practiced. However, the fermentation of fish is a time consuming practise and only by accelerating the process would it be possible to ensure the production of a more cost effective and readily available safe end-product. The quality of the fermented fish product is partially determined by the fermentation conditions and the metabolic activity of the microbes present. The rapid identification of the microbes present during the fermentation would enable the selection of possible starters to ensure an accelerated production of high quality fermented fish products. This study was thus undertaken to develop identification fingerprints for bacteria isolated from fermented fish products. A 1300 bp fragment of the 16S rRNA genes of each of the bacteria previously isolated was successfully amplified using the PCR technique. The isolates included strains of the genera Bacillus, Staphylococcus, Sphingomonas, Kocuria, Brevibacillus, Cryseomonas, Vibrio, Stenotrophomonas and Agrobacterium. The data obtained can, therefore, be used in the identification of these microbes isolated from other similar fermented fish products. The fingerprints could also be used to assist in determining the dominant microbial populations responsible for the characteristic qualitative changes occurring in the fish product during fermentation. The microbial composition of a fermenting fish product partially determines the quality of the end-product, therefore, the use of selected bacterial starters could result in the accelerated production of a microbial safe fermented fish product. A further objective of this study was to accelerate the production of a fermented fish product by inoculating macerated trout with either selected lactic acid bacteria (LAB) or with selected bacteria with high proteolytic activity over a 30 day fermentation period. The LAB included a combination of Lactobacillus plantarum, Lactococcus diacetylactis and Pediococcus cerevisiae strains, whereas the bacteria with high proteolytic activity included strains of Kocuria varians, Bacillus subtilis, two strains of B. amyloliquefaciens and a combination of these bacterial species. The quality of the fermented product was determined using changes in product pH, titratable acidity (%TA) and free amino nitrogen (FAN) formation as efficiency parameters. The data obtained during the fermentation of the macerated trout showed that the selected starters did not have a significant effect on the pH decrease in the product over a 30 day fermentation period. The LAB strains did not have a significant effect on the %TA of the fermenting fish product, yet the presence of these bacteria appeared to limit the FAN production in the product. The bacteria with high proteolytic activity resulted in slightly enhanced %TA values and a higher FAN content in the fermented product. It was also determined that the LAB and Kocuria varians, in contrast to the Bacillus spp. inoculums, did not survive the fermentation conditions well, possibly due to the low pH environment. The presence of the starter bacteria in the fermenting fish mixture at the end of the fermentation was also successfully determined with the use of the PCR-RFLP technique. The fermented fish product, obtained at the end of the fermentation period, had a good aroma and compared favourably to similar commercially available fermented fish products. The use of different microbial starters could in future enable the production of a diverse range of high quality products, which could be produced and marketed locally.
AFRIKAANSE OPSOMMING: Die preservering van ‘n verskeidenheid vars vis produkte word bereik deur die hoogs bederfbare produk te rook, te sout, te blik, te vries of te fermenteer. Aangesien baie van hierdie fasiliteite nie geredelik beskikbaar is nie, is die gebruik van fermentasie as ‘n preserverings metode al ekstensief beoefen. Die fermentasie van vis is egter 'n tydsame proses en slegs deur die versnelling van die proses sal dit moontlik wees om die produksie van ‘n meer koste effektiewe en geredelike beskikbare veilige eindproduk te verseker. Die kwaliteit van die gefermenteerde vis produk word gedeeltelik bepaal deur die fermentasie kondisies en die metaboliese aktiwiteit van die mikrobes teenwoordig. Die vinnige identifikasie van die mikrobes teenwoordig gedurende die fermentasie sal die seleksie van moontlike suursels om die versnelde produksie van hoë kwaliteit gefermenteerde vis produkte moontlik maak. Hierdie studie is dus onderneem om identifikasie vingerafdrukke vir bakteriee wat gei'soleer is van gefermenteerde vis produkte moontlik te maak. ‘n 1300 bp fragment van die 16S rRNA gene van elkeen van die bakteriee wat voorheen gei'soleer is, is suksesvol geamplifiseer deur die PCR tegniek. Die isolate sluit in stamme van die genera Bacillus, Staphylococcus, Sphingomonas, Kocuria, Brevibacillus, Cryseomonas, Vibrio, Stenotrophomonas en Agrobacterium. Die data kan dus gebruik word in die identifikasie van hierdie mikrobes as dit gei'soleer word van ander gefermenteerde vis produkte. Die vingerafdrukke kan ook gebruik word om die dominante mikrobiese populasies wat verantwoordelik is vir die kenmerklike kwalitatiewe veranderinge wat plaasvind in die vis produk gedurende die fermentasie, te identifiseer. Die mikrobiese samestelling van ‘n fermenterende vis produk bepaal gedeeltelik die kwaliteit van die eindproduk, daarom kan die gebruik van geselekteerde bakteriese suursels die versnelde produksie van ‘n mikrobies veilige gefermenteerde vis produk teweeg bring. ‘n Verdere doel van hierdie studie was om die produksie van ‘n gefermenteerde vis produk te versnel deur fyngemaakte forel met of geselekteerde melksuurbakteriee of met geselekteerde bakteriee met hoë proteolitiese aktiwiteit te inokuleer oor ‘n 30 dag fermentasie periode. Die melksuurbakteriee het ingesluit ‘n kombinasie van Lactobacillus plantarum, Lactococcus diacetylactis en Pediococcus cerevisiae, terwyl die bakterieë met hoë proteolitiese aktiwiteit stamme van Kocuria varians, Bacillus subtilis, twee stamme van Bacillus amyloliquefaciens en ‘n kombinasie van hierdie bakteriese stamme ingesluit het. Die kwaliteit van die gefermenteerde produk is bepaal deur die veranderinge in die pH, titreerbare suur (%TS) en vrye amino stikstof (VAS) vorming van die produk as effektiwiteits parameters te gebruik. Die data wat verkry is gedurende die fermentasie van die fyngemaakte forel het gedui daarop dat die geselekteerde suursels nie ‘n merkwaardige effek op die afname in pH in die produk oor ‘n 30 dag fermentasie periode het nie. Die melksuurbakteriee het nie ‘n merkwaardige effek op die %TS van die gefermenteerde vis produk gehad nie, terwyl dit geblyk het dat die teenwoordigheid van hierdie bakterieë die produksie van VAS in die produk belemmer het. Die bakteriee met hoe proteolitiese aktiwiteit het ‘n effense verhoogde %TS en ‘n hoër VAS inhoud in die gefermenteerde produk veroorsaak. Dit is ook bepaal dat die melksuurbakteriee en Kocuria varians, in teenstelling met die Bacillus spp. inokulums, nie die fermentasie kondisies goed oorleef het nie, moontlik as gevolg van die lae pH omgewing. Die teenwoordigheid van die suursel bakteriee in die fermenterende vis mengsel aan die einde van die fermentasie is ook suksesvol bepaal met die PKR-RFLP tegniek. Die gefermenteerde vis produk, verkry aan die einde van die fermentasie periode, het ‘n goeie aroma gehad en het goed vergelyk met soortgelyke kommersieel beskikbare gefermenteerde vis produkte. Die gebruik van verskillende mikrobiese suursels kan in die toekoms die produksie van ‘n diverse reeks hoë kwaliteit produkte wat plaaslik geproduseer en bemark kan word moontlik maak.
Książki na temat "PCR-RFLP"
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A. Zakeri i S.A. Pourbakhsh. Differential diagnosis between ts-11 vaccine strain and field Mycoplasma gallisepticum isolates in clinical samples by PCR-RFLP. Verlag Eugen Ulmer, 2017. http://dx.doi.org/10.1399/eps.2017.195.
Pełny tekst źródłaCzęści książek na temat "PCR-RFLP"
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Frank, J. Howard, J. Howard Frank, Michael C. Thomas, Allan A. Yousten, F. William Howard, Robin M. Giblin-davis, John B. Heppner i in. "PCR-RFLP". W Encyclopedia of Entomology, 2766. Dordrecht: Springer Netherlands, 2008. http://dx.doi.org/10.1007/978-1-4020-6359-6_2812.
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Atoui, Ali, i André El Khoury. "PCR-RFLP for Aspergillus Species". W Methods in Molecular Biology, 313–20. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-6707-0_20.
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Aguilar, Fernando, i Peter Cerutti. "Genotypic Mutation Assay (RFLP/PCR)". W Technologies for Detection of DNA Damage and Mutations, 431–38. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4899-0301-3_31.
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Nakashima, Hitoshi, Mitsuteru Akahoshi i Yosuke Tanaka. "Mutation Detection Using RT-PCR-RFLP". W PCR Protocols, 319–22. Totowa, NJ: Humana Press, 2003. http://dx.doi.org/10.1007/978-1-4612-0055-0_46.
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Rousseaux, Sandrine, i Michèle Guilloux-Bénatier. "PCR ITS-RFLP for Penicillium Species and Other Genera". W Methods in Molecular Biology, 321–33. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-6707-0_21.
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Martini, Marta, i Ing-Ming Lee. "PCR and RFLP Analyses Based on the Ribosomal Protein Operon". W Methods in Molecular Biology, 173–88. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-62703-089-2_15.
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Maramis, Christos, Evangelia Minga i Anastasios Delopoulos. "An Application for Semi-automatic HPV Typing of PCR-RFLP Images". W Lecture Notes in Computer Science, 173–84. Berlin, Heidelberg: Springer Berlin Heidelberg, 2010. http://dx.doi.org/10.1007/978-3-642-13775-4_18.
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Duduk, Bojan, Samanta Paltrinieri, Ing-Ming Lee i Assunta Bertaccini. "Nested PCR and RFLP Analysis Based on the 16S rRNA Gene". W Methods in Molecular Biology, 159–71. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-62703-089-2_14.
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Scarano, M. T., L. Abbate, S. Ferrante, S. Lucretti i N. Tusa. "Molecular Characterization of Citrus Symmetric and Asymmetric Somatic Hybrids by Means of ISSR-PCR and PCR-RFLP". W Plant Biotechnology 2002 and Beyond, 549–50. Dordrecht: Springer Netherlands, 2003. http://dx.doi.org/10.1007/978-94-017-2679-5_113.
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Heinze, B. "PCR-RFLP analysis of introns of nuclear genes in Populus and Prunus". W Genetic Response of Forest Systems to Changing Environmental Conditions, 117–27. Dordrecht: Springer Netherlands, 2001. http://dx.doi.org/10.1007/978-94-015-9839-2_10.
Pełny tekst źródłaStreszczenia konferencji na temat "PCR-RFLP"
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Chuang, Li-Yeh, Yu-Da Lin, Hsueh-Wei Chang i Cheng-Hong Yang. "An Improved Natural PCR-RFLP Primer Design Method". W Bioengineering (BIBE). IEEE, 2011. http://dx.doi.org/10.1109/bibe.2011.21.
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Maramis, Christos, i Anastasios Delopoulos. "Efficient Quantitative Information Extraction from PCR-RFLP Gel Electrophoresis Images". W 2010 20th International Conference on Pattern Recognition (ICPR). IEEE, 2010. http://dx.doi.org/10.1109/icpr.2010.627.
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Llerena, S. Espezua, i C. D. Maciel. "Exploratory Visualization of RFLP-PCR Genomic Data Using Multidimensional Scaling". W 2008 XXI Brazilian Symposium on Computer Graphics and Image Processing (SIBGRAPI). IEEE, 2008. http://dx.doi.org/10.1109/sibgrapi.2008.42.
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Maramis, Christos F., Anastasios N. Delopoulos, Alexandros F. Lambropoulos i Sokratis P. Katafigiotis. "A system for automatic HPV typing via PCR-RFLP gel electrophoresis". W 2011 IEEE International Conference on Automation Science and Engineering (CASE 2011). IEEE, 2011. http://dx.doi.org/10.1109/case.2011.6042466.
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Yang, Cheng-Hong, Yu-Huei Cheng, Li-Yeh Chuang i Hsueh-Wei Chang. "A mismatch PCR-RFLP primer design for SNP genotyping using genetic algorithm". W 2010 9th IEEE International Conference on Cognitive Informatics (ICCI). IEEE, 2010. http://dx.doi.org/10.1109/coginf.2010.5599752.
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Maramis, Christos F., Anastasios N. Delopoulos i Alexandros F. Lambropoulos. "Analysis of PCR-RFLP gel electrophoresis images for accurate and automated HPV typing". W 2010 10th IEEE International Conference on Information Technology and Applications in Biomedicine (ITAB 2010). IEEE, 2010. http://dx.doi.org/10.1109/itab.2010.5687732.
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Chuang, Li-Yeh, Yu-Da Lin, Hsueh-Wei Chang i Cheng-Hong Yang. "PCR-RFLP Primer Design Using Particle Swarm Optimization Combined with Chaotic Logistic Map". W 2012 IEEE Workshops of International Conference on Advanced Information Networking and Applications (WAINA). IEEE, 2012. http://dx.doi.org/10.1109/waina.2012.208.
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Cheng, Yu-Huei, Li-Yeh Chuang i Cheng-Hong Yang. "Apply genetic algorithm with an adaptive stopping criterion to PCR-RFLP Primer Design". W 2012 11th IEEE International Conference on Cognitive Informatics & Cognitive Computing (ICCI*CC). IEEE, 2012. http://dx.doi.org/10.1109/icci-cc.2012.6311176.
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Javadi, Alireza, Masoud Zarei, Masoud Shamaei, Arda Kiani, Atefeh Abedini, Makan Sadr i Mihan Pourabdollah. "The first designed PCR-RFLP technique for butyrophilin-like 2 rs2076530 polymorphism in sarcoidosis". W ERS International Congress 2016 abstracts. European Respiratory Society, 2016. http://dx.doi.org/10.1183/13993003.congress-2016.pa884.
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Eltai, Nahla O., Sara H. Al-Hadidi, Asmaa A. Al Than, Sanjay H. Doiphode i Hadi M. Yassine. "Salmonellosis among Pediatric Population in Qatar: Prevalence, Antibiotic Resistance and Molecular Epidemiology". W Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0126.
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Objectives: This study aims to characterize at the molecular level the genes encoding resistance in Salmonella and explain the molecular mechanisms underlying resistance to ceftriaxone, cefepime, amoxicillin-clavulanate, tetracycline, trimethoprim-sulfamethoxazole, chloramphenicol, colistin and azithromycin in Salmonella. It aims as well to characterize the 16S rRNA gene region by restriction fragment length polymorphism (RFLP) to investigate if this region constitutes an appropriate ‘coincidental’ marker to distinguish important pathogenic Salmonella species. Finally, determine the lineages of Salmonella species and evolutionary relationships among bacteria classified within the same genus. Methodology: 246 Salmonella isolates were collected from children under 16 years old during Jan. 2018 - Dec 2019, presented with gastroenteritis at Hamad Medical Corporation. Isolates were tested for antibiotic susceptibility against nineteen relevant antibiotics using E-test. Isolates that harbor antibiotic resistance were confirmed using PCR specific primers for 38 genes. In addition, the variable region of class 1 and 2 integrons were identified by PCR among amoxicillin-clavulanate (AMC) resistant samples. RFLP targeting16S rRNAwas performed using seven restriction enzymes including AluI, Bgl I, Bgl II, EcoR I, SmaI, Hinf I & Hae III. Results: Resistance was detected against 15 antibiotics and (38.2%) of isolates were resistant to at least one antibiotic. Overall, high resistance was reported to tetracycline (23.9%), ampicillin (21.1%), AMC (18.7%) and sulfamethoxazoletrimethoprim (13%). Further, 22.4% of the isolates were multidrug-resistant (MDR), with 4.1% being ESBL producers. 90 % of ESBL producers had one of bla CTX-M-Group. Class (1) AMC resistant samples showed the highest resistance to different antibiotics. 16S rRNA-RFLP analysis divided Salmonella isolates into two main groups. Conclusion: Our results indicate a high antimicrobial resistance pattern of Salmonella, which necessities the development of regulatory programs to combats antimicrobial resistance. In particular, our results showed high resistance to Class (1) AMC cassette that involves the transmission and expression of the resistance. This might lead to a concern of increased multidrug resistance in the future. This study provides evidence guidance to activate and implement the pillars of an antimicrobial stewardship program in animal and human health to reduce MDR salmonellosis.
Raporty organizacyjne na temat "PCR-RFLP"
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Levisohn, Sharon, Mark Jackwood i Stanley Kleven. New Approaches for Detection of Mycoplasma iowae Infection in Turkeys. United States Department of Agriculture, luty 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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Michel Jr., Frederick C., Harry A. J. Hoitink, Yitzhak Hadar i Dror Minz. Microbial Communities Active in Soil-Induced Systemic Plant Disease Resistance. United States Department of Agriculture, styczeń 2005. http://dx.doi.org/10.32747/2005.7586476.bard.
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Induced Systemic Resistance (ISR) is a highly variable property that can be induced by compost amendment of potting media and soils. For example, previous studies showed that only 1 of 79 potting mixes prepared with different batches of mature composts produced from several different types of solid wastes were able to suppress the severity of bacterial leaf spot of radish caused by Xanthomonas campestris pv. armoraciae compared with disease on plants produced in a nonamended sphagnum peat mix. In this project, microbial consortia in the rhizosphere of plants grown in ISR-active compost-amended substrates were characterized. The plants used included primarily cucumber but also tomato and radish. Rhizosphere microbial consortia were characterized using multiple molecular tools including DGGE (Israel) and T -RFLP (Ohio) in both ISR-active field plots and potting media. Universal as well as population-specific bacterial and fungal PCR primers were utilized. T -RFLP analyses using universal bacterial primers showed few significant differences in overall bacterial community composition in ISR-active and inactive substrates (Ohio). In addition, the community members which were significantly different varied when different ISR-activecomposts were used (Ohio). To better characterize the shifts in microbial community structure during the development of ISR, population specific molecular tools were developed (Israel, Ohio).-PCR primers were designed to detect and quantify bacterial groups including Pyrenomycetes, Bacillus, Pan toea, Pseudomonas, Xanthomonas and Streptomyces as well as Trichoderma and Fusarium; two groups of fungi that harbor isolates which are ISR active (Isreal and Ohio). Bacterial consortia associated with cucumber plants grown in compost-amended potting mixtures were shown to be dominated by the phylogenetic taxon Bacteroidetes, including members of the genus Chryseobacterium, which in some cases have been shown to be involved in biocontrol (Israel). Nested-PCR-DGGE analyses coupled with long l6S rDNA sequencing, demonstrated that the Chryseobacteriumspp. detected on seed and the root in compost-amended treatments were derived from the compost itself. The most effective ISR inducing rhizobacterial strains were identified as Bacillus sp. based on partial sequencing of l6S rDNA. However, these strains were significantly less effective in reducing the severity of disease than Trichoderma hamatum382 (T382). A procedure was developed for inoculation of a compost-amended substrate with T -382 which consistently induced ISR in cucumber against Phytophthora blight caused by Phytophthora capsiciand in radish against bacterial spot (Ohio). Inoculation of compost-amended potting mixes with biocontrol agents such as T -382 and other microbes that induce systemic resistance in plants significantly increased the frequency of systemic disease control obtained with natural compost amendments.
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Gelb, Jr., Jack, Yoram Weisman, Brian Ladman i Rosie Meir. Identification of Avian Infectious Brochitis Virus Variant Serotypes and Subtypes by PCR Product Cycle Sequencing for the Rational Selection of Effective Vaccines. United States Department of Agriculture, grudzień 2003. http://dx.doi.org/10.32747/2003.7586470.bard.
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Objectives 1. Determine the serotypic identities of 40 recent IBV isolates from commercial chickens raised in the USA and Israel. 2. Sequence all IBV field isolates using PCR product cycle sequencing and analyze their S 1 sequence to detennine their homology to other strains in the Genbank and EMBL databases. 3. Select vaccinal strains with the highest S 1 sequence homology to the field isolates and perform challenge of immunity studies in chickens in laboratory trials to detennine level of protection afforded by the vaccines. Background Infectious bronchitis (IB) is a common, economically important disease of the chicken. IB occurs as a respiratory form, associated with airsacculitis, condemnation, and mortality of meat-type broilers, a reproductive form responsible for egg production losses in layers and breeders, and a renal form causing high mortality in broilers and pullets. The causative agent is avian coronavirus infectious bronchitis virus (IBV). Replication of the virus' RNA genome is error-prone and mutations commonly result. A major target for mutation is the gene encoding the spike (S) envelope protein used by the virus to attach and infect the host cell. Mutations in the S gene result in antigenic changes that can lead to the emergence of variant serotypes. The S gene is able to tolerate numerous mutations without compromising the virus' ability to replicate and cause disease. An end result of the virus' "flexibility" is that many strains of IBV are capable of existing in nature. Once formed, new mutant strains, often referred to as variants, are soon subjected to immunological selection so that only the most antigenically novel variants survive in poultry populations. Many novel antigenic variant serotypes and genotypes have been isolated from commercial poultry flocks. Identification of the field isolates of IBV responsible for outbreaks is critical for selecting the appropriate strain(s) for vaccination. Reverse transcriptase polymerase chain reaction (RT-PCR) of the Sl subunit of the envelope spike glycoprotein gene has been a common method used to identify field strains, replacing other time-consuming or less precise tests. Two PCR approaches have been used for identification, restriction fragment length polymorphism (RFLP) and direct automated cycle sequence analysis of a diagnostically relevant hypervariab1e region were compared in our BARD research. Vaccination for IB, although practiced routinely in commercial flocks, is often not protective. Field isolates responsible for outbreaks may be unrelated to the strain(s) used in the vaccination program. However, vaccines may provide varying degrees of cross- protection vs. unrelated field strains so vaccination studies should be performed. Conclusions RFLP and S1 sequence analysis methods were successfully performed using the field isolates from the USA and Israel. Importantly, the S1 sequence analysis method enabled a direct comparison of the genotypes of the field strains by aligning them to sequences in public databases e.g. GenBank. Novel S1 gene sequences were identified in both USA and Israel IBVs but greater diversity was observed in the field isolates from the USA. One novel genotype, characterized in this project, Israel/720/99, is currently being considered for development as an inactivated vaccine. Vaccination with IBV strains in the US (Massachusetts, Arkansas, Delaware 072) or in Israel (Massachusetts, Holland strain) provided higher degrees of cross-protection vs. homologous than heterologous strain challenge. In many cases however, vaccination with two strains (only studies with US strains) produced reasonable cross-protection against heterologous field isolate challenge. Implications S1 sequence analysis provides numerical similarity values and phylogenetic information that can be useful, although by no means conclusive, in developing vaccine control strategies. Identification of many novel S1 genotypes of IBV in the USA is evidence that commercial flocks will be challenged today and in the future with strains unrelated to vaccines. In Israel, monitoring flocks for novel IBV field isolates should continue given the identification of Israel/720/99, and perhaps others in the future. Strains selected for vaccination of commercial flocks should induce cross- protection against unrelated genotypes. Using diverse genotypes for vaccination may result in immunity against unrelated field strains.
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Levisohn, Sharon, Maricarmen Garcia, David Yogev i Stanley Kleven. Targeted Molecular Typing of Pathogenic Avian Mycoplasmas. United States Department of Agriculture, styczeń 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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Sink, Ken, Shamay Izhar i Abraham Nachmias. Asymmetric Somatic Hybridization: Developing a Gene Transfer System for Solanaceous Vegetable Crops. United States Department of Agriculture, luty 1996. http://dx.doi.org/10.32747/1996.7613010.bard.
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Highly asymmetric somatic hybrid plants were obtained by PEG/DMSO fusion of gamma irradiated (100, 250, 7500 and 1000 Gy) protoplasts of a (KmR-) interspecific hybrid Lycopersicon esculentum x L. pennellii (EP) with protoplasts of eggplant (E). Somatic hybrid calli were selected based on kanamycin resistance and verified by PCR of the NptII gene, RAPD's and Southern's using potato rDNA pTHG2 probes. Flow cytometry indicated all hybrid calli that did not regenerate shoots were 5-9n. Three asymmetric plants regenerated only from callus close to 4n and such calli oly occurred when EP received 100 Gy. The asymmetric plants had eggplant morphology and regenerated from one hybrid callus with 6.29 average size tomato chromosomes. Limited amounts of EP DNA were found in the three somatic hybrid plants H18-1 to -3 by dot-blot hybridization with probe pTHG2, to be equivalent to 6.23, 5.41, and 5.95 % EP, respectively. RFLP analysis of Lycopersicon esculentum and L. pennellii specific chromosomes revealed that only fragments of 8 to 10 out of the 24 EP chromosomes are present in the asymmetric plants. Transgenic plants 2-3, 2-4 and 10-3 were found resistant to verticillium; suggesting successful transfer of the Ve complex from S. torvum to eggplant.
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Dubcovsky, Jorge, Tzion Fahima i Ann Blechl. Positional cloning of a gene responsible for high grain protein content in tetraploid wheat. United States Department of Agriculture, wrzesień 2003. http://dx.doi.org/10.32747/2003.7695875.bard.
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High Grain Protein Content (GPC) is a desirable trait in breadmaking and pasta wheat varieties because of its positive effects on quality and nutritional value. However, selection for GPC is limited by our poor understanding of the genes involved in the accumulation of protein in the grain. The long-term goal of this project is to provide a better understanding of the genes controlling GPC in wheat. The specific objectives of this project were: a) to develop a high-density genetic map of the GPC gene in tetraploid wheat, b) to construct a T. turgidum Bacterial Artificial Chromosome (BAC) library, c) to construct a physical map of the GPC gene and identify a candidate for the GPC gene. A gene with a large effect on GPC was detected in Triticum turgidum var. dicoccoides and was previously mapped in the short arm of chromosome 6B. To define better the position of the Gpc-B1 locus we developed homozygous recombinant lines with recombination events within the QTL region. Except for the 30-cM region of the QTL these RSLs were isogenic for the rest of the genome minimizing the genetic variability. To minimize the environmental variability the RSLs were characterized using 10 replications in field experiments organized in a Randomized Complete Block Design, which were repeated three times. Using this strategy, we were able to map this QTL as a single Mendelian locus (Gpc-B1) on a 2.6-cM region flanked by RFLP markers Xcdo365 and Xucw67. All three experiments showed that the lines carrying the DIC allele had an average absolute increase in GPC of 14 g/kg. Using the RFLP flanking markers, we established the microcolinearity between a 2.l-cM region including the Gpc-B1 gene in wheat chromosome 6BS and a 350-kb region on rice chromosome 2. Rice genes from this region were used to screen the Triticeae EST collection, and these ESTs were used to saturate the Gpc-B1 region with molecular markers. With these new markers we were able to map the Gpc-B1 locus within a 0.3-cM region flanked by PCR markers Xucw83 and Xucw71. These flanking markers defined a 36-kb colinear region with rice, including one gene that is a potential candidate for the Gpc-B1 gene. To develop a physical map of the Gpc-B1 region in wheat we first constructed a BAC library of tetraploid wheat, from RSL#65 including the high Gpc-B1 allele. We generated half- million clones with an average size of l3l-kb (5.1 X genome equivalents for each of the two genomes). This coverage provides a 99.4% probability of recovering any gene from durum wheat. We used the Gpc-BI flanking markers to screen this BAC library and then completed the physical map by chromosome walking. The physical map included two overlapping BACs covering a region of approximately 250-kb, including two flanking markers and the Gpc-B1 gene. Efforts are underway to sequence these two BACs to determine if additional wheat genes are present in this region. Weare also developing new RSLs to further dissect this region. We developed PCR markers for flanking loci Xucw79andXucw71 to facilitate the introgression of this gene in commercial varieties by marker assisted selection (httQ://maswheat.ucdavis.edu/ orotocols/HGPC/index.hlm). Using these markers we introgressed the Gpc-B1 gene in numerous pasta and common wheat breeding lines.
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Reisch, Bruce, Pinhas Spiegel-Roy, Norman Weeden, Gozal Ben-Hayyim i Jacques Beckmann. Genetic Analysis in vitis Using Molecular Markers. United States Department of Agriculture, kwiecień 1995. http://dx.doi.org/10.32747/1995.7613014.bard.
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Genetic analysis and mapping in grapes has been difficult because of the long generation period and paucity of genetic markers. In the present work, chromosome linkage maps were developed with RAPD, RFLP and isozyme loci in interspecific hybrid cultivars, and RAPD markers were produced in a V. vinifera population. In three cultivars, there were 19 linkage groups as expected for a species with 38 somatic chromosomes. These maps were used to locate chromosome regions with linkages to important genes, including those influencing powdery mildew and botrytis bunch rot resistance; flower sex; and berry shape. In V. vinifera, the occurrence of specific markers was correlated with seedlessness, muscat flavor and fruit color. Polymorphic RAPD bands included single copy as well as repetitive DNA. Mapping procedures were improved by optimizing PCR parameters with grape DNA; by the development of an efficient DNA extraction protocol; and with the use of long (17- to 24-mer) primers which amplify more polymorphic loci per primer. DNA fingerprint analysis with RAPD markers indicated that vinifera cultivars could be separated readily with RAPD profiles. Pinot gris, thought to be a sort of Pinot noir, differed by 12 bands from Pinot noir. This suggests that while Pinot gris may be related to Pinot noir, it is not likely to be a clone. The techniques developed in this project are now being further refined to use marker-assisted selection in breeding programs for the early selection of elite seedlings. Furthermore, the stage has been set for future attempts to clone genes from grapes based upon map locations.
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Paran, Ilan, i Molly Jahn. Genetics and comparative molecular mapping of biochemical and morphological fruit characters in Capsicum. United States Department of Agriculture, marzec 2005. http://dx.doi.org/10.32747/2005.7586545.bard.
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Original objectives: The overall goal of our work was to gain information regarding the genetic and molecular control of pathways leading to the production of secondary metabolites determining major fruit quality traits in pepper and to develop tools based on this information to assist in crop improvement. The specific objectives were to: (1) Generate a molecular map of pepper based on simple sequence repeat (SSR) markers. (2) Map QTL for capsaicinoid (pungency) content (3) Determine possible association between capsaicinoid and carotenoid content and structural genes for capsaicinoid and carotenoid biosynthesis. (4) Map QTL for quantitative traits controlling additional fruit traits. (5) Map fruit-specific ESTs and determine possible association with fruit QTL (6) Map the C locus that determines the presence and absence of capsaicinoid in pepper fruit and identify candidate genes for C.locus. Background: Pungency, color, fruit shape and fruit size are among the most important fruit quality characteristics of pepper. Despite the importance of the pepper crop both in the USA and Israel, the genetic basis of these traits was poorly understood prior to the studies conducted in the present proposal. In addition, molecular tools for use in pepper improvement were lacking. Major conclusions and achievements: Our studies enabled the development of a saturated genetic map of pepper that includes numerous SSR markers. This map has been integrated with a number of other independent maps resulting in the publication of a single resource map consisting of more than 2000 markers. Unlike previous maps based primarily on tomato-originated RFLP markers, the new maps are based on PCR markers that originate in Capsicum providing a comprehensive and versatile resource for marker-assisted selection in pepper. We determined the genetic and molecular bases of qualitative and quantitative variation of pungency, a character unique to pepper fruit. We mapped and subsequently cloned the Pun1 gene that serves as a master regulatoar for capsaicinoid accumulation and showed that it is an acyltransferase. By sequencing the Pun1 gene in pungent and non-pungent cultivars we identified a deletion that abolishes the expression of the gene in the latter cultivars. We also identified QTL that control capsaicinoid content and therefore pungency level. These genes will allow pepper breeders to manipulate the level of pungency for specific agricultural and industrial purposes. In addition to pungency we identified genes and QTL that control other key developmental processes of fruit development such as color, texture and fruit shape. The A gene controlling anthocyanin accumulation in the immature fruit was found as the ortholog of the petunia transcription factor Anthocyanin2. The S gene required for the soft flesh and deciduous fruit nature typical of wild peppers was identified as the ortholog of tomato polygalacturonase. We identified two major QTL controlling fruit shape, fs3.1 and fs10.1, that differentiate elongated and blocky and round fruit shapes, respectively. Scientific and agricultural implications: Our studies allowed significant advances in our understanding of important processes of pepper fruit development including the isolation and characterization of several well known genes. These results also provided the basis for the development of molecular tools that can be implemented for pepper improvement. A total of eleven refereed publications have resulted from this work, and several more are in preparation.