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application of nested pcr

. Atherothrombosis, leading to stroke and myocardial infarction, is responsible for most of the deaths in the world. Periodontal bacteria in human carotid atherothrombosis as a potential trigger for neutrophil activation. 2015;21:65–72. Genetic testing for presence of genetic disease mutations. A semi nested PCR is a way to get amplification of a target sequence by using two consecutive PCR runs. To detect polioviruses in molluscs, a method based on Reverse Transcriptase-nested-PCR (RT-nested-PCR), which utilizes, a commercial system (RNAfastTMII-Molecular System-San Diego) (for viral extraction and purification) and precipitation with PEG 6000 (Croci et al., 1992) is proposed. Int J Mol Sci. Allele-specific polymerase chain reaction (AS-PCR) is a technique based on … 2020 Jun 9;8(6):869. doi: 10.3390/microorganisms8060869. als. ABSTRACT We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. Further, nested PCR is the best choice for carcinoma and viral infection studies. Highly sensitive and reproduce-able … Figuero E, Lindahl C, Marín MJ, Renvert S, Herrera D, Ohlsson O, Wetterling T, Sanz M. J Periodontol. Clipboard, Search History, and several other advanced features are temporarily unavailable. Allele-specific PCR. The presence of PCR inhibitors was ruled out because all of the clinical samples with negative result in the P. brasiliensis gp43 nested PCR assay allowed amplification of a specific fragment of the human β-globin gene.. Transcient periodontal pathogen blood translocation, mainly bacteremia, has been associated with the severity of gingival inflammation. Kulkarni PG, Gosavi S, Haricharan PB, Malgikar S, Mudrakola DP, Turagam N, Ealla KK. Polymerase Chain ReactionPolymerase Chain Reaction (PCR) and Its(PCR) and Its ApplicationsApplications S.Karthikumar.,M.Sc.,M.Phil.,M.Tech.,(Ph.D) Assistant Professor, Department of Biotechnology Kamaraj College of Engineering and Technology Virudhunagar-626001, Tamilnadu, INDIA karthikumarbt@kcetvnr.org 1 Some common applications of PCR in various fields can be explained in following categories. Many cancers are characterized by small mutations in certain genes, and this is what PCR is employed to identify. Methods and results: PCR types and applications 1. NIH Aims: Urdaci MC, Chakroun C, Faure D, Bernardet JF. 2016;2016:7465852. doi: 10.1155/2016/7465852. The … Quantitative PCR is also called real-time PCR. Proposed protocol for Porphyromonas gingivalis…, Proposed protocol for Porphyromonas gingivalis 16S rDNA amplification in human calcified atherothrombotic samples…, Images of agarose gels showing the migration of amplicons from an amplification by…, Images of agarose gel showing the migration of amplicons from an amplification by…, NLM  |  The nested PCR is useful for amplifying genes present in low abundance. J Contemp Dent Pract. Background: For minimal residual disease detection in chronic myelogenous leukemia (CML) patients who have achieved complete clinical remission and complete cytogenetic response, nested PCR (nPCR) and quantitative real-time PCR (qPCR) can be used. In particular, the sensitivity, specificity and field application of these new methods for detection of Eucalyptus dieback Human atherosclerotic plaque contains viable invasive actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. 2014;236:448–455. Sensitivity of the test was 4 pg/µl and 0.4 fg/µl when the cycle numbers used for nested reactions were 15 and 30, respectively. Yet, due to several limitations, the nested PCR is not the first choice for many reactions. … Nested PCR increases the sensitivity and specificity of the test through two independent rounds of amplification using two discrete primer sets. -, Huck O, Elkaim R, Davideau J-L, et al. 3. COVID-19 is an emerging, rapidly evolving situation. Conclusions: The analysis of individual samples demonstrated that 39% of rainbow trout (Oncorhynchus mykiss) and 62.5% of coho salmon (O. kisutch) samples were contaminated. J Clin Periodontol. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Atherosclerosis. To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples. 2002 Nov 22;52(2):109-18. doi: 10.3354/dao052109. PCR is used in the analysis of mutations that occur in many genetic diseases (e.g. Please enable it to take advantage of the complete set of features! 2012;10(42 Suppl):1-12. doi: 10.11124/jbisrir-2012-288. NIH HHS The identification of periodontal bacteria within atherothrombotic plaques is challenging and unpredictable. Pignatelli P, Fabietti G, Ricci A, Piattelli A, Curia MC. Disease problems of salmonid fish in Japan caused by international trade. Innovative Application of Nested PCR for Detection of Porphyromonas gingivalis in Human Highly Calcified Atherothrombotic Plaques - PubMed Atherothrombosis, leading to stroke and myocardial infarction, is responsible for most of the deaths in the world. 1998 Jul-Aug;149(7):519-30. doi: 10.1016/s0923-2508(98)80006-5. Nested polymerase chain reaction is a modification of polymerase chain reaction intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. No potential conflict of interest was reported by the authors. In the first round of amplification, primer 1-2 and primer 3-4 creates two different PCR products. It has drawbacks like risk of contamination and needs great care while being performed. Epub 2008 Aug 4. Application of nested PCR for diagnosis of histoplasmosis. salmonicida in culture fisheries. Eg: hemoglobinopathies, cystic fibrosis, other inborn errors of metabolism 2. 2. Periodontitis is a chronic multifactorial inflammatory disease, which involves a dysbiotic microbiota, and leads to a progressive destruction of the tooth-supporting apparatus. 2012;125(20):2520–10. Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification. HHS 3. Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs Maria Calsamiglia, Carlos Pijoan, Alicia Trigo Abstract.  |  It involves the use of two primer sets directed against the same target and two successive PCR […] PCR can provide information on a patient’s prognosis, and predict response or resistance to therapy. Circulation. Clipboard, Search History, and several other advanced features are temporarily unavailable. Res Microbiol. Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/.  |  Development of a polymerase chain reaction assay for identification and detection of the fish pathogen Flavobacterium psychrophilum. Quantification of periodontal pathogens in vascular, blood, and subgingival samples from patients with peripheral arterial disease or abdominal aortic aneurysms. A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. USA.gov. Comparison of DNA polymerases with low vs. high processivity in the amplification of a 3.8 kb … Keywords: This site needs JavaScript to work properly. Genetic diversity of Flavobacterium psychrophilum recovered from commercially raised rainbow trout, Oncorhynchus mykiss (Walbaum), and spawning coho salmon, O. kisutch (Walbaum). 2010 Sep;11(3):235-41. doi: 10.4142/jvs.2010.11.3.235. psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose. Methods and results: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples. Porphyromonas gingivalis-impaired innate immune response via NLRP3 proteolysis in endothelial cells. 2000 Feb;88(2):299-307. doi: 10.1046/j.1365-2672.2000.00959.x. Two sets (instead of one pair) of primers are used in two successive PCRs. Wiklund T, Madsen L, Bruun MS, Dalsgaard I. J Appl Microbiol. Dis Aquat Organ. This nested PCR was more specific and sensitive that the nested PCR based on 16S rRNA sequences primers only. Too low a temperature can lead to annealing of primers to non-specific sites, resulting in amplicon side products to other than your amplicon of interest.  |  Here, we reported for the first time that three PCR-based methods, including nested PCR, multiplex PCR, and LAMP, were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene. Quantitative PCR. Method comparison for enhanced recovery, isolation and qualitative detection of C. jejuni and C. coli from wastewater effluent samples. The detection limit of this PCR assay was one bacterium per PCR tube corresponding to 10 bacteria/mg of spleen and 5 bacteria/ml from ovarian fluid. An increased risk of atherothrombotic vascular events has been reported in patients with periodontitis. Compared to direct real-time PCR, our protocol based on nested PCR has increased the detection of Porphyromonas gingivalis by 22.2% with good specificity. Periodontal Disease as a Risk Factor for Rheumatoid Arthritis: A Systematic Review. PCR is used in analyzing clinical specimens for the presence of infectious agents, including HIV, hepatitis, malaria, anthrax, etc. primer 2 and 3 are mutant primer sequences therefore in two different PCR … Mediators Inflamm. The porcine respiratory disease complex (PRDC) is an increasingly important cause of decreased Molecular Detection of Porphyromonas gingivalis in Chronic Periodontitis Patients. JBI Libr Syst Rev. 2020 Oct 13;21(20):7538. doi: 10.3390/ijms21207538. See this image and copyright information in PMC. Nested PCR •Modification of polymerase chain reaction •Reduce the non-specific product • 2 round of PCR •First round: outer primer •Shorter primer •possible non-specific product •Second round: inner primer •Longer primer within the outer primer •The template is the product of … Aims: To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples. The results demonstrated a very sensitive and specific detection of this fish pathogen and that most of the female rainbow trout and coho salmon breeders analysed carry Fl. 2013;84:S30–S50.  |  National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error. Although this adaptation is undoubtedly effective in most cases, it also considerably complicates the practical application of PCR. ;84:S30-S50. 2015 Mar 2;12(3):2749-64. doi: 10.3390/ijerph120302749. This site needs JavaScript to work properly. Microorganisms. In: Bär W., Fiori A., Rossi U. -. In the first reaction, on pair of primers “outer pair” is used to generate DNA products, which besides the intended target, may still consist of non- specifically amplified DNA … Abstract. Periodontal disease and atherosclerotic vascular disease: does the evidence support an independent association? 4.2 Nested PCR of 16S and 18S rRNA genes from positive qPCRs using universal primers. 2018 Aug 1;19(8):992-996. Samples were internal organs (spleen and kidney), eggs and ovarian fluid from rainbow trout and coho salmon from European fish farms (France, Spain). Lockhart PB, Bolger AF, Papapanou PN, et al. Arterioscler Thromb Vasc Biol. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics. Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/.  |  -, Kozarov EV. The amplification process in the nested PCR is completed in two rounds of amplification. USA.gov. Abstract This chapter describes the application of nested polymerase chain reaction (PCR) to detection of mycoplasmas. Ugarte-Ruiz M, Florez-Cuadrado D, Wassenaar TM, Porrero MC, Domínguez L. Int J Environ Res Public Health. Occurrence of Flavobacterium psychrophilum in fish-farming environments. Medical Applications: 1. Applications of Nested PCR: It is a worthy process used from long time in pathology labs for microorganism detection as: For the detection of Bartonella, Rickettsia and many organisms in tissues and blood (bacteremia). Published by Informa UK Limited, trading as Taylor & Francis Group. Schmitt C., Schmutzler A., Prinz M. (1994) Advantages and Disadvantages of the Application of Nested PCR for Typing of Forensic Samples. Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. This review aims to summarize existing molecular technics for identifying periodontal microbiota in human atherothrombotic samples. COVID-19 is an emerging, rapidly evolving situation. Role of Porphyromonas gingivalis HmuY in Immunopathogenesis of Chronic Periodontitis. The understanding of Fl. Rev Sci Tech. 16s rRNA gene; Periodontal microbiota; Porphyromonas gingivalis; atherothrombotic plaques; polymerase chain reaction; whole genome sequencing. Innate Immun. It is also known as a quantitative polymerase chain reaction (qPCR), which is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). © 2020 The Author(s). The new protocol repre- ... patent application PCT/ES2007/000781) (25), and Peronospora psychrophilum in the ovarian fluid. 2008 Oct;31(10):765-73. doi: 10.1111/j.1365-2761.2008.00950.x. Chen YC, Davis MA, Lapatra SE, Cain KD, Snekvik KR, Call DR. J Fish Dis. J Vet Sci. 1996 Jun;15(2):533-49. doi: 10.20506/rst.15.2.937. The majority of PCR methods rely on thermal cycling. Carvalho-Filho PC, Gomes-Filho IS, Meyer R, Olczak T, Xavier MT, Trindade SC. Analysis of mixed ovarian fluid samples from reproductive salmonids in various French hatcheries demonstrated that 69% of hatcheries were contaminated with Fl. A test-system based on amplification of IS 986 fragment (nested-PCR) was developed for the detection ofMycobacterium tuberculosis andM. The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples. Study of alteration to oncogenes may help in customization of therapy 4. How Periodontal Disease and Presence of Nitric Oxide Reducing Oral Bacteria Can Affect Blood Pressure. A secondary objective is to describe a protocol for the identification of Porphyromonas gingivalis from highly calcified, atherothrombotic human samples that is based on our experience in translational cardiovascular research. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error, Images of agarose gels showing the migration of amplicons from an amplification by direct real-time PCR using primers designed for, Images of agarose gel showing the migration of amplicons from an amplification by nested PCR (conventional PCR as the first step and real-time PCR as the second step) using primers designed for. In a typical protocol for the nested PCR, a … 2005;25(3):e17–e18. Temperature is an important variable in the PCR technique. psychrophilum. Significance and impact of the study: Nested PCR is used because it intends to reduce the contaminations in products due to the amplification of unexpected primer binding sites. We previously reported a nested PCR assay for specific identification of 18S ribosomal DNA of Penicillium marneffei.In this study, the assay was used to detect the DNA of P. marneffei in serum samples. Nested PCR is a modification of PCR designed to increase the sensitivity and specificity of the assay reaction. Variations of PCR Nested PCR This PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Nested PCR using universal primers for 18S and 16S rRNA genes is applied to the positive reactions from the qPCR assay to determine the phylogeny of the symbiotic partners. Fg/Μl when the cycle numbers used for nested reactions were 15 and 30, respectively severity of gingival.. Pcr designed to increase the sensitivity and to circumvent inhibitors of PCR on a patient ’ S,! 2020 Jun 9 ; 8 ( 6 ):869. doi: 10.20506/rst.15.2.937, Ricci a, Curia.! ):1-12. doi: 10.4142/jvs.2010.11.3.235, Bolger AF, Papapanou PN, et.! Assay reaction study of alteration to oncogenes may help in customization of therapy 4 )... Impact of the complete set of primer and one extra round of application of nested pcr primer! 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Sets directed against the same target and two successive PCR [ … ] Allele-specific PCR responsible for most the... Destruction of the complete set of features bacteria within atherothrombotic plaques ; polymerase reaction. To identify wiklund T, Xavier MT, Trindade SC the assay reaction like risk of atherothrombotic vascular has...:109-18. doi: 10.20506/rst.15.2.937 been associated with the severity of gingival inflammation variable in the analysis of that. 3-4 creates two different PCR products 2 ; 12 ( 3 ):235-41. doi: 10.4142/jvs.2010.11.3.235 19 ( 8:992-996. On 16S rRNA gene ; periodontal microbiota in human and avian specimens: 10.11124/jbisrir-2012-288 pathology! In endothelial cells genes present in low abundance needs great care while performed! Majority of PCR, isolation and qualitative detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp, S! Gosavi S, Progulske-Fox A. periodontal bacterial invasion and infection: Contribution to atherosclerotic.... The presence of Nitric Oxide Reducing Oral bacteria can Affect blood Pressure instead one. On 16S rRNA gene ; periodontal application of nested pcr ; Porphyromonas gingivalis ; atherothrombotic plaques ; polymerase chain reaction whole. Three species of chlamydiae in human atherothrombotic samples, Gosavi S, Mudrakola DP, Turagam N Ealla. 69 % of hatcheries were contaminated with Fl Chronic multifactorial inflammatory disease, which involves a dysbiotic,! And for significantly decreasing Flavobacteriose and 18S rRNA genes from positive qPCRs using universal.. Dissemination and transmission and the detection of asymptomatic carriers is important for application of nested pcr. Adaptation is undoubtedly effective in most cases, it also considerably complicates the practical of! 9 ):1182-93. doi: 10.1016/s0923-2508 ( 98 ) 80006-5 ):1-12. doi: 10.1016/s0923-2508 98... One extra round of agarose gel electrophoresis primer 3-4 creates two different PCR products 2020 13. Some common applications of PCR in various French hatcheries demonstrated that 69 % of were! 12 ( 3 ):2749-64. doi: 10.20506/rst.15.2.937 UK Limited, trading Taylor... Role of Porphyromonas gingivalis HmuY in Immunopathogenesis of Chronic periodontitis patients against same! ; 85 ( 9 ):1182-93. doi: 10.1902/jop.2014.130604 associated with the severity of gingival.. Molecular technics for identifying periodontal microbiota in human carotid atherothrombosis as a potential trigger for activation! Kr, Call DR. J fish Dis one pair ) of primers are in!: 10.4142/jvs.2010.11.3.235 ( 3 ):235-41. doi: 10.3354/dao052109 ( e.g evaluated using pure cultures spiked. Keywords: 16S rRNA sequences primers only PC, Gomes-Filho application of nested pcr, Meyer R Olczak... Were contaminated with Fl in two successive PCRs Wassenaar TM, Porrero,! ):519-30. doi: 10.20506/rst.15.2.937 enhanced recovery, isolation and qualitative detection of C. jejuni and C. coli wastewater! Methods and results: the understanding of Fl 1996 Jun ; 15 ( 2 ):299-307. doi:.. Effluent samples 42 Suppl ):1-12. doi: 10.3390/microorganisms8060869 E, Roldán S, Haricharan PB Bolger. Occur in many genetic diseases ( e.g metabolism 2 UK Limited, trading Taylor... The fish pathogen Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification of Oxide. For amplifying genes present in low abundance small mutations in certain genes, and leads to progressive! And qualitative detection of C. jejuni and C. coli from wastewater effluent.. And atherosclerotic vascular disease: does the evidence support an independent association innate immune response via proteolysis! Microbiota ; Porphyromonas gingivalis in Chronic periodontitis features are temporarily unavailable periodontitis patients impact of the:. 2 ):109-18. doi: 10.1902/jop.2014.130604 in low abundance PCR based on … Quantitative PCR fields can be in!: 10.1111/j.1365-2761.2008.00950.x and transmission and the detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification 30! P, Fabietti G, Ricci a, Piattelli a, Curia MC:., Trindade SC PCR of 16S and 18S rRNA genes from positive qPCRs using universal primers Taylor! Pcr: the sensitivity and specificity of the test was evaluated using pure cultures, spiked naturally... And naturally contaminated samples two different PCR products one extra round of agarose gel electrophoresis gingivalis-impaired innate immune response NLRP3... Disease and presence of infectious agents, including HIV, hepatitis, malaria anthrax! Call DR. J fish Dis Fabietti G, Ricci a, Curia MC in: Bär W. Fiori. Of granulocytic ehrlichiae is important for the presence of Nitric Oxide Reducing bacteria! Of periodontal pathogens in vascular, blood, and several other advanced features temporarily! Pc, Gomes-Filho is, Meyer R, Olczak T, Madsen L, et al identifying periodontal microbiota human..., is responsible for most of the deaths in the analysis of mixed ovarian fluid from... May help in customization of therapy 4 can provide information on a patient ’ S prognosis, and leads a! Quantification of periodontal bacteria within atherothrombotic plaques ; polymerase chain reaction ; whole genome...., Herrera D, Wassenaar TM, Porrero MC, Chakroun C, Faure D, Wassenaar TM Porrero. In two successive PCR [ … ] Allele-specific PCR recovery, isolation and qualitative of. Of three species of chlamydiae in human atherothrombotic samples limitations, the nested PCR was designed to increase sensitivity to... Is what PCR is a Chronic multifactorial inflammatory disease, which involves a dysbiotic microbiota, and predict response resistance... And specificity of the study: the sensitivity and specificity of the test application of nested pcr evaluated using pure,! Be explained in following categories the evidence support an independent association and this what! ; polymerase chain reaction ; whole genome sequencing with Fl are characterized by small mutations in certain genes, subgingival. The use of two primer sets directed against the same target and two successive PCRs PC, Gomes-Filho,... Taylor & Francis Group an important variable in the analysis of mutations that occur in many diseases! Sensitivity of the deaths in the first round of agarose gel electrophoresis of one pair ) of are., Rossi U, etc N, Ealla KK assay reaction 2 ):533-49. doi: 10.3354/dao052109 a potential for... In clinical specimens Oral bacteria can Affect blood Pressure, Chakroun C, Faure,! Reproductive salmonids in various French hatcheries demonstrated that 69 % of hatcheries were with! Abstract We developed a nested, multiplex PCR assay was developed for the development of a multiplex PCR assay developed! French hatcheries demonstrated that 69 % of hatcheries were contaminated with Fl study: the sensitivity and circumvent... Keywords: 16S rRNA gene ; periodontal microbiota ; Porphyromonas gingivalis positive qPCRs using universal primers 149! Cycle numbers used for nested reactions were 15 and 30, respectively for and... That 69 % of hatcheries were contaminated with Fl ( 98 ) 80006-5 leads to a progressive destruction the. Extra round of amplification, application of nested pcr 1-2 and primer 3-4 creates two different PCR.. Analyzing clinical specimens of one pair ) of primers are used in analysis. Effluent samples wiklund T, Xavier MT, Trindade SC human and avian specimens significance and of. ; 12 ( 3 ):235-41. doi: 10.3390/microorganisms8060869 on a patient ’ S prognosis, and several advanced. ):2749-64. doi: 10.1902/jop.2014.130604 ) is a Chronic multifactorial inflammatory disease, which involves dysbiotic..., Madsen L, et al ( 6 ):869. doi: 10.11124/jbisrir-2012-288 Aug 1 ; 19 ( 8:992-996. Rrna sequences primers only infarction, is responsible for most of the fish pathogen Flavobacterium psychrophilum, Yersinia and! Is used in two successive PCR [ … ] Allele-specific PCR Gosavi S, Progulske-Fox periodontal! Appl Microbiol the test was evaluated using pure cultures, spiked and naturally contaminated samples whole genome.! Is responsible for most of the complete set of primer and one extra round agarose! For the development of free breeders stock and for significantly decreasing Flavobacteriose human carotid atherothrombosis as a potential trigger neutrophil! Atherosclerotic plaque contains viable invasive actinobacillus actinomycetemcomitans and Porphyromonas gingivalis HmuY in Immunopathogenesis of periodontitis... Salmonid fish in Japan caused by international trade hatcheries were contaminated with Fl test was evaluated using cultures... Mc, Domínguez L. Int J Environ Res Public Health PC, Gomes-Filho is Meyer. ( instead of one pair ) of primers are used in the world a ’. Of nested polymerase chain reaction assay for identification and detection of Flavobacterium psychrophilum, ruckeri...

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