PCR Multiplex fluorescente para detecção de bactérias em sêmen bovino
Francisca Elda Ferreira DiasCáris Maroni NunesTânia Vasconcelos CavalcanteHelciléia Dias SantosSílvia MinharroJosé Fernando Garcia
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Abstract:
Este estudo teve como objetivo avaliar o limiar de detecção da técnica de PCR multiplex fluorescente aliada a eletroforese capilar na detecção de agentes infecciosos em amostras de sêmen experimentalmente contaminadas com concentrações decrescentes das bactérias Brucella abortus, Leptospira interrogans sorovar pomona, Campylobacter fetus e Haemophilus somnus. Amostras de sêmen bovino foram experimentalmente contaminadas com concentrações decrescentes de bactérias obtidas através de diluições seriadas na base 10 de modo a obter-se amostras contendo desde 1 vez até 10-7 bactérias/mL a partir da concentração inicial de Leptospira pomona, Brucella abortus, Campylobacter fetus e Haemophilus somnus. As diluições foram efetuadas individualmente para cada bactéria, bem como nas diferentes concentrações necessárias para a padronização do teste de multiplex PCR. As extrações de DNA de todas as soluções contendo espermatozóides e bactérias analisadas no presente estudo foram realizadas segundo protocolo descrito por Heinemann et al. (2000). Os produtos de PCR multiplex foram avaliados por eletroforese em gel de poliacrilamida 8% e separação eletroforética por sistema capilar em equipamento automático de análise de fragmentos de DNA MegaBace. Observou-se a amplificação de fragmentos de 193pb, 330pb, 400pb e 415pb a partir do DNA de B. abortus, L. pomona, H. somnus, C. fetus, respectivamente. Na análise por eletroforese capilar de produtos da PCR multiplex do DNA para detecção simultânea dos quatro patógenos observou-se a sinal de positividade até a diluição de 10-3 bactérias/mL vezes da concentração inicial da solução estoque de cada bactéria. A técnica de PCR multiplex aliada à eletroforese capilar foi usada pela primeira vez para o diagnóstico direto de quatro bactérias patogênicas no sêmen, demonstrando ser um método rápido na detecção de bactérias causadoras de doenças reprodutivas.Keywords:
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In this study, three molecular assays (real-time multiplex polymerase chain reaction [PCR], merozoite surface antigen gene [ MSP ]-multiplex PCR, and the PlasmoNex Multiplex PCR Kit) have been developed for diagnosis of Plasmodium species. In total, 52 microscopy-positive and 20 malaria-negative samples were used in this study. We found that real-time multiplex PCR was the most sensitive for detecting P. falciparum and P. knowlesi . The MSP -multiplex PCR assay and the PlasmoNex Multiplex PCR Kit were equally sensitive for diagnosing P. knowlesi infection, whereas the PlasmoNex Multiplex PCR Kit and real-time multiplex PCR showed similar sensitivity for detecting P. vivax . The three molecular assays displayed 100% specificity for detecting malaria samples. We observed no significant differences between MSP -multiplex PCR and the PlasmoNex multiplex PCR kit (McNemar's test: P = 0.1489). However, significant differences were observed comparing real-time multiplex PCR with the PlasmoNex Multiplex PCR Kit (McNemar's test: P = 0.0044) or real-time multiplex PCR with MSP -multiplex PCR (McNemar's test: P = 0.0012).
Genetic algorithm
Plasmodium (life cycle)
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Objective To evaluate the application of fluorescent multiplex amplification of short tandem repeat(STR)for monitoring survival of engraftment after bone marrow transplantation(BMT).Methods Three STR loci named D12S391,D18S865 and D20S161 in 56 cases were detected by fluorescent multiplex amplification.PCR products were separated and typed by DNA Sequencer.Results The genotypes of STR in 52 recipient after bone marrow transplantation were completely identical with those of the donors.In another 4 cases the evidences of mixed chimerism were observed.Conclusion The system of fluorescent multiplex amplification of STRs exhibited high capacity of discrimination and low cost.Its application in the detection of STR after BMT is reliable,sensitive and simple.Combined with the clinical manifestation it can be used to evaluate the effect of BMT.
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This is the second review in a series papers of application of modern biotechnology to evironmental microbiology.It deals with polymerase chain reaction and contains the steps of PCR, PCR detection of a specific gene, RT-PCR, ICC-PCR,multiplex and seminested PCR and PCR fingerprinting.
Applications of PCR
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Identification
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By simultaneously amplifying several loci in the same reaction, multiplex PCR has been used in gene mapping and DNA typing with polymorphic short tandem repeat loci. Previous studies have discussed in detail the various parameters and conditions that influence the quantity of individual products generated by multiplex PCR. In practice, when a primer pair fails to amplify in a multiplex PCR for some individuals, singleplex PCR is often employed as a supplement to amplify the primer pair. However, the reliability of this procedure is unknown. In this study, we used six primer pairs from ABI PRISMTM Linkage Mapping Set version 2 to perform multiplex and singleplex reactions. The fluorescence-labeled amplification products were separated and detected on ABI PRISM 310 Genetic Analyzer. We found that for the marker D1S468, multiplex and singleplex reactions for the majority of individuals yielded reactions of different sizes. Therefore, the potential size difference between multiplex and singleplex reactions needs to be investigated. This investigation is essential to employ multiplex PCR supplemented with singleplex PCR in gene mapping and DNA typing.
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STR multiplex system
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Species Identification
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We describe a sensitive, reliable and reproducible method, based on three multiplex PCR assays, for the rapid detection of seven common α‐thalassaemia deletions and one α‐globin gene triplication. The new assay detects the α 0 deletions – – SEA , – (α) 20.5 , – – MED , – – FIL and – – THAI in the first multiplex PCR, the second multiplex detects the –α 3.7 deletion and ααα anti3.7 variant, the third multiplex detects the –α 4.2 deletion. This simple multiplex method should greatly facilitate the genetic screening and molecular diagnosis of these determinants in populations where α‐thalassaemias are prevalent.
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India contributes about 80% of the global leprosy case load including case of fresh infection and reinfection. Due to lack of gold standard, diagnosis is done mainly based on routine clinical signs and symptoms, smear and histopathological evidences. There is a lot of lacunae in early confirmatory diagnosis in terms of sensitivity and specificity, especially in paucibacillary tuberculoid type. Moreover, the classification of different classes of leprosy is very important for selection of proper therapeutic schedule. Hence this study was undertaken to develop a multiplex polymerase chain reaction for the diagnosis and strain differentiation of M leprae. A multiplex polymerase chain reaction was developed using the primers R1 and R2 (a) amplifying 372bp DNA target from a repetitive sequence of M leprae and this repetitive sequence (372bp) that was used as a target DNA for amplification was reported to be specific for M leprae was not present in 20 mycobacterium species other than M leprae and primers TTCA and TTCB (b) amplifying (201bp) DNA target of variable sizes from the regions flanking TTC repeats of M leprae genome. This multiplex polymerase chain reacton developed in our laboratory revealed that the number of repeats at each locus might be variable among M leprae but they are found mostly in multibacillary (as the bacterial load is higher in multibacillary) type.
Mycobacterium leprae
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