scholarly journals Multiplex PCR-based titration (MPBT) assay for determination of infectious titers of the three Sabin strains of live poliovirus vaccine

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Hasmik Manukyan ◽  
Elvira Rodionova ◽  
Tatiana Zagorodnyaya ◽  
Tsai-Lien Lin ◽  
Konstantin Chumakov ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Clinical Trials ◽  
Cell Cultures ◽  
Multiplex Polymerase Chain Reaction ◽  
Cytopathic Effect ◽  
Oral Poliovirus Vaccine ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Titration Assay ◽  
Serial Dilutions

Abstract Background Conventional assays to titrate polioviruses usually test serial dilutions inoculated into replicate cell cultures to determine a 50% cytopathic endpoint, a process that is both time-consuming and laborious. Such a method is still used to measure potency of live Oral Poliovirus Vaccine during vaccine development and production and in some clinical trials. However, the conventional method is not suited to identify and titrate virus in the large numbers of fecal samples generated during clinical trials. Determining titers of each of the three Sabin strains co-existing in Oral Poliovirus Vaccine presents an additional challenge. Results A new assay using quantitative multiplex polymerase chain reaction as an endpoint instead of cytopathic effect was developed to overcome these limitations. In the multiplex polymerase chain reaction-based titration assay, cell cultures were infected with serial dilutions of test samples, lysed after two-day incubation, and subjected to a quantitative multiplex one-step reverse-transcriptase polymerase chain reaction. All three serotypes of poliovirus were identified in single samples and titers calculated. The multiplex polymerase chain reaction-based titration assay was reproducible, robust and sensitive. Its lower limits of titration for three Sabin strains were 1–5 cell culture 50% infectious doses per ml. We prepared different combinations of three Sabin strains and compared titers obtained with conventional and multiplex polymerase chain reaction-based titration assays. Results of the two assays correlated well and showed similar results and sensitivity. Multiplex polymerase chain reaction-based titration assay was completed in two to 3 days instead of 10 days for the conventional assay. Conclusions The multiplex polymerase chain reaction-based titration (MPBT) is the first quantitative assay that identifies and titrates each of several different infectious viruses simultaneously in a mixture. It is suitable to identify and titrate polioviruses rapidly during the vaccine manufacturing process as a quality control test, in large clinical trials of vaccines, and for environmental surveillance of polioviruses. The MPBT assay can be automated for high-throughput implementation and applied for other viruses including those with no cytopathic effect.

scholarly journals Isolamento e avaliação do comportamento do vírus ORF em células de córnea fetal caprina e identificação pela Reação em Cadeia da Polimerase

2021 ◽  
Vol 4 (4) ◽  
pp. 107-115
Author(s):  
Rosana Leo da Santana ◽  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cell Cultures ◽  
Cytopathic Effect ◽  
Clinical Symptoms ◽  
Small Variation ◽  
Clinical Samples ◽  
Vaccine Production ◽  
Chain Reaction ◽  
Cytoplasmic Vacuolization ◽  
Polymerase Chain

The control of the infection in endemic regions is performed with vaccines, however there are limitations in the vaccine production due to the difficulties in replicating the virus in cell cultures. This work was conducted so as to isolate and evaluate the behavior of ECV samples in fetal goat cell line cornea (CorFC) naturally immortalized. Crust samples from 22 sheep and seven goats presenting the clinical symptoms of EC from the states of Pernambuco, Bahia, Sergipe and Paraíba, were inoculated in CorFC monolayers and identified by polymerase chain reaction (PCR) using primers for amplification of a fragment of235 bp of gene B2L envelope ECV. Eleven samples were submitted to seven consecutive passes, at weekly intervals. Cytopathic effect (CPE) was observed in all passages, after24 hours post-infection, characterized by cell rounding, syncytial fusion with training, inclusion and cytoplasmic vacuolization. Thus, CorFC cell cultures proved highly permissible to ECV replication with small variation among viral samples. The PCR technique can be an efficient method used for confirmation of ECV infection in clinical samples.

Virus respiratoires dans les pneumonies associées aux soins

2018 ◽  
Vol 27 (3) ◽  
pp. 217-227
Author(s):  
P. Loubet ◽  
G. Voiriot ◽  
M. Neuville ◽  
B. Visseaux ◽  
J.-F. Timsit
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Biologie Moléculaire ◽  
Mise En Œuvre ◽  
Polymerase Chain ◽  
Infection Bactérienne

Les pneumonies acquises à l’hôpital (PAH) sont fréquentes. À l’ère des techniques diagnostiques de biologie moléculaire (multiplex polymerase chain reaction), les rares données disponibles estiment que les virus respiratoires sont impliqués dans 22 à 32 % des épisodes. Les patients immunodéprimés constituent probablement la population la plus à risque. La présentation clinique et radiologique ne diffère pas entre pneumonies bactériennes, virales et mixtes (virus–bactérie). L’excrétion prolongée de virus respiratoires dans les voies aériennes a été rapportée chez les patients immunodéprimés. Elle pourrait promouvoir la co-infection bactérienne, associée à des durées d’hospitalisation prolongées. L’acquisition intrahospitalière a été démontrée chez tous les virus respiratoires. Elle encourage la mise en œuvre et le respect des mesures d’hygiène et de confinement, dans l’objectif de protéger soignants, visiteurs et patients. De nombreux points restent largement méconnus, relatifs aux interactions entre virus respiratoires et pathogènes non viraux, aux périodes d’incubation, ou encore aux durées d’excrétion virale. L’amélioration des techniques diagnostiques et l’accumulation de données épidémiologiques et cliniques devraient permettre de mieux appréhender le rôle des virus respiratoires dans les PAH. Cette meilleure connaissance aidera à rationaliser l’utilisation des tests de détection et facilitera l’interprétation de leurs résultats. Elle guidera aussi le clinicien dans l’utilisation future des nombreuses molécules antivirales actuellement en développement clinique chez l’homme.

Molecular Analysis of Uropathogenic E.coli Isolates from Urinary Tract Infections

2019 ◽  
Vol 19 (3) ◽  
pp. 322-326 ◽  
Author(s):  
Hassan Valadbeigi ◽  
Elham Esmaeeli ◽  
Sobhan Ghafourian ◽  
Abbas Maleki ◽  
Nourkhoda Sadeghifard
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Multiplex Polymerase Chain Reaction ◽  
Virulence Genes ◽  
Uropathogenic Escherichia Coli ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tract Infections

Introduction: The aim of the current study was to investigate the prevalence of virulence genes in uropathogenic Escherichia coli (UPEC) isolates in Ilam. Materials and Methods: For this purpose, a total of 80 UPEC isolates were collected for patients with UTIs during a 6 months period. The multiplex polymerase chain reaction (multiplex PCR) was used to detect the papEF, fimH, iucD, hlyA, fyuA, and ompT genes. Results: The prevalence of fimH, papEF, iucD, fyuA, hlyA, hlyA, and ompT genes were 87.5%, 47.5%, 60%, 67.5%, 27.5%, 47.5% and 71.2%, respectively. Among all of the isolates, 27 profiles were obtained. Conclusion: Our findings demonstrated that the most prevalence was found for fimH, and different distribution of virulence genes suggested different ability of pathogenicity.

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