Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens.
Vera Gouvea,Roger I. Glass,P. A. Woods,Koki Taniguchi,H. F. Clark,Barbara Forrester,Zhao-Yin Fang +6 more
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TLDR
A PCR typing method was devised in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels, which provided a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.Abstract:
The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.read more
Citations
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Journal ArticleDOI
Changing Patterns of Rotavirus Genotypes in Turkey
TL;DR: Distribution of rotavirus genotypes exhibited distinctive changes in this study, and ongoing surveillance studies are important before the inclusion ofRotavirus vaccines in national immunization program of Turkey.
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First detection of a reassortant G3P[8] rotavirus A strain in Italy: a case report in an 8-year-old child
Susanna Esposito,Barbara Camilloni,Sonia Bianchini,Giovanni Ianiro,Ilaria Polinori,Edoardo Farinelli,Marina Monini,Nicola Principi +7 more
TL;DR: This case shows the first detection in Italy of a reassortant G3P[8] RVA associated with a severe AGE, which is unusual in a school-age child without any known severe underlying problems and highlights the importance of continuously monitoring the RVA strains circulating in paediatric age in order to detect novel viral variants able to spread in the general population.
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Norovirus-specific immunoglobulin A in breast milk for protection against norovirus-associated diarrhea among infants.
Hannah Karen Mina Labayo,Monica J. Pajuelo,Kentaro Tohma,Lauren A. Ford-Siltz,Robert H. Gilman,Lilia Cabrera,Holger Mayta,Gerardo Sanchez,Anniuska Toledo Cornejo,Caryn Bern,Clyde Dapat,Tomonori Nochi,Gabriel I. Parra,Hitoshi Oshitani,Mayuko Saito +14 more
TL;DR: Mothers with high positivity rates and titers of NV-IgA in breast milk had NV infected infants with reduced diarrheal symptoms, and antigenic relatedness to the genetic diversity of human norovirus was suggested.
Journal Article
National Rotavirus Surveillance Program annual report, 2004-05.
TL;DR: The serotypes of rotavirus strains responsible for the hospitalisation of children with acute gastroenteritis during the period 1 July 2004 to 30 June 2005 are described.
Journal ArticleDOI
Increasing predominance of G8P[8] species A rotaviruses in children admitted to hospital with acute gastroenteritis in Thailand, 2010-2013
Arpaporn Yodmeeklin,Pattara Khamrin,Kattareeya Kumthip,Rungnapa Malasao,Nuthapong Ukarapol,Hiroshi Ushijima,Niwat Maneekarn +6 more
TL;DR: Overall, the data indicated a high degree of diversity of RVA genotypes, with the emergence of several uncommon RVA strains in children with acute gastroenteritis in Thailand.
References
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