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Genotyping of Rotavirus by Using RT-PCR Methods Nirwati, Hera; Wibawa, Tri; Aman, Abu Tholib; Soenarto, Yati
Indonesian Journal of Biotechnology Vol 18, No 1 (2013)
Publisher : Universitas Gadjah Mada

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There is a great diversity of rotavirus genotypes circulating worldwide, with dominant genotypes changing from year to year. Rotavirus genotyping was performed by using reverse transcription PCR with type-specifi c-primers. Since rotavirus is a RNA virus that has high mutation rate, there was a possibility of technical diffi culty in genotyping due to mutation in the primer binding sites. During Indonesian rotavirus surveillance study 2006-2009, it was reported that 17% of samples subjected for G type and 21% of samplessubjected for P type were untypeable. The objective of this study was to identify genotypes of the samples that were untypeable previously using RT-PCR based on the method described by Das et al. (1994) and Gentsch et al. (1992). There were 30 samples subjected to G type and 61 samples subjected to P type to be re-typed using method described by Gouvea et al. (1990) and Simmond et al. (2008) for G and P typing, respectively. By using another set of primer, the genotype of all samples was identifi ed. This study highlights the importance of a constant reconsideration of primer sequences employed for the molecular typing of rotaviruses.Key words: rotavirus, G typing, P typing

High Frequency Spontaneous Deletions within the IcaADBC Operon of Clinical Staphylococcus epidermidis Isolates. Nuryastuti, Titik; van der Mei, Henny C.; Busscher, Henk J.; Kuijer, Roel; Aman, Abu Tholib; P. Krom, Bastiaan
Indonesian Journal of Biotechnology Vol 17, No 2 (2012)
Publisher : Universitas Gadjah Mada

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Staphylococcus epidermidis has been shown to undergo a phase variation correlating with expression of the icaADBC operon which contributes to biofilm formation. Biofilm formation of Enterococcus faecalis is related to heterogeneity in electrophoretic mobility. Here the relationship between phase variants of clinical isolates of S. epidermidis, icaADBC presence and electrophoretic mobility distributions is investigated. Of 105 S. epidermidis clinical isolates, 5 showed phase variation on Congo Red agar plate. Biofilm forming capability of the blackcolonies and inability of the red colonies were confirmed using a microtiter plate assay and confocal laser scanning microscopy. Upon analysis of electrophoretic mobility distributions, the black colonies displayed heterogeneity at pH 2 which was absent in the red colonies of the same strain. Surprisingly, it was shown that in all red colonies had lost the icaADBC genes. Determination of gene copy number using Real Time PCR targeting icaA showed reduction of gene copy within a culture with phase variation. In conclusion, using three fundamentally different approaches phase variation of the five clinical isolates was observed. Variants appeared through loss of icaA and icaC gens. To our knowledge this is the first report indicating S. epidermidis strains irreversible switching from biofilm + to biofilm – phenotype by deletion of ica genes. Key words: deletion, ica genes, Staphylococcus epidermidis, IcaADBC operon

High Frequency Spontaneous Deletions within the IcaADBC Operon of Clinical Staphylococcus epidermidis Isolates. Titik Nuryastuti; Henny C. van der Mei; Henk J. Busscher; Roel Kuijer; Abu Tholib Aman; Bastian P. Krom
Indonesian Journal of Biotechnology Vol 17, No 2 (2012)
Publisher : Universitas Gadjah Mada

Staphylococcus epidermidis has been shown to undergo a phase variation correlating with expression of the icaADBC operon which contributes to biofilm formation. Biofilm formation of Enterococcus faecalis is related to heterogeneity in electrophoretic mobility. Here the relationship between phase variants of clinical isolates of S. epidermidis, icaADBC presence and electrophoretic mobility distributions is investigated. Of 105 S. epidermidis clinical isolates, 5 showed phase variation on Congo Red agar plate. Biofilm forming capability of the blackcolonies and inability of the red colonies were confirmed using a microtiter plate assay and confocal laser scanning microscopy. Upon analysis of electrophoretic mobility distributions, the black colonies displayed heterogeneity at pH 2 which was absent in the red colonies of the same strain. Surprisingly, it was shown that in all red colonies had lost the icaADBC genes. Determination of gene copy number using Real Time PCR targeting icaA showed reduction of gene copy within a culture with phase variation. In conclusion, using three fundamentally different approaches phase variation of the five clinical isolates was observed. Variants appeared through loss of icaA and icaC gens. To our knowledge this is the first report indicating S. epidermidis strains irreversible switching from biofilm + to biofilm – phenotype by deletion of ica genes. Key words: deletion, ica genes, Staphylococcus epidermidis, IcaADBC operon

Genotyping of Rotavirus by Using RT-PCR Methods Hera Nirwati; Tri Wibawa; Abu Tholib Aman; Yati Soenarto
Indonesian Journal of Biotechnology Vol 18, No 1 (2013)
Publisher : Universitas Gadjah Mada

There is a great diversity of rotavirus genotypes circulating worldwide, with dominant genotypes changing from year to year. Rotavirus genotyping was performed by using reverse transcription PCR with type-specifi c-primers. Since rotavirus is a RNA virus that has high mutation rate, there was a possibility of technical diffi culty in genotyping due to mutation in the primer binding sites. During Indonesian rotavirus surveillance study 2006-2009, it was reported that 17% of samples subjected for G type and 21% of samplessubjected for P type were untypeable. The objective of this study was to identify genotypes of the samples that were untypeable previously using RT-PCR based on the method described by Das et al. (1994) and Gentsch et al. (1992). There were 30 samples subjected to G type and 61 samples subjected to P type to be re-typed using method described by Gouvea et al. (1990) and Simmond et al. (2008) for G and P typing, respectively. By using another set of primer, the genotype of all samples was identifi ed. This study highlights the importance of a constant reconsideration of primer sequences employed for the molecular typing of rotaviruses. Key words: rotavirus, G typing, P typing

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