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Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription.

Endoh D, Mizutani T, Kirisawa R, Maki Y, Saito H, Kon Y, Morikawa S, Hayashi M - Nucleic Acids Res. (2005)

Bottom Line: We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA.A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides.In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Veterinary Radiology, School of Veterinary Medicine, Rakuno Gakuen University Ebetsu 069-8501, Japan. dendoh@rakuno.ac.jp

ABSTRACT
A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)(+) RNAs but not for poly(A)(-) RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses.

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Agarose gel electrophoresis of RDA products with PCR products used for probes for hybridization (A) and a hybridized fluorogram (B). RNA was extracted from SARS-CoV-infected cells and subjected to RDA according to the method described in Materials and Methods. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gels and blotted on a Nylon membrane. The membrane was then cut into slits that contained the lane showing the presence of DNA. On the other hand, the PCR fragments predicted to be amplified in the RDA reaction were amplified and subsequently ascertained by agarose gel electrophoresis (A). The amplified genomic fragments of SARS-CoV were Dig-labelled and used as probes for hybridization to each slit of the Nylon membrane containing the RDA product. Hybridization was performed in separate hybridization bags. After washing with 1× SSC and 0.1% SDS solution, the hybridized probes were detected on a fluorogram (B). Positions and sizes (bp) of markers are present on the left.
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fig5: Agarose gel electrophoresis of RDA products with PCR products used for probes for hybridization (A) and a hybridized fluorogram (B). RNA was extracted from SARS-CoV-infected cells and subjected to RDA according to the method described in Materials and Methods. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gels and blotted on a Nylon membrane. The membrane was then cut into slits that contained the lane showing the presence of DNA. On the other hand, the PCR fragments predicted to be amplified in the RDA reaction were amplified and subsequently ascertained by agarose gel electrophoresis (A). The amplified genomic fragments of SARS-CoV were Dig-labelled and used as probes for hybridization to each slit of the Nylon membrane containing the RDA product. Hybridization was performed in separate hybridization bags. After washing with 1× SSC and 0.1% SDS solution, the hybridized probes were detected on a fluorogram (B). Positions and sizes (bp) of markers are present on the left.

Mentions: Similar to BPI3, cDNA fragments derived from SARS-CoV were also amplified from SARS-CoV-infected cells (Figure 5). Viral origin of the amplified fragments was confirmed by hybridization (Figure 5B) and PCR amplification by SARS-CoV-specific primers (Figure 5A). These results indicate that genomic fragments of SARS-CoV can also be isolated by this method.


Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription.

Endoh D, Mizutani T, Kirisawa R, Maki Y, Saito H, Kon Y, Morikawa S, Hayashi M - Nucleic Acids Res. (2005)

Agarose gel electrophoresis of RDA products with PCR products used for probes for hybridization (A) and a hybridized fluorogram (B). RNA was extracted from SARS-CoV-infected cells and subjected to RDA according to the method described in Materials and Methods. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gels and blotted on a Nylon membrane. The membrane was then cut into slits that contained the lane showing the presence of DNA. On the other hand, the PCR fragments predicted to be amplified in the RDA reaction were amplified and subsequently ascertained by agarose gel electrophoresis (A). The amplified genomic fragments of SARS-CoV were Dig-labelled and used as probes for hybridization to each slit of the Nylon membrane containing the RDA product. Hybridization was performed in separate hybridization bags. After washing with 1× SSC and 0.1% SDS solution, the hybridized probes were detected on a fluorogram (B). Positions and sizes (bp) of markers are present on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1074749&req=5

fig5: Agarose gel electrophoresis of RDA products with PCR products used for probes for hybridization (A) and a hybridized fluorogram (B). RNA was extracted from SARS-CoV-infected cells and subjected to RDA according to the method described in Materials and Methods. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gels and blotted on a Nylon membrane. The membrane was then cut into slits that contained the lane showing the presence of DNA. On the other hand, the PCR fragments predicted to be amplified in the RDA reaction were amplified and subsequently ascertained by agarose gel electrophoresis (A). The amplified genomic fragments of SARS-CoV were Dig-labelled and used as probes for hybridization to each slit of the Nylon membrane containing the RDA product. Hybridization was performed in separate hybridization bags. After washing with 1× SSC and 0.1% SDS solution, the hybridized probes were detected on a fluorogram (B). Positions and sizes (bp) of markers are present on the left.
Mentions: Similar to BPI3, cDNA fragments derived from SARS-CoV were also amplified from SARS-CoV-infected cells (Figure 5). Viral origin of the amplified fragments was confirmed by hybridization (Figure 5B) and PCR amplification by SARS-CoV-specific primers (Figure 5A). These results indicate that genomic fragments of SARS-CoV can also be isolated by this method.

Bottom Line: We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA.A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides.In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Veterinary Radiology, School of Veterinary Medicine, Rakuno Gakuen University Ebetsu 069-8501, Japan. dendoh@rakuno.ac.jp

ABSTRACT
A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)(+) RNAs but not for poly(A)(-) RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses.

Show MeSH
Related in: MedlinePlus