Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates

Taina Jalava, Päivi Lehtovaara, A. Kallio, Marjut Ranki, Hans Söderlund

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Present methods for quantification of hepatitis B virus (HBV) particles from serum samples are not sensitive enough for some recent clinical applications. We describe a test that allows quantification of HBV DNA in a broad dynamic range from less than 40 to 10(6) molecules based on competitive PCR. The specimen DNA and a known amount of an internal standard (IS) are co-amplified in the same tube with the same primers, one of which is biotinylated. The two biotinylated products can be quantified by hybridization on microplates coated with streptavidin, because their internal sequences are nonhomologous. An adequate standard curve is obtained by amplifying HBV DNA from a plasmid clone together with an IS. The ratio of amplified HBV DNA to IS DNA enables quantification of the original amount of HBV without tedious titrations of each sample with competitor. The lower limit for quantitative analysis with radioactive probes was between 4 and 40 virus particles in a 10-microliters serum samples.
Original languageEnglish
Pages (from-to)134 - 139
Number of pages6
JournalBioTechniques
Volume15
Issue number1
Publication statusPublished - 1993
MoE publication typeA1 Journal article-refereed

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Viruses
Hepatitis B virus
Amplification
DNA
Virion
Streptavidin
Serum
Titration
Plasmids
Clone Cells
Polymerase Chain Reaction
Molecules
Chemical analysis

Cite this

Jalava, T., Lehtovaara, P., Kallio, A., Ranki, M., & Söderlund, H. (1993). Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates. BioTechniques, 15(1), 134 - 139.
Jalava, Taina ; Lehtovaara, Päivi ; Kallio, A. ; Ranki, Marjut ; Söderlund, Hans. / Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates. In: BioTechniques. 1993 ; Vol. 15, No. 1. pp. 134 - 139.
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abstract = "Present methods for quantification of hepatitis B virus (HBV) particles from serum samples are not sensitive enough for some recent clinical applications. We describe a test that allows quantification of HBV DNA in a broad dynamic range from less than 40 to 10(6) molecules based on competitive PCR. The specimen DNA and a known amount of an internal standard (IS) are co-amplified in the same tube with the same primers, one of which is biotinylated. The two biotinylated products can be quantified by hybridization on microplates coated with streptavidin, because their internal sequences are nonhomologous. An adequate standard curve is obtained by amplifying HBV DNA from a plasmid clone together with an IS. The ratio of amplified HBV DNA to IS DNA enables quantification of the original amount of HBV without tedious titrations of each sample with competitor. The lower limit for quantitative analysis with radioactive probes was between 4 and 40 virus particles in a 10-microliters serum samples.",
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Jalava, T, Lehtovaara, P, Kallio, A, Ranki, M & Söderlund, H 1993, 'Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates', BioTechniques, vol. 15, no. 1, pp. 134 - 139.

Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates. / Jalava, Taina; Lehtovaara, Päivi; Kallio, A.; Ranki, Marjut; Söderlund, Hans.

In: BioTechniques, Vol. 15, No. 1, 1993, p. 134 - 139.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates

AU - Jalava, Taina

AU - Lehtovaara, Päivi

AU - Kallio, A.

AU - Ranki, Marjut

AU - Söderlund, Hans

N1 - Project code: -

PY - 1993

Y1 - 1993

N2 - Present methods for quantification of hepatitis B virus (HBV) particles from serum samples are not sensitive enough for some recent clinical applications. We describe a test that allows quantification of HBV DNA in a broad dynamic range from less than 40 to 10(6) molecules based on competitive PCR. The specimen DNA and a known amount of an internal standard (IS) are co-amplified in the same tube with the same primers, one of which is biotinylated. The two biotinylated products can be quantified by hybridization on microplates coated with streptavidin, because their internal sequences are nonhomologous. An adequate standard curve is obtained by amplifying HBV DNA from a plasmid clone together with an IS. The ratio of amplified HBV DNA to IS DNA enables quantification of the original amount of HBV without tedious titrations of each sample with competitor. The lower limit for quantitative analysis with radioactive probes was between 4 and 40 virus particles in a 10-microliters serum samples.

AB - Present methods for quantification of hepatitis B virus (HBV) particles from serum samples are not sensitive enough for some recent clinical applications. We describe a test that allows quantification of HBV DNA in a broad dynamic range from less than 40 to 10(6) molecules based on competitive PCR. The specimen DNA and a known amount of an internal standard (IS) are co-amplified in the same tube with the same primers, one of which is biotinylated. The two biotinylated products can be quantified by hybridization on microplates coated with streptavidin, because their internal sequences are nonhomologous. An adequate standard curve is obtained by amplifying HBV DNA from a plasmid clone together with an IS. The ratio of amplified HBV DNA to IS DNA enables quantification of the original amount of HBV without tedious titrations of each sample with competitor. The lower limit for quantitative analysis with radioactive probes was between 4 and 40 virus particles in a 10-microliters serum samples.

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Jalava T, Lehtovaara P, Kallio A, Ranki M, Söderlund H. Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates. BioTechniques. 1993;15(1):134 - 139.