Diagnostic assays for parvovirus B19
AssigneeChiron Corporation
InventorsSergio Pichuantes, Venkatakrishna Shyamala
Life of this patent
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Abstract
Human parvovirus B19 primers and probes derived from conserved regions of the parvovirus B19 genome are disclosed. Also disclosed are nucleic acid-based assays using the primers and probes.
Claims83
A method of detecting human parvovirus B19 infection in a biological sample, said method comprising:
- (a) isolating nucleic acid from a biological sample suspected of containing human parvovirus B19 DNA, wherein said nucleic acid comprises an RNA target sequence;
- (b) reacting the isolated parvovirus B19 nucleic acid with a first oligonucleotide which comprises a first primer comprising a complexing sequence sufficiently complementary to the 3′-terminal portion of the RNA target sequence to complex therewith, wherein said first primer further comprises a promoter for a DNA-dependent RNA polymerase 5′ and operably linked to the complexing sequence, wherein said reacting is done under conditions that provide for the formation of an oligonucleotide/target sequence complex and initiation of DNA synthesis;
- (c) extending the first primer in an extension reaction using the RNA target sequence as a template to give a first DNA primer extension product complementary to the RNA target sequence;
- (d) separating the first DNA primer extension product from the RNA target sequence using an enzyme which selectively degrades the RNA target sequence;
- (e) treating the DNA primer extension product with a second oligonucleotide which comprises a second primer comprising a complexing sequence sufficiently complementary to the 3′-terminal portion of the DNA primer extension product to complex therewith under conditions that provide for the formation of an oligonucleotide/target sequence complex and initiation of DNA synthesis;
- (f) extending the 3′-terminus of the second primer in a DNA extension reaction to give a second DNA primer extension product, thereby producing a template for the DNA-dependent RNA polymerase;
- (g) using the template to produce multiple RNA copies of the target sequence using a DNA-dependent RNA polymerase which recognizes the promoter sequence;
- and
- (h) using the RNA copies of step (g), autocatalytically repeating steps (b) to (g) to amplify the target sequence.
A method of detecting human parvovirus B19 infection in a biological sample, said method comprising:
- (a) isolating nucleic acid from a biological sample suspected of containing human parvovirus B19 DNA, wherein said nucleic acid comprises an RNA target sequence;
- (b) reacting the isolated parvovirus B19 nucleic acid with a first oligonucleotide which comprises a first primer comprising a complexing sequence sufficiently complementary to the 3′-terminal portion of the RNA target sequence to complex therewith, wherein said first primer further comprises a promoter for a DNA-dependent RNA polymerase 5′ and operably linked to the complexing sequence, wherein said first primer comprises a sequence derived from the polynucleotide sequence depicted in any one of FIGS. 2A-2U or FIGS. 11A-11Z and said reacting is done under conditions that provide for the formation of an oligonucleotide/target sequence complex and initiation of DNA synthesis;
- (c) extending the first primer in an extension reaction using the RNA target sequence as a template to give a first DNA primer extension product complementary to the RNA target sequence;
- (d) separating the first DNA primer extension product from the RNA target sequence using an enzyme which selectively degrades the RNA target sequence;
- (e) treating the DNA primer extension product with a second oligonucleotide which comprises a second primer comprising a complexing sequence sufficiently complementary to the 3′-terminal portion of the DNA primer extension product to complex therewith, wherein said second primer is derived from the polynucleotide sequence depicted in any one of FIGS. 2A-2U or FIGS. 11A-11Z and said treating is done under conditions that provide for the formation of an oligonucleotide/target sequence complex and initiation of DNA synthesis;
- (f) extending the 3′-terminus of the second primer in a DNA extension reaction to give a second DNA primer extension product, thereby producing a template for the DNA-dependent RNA polymerase;
- (g) using the template to produce multiple RNA copies of the target sequence using a DNA-dependent RNA polymerase which recognizes the promoter sequence;
- and
- (h) using the RNA copies of step (g), autocatalytically repeating steps (b) to (g) to amplify the target sequence;
- (i) adding an acridinium ester-labeled oligonucleotide probe to the product of step (h), wherein said oligonucleotide probe is complementary to a portion of said target sequence and said probe is derived from the polynucleotide sequence depicted in any one of FIGS. 2A-2U or FIGS. 11A-11Z, wherein said probe is added under conditions that provide for the hybridization of said probe with said target sequence to form a probe:target complex;
- and
- (j) detecting the presence or absence of label as an indication of the presence or absence of the target sequence.
A method for amplifying a target parvovirus B19 nucleotide sequence, said method comprising:
- (a) isolating nucleic acid from a biological sample suspected of containing human parvovirus B19 DNA, wherein said nucleic acid comprises an RNA target sequence;
- (b) adding one or more primers capable of hybridizing to the RNA target sequence, wherein said one or more primers are derived from the polynucleotide sequences depicted in any one of FIGS. 2A-2U and FIGS. 11A-11Z;
- (c) adding an oligonucleotide probe capable of hybridizing to the RNA target sequence 3′ relative to the one or more primers;
- (d) extending the one or more primers using a polymerase.
A polynucleotide comprising a nucleotide sequence comprising any one of the nucleotide sequences depicted in FIGS. 2A-2U or FIGS. 11A-11Z.
A polynucleotide comprising a nucleotide sequence comprising any one of the nucleotide sequences depicted in FIGS. 3A-3C or 4A-4C.
An oligonucleotide primer consisting of a promoter region recognized by a DNA-dependent RNA polymerase operably linked to a human parvovirus B19-specific complexing sequence of about 10 to about 75 nucleotides.
An oligonucleotide primer consisting of a T7 promoter operably linked to a human parvovirus B19-specific complexing sequence of about 10 to about 75 nucleotides, wherein said human parvovirus B19-specific complexing sequence is derived from the polynucleotide sequence depicted in any one of FIGS. 2A-2U or FIGS. 11A-11Z.
An oligonucleotide probe comprising a parvovirus B19-specific hybridizing sequence of about 10 to about 50 nucleotides linked to an acridinium ester label.
A method for detecting human parvovirus B19 infection in a biological sample, said method comprising:
- (a) isolating nucleic acid from a biological sample suspected of containing human parvovirus B19 DNA, wherein said nucleic acid comprises a target sequence;
- (b) reacting the isolated parvovirus B19 nucleic acid with a detectably labeled probe sufficiently complementary to and capable of hybridizing with the target sequence, wherein the probe is derived from the polynucleotide sequences depicted in any one of FIGS. 2A-2U and FIGS. 11A-11Z, and further wherein said reacting is done under conditions that provide for the formation of a probe/target sequence complex;
- and
- (c) detecting the presence or absence of label as an indication of the presence or absence of the target sequence.
Dependencies are inferred from the claim text.