US 5,853,981 A · Oligonucleotides, methods and kits for amplifying and detecting a nucleic acid of cytomegalovirus (CMV) using nucleic acid sequence .beta.2.7

An oligonucleotide for amplifying a nucleic acid of cytomegalovirus (CMV) utilizing a nucleic acid sequence (.beta.2.7), said oligonucleotide containing a nucleic acid sequence consisting of at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1.

A method for amplifying a nucleic acid of cytomegalovirus (CMV) utilizing a nucleic acid sequence (.mu.2.7), which method comprises

(a) Providing a target nucleic acid consisting of RNA (+);
(b) hybridizing a first primer for amplifying a nucleic acid of cytomegalovirus (CMV) which first primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal with the RNA (+) as a template to elongate the primer by the RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(c) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrids whereupon a single-stranded DNA is obtained,
(d) hybridizing a second primer for amplifying a nucleic acid of cytomegalovirus (CMV) which second primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase,_ whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(e) transcribing a single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (d),
(f) hybridizing the second primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single stranded RNA (-) obtained in step (e) as a template to perform a DNA elongation reaction by a RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(g) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrids whereupon a single-stranded DNA is obtained,
(h) hybridizing the first primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(i) transcribing the single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (h), and
(j) optionally, repeating steps (f) to (i) using the resulting single stranded RNA (-) as a template in optionally repeated step (f).

7 more elements

A method for amplifying a nucleic acid of cytomegalovirus (CMV) utilizing a nucleic acid sequence (.mu.2.7), which method comprises:

(a) providing a target nucleic acid consisting of RNA (+);
(b) hybridizing a first primer for amplifying a nucleic acid of cytomegalovirus (CMV) which first primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1, and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal with the RNA (+) as a template to elongate the primer by the RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(c) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained,
(d) hybridizing a second primer for amplifying a nucleic acid of cytomegalovirus (CMV) which second primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(e) transcribing a single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (d),
(f) hybridizing the second primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single stranded RNA (-) obtained in step (e) as a template to perform a DNA elongation reaction by a RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(g) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained,
(h) hybridizing the first primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of both terminals is produced,
(i) transcribing the single-stranded RNA(-) and RNA(+) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (h), and
(j) optionally, repeating steps (f) to (i) using the resulting single stranded RNA(-) and RNA(+) as a template in optionally repeated step (f).

7 more elements

A method for detecting and/or capturing a nucleic acid of the cytomegalovirus (CMV) utilizing a nucleic acid sequence (.mu.2.7), which comprises:

(a) providing a target nucleic acid consisting of RNA (+);
(b) hybridizing a first primer for amplifying a nucleic acid of cytomegalovirus (CMV) which first primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal with the RNA (+) as a template to elongate the primer by the RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(c) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid. whereupon a single-stranded DNA is obtained,
(d) hybridizing a second primer for amplifying a nucleic acid of cytomegalovirus (CMV) which second primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(e) transcribing a single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (d),
(f) hybridizing the second primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single stranded RNA (-) obtained in the above step (e) as a template to perform a DNA elongation reaction by a RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(g) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained,
(h) hybridizing the first Primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(i) transcribing the single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (h).
(j) optionally. repeating steps (f) to (i) using the resulting single stranded RNA (-) as a template in optionally repeated step (f), and
(k) detecting and/or capturing the resulting amplified nucleic acids with a probe for detecting and/or capturing a nucleic acid of the cytomegalovirus (CMV) which probe contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6, and/or SEQ ID NO:7, or a complementary nucleic acid thereof, wherein the nucleic acid sequence optionally may be modified by a label.

8 more elements

A method for detecting a nucleic acid of the cytomegalovirus (CMV) utilizing a nucleic acid sequence (.mu.2.7), which method comprises

(a) providing a target nucleic acid consisting of RNA
(b) hybridizing a first primer for amplifying a nucleic acid of cytomegalovirus (CMV) which first primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1 and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal with the RNA (+) as a template to elongate the primer by the RNA-dependent DNA polymerase. whereupon an elongated RNA/DNA hybrid is obtained,
(c) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained,
(d) hybridizing a second primer for amplifying a nucleic acid of cytomegalovirus (CMV) which second primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(e) transcribing a single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (d),
(f) hybridizing the second primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single stranded RNA (-) obtained in step (e) as a template to perform a DNA elongation reaction by a RNA-dependent DNA polymerase. whereupon an elongated RNA/DNA hybrid is obtained.
(g) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid. whereupon a single-stranded DNA is obtained.
(h) hybridizing the first primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase. whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(i) transcribing the single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (h),
(j) optionally. repeating steps (f) to (i) using the resulting single stranded RNA (-) as a template in optionally repeated step (f).
(k) hybridizing the resulting nucleic acids with
a capturing probe absorbed to or bound with a solid support which probe contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6, and/or SEQ ID NO:7, and which optionally may be modified and
a detecting probe containing a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6, and/or SEQ ID NO:7, and which is modified by a labels
separating any unreacted detecting probe, and
(m) detecting the label of the optionally labeled capturing probe, the amplified nucleic acids and the detecting probe.

12 more elements

A method for detecting and/or capturing a nucleic acid of the cytomegalovirus (CMV) utilizing a nucleic acid sequence (.beta.2.7), which comprises:

(a) providing a target nucleic acid consisting of RNA (+);
(b) hybridizing a first primer for amplifying a nucleic acid of cytomegalovirus (CMV) which first primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1, and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal with the RNA (+) as a template to elongate the primer by the RNA-dependent DNA polymerase, whereupon an elongated RNAIDNA hybrid is obtained.
(c) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNAIDNA hybrid. whereupon a single-stranded DNA is obtained.
(d) hybridizing a second primer for amplifying a nucleic acid of cytomegalovirus (CMV) which second primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase. whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(e) transcribing a single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA Polymerase which is capable of recognizing the functional promoter sequence of step (d),
(f) hybridizing the second primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single stranded RNA (-) obtained in step (e) as a template to perform a DNA elongation reaction by a RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(g) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained,
(h) hybridizing the first primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of both terminals is produced,
(i) transcribing the single-stranded RNA(-) and RNA(+) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (h), and
(j) optionally. repeating steps (f) to (i) using the resulting single stranded RNA(-) and RNA(+) as a template in optionally repeated step (f), and
(k) detecting and/or capturing the resulting nucleic acids with a probe for detecting and/or capturing a nucleic acid of the cytomegalovirus (CMV) which probe contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6, and/or SEQ ID NO:7, or a complementary nucleic acid thereof, wherein the nucleic acid sequence optionally may be modified by a label.

8 more elements

A method for detecting a nucleic acid of the cytomegalovirus (CMV) utilizing a nucleic acid sequence (.beta.2.7), which method comprises:

(a) providing a target nucleic acid consisting of RNA
(b) hybridizing a first primer for amplifying a nucleic acid of cytomegalovirus (CMV) which first primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1, and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal with the RNA (+) as a template to elongate the Primer by the RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(c) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained.
(d) hybridizing a second primer for amplifying a nucleic acid of cytomegalovirus (CMV) which second primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase. whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced.
(e) transcribing a single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (d).
(f) hybridizing the second primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single stranded RNA (-) obtained in the above step (e) as a template to perform a DNA elongation reaction by a RNA-dependent DNA polymerase. whereupon an elongated RNA/DNA hybrid is obtained,
(g) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained.
(h) hybridizing the first primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA- dependent DNA polymerase. whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of both terminals is produced,
(i) transcribing the single-stranded RNA(-) and RNA(+) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (h). and
(j) optionally, repeating steps (f) to (i) using the resulting single stranded RNA(-) and RNA(+) as a template in optionally repeated step (f),
(k) hybridizing the resulting nucleic acids with
a capturing probe absorbed to or bound with a solid support which probe contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6, and/or SEQ ID NO:7, and which optionally may be modified and
a detecting probe containing a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6, and/or SEQ ID NO:7, and which is modified by a label,
(l) separating any unreacted detecting probe and
(m) measuring the label of the optionally labeled capturing probe, the amplified nucleic acids and the detecting probe.

12 more elements

A method for amplifying a nucleic acid of cytomegalovirus (CMV) utilizing a nucleic acid sequence (.beta.2.7), which method comprises:

(a) providing a target nucleic acid consisting of
(b) hybridizing a first primer for amplifying a nucleic acid of cytomegalovirus (CMV) which first primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:3, OR SEQ ID NO:4, and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal, with the RNA (+) as a template to elongate the primer by the RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(c) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained,
(d) hybridizing a second primer for amplifying a nucleic acid of cytomegalovirus (CMV) which second primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1, with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(e) transcribing a single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (d),
(f) hybridizing the second primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single stranded RNA (-) obtained in step (e) as a template to perform a DNA elongation reaction by a RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(g) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained,
(h) hybridizing the first primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(i) transcribing the single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (h), and
(j) optionally, repeating steps (f) to (i) using the resulting single stranded RNA (-) as a template in optionally repeated step (f).

7 more elements

A method for detecting and/or capturing a nucleic acid of the cytomegalovirus (CMV) utilizing a nucleic acid sequence (.beta.2.7), which comprises:

(a) providing a target nucleic acid consisting of RNA (+);
(b) hybridizing a first primer for amplifying a nucleic acid of cytomegalovirus (CMV) which first primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:3, OR SEQ ID NO:4, and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal, with the RNA (+) as a template to elongate the primer by the RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(c) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained,
(d) hybridizing a second primer for amplifying a nucleic acid of cytomegalovirus (CMV) which second primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1, with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(e) transcribing a single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (d),
(f) hybridizing the second primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single stranded RNA (-) obtained in step (e) as a template to perform a DNA elongation reaction by a RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(g) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained,
(h) hybridizing the first primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of both terminals is produced,
(i) transcribing the single-stranded RNA(-) and RNA(+) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (h), and
(j) optionally, repeating steps (f) to (i) using the resulting single stranded RNA(-) and RNA(+) as a template in optionally repeated step (f), and (k) detecting and/or capturing the resulting nucleic acids with a probe for detecting and/or capturing a nucleic acid of the cytomegalovirus (CMV) which probe contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6, and/or SEQ ID NO:7, or a complementary nucleic acid thereof, wherein the nucleic acid sequence optionally may be modified by a label.

7 more elements

A method for detecting a nucleic acid of the cytomegalovirus (CMV) utilizing a nucleic acid sequence (.beta.2.7), which method comprises:

(a) providing a target nucleic acid consisting of RNA
(b) hybridizing a first primer for amplifying a nucleic acid of cytomegalovirus (CMV) which first primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:3, OR SEQ ID NO:4, and has a promoter sequence of a DNA-dependent RNA polymerase at the 5'-terminal, with the RNA (+) as a template to elongate the primer by the RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(c) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained,
(d) hybridizing a second primer for amplifying a nucleic acid of cytomegalovirus (CMV) which second primer contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1, with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA-dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of the 5'-terminal is produced,
(e) transcribing a single-stranded RNA (-) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (d),
(f) hybridizing the second primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single stranded RNA (-) obtained in the above step (e) as a template to perform a DNA elongation reaction by a RNA-dependent DNA polymerase, whereupon an elongated RNA/DNA hybrid is obtained,
(g) digesting the RNA in the elongated RNA/DNA hybrid by a ribonuclease H which specially digests only the RNA of the elongated RNA/DNA hybrid, whereupon a single-stranded DNA is obtained,
(h) hybridizing the first primer for amplifying a nucleic acid of cytomegalovirus (CMV) with the single-stranded DNA as a template to perform a DNA elongation reaction by a DNA- dependent DNA polymerase, whereupon a double-stranded DNA intermediate having a functional promoter sequence upstream of both terminals is produced,
(i) transcribing the single-stranded RNA(-) and RNA(+) increasingly from the double-stranded DNA intermediate by the DNA-dependent RNA polymerase which is capable of recognizing the functional promoter sequence of step (h), and
(j) optionally, repeating steps (f) to (i) using the resulting single stranded RNA(-) and RNA(+) as a template in optionally repeated step (f),
(k) hybridizing the resulting nucleic acids with
a capturing probe absorbed to or bound with a solid support which probe contains a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6, and/or SEQ ID NO:7, and which optionally may be modified and
a detecting probe containing a nucleic acid sequence comprising at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6, and/or SEQ ID NO:7, and which is modified by a label,
(l) separating any unreacted detecting probe and
(m) measuring the label of the optionally labeled capturing probe, the amplified nucleic acids and the detecting probe.

12 more elements

Oligonucleotides, methods and kits for amplifying and detecting a nucleic acid of cytomegalovirus (CMV) using nucleic acid sequence .beta.2.7

Life of this patent

Oligonucleotides, methods and kits for amplifying and detecting a nucleic acid of cytomegalovirus (CMV) using nucleic acid sequence .beta.2.7

Life of this patent

Abstract

Claims32