Universal multi-variant detection system
First Claim
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1. A method for determining the presence of a viral target nucleic acid molecule in a sample containing a virus, the method comprising:
- (a) hybridizing a reverse primer to the viral target nucleic acid molecule under conditions suitable for carrying out a primer extension chain reaction;
(b) extending the reverse primer using the viral target nucleic acid molecule as a template to form a reverse primer extension product, wherein the reverse primer joined to the reverse primer extension product constitutes a reverse primer amplification product;
(c) denaturing the reverse primer amplification product from its template;
(d) hybridizing a forward primer to;
(i) a nucleic acid molecule which is complementary to the viral target nucleic acid molecule, if present;
or(ii) the reverse primer amplification product;
(e) extending the forward primer using the complementary target nucleic acid molecule, if present, or the reverse primer amplification product, as a template, wherein the forward primer joined to the forward primer extension product constitutes a forward primer amplification product;
(f) denaturing the forward primer amplification product from its template;
(g) hybridizing the reverse primer to the forward primer amplification product;
(h) extending the reverse primer using the forward primer amplification product as a template to form an additional reverse primer extension product, wherein the reverse primer joined to the additional reverse primer extension product constitutes an additional reverse primer amplification product;
(i) denaturing the additional reverse primer amplification product from its template;
(j) hybridizing the forward primer to the reverse primer amplification product;
(k) extending the forward primer using the reverse primer amplification product as a template to form an additional forward primer extension product, wherein the forward primer joined to the additional forward primer extension product constitutes an additional forward primer amplification product;
(l) denaturing the additional forward primer amplification product from its template;
(m) repeating steps (g) through (I), using the additional reverse primer amplification product and the additional forward primer amplification product as templates for the forward primer and the reverse primer, respectively, a sufficient number of times to produce a detectable quantity of additional reverse primer amplification product or of additional forward primer amplification product; and
(n) detecting the presence of the additional reverse primer amplification product or the additional forward primer amplification product;
the improvement wherein the nucleotide at the 3′
end of the reverse primer hybridizes with;
(i) the nucleotide at the 5′
end of the forward primer extension product or of the additional forward primer extension product;
or(ii) a nucleotide separated from the nucleotide at the 5′
end of the forward primer extension product or of the additional forward primer extension product by a gap of two nucleotides, wherein the gap comprises a sequence known to be highly conserved; and
wherein the nucleotide at the 3′
end of the forward primer hybridizes with;
(i) the nucleotide at the 5′
end of the reverse primer extension product or of the additional reverse primer extension product;
or(ii) a nucleotide separated from the nucleotide at the 5′
end of the reverse primer extension product or of the additional reverse primer extension product by a gap of two nucleotides, wherein the gap comprises a sequence known to be highly conserved.
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Abstract
The present invention provides a method to diagnostically detect the variants of a given pathogen, such as HIV, hepatitis C, hepatitis B (HBV), Parvovirus B19, etc., with the use of a single detection probe.
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Citations
32 Claims
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1. A method for determining the presence of a viral target nucleic acid molecule in a sample containing a virus, the method comprising:
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(a) hybridizing a reverse primer to the viral target nucleic acid molecule under conditions suitable for carrying out a primer extension chain reaction; (b) extending the reverse primer using the viral target nucleic acid molecule as a template to form a reverse primer extension product, wherein the reverse primer joined to the reverse primer extension product constitutes a reverse primer amplification product; (c) denaturing the reverse primer amplification product from its template; (d) hybridizing a forward primer to; (i) a nucleic acid molecule which is complementary to the viral target nucleic acid molecule, if present;
or(ii) the reverse primer amplification product; (e) extending the forward primer using the complementary target nucleic acid molecule, if present, or the reverse primer amplification product, as a template, wherein the forward primer joined to the forward primer extension product constitutes a forward primer amplification product; (f) denaturing the forward primer amplification product from its template; (g) hybridizing the reverse primer to the forward primer amplification product; (h) extending the reverse primer using the forward primer amplification product as a template to form an additional reverse primer extension product, wherein the reverse primer joined to the additional reverse primer extension product constitutes an additional reverse primer amplification product; (i) denaturing the additional reverse primer amplification product from its template; (j) hybridizing the forward primer to the reverse primer amplification product; (k) extending the forward primer using the reverse primer amplification product as a template to form an additional forward primer extension product, wherein the forward primer joined to the additional forward primer extension product constitutes an additional forward primer amplification product; (l) denaturing the additional forward primer amplification product from its template; (m) repeating steps (g) through (I), using the additional reverse primer amplification product and the additional forward primer amplification product as templates for the forward primer and the reverse primer, respectively, a sufficient number of times to produce a detectable quantity of additional reverse primer amplification product or of additional forward primer amplification product; and (n) detecting the presence of the additional reverse primer amplification product or the additional forward primer amplification product; the improvement wherein the nucleotide at the 3′
end of the reverse primer hybridizes with;(i) the nucleotide at the 5′
end of the forward primer extension product or of the additional forward primer extension product;
or(ii) a nucleotide separated from the nucleotide at the 5′
end of the forward primer extension product or of the additional forward primer extension product by a gap of two nucleotides, wherein the gap comprises a sequence known to be highly conserved; andwherein the nucleotide at the 3′
end of the forward primer hybridizes with;(i) the nucleotide at the 5′
end of the reverse primer extension product or of the additional reverse primer extension product;
or(ii) a nucleotide separated from the nucleotide at the 5′
end of the reverse primer extension product or of the additional reverse primer extension product by a gap of two nucleotides, wherein the gap comprises a sequence known to be highly conserved. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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Specification