Compositions and methods for detection of viral pathogens in samples
This disclosure concerns amplification primers, hybridization assay probes, compositions containing such primers and probes, and associated reagents, kits, and methods, that can be used to analyze samples for the presence of Influenza A virus, Influenza B virus, Respiratory Syncytial Virus A, and/or Respiratory Syncytial Virus B target nucleic acids.
1 . A kit for analysis of one or more Influenza A (Flu A) target nucleic acid molecule species that may be present in a biological sample, comprising:
(a) a first Flu A primer pair for generating a first Flu A amplicon if Flu A is present in the biological sample, the first Flu A primer pair comprising a first Flu A primer and a second Flu A primer, wherein:
(i) the first Flu A primer comprises a target hybridizing sequence consisting of the nucleic acid sequence of SEQ ID NO:23; and
(ii) the second Flu A primer comprises a target hybridizing sequence consisting of the nucleic acid sequence of SEQ ID NO:25;
(b) a second Flu A primer pair for generating a second Flu A amplicon if Flu A is present in the biological sample, the second Flu A primer pair comprising a third Flu A primer and a fourth Flu A primer, wherein:
(i) the third Flu A primer comprises a target hybridizing sequence consisting of the nucleic acid sequence of SEQ ID NO:24; and
(ii) the fourth Flu A primer comprises a target hybridizing sequence consisting of the nucleic acid sequence of SEQ ID NOs: 26 or 27;
(c) a first Flu A probe molecule species that is substantially complementary to a sequence in the first Flu A amplicon and comprises an oligonucleotide sequence comprising the nucleotide sequence of SEQ ID NO:7 or 12, wherein the first Flu A probe molecule species is linked to a fluorescent label; and
(d) a second Flu A probe molecule species that is substantially complementary to a sequence in the second Flu A amplicon and comprises an oligonucleotide sequence comprising the nucleotide sequence of SEQ ID NO:18, wherein the first Flu A probe molecule species is linked to a fluorescent label.
2 . The kit of claim 1 , wherein one or more of the Flu A primers comprises a primer upstream region having a nucleotide sequence that is not complementary to a sequence present in the Flu A target nucleotide sequence.
3 . The kit according to claim 1 , further comprising one or more of:
(a) a Flu B primer pair for generating a Flu B amplicon if Flu B is present in the biological sample, the Flu B primer pair comprising a first Flu B primer and a second Flu B primer, wherein:
(i) the first Flu B primer comprises a target hybridizing sequence consisting of the nucleic acid sequence of SEQ ID NO:29 or 67; and
(ii) the second Flu B primer comprises a target hybridizing sequence consisting of the nucleic acid sequence of SEQ ID NO:68 or 70;
(b) an RSV A primer pair for generating an RSV A amplicon if RSV A is present in the biological sample, the RSV A primer pair comprising a first RSV A primer and a second RSV A primer, wherein:
(i) the first RSV A primer comprises a target hybridizing sequence consisting of a nucleic acid sequence of SEQ ID NO:79 or 88; and
(ii) the second RSV A primer comprises a target hybridizing sequence consisting of a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 72-74 and 92-96; and
(c) an RSV B primer pair for generating an RSV B amplicon if RSV B is present in the biological sample, the RSV B primer pair comprising a first RSV B primer and a second RSV B primer, wherein:
(i) the first RSV B primer comprises a target hybridizing sequence consisting of the nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 99 to 101 and 106; and
(ii) the second RSV B primer comprises a target hybridizing sequence consisting of nucleic acid sequence of selected from the group consisting of: SEQ ID NOs: 104-106 and 115.
4 . The kit of claim 3 , wherein the kit contains
(a) the Flu B primer pair;
(b) the RSV A primer pair;
(c) the RSV B primer pair;
(d) the Flu B primer pair and the RSV A primer pair;
(e) the Flu B primer pair and the RSV B primer pair;
(g) the RSV A primer pair and the RSV B primer pair; or
(h) the Flu B primer pair, the RSV A primer pair, and the RSV B primer pair.
5 . The kit of claim 4 , wherein:
(c) if the kit contains the Flu B primer pair, then the kit further comprises a Flu B probe molecule species that is substantially complementary to a sequence in the Flu B amplicon, is about 17 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence of any one of SEQ ID NOs: 30-66;
(d) if the kit contains the RSV A primer pair, then the kit further comprises a RSV A probe molecule species that is substantially complementary to a sequence in the RSV A amplicon, is about 17 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence of any one of SEQ ID NOs: 71, 75-78, 80-87, 89-91, and 97-98; and
(e) if the kit contains the RSV B primer pair, then the kit further comprises a RSV B probe molecule species that is substantially complementary to a sequence in the RSV B amplicon, is about 17 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence of any one of SEQ ID NOs: 102, 103 and 107-114.
6 . The kit of claim 4 , wherein one or more of the primers comprises a primer upstream region having a nucleotide sequence that is not complementary to the primer's target nucleotide sequence.
7 . The kit of claim 1 , wherein the kit further comprises reagents to perform at least one nucleic acid amplification reaction.
8 . The kit of claim 7 , wherein the kit further comprises instructions for using the primer pair species and the reagents to perform the at least one nucleic acid amplification.
9 . The kit of claim 1 , wherein at least one primer or at least one probe molecule species is present in the kit as a lyophilized reagent.
10 . The kit of claim 7 , wherein the kit comprises one or more of: a reverse transcriptase, a DNA polymerase, a buffer, and dNTPs.
11 . The kit of claim 10 , wherein the kit comprises a reverse transcriptase, a DNA polymerase, a buffer, and dNTPs.
12 . The kit of claim 11 , wherein the reverse transcriptase, the DNA polymerase, the buffer, and the dNTPs are present in the kit as a lyophilized reagent.
13 . The kit of claim 12 , further comprising a rehydration reagent comprising a salt.
14 . The kit of claim 13 , wherein the salt is a magnesium salt.
15 . The kit of claim 1 , wherein the first Flu A primer comprises a nucleic acid sequence consisting of the nucleic acid sequence of SEQ ID NO:23, the second Flu A primer comprises a nucleic acid sequence consisting of the nucleic acid sequence of SEQ ID NO: 25, the third Flu A primer comprises a nucleic acid sequence consisting of the nucleic acid sequence of SEQ ID NO:24, and the fourth Flu A primer comprises a nucleic acid sequence consisting of the nucleic acid sequence of SEQ ID NO:26 or 27.
16 . The kit of claim 5 , wherein each probe molecule species is labeled with one or more detectable labels selected from the group consisting of: a chemiluminescent moiety, a fluorophore moiety, a quencher moiety, and both a fluorophore moiety and a quencher moiety.
17 . The kit of claim 16 , wherein at least one of the probe molecule species is distinguishably labeled such that the at least one probe molecule species can be distinguished from the other probe molecule species.
18 . The kit of claim 1 , wherein the one or more of the probe molecule species is detectably labeled with a donor/acceptor label pair.
19 . The kit of claim 1 , wherein at least one primer and/or at least one probe molecule species contains one or more methylated cytosine bases.
20 . A method for determining the presence or absence of a Flu A target nucleic acid, and one or more of a Flu B target nucleic acid, an RSV A target nucleic acid, and an RSV B target nucleic acid in a biological sample, the method comprising the steps of:
(a) providing the kit of claim 5 and contacting the biological sample with the first Flu A primer pair, second Flu A primer pair, the first Flu A probe molecule species, the second Flu A probe molecule species, the one of more of the Flu B primer pair, the RSV A primer pair, and the RSV B primer pair, and the Flu B probe molecule species, the RSV A probe molecule species, and/or the RSV B probe molecule species of claim 5 ;
(b) performing an in vitro nucleic acid amplification reaction wherein:
(i) the Flu A target nucleic acid, if present in the biological sample, is used by the first Flu A primer pair to generate the first Flu A amplicon and/or the second Flu A primer pair to generate the second Flu A amplicon;
(ii) the Flu B target nucleic acid, if present in the biological sample, is used by the Flu B primer pair to generate the Flu B amplicon;
(iii) the RSV A target nucleic acid, if present in the biological sample, is used by the RSV A primer pair to generate the RSV A amplicon; and
(iv) the RSV B target nucleic acid, if present in the biological sample, is used by the RSV B primer pair to generate the RSV B amplicon; and
(c) detecting the presence of absence of the first Flu A amplicon, the second Flu A amplicon, the Flu B amplicon, the RSV A amplicon, and/or the RSV B amplicon, wherein detecting the presence of an amplicon indicates the presence of the corresponding target nucleic acid in the biological sample.
21 . The method of claim 20 , wherein the detecting is performed in real time.
22 . A reaction mixture comprising a first Flu A primer pair, a second Flu A primer pair, a first Flu A probe molecule species and a second Flu A probe molecule species wherein:
(a) the first Flu A primer pair generates a first Flu A amplicon if Flu A is present in a biological sample, the first Flu A primer pair comprises:
(i) a first Flu A primer comprising a target hybridizing sequence consisting of the nucleic acid sequence of SEQ ID NO:23; and
(ii) a second Flu A primer comprising a target hybridizing sequence consisting of the nucleic acid sequence of SEQ ID NO:25;
(b) the second Flu A primer pair generates a second Flu A amplicon if Flu A is present in the biological sample, the second Flu A primer pair comprises:
(i) a third Flu A primer comprising a target hybridizing sequence consisting of the nucleic acid sequence of SEQ ID NO:24; and
(ii) a fourth Flu A primer comprising a target hybridizing sequence consisting of the nucleic acid sequence of SEQ ID NOs: 26 or 27;
(c) a first Flu A probe molecule species that is substantially complementary to a sequence in the first Flu A amplicon and comprises an oligonucleotide sequence comprising the nucleotide sequence of SEQ ID NO:7 or 12, wherein the first Flu A probe molecule species is linked to a fluorescent label; and
(d) a second Flu A probe molecule species that is substantially complementary to a sequence in the second Flu A amplicon and comprises an oligonucleotide sequence comprising the nucleotide sequence of SEQ ID NO:18, wherein the first Flu A probe molecule species is linked to a fluorescent label.
23 . The kit of claim 6 , wherein the first Flu A probe molecule species comprises a first probe comprising an oligonucleotide sequence comprising the nucleotide sequence of SEQ ID NO:7 and a second probe comprising an oligonucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 12.
24 . The reaction mixture of claim 22 , wherein the first Flu A probe molecule species comprises a first probe comprising an oligonucleotide sequence comprising the nucleotide sequence of SEQ ID NO:7 and a second probe comprising an oligonucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 12.