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US 2020/0048721 A1Published application
Compositions and methods for detection of viral pathogens in samples
AssigneeGen-Probe Incorporated
InventorsMatthias JOST, Pamela DOUGLASS, Daniel P. KOLK, Mehrdad R. MAJLESSI
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Abstract
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.
Claims41
A kit for analysis of one or more of a plurality of pathogen-associated 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 that is substantially complementary to a first Flu A target nucleic acid sequence, is about 18 to about 100 contiguous bases in length and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:23;
- and
- (ii) the second Flu A primer that is substantially complementary to a second Flu A target nucleic acid, is about 18 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NO:25 and SEQ ID NO:28;
- and/or
- (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 that is substantially complementary to a third Flu A target nucleic acid sequence, is about 19 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS;
- 2 to 5 and 24;
- and
- (ii) the fourth Flu A primer that is substantially complementary to a fourth Flu A target nucleic acid sequence, is about 20 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:26 & 27;
- and/or
- (c) 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 that is substantially complementary to a first Flu B target nucleic acid sequence, is about 22 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting SEQ ID NOS;
- 29 & 67;
- and
- (ii) the second Flu B primer that is substantially complementary to a second Flu B target nucleic acid sequence, is about 21 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:68 to 70;
- and/or
- (d) 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 (RSV A) primer that is substantially complementary to a first RSV A target nucleic acid sequence, is about 26 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:79 & 88;
- and
- (ii) the second RSV A primer that is substantially complementary to a second RSV A target nucleic acid sequence, is about 22 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:72 to 74 & 92 to 96;
- and/or
- (e) 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 (RSV B) primer is substantially complementary to a first RSV B target nucleic acid sequence, is about 17 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:99 to 101 and 106;
- and
- (ii) the second RSV B primer is substantially complementary to a second RSV B target nucleic acid sequence, is about 17 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:104 to 106 &115.
23 more elements
The kit of any one of the preceding claims, wherein at least one primer is present in the kit as a lyophilized reagent.
The kit of any one of the preceding claims, wherein at least one probe is present in the kit as a lyophilized reagent.
A method of determining whether a biological sample contains Influenza A (Flu A), Influenza B (Flu B), Respiratory Syncytial Virus A (RSV A), and/or Respiratory Syncytial Virus B (RSV B), comprising:
- (a) contacting under stringent hybridization conditions target nucleic acid molecules from the biological sample with one or more distinguishably labeled probe molecule species to form probe:target duplexes, wherein:
- (i) to determine if a the biological sample contains Flu A, using a distinguishably labeled first Flu A probe molecule species and/or a distinguishably labeled second Flu A probe molecule species, wherein (A) the distinguishably labeled first Flu A probe molecule species is substantially complementary to a first Flu A probe target nucleic acid sequence, is about about 17 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:6 to 17, and (B) the distinguishably labeled second Flu A probe molecule species is substantially complementary to a second Flu A probe target nucleic acid sequence, is about 17 to about 100 contiguous bases in length and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:18 to 22;
- and/or
- (ii) to determine if a the biological sample contains Flu B, using a distinguishably labeled Flu B probe molecule species that is substantially complementary to a Flu B probe target nucleic acid sequence, is about 17 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:30 to 66;
- and/or
- (iii) to determine if a the biological sample contains RSV A, using a distinguishably labeled RSV A probe molecule species that is substantially complementary to an RSV A probe target nucleic acid sequence, is about 17 to about 100 contiguous bases in length and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:71, 75 to 78, 80 to 87, 89 to 91, 97, & 98;
- and/or
- (iv) to determine if a the biological sample contains RSV B, using a distinguishably labeled RSV B probe molecule species is substantially complementary to an RSV probe target nucleic acid sequence, is about 17 to about 100 contiguous bases in length and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:102, 103, & 107 to 114;
- and
- (b) detecting, if stable, distinguishably labeled probe:target duplexes are formed in step (a);
- and, if so,
- (c) based on whether the first, second, third, fourth, and/or fifth distinguishable labels are detected, determining that the biological sample contains Flu A, Flu B, RSV A, and/or RSV B.
9 more elements
A composition comprising one more nucleic acid molecules, each independently selected from the group consisting of:
- (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 that is substantially complementary to a first Flu A target nucleic acid sequence, is about 18 to about 100 contiguous bases in length and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:23;
- and
- (ii) the second Flu A primer that is substantially complementary to a second Flu A target nucleic acid, is about 18 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NO:25 and SEQ ID NO:28;
- and/or
- (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 that is substantially complementary to a third Flu A target nucleic acid sequence, is about 19 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS;
- 2 to 5 and 24;
- and
- (ii) the fourth Flu A primer that is substantially complementary to a fourth Flu A target nucleic acid sequence, is about 20 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:26 & 27;
- and/or
- (c) 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 that is substantially complementary to a first Flu B target nucleic acid sequence, is about 22 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting SEQ ID NOS;
- 29 & 67;
- and
- (ii) the second Flu B primer that is substantially complementary to a second Flu B target nucleic acid sequence, is about 21 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:68 to 70;
- and/or
- (d) 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 (RSV A) primer that is substantially complementary to a first RSV A target nucleic acid sequence, is about 26 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:79 & 88;
- and
- (ii) the second RSV A primer that is substantially complementary to a second RSV A target nucleic acid sequence, is about 22 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:72 to 74 & 92 to 96;
- and/or
- (e) 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 (RSV B) primer is substantially complementary to a first RSV B target nucleic acid sequence, is about 17 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:99 to 101 and 106;
- and
- (ii) the second RSV B primer is substantially complementary to a second RSV B target nucleic acid sequence, is about 17 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:104 to 106 &115;
- (f) a probe molecule species that is substantially complementary to a first Flu A probe target nucleic acid sequence, is about 17 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:6 to 17;
- and/or
- (g) a probe molecule species that is substantially complementary to a second Flu A probe target nucleic acid sequence, is about 17 to about 100 contiguous bases in length and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:18 to 22;
- and/or
- (h) a probe molecule species that is substantially complementary to a Flu B probe target nucleic acid sequence, is about 17 to about 100 contiguous bases in length, and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:30 to 66;
- and/or
- (i) a probe molecule species that is substantially complementary to an RSV A probe target nucleic acid sequence, is about 17 to about 100 contiguous bases in length and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:71, 75 to 78, 80 to 87, 89 to 91, 97, & 98;
- and/or
- (j) a probe molecule species that is substantially complementary to an RSV probe target nucleic acid sequence, is about 17 to about 100 contiguous bases in length and comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOS:102, 103, & 107 to 114;
- (k) an Influenza A (Flu A) amplicon, which amplicon is generated by a nucleic acid amplification process that utilizes a Flu A primer pair according to part (a);
- (l) an Influenza A (Flu A) amplicon, which amplicon is generated by a nucleic acid amplification process that utilizes a Flu A primer pair according to part (b);
- (m) an Influenza B (Flu B) amplicon, which amplicon is generated by a nucleic acid amplification process that utilizes a Flu B primer pair according to part (c);
- (n) a Respiratory Syncytial Virus A (RSV A) amplicon, which amplicon is generated by a nucleic acid amplification process that utilizes a RSV A primer pair according to part (d);
- and
- (o) a Respiratory Syncytial Virus B (RSV B) amplicon, which amplicon is generated by a nucleic acid amplification process that utilizes a RSV B primer pair according to part (e).
38 more elements
Dependencies are inferred from the claim text.