# US 11,306,366 B2 · Methods of detecting influenza

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- Number: US 11,306,366 B2
- Document ID: US11306366B2
- Status: Granted
- Kind: B2
- Application number: 16/595,595
- Filed: 2019-10-08
- Published: 2020-04-02
- Granted: 2022-04-19
- Earliest claimed: 2019-10-08
- Assignees: Cepheid
- Applicants: Cepheid
- Inventors: Anupama Mokkapati; Bradley Brown; Robert Jones
- Web page: https://patentgrep.com/patent/US11306366B2

## CPC

- C12Q 1/701: {Specific hybridization probes}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Compositions and methods for detecting influenza are provided.

## Claims

### Claim 1

A method of detecting the presence of influenza in a sample from a subject comprising (A) obtaining a sample from the subject; and (B) amplifying nucleic acids from the sample with primer pairs for detecting the presence of influenza A polymerase acidic (PA), influenza A polymerase basic 2 (PB2), influenza A matrix protein (MP), and avian influenza MP gene in the sample, wherein
(a) the first primer pair for detecting influenza A PA comprises a first and second primer, wherein the first primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 20 or SEQ ID NO: 2; and the second primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 21 or 90% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 2;
(b) the second primer pair for detecting influenza A PB2 comprises a third and fourth primer, wherein the third primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 17 or SEQ ID NO: 1, and the fourth primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 18 or 90% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 1;
(c) the third primer pair for detecting influenza A MP comprises a fifth and sixth primer, wherein the fifth primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 23 or SEQ ID NO: 3, and the sixth primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 24 or 90% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 3; and
(d) the fourth primer pair for detecting avian influenza MP comprises a seventh and eighth primer, wherein the seventh primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 26 or SEQ ID NO: 4, and the eighth primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 27 or 90% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 4; and
wherein detection of the presence of at least one influenza gene indicates the presence of influenza in the sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the method further comprises detecting the presence of at least one influenza nonstructural (NS) gene, wherein the NS gene is an influenza B NS gene.

### Claim 3 (depends on 1)

The method of claim 1, wherein the method further comprises detecting the presence of at least one influenza hemagglutinin (HA) gene, wherein the HA gene is an influenza A HA gene and/or an avian influenza HA gene.

### Claim 4 (depends on 3)

The method of claim 3, wherein the method comprises detecting the presence of an influenza A PA gene, an influenza A PB2 gene, an influenza A MP gene, an avian influenza MP gene, and an avian influenza HA gene, wherein the sequence of the influenza A PA gene is at least 95% identical to SEQ ID NO: 2, the sequence of the influenza A PB2 gene is at least 95% identical to SEQ ID NO: 1, the sequence of the influenza A MP gene is at least 95% identical to SEQ ID NO: 3, the sequence of the avian influenza MP gene is at least 95% identical to SEQ ID NO: 4, and the sequence of the avian influenza HA gene is at least 95% identical to SEQ ID NO: 5.

### Claim 5 (depends on 1)

The method of claim 1, wherein the method further comprises detecting the presence of an influenza B MP gene and an influenza B NS gene.

### Claim 6 (depends on 1)

The method of claim 1, wherein the method further comprises detecting the presence of respiratory syncytial virus (RSV) A and/or RSV B in a sample from the subject.

### Claim 7 (depends on 1)

The method of claim 1, wherein the method comprises detecting an exogenous control and/or an endogenous control.

### Claim 8 (depends on 1)

The method of claim 1, wherein the method comprises PCR and the PCR reaction takes less than 2 hours from an initial denaturation step through a final extension step.

### Claim 9 (depends on 1)

The method of claim 1, wherein
(a) the first primer pair comprises a first and second primer, wherein the first primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 20; and the second primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 21;
(b) the second primer pair comprises a third and fourth primer, wherein the third primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 17, and the fourth primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 18;
(c) the third primer pair comprises a fifth and sixth primer, wherein the fifth primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 23, and the sixth primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 24; and
(d) the fourth primer pair comprises a seventh and eighth primer, wherein the seventh primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 26, and the eighth primer comprises a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 27.

### Claim 10 (depends on 9)

The method of claim 9, wherein the first primer comprises the sequence of SEQ ID NO: 20, the second primer comprises the sequence of SEQ ID NO: 21, the third primer comprises the sequence of SEQ ID NO: 17, and the fourth primer comprises the sequence of SEQ ID NO: 18.

### Claim 11 (depends on 9)

The method of claim 9 wherein the method comprises amplifying nucleic acids from the sample with at least one additional primer pair for detecting an avian influenza HA gene, wherein the additional primer pair comprises a ninth and tenth primer independently selected from:
(a) a ninth primer comprising a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 29, and a tenth primer comprising a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 30; or
(b) a ninth primer comprising a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 5, and a tenth primer comprising a sequence that is at least 90% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 5.

### Claim 12 (depends on 11)

The method of claim 11, wherein the method comprises amplifying nucleic acids from the sample with at least one additional primer pair, wherein each of the additional primer pairs is for detecting a different influenza gene selected from an influenza B MP gene and an influenza B NS gene, wherein the additional primer pair comprises an eleventh and twelfth primer independently selected from:
(a) an eleventh primer comprising a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 6, and twelfth primer comprising a sequence that is at least 90% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 6;
(b) an eleventh primer comprising a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 7, and twelfth primer comprising a sequence that is at least 90% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 7;
(c) an eleventh primer comprising a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 32, and twelfth primer comprising a sequence that is at least 90% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 33; and
(d) an eleventh primer comprising a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 35, and twelfth primer comprising a sequence that is at least 90% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 36.

### Claim 13 (depends on 12)

The method of claim 12, wherein the method comprises amplifying nucleic acids from the sample with at least one additional primer pair, wherein each of the additional primer pairs is for detecting RSV A or RSV B, wherein the additional primer pair comprises a thirteenth and fourteenth primer independently selected from:
(a) a thirteenth primer comprising a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 38, and a fourteenth primer comprising a sequence that is at least 90% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 39; and
(b) a thirteenth primer comprising a sequence that is at least 90% identical to at least 15 contiguous nucleotides of SEQ ID NO: 41, and a fourteenth primer comprising a sequence that is at least 90% complementary to at least 15 contiguous nucleotides of SEQ ID NO: 42.

### Claim 14 (depends on 1)

The method of claim 1, wherein the method comprises forming an influenza A PA amplicon, an influenza A PB2 amplicon, an influenza A MP amplicon, and an avian influenza MP amplicon.

### Claim 15 (depends on 13)

The method of claim 13, wherein the method comprises forming (i) an influenza A PA amplicon, an influenza A PB2 amplicon, an influenza A MP amplicon, an avian influenza MP amplicon, and an avian influenza HA amplicon, (ii) an influenza B MP amplicon and/or an influenza B NS amplicon, and (iii) a RSV A amplicon and/or a RSV B amplicon.

### Claim 16 (depends on 14)

The method of claim 14, wherein the method comprises contacting the amplicons with an influenza A PA probe, an influenza A PB2 probe, an influenza A MP probe, and an avian influenza MP probe.

### Claim 17 (depends on 16)

The method of claim 16, wherein the influenza A PA probe comprises a sequence that is at least 90% identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 22, the influenza A PB2 probe comprises a sequence that is at least 90% identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 19, the influenza A MP probe comprises a sequence that is at least 90% identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 25, and the avian influenza MP probe comprises a sequence that is at least 90% identical or complementary to at least 15 contiguous nucleotides of SEQ ID NO: 28.

### Claim 18 (depends on 15)

The method of claim 15, wherein the method comprises contacting the amplicons with (i) an influenza A PA probe, an influenza A PB2 probe, an influenza A MP probe, an avian influenza MP probe, and an avian influenza HA probe, (ii) an influenza B MP probe and/or an influenza B NS probe, and (iii) a RSV A probe and/or a RSV B probe.

### Claim 19 (depends on 7)

The method of claim 7, wherein the method comprises detecting an exogenous control, and wherein the exogenous control is a sample processing control.

### Claim 20 (depends on 19)

The method of claim 19, wherein the exogenous control comprises RNA packaged in a bacteriophage protective coat.