# US 8,785,133 B2 · Oligonucleotides and process for detection of swine flu virus

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- Number: US 8,785,133 B2
- Document ID: US8785133B2
- Status: Granted
- Kind: B2
- Application number: 13/509,535
- Filed: 2010-11-11
- Published: 2012-09-13
- Granted: 2014-07-22
- Earliest claimed: 2009-11-12
- Assignees: Defence Research & Development Organisation
- Applicants: Manmohan Parida; Jyoti Shukla; Santhosh Sannarangiah; Sashi Sharma; Venkata Lakshmana Putcha Rao; Rajagopalan Vijayaraghavan
- Inventors: Manmohan Parida; Jyoti Shukla; Santhosh Sannarangiah; Sashi Sharma; Venkata Lakshmana Putcha Rao; Rajagopalan Vijayaraghavan
- Web page: https://patentgrep.com/patent/US8785133B2

## CPC

- C12Q 1/70: involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)}
- 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

A process of detection of pandemic swine flu virus H1N1 type in a sample is provided herein. Also provided are highly specific oligonucleotides useful for rapid detection of swine flu virus in a sample, as well as swine flu virus specific isothermal gene amplification assay for early clinical diagnosis of H1N1 human patients.

## Claims

### Claim 1

A process for detecting pandemic Swine flu virus H1N1 type in a sample, said process comprises providing the sample, mixing the sample with the oligonucleotides as set forth in SEQ ID NO: 1 to SEQ ID NO: 8 and buffer comprising reagents used for RNA amplification to obtain a reaction mixture, incubating said reaction mixture at a temperature of about 60° C. to about 65° C. for a period of about 15 minutes to about 45 minutes and detecting amplified product, wherein presence of the amplified products confirms presence of the virus.

### Claim 2

A process of detecting pandemic Swine Flu Virus H1N1 type in a sample, wherein said process comprises providing the sample, obtaining cDNA from the sample using oligonucleotides as set forth in SEQ ID NO: 1 and SEQ ID NO: 2, mixing the cDNA obtained with the oligonucleotides as set forth in SEQ ID NO: 3 to SEQ ID NO: 8 and buffer comprising reagents used for DNA amplification to obtain a reaction mixture, incubating said reaction mixture at a temperature of about 60° C. to about 65° C. for a period of about 15 minutes to about 45 minutes and detecting amplified product, wherein presence of the amplified products confirms presence of the virus.

### Claim 3 (depends on 1, 2)

The process as claimed in claim 1 or 2, wherein the temperature is about 63° C.

### Claim 4 (depends on 1, 2)

The process as claimed in claim 1 or 2, wherein the period is about 30 minutes.

### Claim 5 (depends on 1, 2)

The process as claimed in claim 1 or 2, wherein the amplified product is detected by checking turbidity of the sample, wherein formation of turbidity indicates the presence of Swine Flu Virus H1N1 type DNA in the sample.

### Claim 6 (depends on 1, 2)

The process as claimed in claim 1 or 2, wherein the amplified product is detected by a SYBR Green I dye assay.

### Claim 7

A primer set for detection of pandemic Swine Flu Virus H1N1 type in a sample comprising:
(a) a forward outer primer (F3) comprising the oligonucleotide of SEQ ID NO:1 and
(b) a backward outer primer (B3) comprising the oligonucleotide of SEQ ID NO:2a forward inner primer (FIP) comprising the oligonucleotide of SEQ ID NO:3, a linker and the oligonucleotide of SEQ ID NO:4;
(c) a backward inner primer (BIP) comprising the oligonucleotide of SEQ ID NO:5, a linker and the oligonucleotide of SEQ ID NO:6;
(d) a forward loop primer (FLP) comprising the oligonucleotide of SEQ ID NO:5; and
(e) a backward loop primer (BLP) comprising the oligonucleotide of SEQ ID NO: 8.

### Claim 8 (depends on 7)

The primer set as claimed in claim 7, wherein the linker is a TTTT tetra linker.