# US 12,018,334 B2 · Controls for nucleic acid assays

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- Number: US 12,018,334 B2
- Document ID: US12018334B2
- Status: Granted
- Kind: B2
- Application number: 17/411,606
- Filed: 2021-08-25
- Published: 2021-12-16
- Granted: 2024-06-25
- Earliest claimed: 2012-08-30
- Assignees: Exosome Diagnostics, Inc.
- Applicants: Exosome Diagnostics, Inc.
- Inventors: Mikkel Noerholm; Susan Belzer; Charlotte Romain; Johan Karl Olov Skog; Leileata M. Russo; Wayne Comper
- Web page: https://patentgrep.com/patent/US12018334B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 2795/18122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2795/18131: Uses of virus other than therapeutic or vaccine, e.g. disinfectant
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/118: Prognosis of disease development
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention features a method and kit for isolating microvesicles or extracting microvesicle nucleic acids from a biological sample by using a control particle. The present invention provides control particles that are viruses or virus-like particles, such as bacteriophages, that contain control nucleic acids that can be detected to assess the accuracy, reliability, and efficiency of the microvesicle isolation or nucleic acid extraction steps. The methods described herein may further comprise the analysis of the presence, absence, or level of at least one biomarker associated with a disease or medical condition for diagnosing, prognosing, or monitoring the disease or medical condition.

## Claims

### Claim 1

A method comprising:
(a) forming a mixture by adding a known quantity of control particles to a biological sample comprising microvesicles, wherein the control particles are Q-beta bacteriophage particles, wherein the Q-beta bacteriophage particles comprise a known copy number of a control nucleic acid, and wherein the microvesicles comprise nucleic acids;
(b) isolating a fraction comprising the microvesicles and the control particles from the mixture;
(c) extracting nucleic acids from the fraction comprising the microvesicles and the control particles, thereby producing extracted nucleic acids;
(d) measuring the amount of the control nucleic acid in the extracted nucleic acids of step (c) by a quantitative polymerase chain reaction (PCR) using specific primers that hybridize to the control nucleic acid;
(e) determining the copy number of the control nucleic acid in the extracted nucleic acids of step (c) by comparing the amount of the control nucleic acid measured in step (d) to a standard curve generated using known different concentrations of the control nucleic acid and known different copy numbers of the control nucleic acid;
(f) determining the amount of the control particles that are present in the fraction from step (b) based on the copy number of the control nucleic acid in the extracted nucleic acids determined in step (e); and
(g) subjecting the extracted nucleic acids from step (c) to further analysis if the amount of the control particles determined in step (f) is within a predetermined range of values.

### Claim 2 (depends on 1)

The method of claim 1, wherein the biological sample is a bodily fluid sample.

### Claim 3 (depends on 2)

The method of claim 2 wherein the bodily fluid sample is urine sample, blood sample, cerebrospinal fluid sample, plasma sample or serum sample.

### Claim 4 (depends on 3)

The method of claim 3, wherein the bodily fluid sample is a urine sample.

### Claim 5 (depends on 3)

The method of claim 3, wherein the bodily fluid sample is a blood sample.

### Claim 6 (depends on 3)

The method of claim 3, wherein the bodily fluid sample is a plasma sample.

### Claim 7 (depends on 1)

The method of claim 1, wherein the control nucleic acid comprises RNA or DNA.

### Claim 8 (depends on 1)

The method of claim 1, wherein the control nucleic acid comprises a Q-beta coat protein gene.

### Claim 9 (depends on 1)

The method of claim 1, wherein the fraction comprising the microvesicles and the control particles is isolated by differential centrifugation, gel permeation chromatography, anion exchange chromatography, a sucrose density gradient, organelle electrophoresis or any combination thereof.

### Claim 10 (depends on 1)

The method of claim 1, wherein the control nucleic acid is RNA and the quantitative PCR is a reverse transcription quantitative PCR.