# US 9,518,901 B2 · Pretreatment method of biological sample, detection method of RNA, and pretreatment kit

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- Number: US 9,518,901 B2
- Document ID: US9518901B2
- Status: Granted
- Kind: B2
- Application number: 13/807,605
- Filed: 2012-06-28
- Published: 2013-11-14
- Granted: 2016-12-13
- Earliest claimed: 2011-06-29
- Assignees: Kabushiki Kaisha Dnaform
- Applicants: Yoshihide Hayashizaki; Kengo Usui; Saori Goda; Kazuhito Nomura; Yuki Kawai
- Inventors: Yoshihide Hayashizaki; Kengo Usui; Saori Goda; Kazuhito Nomura; Yuki Kawai
- Web page: https://patentgrep.com/patent/US9518901B2

## CPC

- G01N 1/34: Purifying; Cleaning {(processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003)}
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

The present invention is to provide a pretreatment method that allows RNA to be detected promptly and simply. RNA degradation activity due to lactoferrin present in the human rhinal mucosa is inhibited, for example, by adding iron ion and carbonate ion to a biological sample that contains the human rhinal mucosa. With the pretreated biological sample, an RNA virus gene can be amplified by a reverse transcriptase. Iron ion and carbonate ion can also inhibit reverse transcriptase inhibition due to lysozyme C contained in the human rhinal mucosa. Further, it is preferable to remove the envelope of the RNA virus by adding SDS to the biological sample that contains the human rhinal mucosa.

## Claims

### Claim 1

A method of detecting RNA in a sample collected from a living body, the sample containing lactoferrin, the method comprising:
applying an inhibition treatment of RNA degradation activity due to lactoferrin to the sample collected from the living body;
applying an inhibition treatment of reverse transcription inhibitory activity due to lysozyme C to the sample collected from the living body; and
an RNA detection step of detecting RNA in the treated sample,
wherein the inhibition treatment of RNA degradation activity due to lactoferrin is applied to the sample collected from the living body by adding an inhibitor of RNA degradation activity due to lactoferrin to the sample collected from the living body,
wherein the inhibitor is at least one selected from the group consisting of copper, nickel, iron, gold, scandium, yttrium, gadolinium, cerium, neodymium, and zinc,
wherein the inhibition treatment of reverse transcription inhibitory activity due to lysozyme C is applied to the sample collected from the living body by adding an inhibitor of reverse transcription inhibitory activity due to lysozyme C to the sample collected from the living body, and
wherein at least one of the RNA degradation activity inhibitor and the inhibitor of reverse transcription inhibitory activity contains an iron ion agent and a carbonate ion agent.

### Claim 2 (depends on 1)

The method according to claim 1,
wherein in the RNA detection step, a surfactant is added to the treated sample.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the surfactant is a nonpolar surfactant.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the sample collected from the living body includes at least one selected from the group consisting of rhinal mucosae, sinus mucosae, tracheal mucosae, saliva, secretions from the mouth or the throat, tears, milk, bile, blood (leukocyte), cervical mucosae, internal and external genitalia mucosae, amniotic fluids, and urine of a subject.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the subject is human.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the sample collected from the living body contains a human rhinal mucosa.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the sample collected from the living body to which the iron ion agent and the carbonate ion agent are added is applied with heat treatment.

### Claim 8 (depends on 1)

The method according to claim 1, wherein a polar surfactant is added to the sample collected from the living body.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the polar surfactant is SDS.