JPWO2019207308A5

JPWO2019207308A5 -

Info

Publication number: JPWO2019207308A5
Application number: JP2020559412A
Authority: JP
Publication date: 2022-04-28
Anticipated expiration: 2039-04-25

Claims

A method for preparing a sample for reverse transcription (RT), PCR, or RT-PCR, optionally for qPCR, and optionally for RT qPCR, wherein the method is closed. A method comprising centrifuging the sample in a container, wherein the container is the same container in which the PCR is subsequently performed.

Any part of the sample
a) before the PCR was performed and / or b) after the PCR was performed, it was not removed from the container.
Optionally, when the sample is added to the container, the material is
The method of claim 1 , wherein a) it is not removed from the container before PCT and / or b) during PCR and / or after c) PCR.

The sample is a crude sample and
Optionally, the crude sample
(A) A sample optionally taken from an organism, optionally a whole blood sample, feces, urine, CSF, or eluent from a cotton swab taken from a subject, optionally. Crude biological samples selectively taken from the eyes, ears, nose, or mouth, and / or
(B) The method of claim 1 or 2 , wherein the sample is optionally a crude environmental sample comprising a food sample, a cotton swab from the surface, and other samples not taken directly from the organism .

The sample is
The method according to any one of claims 1 to 3 , wherein a) a cotton swab and / or b) an eluate collected from washing the swab.

Optionally, the sample may contain one or more pathogens.
a) A group of viruses, bacteria, fungi, and / or b) Class 4 or 3 pathogens and / or pathogens.
c) Viral hemorrhagic fever selected from the group consisting of Ebola hemorrhagic fever, Lassa fever, Marburg virus disease, Rift Valley fever, Congo fever, and yellow fever, and / or d) From the group consisting of Japanese encephalitis, dengue fever, deer, and chikunnia. The method according to any one of claims 1 to 4 , which is a sample, which may contain a pathogen selected from pathogens, which causes the disease of choice.

The method according to any one of claims 1 to 5 , wherein the sample contains a particulate or cellular object.

The method according to any one of claims 1 to 6 , wherein the sample inhibits PCR, contains a substance, or releases a substance, and optionally, when heated, inhibits PCR, releases a substance.

The centrifugation was performed at a rate and duration such that the first fraction of the sample was pelleted while leaving the second fraction of the sample in the supernatant.
Optionally, if the sample is blood, the first fraction comprises red blood cells and the second fraction contains leukocytes and any virus or bacterium present in the sample. Item 8. The method according to any one of Items 1 to 7 .

The centrifugation is performed at less than 1000 g, optionally 200 g-1000 g, 300 g-900 g, 400 g-800 g, 500 g-700 g, optionally 600 g, and the centrifugation is less than 60 seconds, optionally. It is selectively executed for 5 to 60 seconds, and optionally 10 to 55, 15 to 50, 20 to 45, 25 to 40, and 30 to 35 seconds.
The method according to any one of claims 1 to 8 , wherein the centrifugation is optionally performed at 500 g for 30 seconds.

Claim 1 to claim 1, wherein the container contains a PCR reaction component, optionally contains any one or more polymerases and / or PCR primers, and optionally the reagent is lyophilized. The method according to any one of 9 .

The centrifugation
a) Before starting RT and PCR
b) During RT and / or PCR,
Optionally, the centrifuge
i) After the RT step (if present), but before PCR, or
ii) Performed after RT and 1-5 cycles of PCR,
c) The method according to any one of claims 1 to 10 , which is performed before the start of PCR and during PCR .

A method of performing RT, PCR, or RT-PCR, wherein the sample is prepared according to any one of claims 1-11 .

The excitation wavelength from 630 nm to which the PCR is qPCR or the RT-PCR is RT-qPCR and is optionally used to excite the phosphor associated with the qPCR. 12. The method of claim 12 , wherein the method is 645 nm, optionally 633 nm to 642 nm, and / or radiation is collected at a wavelength of 650 nm to 750 nm.

13 . the method of.

A closed tube method in which PCR is performed, wherein the sample is prepared according to any one of claims 1-11 , and optionally the presence of the PCR product removes any material from the container. A method that has been detected without.

The sample is at least 5%, optionally at least 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16% of the PCR reaction volume. , 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33. The method of any of claims 12-15 , comprising%, 34% or 35% or more and optionally 13 % of the reaction volume.

A method for detecting a pathogen, wherein the method comprises the method according to any one of claims 1 to 16 .

The method comprises detecting the presence of an amplification product.
17. The method of claim 17 , wherein the detection of the amplification product optionally indicates the presence of the pathogen .

A method for diagnosing a subject to be infected with a target pathogen, wherein the method comprises the method according to any one of claims 1-18 .