Method for detecting microorganisms

Patent metadata

Field	Value
Publication number	US-12473591-B2
Application number	US-202217687490-A
Country	US
Kind code	B2
Filing date	Mar 4, 2022
Priority date	Jun 7, 2019
Publication date	Nov 18, 2025
Grant date	Nov 18, 2025

Abstract

The present invention relates to a method for simultaneously detecting a plurality of pathogens from biologically-derived samples, and a kit for carrying out the method. Specifically, the present invention relates to a method for simultaneously detecting a plurality of pathogens that cause infectious uveitis, one of eye infections from samples such as anterior chamber fluid or vitreous by polymerase chain reaction (PCR), and a kit for carrying out the method.

First claim

The invention claimed is:

1 . A method for detecting microorganisms in a sample, comprising
(1) a step of mixing the sample with a PCR buffer comprising a nonionic surfactant; Tris buffer, containing KCl and MgCl 2 ; and a dNTP mix of dATP, dGTP, dCTP and dTTP to obtain a mixed solution; (2) a step of adding the mixed solution in step (1) to a plurality of tubes wherein each tube of the plurality of tubes comprises a solid composition for PCR reaction comprising a DNA polymerase and two types of PCR primer pairs which are selected from the group consisting of:
(i) a Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene detection primer pair having the sequences of SEQ ID NO:1 and 2 and a TATA-binding protein (TBP) gene detection primer pair having the sequences of SEQ ID NO:3 and 4,
(ii) a HSV-1 detection primer pair having the sequences of SEQ ID NO: 5 and 6 and a VZV detection primer pair having the sequences of SEQ ID NO: 7 and 8,
(iii) a HSV-2 detection primer pair having the sequences of SEQ ID NO: 9 and 10 and a HHV-6 detection primer pair having the sequences of SEQ ID NO: 11 and 12,
(iv) a EBV detection primer pair having the sequences of SEQ ID NO: 13 and 14 and a CMV detection primer pair having the sequences of SEQ ID NO: 15 and 16, and
(v) a HTLV-1 detection primer pair having the sequences of SEQ ID NO: 17 and 18 and a Treponema pallidum detection primer pair having the sequences of SEQ ID NO: 19 and 20 and a toxoplasma detection primer pair having the sequences of SEQ ID NO:21 and 22; and
(3) a step of detecting the PCR product generated in each of the plurality of tubes; wherein the sample is anterior chamber fluid or vitreous.

2 . The method according to claim 1 , wherein the amount of the sample is 12-20 μL.

3 . The method according to claim 1 , wherein the microorganism is selected from the group consisting of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6), cytomegalovirus (CMV), human adult T-cell leukemia virus (HTLV-1), Treponema pallidum and toxoplasma.

4 . The method according to claim 1 , wherein in the step (2), the plurality of tubes comprises 2-12 tube strips.

5 . The method according to claim 1 , wherein in the step (2), the solid composition for PCR reaction contains an oligonucleotide probe labeled with one or more types of fluorescent dyes for fluorescent detection of PCR amplification products.

6 . The method according to claim 5 , wherein the fluorescent dyes are selected from the group consisting of FAM (6-carboxyfluorescein), ROX (6-carboxy-X-rhodamine), Cy5 (Cyanine dye) and HEX (4,7,2′,4′,5′,7′-hexachlorofluor-6-carboxyfluorescein).

7 . The method according to claim 1 , wherein in the step (2), the solid composition for PCR reaction is in a lyophilized form.

8 . The method according to claim 1 , wherein in the step (3), the PCR products are detected by real-time determination.

Assignees

• Univ Nat Corp Tokyo Medical & Dental
• Nihon Techno Service Co Ltd
• National Univ Corporation Oita Univ
• Riken
• Shimadzu Corp
• National Univ Corp Tokyo Medical And Dental Univ
• National Univ Corp Oita Univ

Inventors

• Shimizu Norio
• Takase Hiroshi
• Mochizuki Manabu
• Tomaru Yasuhiro
• Nakano Satoko
• Sugita Sunao
• Shikata Masamitsu

Classifications

• C12Q1/6893

  for protozoa · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/32

  involving dehydrogenase · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• G01N21/6486

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title