# US 10,245,590 B2 · High-speed real-time PCR device based on lab-on-a-chip for detecting food-borne bacteria to agrifood, and methods for detecting food-borne bacteria to agrifood using the same

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- Number: US 10,245,590 B2
- Document ID: US10245590B2
- Status: Granted
- Kind: B2
- Application number: 15/109,078
- Filed: 2014-12-30
- Published: 2017-06-08
- Granted: 2019-04-02
- Earliest claimed: 2013-12-31
- Assignees: Nanobiosys Inc.
- Applicants: Nanobiosys Inc.
- Inventors: Sung Woo Kim; Mi-Ree Kim
- Web page: https://patentgrep.com/patent/US10245590B2

## CPC

- B01L 7/5255: {by moving sample containers}
- B01L 3/50851: {specially adapted for heating or cooling samples}
- B01L 3/50853: {with covers or lids (closures for test tubes B01L3/50825)}
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/689: for bacteria
- B01L 2200/0684: Venting, avoiding backpressure, avoid gas bubbles
- B01L 2200/10: Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
- B01L 2300/046: Function or devices integrated in the closure
- B01L 2300/0654: Lenses; Optical fibres
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0829: Multi-well plates; Microtitration plates
- C12Q 1/6851: Quantitative amplification
- C12Q 2600/158: Expression markers
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- 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 relates to an ultra-high speed real-time PCR device on the basis of a lab-on-a-chip for detecting bacteria that causes food poisoning pertaining to agricultural food and a food poisoning detection method using the same. The present invention can provide a micro PCR chip which can simultaneously accommodate a plurality of small-volume samples and concurrently secure maximum thermal contact efficiency with a heating block so as to secure rapid results, and accurately measure an optical signal emitted from a nucleic acid amplification product even without any separate filtering or processing. Further, on the basis of the PCR chip, the present invention can provide a real-time PCR device which can rapidly obtain a nucleic acid amplification result of which the reliability is secured even without a complicated light-signal measuring module.

## Claims

### Claim 1

A micro Polymerase Chain Reaction (PCR) chip, comprising: a reaction chamber having an opened upper surface intended to receive a fluid; and a cover intended to seal the reaction chamber, the cover comprising a light-transmitting portion of a light transmitting material which is faced with the opened upper surface of the PCR reaction chamber to close the open upper surface, the light-transmitting portion having a protruding part that extends toward the interior of the reaction chamber from regions of closed surfaces faced on the opened upper surface of the chamber, and the end of the protruding part being adjacent to or in contact with the surface of said fluid, wherein the protruding part of the light-transmitting portion is located in an optical path between a light-emitting module and a light-detecting module for detecting optical signals in said fluid.

### Claim 2 (depends on 1)

The micro PCR chip according to claim 1, wherein the reaction chamber comprises one or more PCR primer sets for detecting food-borne bacteria.

### Claim 3 (depends on 2)

The micro PCR chip according to claim 2, wherein the one or more PCR primer sets are selected from the group consisting of:
a primer set for detecting Salmonella spp. gene, which consists of a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 1 and a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 2;
a primer set for detecting Listeria monocytogenes gene consists of a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 3 and a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 4;
a primer set for detecting Staphylococcus aurens gene which consists of a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 5 and a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 6; and
a primer set for detecting Escherichia coli gene which consists of a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 7 and a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 8.

### Claim 4 (depends on 2)

A method for detecting Salmonella spp., comprising applying the micro PCR chip of claim 2 in a real-time PCR assay using the primer set consisting of a primer comprising 15 or more consecutive nucleotides of the base sequence of HQ ID NO: 1 and a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 2, thereby detecting said bacteria.

### Claim 5 (depends on 2)

A method for detecting Listeria monocytogenes, comprising applying the micro PCR chip of claim 2 in a real-time PCR assay using the primer set consisting of a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 3 and a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 4, thereby detecting said bacteria.

### Claim 6 (depends on 2)

A method for detecting Staphylococcus aurens, comprising applying the micro PCR chip of claim 2 in a real-time PCR assay using the primer set consisting of a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 5 and a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 6, thereby detecting said bacteria.

### Claim 7 (depends on 2)

A method for detecting Escherichia coli, comprising applying the micro PCR chip of claim 2 in a real-time PCR assay using the primer set consisting of a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ ID NO: 7 and a primer comprising 15 or more consecutive nucleotides of the base sequence of SEQ. ID NO: 8, thereby detecting said bacteria.

### Claim 8 (depends on 1)

The micro PCR chip according to claim 1, wherein said fluid received in the reaction chamber has a volume of 10 μl or less.

### Claim 9 (depends on 8)

The micro PCR chip according to claim 8, wherein said fluid received in the reaction chamber has a volume ranging from around 5 μl to around 8 μl.

### Claim 10 (depends on 1)

The micro PCR chip according to claim 1, wherein the light-transmitting portion is positioned at the center of the cover and the protruding part of the light-transmitting portion is positioned at the interior center of the reaction chamber.

### Claim 11 (depends on 10)

The micro PCR chip according to claim 10, wherein the cover and the opening of the reaction chamber have a circular shape and the light-transmitting portion has a cylindrical shape.

### Claim 12 (depends on 1)

A real-time PCR plate, comprising a plurality of the micro PCR chips according to claim 1.

### Claim 13 (depends on 12)

The real-time PCR plate according to claim 12, comprising:
a first plate in a flat-plate shape, being a support for said real-time PCR plate;
a second plate in a flat-plate shape, comprising the reaction chambers of the micro PCR chips and disposed on the first plate; and
a third plate in a flat-plate shape, comprising the covers of the micro PCR chips, the covers configured to respectively seal the corresponding reaction chambers when the third plate is disposed on the second plate.

### Claim 14 (depends on 13)

The real-time PCR plate according to claim 13, further comprising a heat-releasing module configured to release the heat from the reaction chambers.

### Claim 15 (depends on 14)

The real-time PCR plate according to claim 14, wherein the reaction chambers comprise one or more PCR primer sets for detecting food-borne bacteria.

### Claim 16 (depends on 14)

A real-time PCR apparatus, comprising:
the real-time PCR plate according to claim 14;
a first heat block configured to heat the real-time PCR plate when in contact therewith;
a second heat block positioned a distance away from the first heat block and configured to heat the real-time PCR plate when in contact therewith;
a light-emitting module;
a light-detecting module; and
a plate-moving means configured to move the real-time PCR plate between the first heat block and the second heat block,
wherein the light-emitting module and the light-detecting module are configure to optically detect an on-going PCR reaction in the reaction chambers when the real-time PCR plate moves between the first heat block and the second heat block by the plate-moving means.

### Claim 17 (depends on 16)

The real-time PCR apparatus according to claim 16, wherein the first heat block has a temperature of 90° C. to 100° C. and the second heat block has a temperature of 55° C. to 75° C.

### Claim 18 (depends on 17)

The real-time PCR apparatus according to claim 17, wherein the first heat block has a temperature of around 95° C. and the second heat block has a temperature of about 72° C.

### Claim 19 (depends on 17)

The real-time PCR apparatus according to claim 17, wherein the reaction chambers comprise one or more PCR primer sets for detecting food-borne bacteria.