# US 2019/0169683 A1 · Detection of an Amplification Product using pH-sensitive Dye

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- Number: US 2019/0169683 A1
- Document ID: US20190169683A1
- Status: Published application
- Kind: A1
- Application number: 16/306,319
- Filed: 2017-05-12
- Published: 2019-06-06
- Earliest claimed: 2016-06-03
- Assignees: New England Biolabs, Inc.
- Applicants: New England Biolabs, Inc.
- Inventors: Yinhua Zhang; Nathan Tanner; Thomas C. Evans, Jr.
- Web page: https://patentgrep.com/patent/US20190169683A1

## CPC

- C12Q 1/6846: {Common amplification features}
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- G01N 33/52: Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)}
- C12Q 2527/119: pH
- C12Q 2527/125: Specific component of sample, medium or buffer
- C12Q 2545/114: involving a quantitation step
- C12Q 2563/173: staining/intercalating agent, e.g. ethidium bromide
- C12Y 207/07007: DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
- 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
- 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)
- C12Y: ENZYMES

## Abstract

Methods are provided for a rapid, low cost approach to monitoring an amplification reaction. This includes monitoring the progress of isothermal or PCR amplification reactions to completion using pH-sensitive dyes that are either colored or fluorescent. Compositions are described that include a mixture of a DNA polymerase, deoxyribonucleotide triphosphate and Tris buffer in the range of 1.5 mM Tris to 5 mM Tris or equivalent.

## Claims

### Claim 1

An amplification method comprising:
(a) setting up a buffered amplification reaction that comprises 1.5 mM to 5 mM Tris or an equivalent thereof and, additionally, a pH sensitive dye;
(b) incubating the amplification reaction under amplification conditions; and,
(c) after or during the incubation of (b), detecting a change in color of the amplification reaction, wherein a change in color of the reaction indicates that an amplification product has been produced.

### Claim 2 (depends on 1)

The amplification method of claim 1, wherein the amplification conditions are isothermal conditions.

### Claim 3 (depends on 1)

The amplification method of claim 1, wherein the amplification reaction is selected from loop-mediated isothermal amplification (LAMP), a helicase dependent amplification (HDA), a strand displacement amplification (SDA), a recombinase polymerase amplification (RPA), rolling circle amplification (RCA), signal mediated amplification of RNA technology (SMART), single primer isothermal amplification (SPIA), nucleic acid sequence based amplification (NASBA) and a nicking enzyme amplification reaction (NEAR).

### Claim 4 (depends on 1)

The amplification method of claim 1, wherein the amplification reaction is a polymerase chain reaction.

### Claim 5 (depends on 1)

The amplification method according to claim 1, wherein the detecting step (c) is done by a naked eye.

### Claim 6 (depends on 5)

The amplification method according to claim 5, wherein the color change of (c) is compared to a control reaction in which no template has been added.

### Claim 7 (depends on 1)

The amplification method according to claim 1, wherein the amplification has a starting pH in the range of pH 7-8.5.

### Claim 8 (depends on 1)

The amplification method according to claim 1, wherein the amplification reaction comprises a biological sample.

### Claim 9 (depends on 1)

The amplification method according to claim 1, wherein the method provides a diagnostic for infectious disease.

### Claim 10 (depends on 1)

The amplification method according to claim 1, wherein the pH sensitive dye is soluble.

### Claim 11 (depends on 10)

The amplification method of claim 10, wherein the soluble pH sensitive dye is a colored dye detectable in visible light.

### Claim 12 (depends on 1)

The amplification method of claim 1, wherein the dye is selected from cresol red, phenol red, m-cresol purple, bromocresol purple, neutral red, naphtholphthalein, thymol blue, and naphtolphthalein.

### Claim 13

An aqueous amplification reaction mix comprising:
(a) a polymerase,
(b) 1.5 mM to 5 mM Tris or an equivalent thereof,
(c) a single pH sensitive dye,
(d) dNTPs,
(e) one or more primers, and
(f) one or more templates;
wherein the reaction mix changes color only during the amplification reaction.

### Claim 14 (depends on 13)

The aqueous amplification reaction mix of claim 13, wherein the aqueous reaction mix has a starting pH at 25° C. in the range of pH 7-8.5.

### Claim 15 (depends on 13)

The aqueous amplification reaction mix according to claim 13, wherein the pH sensitive dye is soluble.

### Claim 16 (depends on 15)

The aqueous amplification reaction mix of claim 15, wherein the soluble pH sensitive dye is a colored dye detectable in visible light.

### Claim 17 (depends on 13)

The aqueous amplification reaction mix of claim 13, wherein the dye is selected from cresol red, phenol red, m-cresol purple, bromocresol purple, neutral red, naphtholphthalein, thymol blue, and naphtolphthalein.