New England Biolabs, Inc.

Yinhua Zhang

Nathan Tanner

Thomas C Evans, Jr

Nicole Nichols

Zhiru Li

Gregory Patton

Guoping Ren

Ivan R Correa, Jr

Clotilde K Carlow

Eric Hunt

Esta Slayton

Andrew Barry

Catherine B Poole

Harriet M Strimpel

Jennifer Ong

Vladimir Potapov

Yanxia Bei

Christopher J Noren

George Tzertzinis

Larry A McReynolds

George R Feehery

Corinna Tuckey

Ece Alpaslan

Compositions and methods are described that are directed to specific and sensitive methods of target nucleic acid detection and more specifically detecting target nucleic acids directly from biological samples. The compositions and methods were developed to be easy to use involving a minimum number of steps and giving rapid and consistent results either at point of care or in high throughput situations. The compositions and methods are directed to labelled probes and their uses in Loop-Mediated Isothermal Amplification (LAMP) diagnostic tests to detect target DNA from the environment or from an individual and also to detect specific variants of the target DNA, both with similar sensitivity. The compositions and methods may use any single improvement or combination of improvements selected from thermolabile enzyme variants, poloxamers, various salts, indicators and one or more LAMP primer sets for detecting single and/or multiple targets, probes for detecting variants of the targets including SARS-CoV-2 variants and lateral flow devices.

Rapid diagnostic test for lamp

Kits and methods are described that are directed to specific and sensitive methods of target nucleic acid detection and more specifically detecting target nucleic acids directly from biological samples. The kits and methods were developed to be easy to use involving a minimum number of steps and giving rapid and consistent results either at point of care or in high throughput situations. The kits and methods utilize in various combinations, reversible inhibitors of kit components, thermolabile enzymes, poloxamers, various salts, indicators and one or more Loop-Mediated Isothermal Amplification (LAMP) primer sets for detecting single and/or multiple targets and variants of the targets including SARS-CoV-2 targets and variants thereof in a single reaction. The kits and methods permit detection of the target nucleic with similar sensitivity regardless of the presence of undefined mutations that may enhance the virulence of cells or viruses containing the undefined mutations.

Rapid diagnostic test for LAMP

Provided herein is a composition comprising an enzyme that releases pyrophosphate from a substrate and a dye of Formula 1. A method for detecting pyrophosphate is also provided. A kit comprising a polymerase that releases pyrophosphate by hydrolysis of nucleoside triphosphates during nucleic acid replication, a divalent manganese salt, and the dye are also provided. The present composition, method and kits provide a way to detect and/or quantify substrates or products of enzyme reacted substrates associated with the release pyrophosphate (e.g., nucleic acid amplification reactions and other reactions that hydrolyze ATP) via a distinct color change without substantially affecting the sensitivity and/or specificity of the reaction.

Compositions and methods for detecting pyrophosphate products of enzyme reactions using pyridylazoaniline dyes

Kits and methods are provided for performing multiplex LAMP reactions. These kits and methods are directed to specific and sensitive methods of target nucleic acid detection and more specifically pathogen diagnostics such as detection of Coronavirus. The kits and methods utilize a plurality of sets of oligonucleotide primers for targeting the viral nucleic acid target.

Rapid diagnostic test using colorimetric lamp

Variants of the bacteriophage B103 DNA polymerase are described herein. The variant has improved properties, that include when compared to wild-type Phi29 DNA polymerase, at least one of the following: increased thermostability, improved reaction rate for DNA amplification, reduced background and a reduction of bias. Methods of using the DNA polymerase variant are also described herein.

Variant DNA polymerases having improved properties and method for improved isothermal amplification of a target DNA

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.

Detection of an amplification product using pH-sensitive dye

Kits and methods are provided for performing multiplex Loop-Mediated Isothermal Amplification (LAMP) reactions. These kits and methods are directed to specific and sensitive methods of target nucleic acid detection and more specifically pathogen diagnostics such as detection of Coronavirus. The kits and methods utilize a plurality of sets of oligonucleotide primers for targeting the viral nucleic acid target.

Rapid diagnostic test using colorimetric LAMP

Kits and methods are provided for performing multiplex LAMP reactions. These kits and methods are directed to specific and sensitive methods of target nucleic acid detection and more specifically pathogen diagnostics such as detection of Coronavirus. The kits and methods utilize a plurality of sets of oligonucleotide primers for targeting different template sequences in a single nucleic acid target. The kits and methods also include guanidium salts that enhance the sensitivity of the assay.

Compositions and methods are provided for improved reverse transcriptases and their uses in reverse transcription where the improvement may include increased temperature, increased salt, increased activity and/or increased dUTP tolerance.

Reverse transcriptase with enhanced properties

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 a weak buffer of less than 1 mM Tris or equivalent or no buffer.

Detection of an amplification reaction product using pH-sensitive dyes

Compositions and methods are provided for quantitative detection of amplification products, the methods being suitable for multiplexing. A first oligonucleotide that includes a primer sequence for priming an amplification reaction and also is labeled with a fluorescent label or quencher is mixed with a second oligonucleotide which has a sequence suitable for hybridizing to a portion of the first oligonucleotide and has a fluorescent label if the first oligonucleotide has a quencher or a quencher if the first oligonucleotide has a fluorescent label; and a third nucleotide which includes some or all the primer sequence contained in the first oligonucleotide but is not labeled, the first and third oligonucleotide being combined in a molar ratio of 2.8 to 8.2.

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North Reading, MA, United States of America

Yinhua Zhang