Rapid Diagnostic Test for LAMP

Abstract

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.

1 - 22 . (canceled) 
     
     
         1 . A method, comprising:
 (a) combining a biological sample from a nasal or buccal swab or from saliva, with a buffer containing a poloxamer in a container to produce a sample;   (b) heating the sample to 95° C.; and   (c) adding one or more primer sets to the sample or adding one or more primer sets to an aliquot of the sample in a reaction buffer.   
     
     
         2 . The method of  claim 1 , wherein the poloxamer is PF68. 
     
     
         3 . The method of  claim 1 , wherein the primer sets are specific for one or more virus nucleic acids selected from a coronavirus, a rhinovirus, a respiratory syncytial virus, an influenza virus, a parainfluenza virus, a metapneumovirus, an adenovirus and a bocavirus. 
     
     
         4 . The method of  claim 3 , wherein the coronavirus is SARS-CoV-2. 
     
     
         5 . The method of  claim 4 , wherein the primer sets are specific for SARS-CoV-2 and influenza virus. 
     
     
         6 . The method of  claim 1 , wherein the primer sets are selected from LAMP primer sets and RT-qPCR primer sets. 
     
     
         7 . The method of  claim 1 , wherein the primer sets are LAMP primer sets, optionally selected from fluorescent LAMP primer sets, DARQ LAMP primer sets, and colorimetric LAMP (cLAMP) primer sets. 
     
     
         8 . The method of  claim 7 , wherein each LAMP primer set comprises a quenched fluorescent dye in a duplex oligonucleotide. 
     
     
         9 . The method of  claim 7 , wherein, in (c), the LAMP primer sets are added to an aliquot of the sample. 
     
     
         10 . The method of  claim 1 , wherein the primer sets are added to an aliquot of the sample, and wherein the reaction buffer comprises one or more reagents selected from a strand displacing polymerase, a reversible inhibitor of the polymerase, and a thermolabile uracil DNA glycosylase (UDG). 
     
     
         11 . The method of  claim 1 , wherein the primer sets are added to the sample, and wherein the resulting mixture further comprises one or more reagents selected from a strand displacing polymerase, a reversible inhibitor of the polymerase, and a thermolabile uracil DNA glycosylase (UDG). 
     
     
         12 . The method of  claim 8 , further comprising:
 (d) unquenching of the quenched fluorescent dye in the presence of the sample to provide a fluorescent signal; and   (e) detecting the fluorescent signal by determining the wavelength of emitted light for each fluorescent signal.   
     
     
         13 . A composition for releasing an RNA from a biological sample for detection by amplification and/or sequencing, comprising a poloxamer and a reducing agent. 
     
     
         14 . The composition of  claim 13 , wherein the composition further comprises a reagent selected from a metal chelating agent and a salt, wherein the salt is optionally selected from lithium chloride, guanidinium hydrochloride, and a salt of C—(NH2)2NH+. 
     
     
         15 . The composition of  claim 13 , wherein the poloxamer is PF68. 
     
     
         16 . A composition, comprising a biological sample combined with a buffer containing a poloxamer and one or more primer sets. 
     
     
         17 . The composition of  claim 16 , wherein the poloxamer is PF68. 
     
     
         18 . The composition of  claim 16 , wherein the one or more primer sets are LAMP primer sets. 
     
     
         19 . A kit, comprising:
 a container for receiving a swab containing a biological sample or a liquid biological sample, the container containing a buffer comprising a poloxamer, or wherein a buffer comprising a poloxamer is contained in a distinct compartment in the container, or wherein a buffer comprising a poloxamer is provided in a separate tube for receiving an aliquot of the biological sample.   
     
     
         20 . The kit of  claim 19 , further comprising a primer set, wherein the primer set is in the container, or in a distinct compartment in the container, or is separate from the container. 
     
     
         21 . The kit of  claim 19 , further comprising a polymerase, wherein the polymerase is in the container, or a distinct compartment in the container, or is separate from the container 
     
     
         22 . The kit of  claim 19 , further comprising a reverse transcriptase, wherein the reverse transcriptase is in the container, or a distinct compartment in the container, or separate from the container.