Rapid diagnostic test using colorimetric lamp

Patent metadata

Field	Value
Publication number	US-11492673-B2
Application number	US-202117178395-A
Country	US
Kind code	B2
Filing date	Feb 18, 2021
Priority date	Mar 12, 2020
Publication date	Nov 8, 2022
Grant date	Nov 8, 2022

Abstract

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.

First claim

What is claimed is:

1. A kit for performing multiplex Loop-Mediated Isothermal Amplification (LAMP), comprising:
(a) at least one set of oligonucleotide primers, for amplifying a template sequence by LAMP;
(b) a strand-displacing DNA polymerase, wherein said strand-displacing DNA polymerase is in a master mix, the master mix further comprising uracil deglycosylase; and
(c) a guanidinium salt; wherein (c) is in a separate container from (a) and/or (b).

2. The kit of claim 1 , wherein the at least one set of primers in (a) comprises 5 or 6 primers and at least one primer contains a sample bar code.

3. The kit according to claim 1 , wherein (a) further comprises one or more sets of oligonucleotide primers for hybridizing to one or more template sequences in each of a first and second nucleic acid target for amplifying the template region of the first and second nucleic acid target by LAMP.

4. The kit according to claim 3 , wherein the first and second nucleic acid targets are a plurality of viral genomes or cDNAs thereof.

5. The kit according to claim 1 , wherein the targeted viral genomes comprise an influenza virus genome and a coronavirus virus genome.

6. The kit according to claim 1 , wherein the master mix further comprises: deoxynucleoside triphosphates; and at least one indicator for detecting an amplification product by a change in color or fluorescence.

7. The kit, according to claim 6 , wherein the indicator is selected from a metallochromic dye, a pH sensitive color dye, an intercalating dye and a fluorescent reporter indicator.

8. The kit according to claim 6 , wherein the master mix further comprises at least one additional reagent selected from the group consisting of: a reverse transcriptase, a reducing agent, a lithium chloride, a detergent and a reversible inhibitor of polymerase activity that is capable of blocking polymerase activity at a temperature below 40° C.

9. The kit according to claim 6 , wherein the master mix further comprises a reverse transcriptase.

10. The kit according to claim 6 , further comprising a reducing agent.

11. The kit according to claim 10 , wherein the reducing agent is Tris(2-carboxyethyl)phosphine hydrochloride (TCEP).

12. The kit according to claim 6 , wherein the master mix is lyophilized.

13. The kit according to claim 1 , wherein the guanidinium salt in (c) is guanidinium chloride.

14. The kit according to claim 1 , wherein if (c) is at 10× concentration then the guanadinium chloride concentration in the range of 200 mM-600 mM.

15. The kit according to claim 1 , further comprising instructions describing how to combine the reagents (a)-(c) in a reaction mix with a nucleic acid target for performing the multiplex Loop-Mediated Isothermal Amplification (LAMP) reaction, wherein the reaction mix comprises the strand displacing polymerase, the plurality of sets of primers and an amount of guanidinium salt for making a reaction mixture containing a final concentration of 20 mM-60 mM guanidinium salt.

16. The kit according to claim 1 , wherein the master mix is suitable for diluting 2 fold, 3 fold, 4 fold or 10 fold in the reaction mix containing purified nucleic acid or a sample obtained from a mammal.

17. A method comprising:
(a) combining reagents (a) and (b) and optionally (c) in the kit according to claim 1 , with a test sample to produce a reaction mix for detecting a one or more nucleic acid targets, wherein the kit comprises a plurality of sets of oligonucleotide primers, wherein each set of primers hybridize to a different template sequence for amplifying different template sequences by Loop-Mediated Isothermal Amplification (LAMP); and
(b) incubating the reaction mix to produce a LAMP product of the one or more nucleic acid target(s) in the test sample.

18. The method of claim 17 , wherein the test sample is a purified nucleic acid, a nucleic acid in a biological fluid or a nucleic acid preparation released from a sample swab or a lysed cell sample into an aqueous fluid.

19. The method according to claim 17 , wherein the test sample is obtained from a human subject or from an environmental sample.

20. The method according to claim 17 , wherein the nucleic acid target is a virus pathogen.

21. The method according to claim 20 , wherein the virus pathogen is an RNA virus or a cDNA copy thereof.

22. The method according to claim 17 , further comprising detecting a plurality of nucleic acid targets wherein a first nucleic acid target is a coronavirus RNA and a second nucleic acid target is an influenza RNA.

23. The method according to claim 17 , further comprising: (c) detecting the amplification product.

24. The method according to claim 23 , further comprising pooling a first set of oligonucleotide primers for a first nucleic acid target and a second set of oligonucleotides for a second nucleic acid target and combining the pooled oligonucleotide primers with the strand displacing polymerase and guanidine salt and one or more nucleic acid samples for detecting the amplification products of the first and second nucleic acid targets in the one or more nucleic acid samples.

25. The method according to claim 22 , wherein any of (a)-(c) is partially or completely automated.

26. The method according to claim 22 , wherein (c) further comprises visualizing a color change by eye, measuring a change of color by means of a spectrophotometer or measuring fluorescence with a fluorescence spectrometer.

27. The method according to claim 17 , wherein the kit further comprises at least one additional reagent selected from the group consisting of: a reverse transcriptase, UDG, dUTP, dATP, dTTP, dGTP, dCTP, an indicator that changes color or fluorescence when amplification has occurred, a reducing agent, lithium chloride and a detergent.

28. The method according to claim 17 , wherein the kit further comprises: a reverse transcriptase if at least one of the nucleic acid targets is RNA, and wherein a step between (a) and (b) comprises reverse transcribing the RNA into DNA.

29. The method according to claim 17 , wherein the guanidinium salt in the kit is guanidinium chloride and the concentration of the guanidinium chloride in the reaction mix is in the range of 20 mM-60 mM.

30. The method according to claim 17 , wherein the kit of claim 1 further comprises a master mix suitable for diluting 2 fold, 3 fold, 4 fold or 10 fold in the reaction mix containing purified nucleic acid or a sample obtained from a mammal.

Assignees

• New England Biolabs Inc

Inventors

• Tanner Nathan
• Zhang Yinhua
• Patton Gregory
• Ren Guoping
• Li Zhiru
• Nichols Nicole

Classifications

• C12Q1/701 Primary

  Specific hybridization probes · CPC title

• G01N21/78

  producing a change of colour · CPC title

• G01N2021/7786

  Fluorescence · CPC title

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title