WO2014003583A4 - Detection of pathogens - Google Patents
Detection of pathogens Download PDFInfo
- Publication number
- WO2014003583A4 WO2014003583A4 PCT/PH2013/000016 PH2013000016W WO2014003583A4 WO 2014003583 A4 WO2014003583 A4 WO 2014003583A4 PH 2013000016 W PH2013000016 W PH 2013000016W WO 2014003583 A4 WO2014003583 A4 WO 2014003583A4
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- primers
- seq
- lamp
- nos
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a methods and diagnostic kits for the rapid detection of different pathogens employing the loop-mediated isothermal amplification platform (LAMP) that may be performed or used even outside the laboratory. The said invention uses novel primer combinations for' sequences within the housekeeping genes of the target organism.
Claims
AMENDED CLAIMS
received by the International Bureau on 18 August 2014 (18.08.14)
1. A method for the detection of a pathogen in a sample by LAMP, comprising: Adding said sample to a reaction tube comprising of a thermostable reaction mixture and primers;
Incubating the reaction tube in a heater; and Detecting the presence of the pathogen in the sample.
2. The method in Claim 1 , wherein the thermostable reaction mixture consists of a forward internal primer, a backward internal primer, a forward outer primer, a backward outer primer, a forward loop primer and a backward loop primer , deoxynucleoside triphosphates, betaine, Tween, MgS04, KC1,
TrisHCl and Bst DNA polymerase.
3. The method in Claims I or 2, wherein the thermostable reaction mixture is lyophilized.
4. The method in Claim 3, wherein the thermostable reaction is further mixed with water.
5. The method in Claim 1 , wherein detection is by means of adding a detection dye to the reaction tube after incubation and observing the corresponding color change in the said reaction tube.
6. The method of Claim 1 , wherein the pathogen is dengue virus. 7, The method of Claim 6, wherein the primers consist of universal LAMP primers containing nucleic acid sequences of SEQ ID NOS. 25 -30.
8. The method of Claim 6, wherein the primers consist of dengue serotype- specific LAMP primers containing nucleic acid sequences of SEQ ID NOS. 1-6 for serotype 1.
9. The method of Claim 6, wherein the primers consist of dengue serotype- specific LAMP primers containing nucleic acid sequences of SEQ ID NOS.
7-12 for serotype 2. 10. The method of Claim 6, wherein the primers consist of dengue serotype- specific LAMP primers containing nucleic acid sequences of SEQ ID NOS. 13-18 for serotype 3. 1 1. The method of Claim 6, wherein the primers consist of dengue serotype- specific LAMP primers containing nucleic acid sequences of SEQ ID NOS. 19-24 for serotype 4. 12. The method of Claim 1 , wherein the pathogen is a Leptospira species. 13. The method of Claim 12, wherein the primers consist of LAMP primers which are complementary to the ompLI region of the Leptospira species containing nucleic acid sequences of SEQ ID NO. 31 , SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 36. 14. The method in Claim 1 , wherein the pathogen is MTB or MOTT. 15. The method of Claim 14, wherein the primers consist of LAMP primers specific for the detection of MTB containing nucleic acid sequences of SEQ ID NOS. 37 - 96. 16. The method of Claim 14, wherein the primers consist of LAMP primers specific for the detection of MOTT containing nucleic acid sequences of SEQ ID NOS. 97 - 156. 17. The method of Claim 1 , wherein the pathogen is Salmonella enterica. 18. The method of Claim 17, wherein the primers consist of universal LAMP primers containing contain nucleic acid sequences of SEQ ID NOS. 157 - 192. 19. The method in Claim 17, wherein the primers consist of LAMP primers which are typhoidal serovar-specific primers containing nucleic acid sequences of SEQ ID NOS. 193 - 222. 20. A kit for the detection of a pathogen in a sample by LAMP, comprising of
primers and a thermostable reaction mixture. 21. A kit as claimed in Claim 20, wherein the thermostable reaction mixture is lyophilized. 22. A kit as claimed in Claim 20, wherein the thermostable reaction mixture is kept at room temperature when not in use. 23. A kit as claimed in Claim 20, wherein the thermostable reaction mixture consists of a pre-mixed mixture of a forward internal primer, a backward internal primer, a forward outer primer, a backward outer primer, a forward loop primer, a backward loop primer, deoxynucleoside triphosphates, betaine. Tween, MgSO, KC1 , TrisHCl and Bst DNA polymerase. 24. A kit as claimed in Claim 20, wherein the kit includes a heater where the primers and the thermostable reaction mixture will be incubated at 63-67* C to achieve a LAMP isothermal temperature.
25. A thermostable reaction mixture for use in LAMP that would detect pathogens upon addition of a sample and primers.
26. A thermostable reaction mixture as claimed in Claim 25, wherein water is further added to detect pathogens. 27. A set of primers to detect dengue by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 25 -30.
28. A set of primers to detect specific serotype of dengue by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 1-6 for serotype 1. SEQ ID NOS. 7-12 for serotype 2, SEQ ID NOS. 13-18 for serotype 3 and SEQ ID NOS. 19-24 for serotype 4.
29. A set of primers to detect Leptospira species by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NO. 31 , SEQ ID NO. 32, SEQ ID NO. 33. SEQ ID NO. 34, SEQ ID N0.35 and SEQ ID NO.
36.
30. A set of primers to detect MTB by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 37 - 96.
31. A set of primers to detect MOTT by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 97 - 156. 32. A set of primers to detect Salmonella enterica by means of LAMP, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 157 - 192,
33. A set of primers to detect typhoidal serovar Salmonella enterica, wherein the primers contain the nucleic acid sequences of SEQ ID NOS. 193 - 222.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PH1/2012/000185A PH12012000185A1 (en) | 2012-06-26 | 2012-06-26 | Detection of pathogens |
| PHPH1-2012-000185 | 2012-06-26 |
Publications (4)
| Publication Number | Publication Date |
|---|---|
| WO2014003583A2 WO2014003583A2 (en) | 2014-01-03 |
| WO2014003583A8 WO2014003583A8 (en) | 2014-05-22 |
| WO2014003583A3 WO2014003583A3 (en) | 2014-08-07 |
| WO2014003583A4 true WO2014003583A4 (en) | 2014-10-02 |
Family
ID=49783971
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/PH2013/000016 Ceased WO2014003583A2 (en) | 2012-06-26 | 2013-06-26 | Detection of pathogens |
Country Status (2)
| Country | Link |
|---|---|
| PH (1) | PH12012000185A1 (en) |
| WO (1) | WO2014003583A2 (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10731227B2 (en) | 2014-12-12 | 2020-08-04 | The Trustees Of The University Of Pennsylvania | Compositions, kits, and methods to detect HIV virus |
| WO2017099801A1 (en) * | 2015-12-11 | 2017-06-15 | The Trustees Of The University Of Pennsylvania | Compositions, kits, and methods to detect hiv virus |
| US11299777B2 (en) | 2016-04-04 | 2022-04-12 | Nat Diagnostics, Inc. | Isothermal amplification components and processes |
| US9617587B1 (en) | 2016-04-04 | 2017-04-11 | Nat Diagnostics, Inc. | Isothermal amplification components and processes |
| CN108531624A (en) * | 2018-04-11 | 2018-09-14 | 重庆高圣生物医药有限责任公司 | Mycobacterium tuberculosis loop-mediated isothermal amplification (LAMP) primer, detection method and kit |
| WO2021216868A1 (en) * | 2020-04-22 | 2021-10-28 | The Regents Of The University Of California | Methods for detecting and sequencing a target nucleic acid |
| WO2021216863A1 (en) * | 2020-04-22 | 2021-10-28 | The Regents Of The University Of California | Universal primers for detection of bacteria, fungi and eukaryotic microorganisms |
| EP4110945A2 (en) * | 2020-08-21 | 2023-01-04 | New England Biolabs, Inc. | A rapid diagnostic test for lamp |
| AU2022242740A1 (en) * | 2021-03-22 | 2023-10-12 | Duke University | Compositions and methods for rapid covid-19 detection |
| US20240240263A1 (en) * | 2023-01-18 | 2024-07-18 | Jincheng Wang | Nucleic acid detection reagent and detection method for mycobacterium tuberculosis |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003199572A (en) * | 2001-12-28 | 2003-07-15 | Eiken Chem Co Ltd | Primer for detection of salmonella and detection method using the same |
| JP4699008B2 (en) * | 2004-11-02 | 2011-06-08 | 旭化成株式会社 | Enzyme reaction reagent |
-
2012
- 2012-06-26 PH PH1/2012/000185A patent/PH12012000185A1/en unknown
-
2013
- 2013-06-26 WO PCT/PH2013/000016 patent/WO2014003583A2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| WO2014003583A2 (en) | 2014-01-03 |
| WO2014003583A8 (en) | 2014-05-22 |
| WO2014003583A3 (en) | 2014-08-07 |
| PH12012000185A1 (en) | 2014-02-24 |
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