WO1992009703A1 - Testing for spirochetal nucleic acid sequences in samples - Google Patents
Testing for spirochetal nucleic acid sequences in samples Download PDFInfo
- Publication number
- WO1992009703A1 WO1992009703A1 PCT/US1991/008737 US9108737W WO9209703A1 WO 1992009703 A1 WO1992009703 A1 WO 1992009703A1 US 9108737 W US9108737 W US 9108737W WO 9209703 A1 WO9209703 A1 WO 9209703A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- borrelia
- gene
- ticks
- detecting
- probe
- Prior art date
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
Definitions
- This invention relates to a method of testing arthropod vectors, such as ticks, or other samples, for the presence or absence of Borrelia burgdorferi-related genetic material to determine whether the ticks are spreaders of Lyme disease.
- Lyme disease has only recently been recognized by the medical profession as a distinct clinical entity. At present in the United States alone, more than six thousand new human incidences of Lyme disease occur each year. Lyme disease has been determined to be global in scope, and is not merely restricted to the Northeastern United States. Symptoms of Lyme disease may be slow to manifest themselves, and may not be diagnosed by even a skilled physician until 'after the infection has been present for many years.
- Lyme disease may only be diagnosed after telltale symptoms have manifested themselves in an infected patient. As noted above, this can entail a considerable period of time after initial infection, and thus can expose the patient to complications from the infection which are painful and even life threatening, in extreme cases.
- Lyme disease is initiated in humans by bites from infected ticks.
- the most common source of Lyme disease, in humans, is the deer tick.
- Deer ticks which carry the Lyme disease-causing parasite spirochete, Borrelia burgdorferi, will infect humans with Lyme disease when bitten.
- the presence of the Lyme spirochetes in a locale will not be realized until after one has been bitten by an infected tick; thus prevention of the disease, until an effective vaccine is developed, is not known to be an option.
- This invention is directed to a procedure for detecting the presence or absence of Borrelia burgdorferi in the tick population.
- ticks found in the locale can be tested on a regular basis so that, if infected ticks are noted, the local populace can take extra precautions against exposure to ticks. Ticks from bitten patients can also obviously be tested for the spirochete. It is a relatively simple procedure to gather ticks without exposing oneself to the danger of being bitten. Since ticks are attracted to changes in surrounding color and temperature, one merely needs to drag a white sheet or the like about one's yard and the sheet will pick up ticks, if there are any in the area.
- the tick can be tested after removal from the skin of the bitten subject.
- the procedure of this invention can be used on dessicated tick bodies, whereby the ticks to be tested can be killed by placing them in a vial containing alcohol, wherein the ticks will be preserved, for subsequent testing.
- the procedure of this invention involves grinding or otherwise rupturing the tick body to release its midgut contents.
- the midgut contents are then subjected to a polymerase chain reaction amplification for the detection of the presence -of Borrelia-specific nucleic acid sequences in the tick midgut contents.
- a positive result indicates that the subject tick is a carrier of the Lyme spirochete, while a negative result suggests the opposite.
- PCR Polymerase chain reaction
- My reagents for PCR amplification consist of oligonucleotide primers and probes encoding the OSP-A (outer surface protein) and flagellin proteins.
- the oligonucleotide primers can be prepared on an Applied Biosystems 320A oligonucleotide synthesizer, desalted in a oligonucleotide purification cartridge, and used in the PCR reactions without further purification.
- sequences of the primers used for the OSP PCR amplification procedure are as follows:
- OSP-A2 5'GTT TTG TAA TTT CAA CTG CTG ACC 3' ;
- OSP-A4 5'CTG CAG CTT GGA ATT CAG GCA CTT C 3'; and the probe
- OSP-A3 5 * GCC ATT TGA GTC GTA TTG TTG TAC TG 3' ; and the Flagellin gene target (1.75 mM MgCl 2 ):
- FLA-l 5'GAT GAT GCT GCT GGC ATG GGA GTT TCT 663';
- FLA-2 5'ATT CAG ACA ACA GAA GGG AAT TTA GAA GTA G 3' .
- tick specimens are air dried on filter paper disks for five minutes.
- Two hundred ⁇ l (dry volume) of 0.5 mm glass beads are pre-treated in 1.0 ml of 1% bovine serum albumin in distilled water at 37 degrees C. for 30 minutes and then washed twice in 1.0 ml distilled water.
- the pre-treatment of the glass beads is necessary to prevent the beads from adsorbing the B. burgdorferi nucleic acid sequences during the PCR procedure.
- the ticks are placed whole into 0.6 ml microcentrifuge tubes containing 20 ⁇ l of a slurry of the pre-treated glass beads.
- tick bodies are then crushed into the beads with a disposable plastic dowel for a time sufficient to liberate the midgut contents, and 25 ⁇ l of PCR buffer (lOmM Tris HCL, pH 8.3; 50 mM KCL; 1.75 mM MgCl 2 ; 0.01% gelatin; 0.5% NP40 detergent; and 0.5% Tween 20 brand detergent) is added.
- PCR buffer lOmM Tris HCL, pH 8.3; 50 mM KCL; 1.75 mM MgCl 2 ; 0.01% gelatin; 0.5% NP40 detergent; and 0.5% Tween 20 brand detergent
- amplification of Borrelia-specific sequences 5 ⁇ l of the tick supernatant prepared as described above, is added to a 50 ⁇ l PCR reaction composition containing: 10 mM Tris HCL, pH 8.3; 50 mM KCL; 0.01% gelatin; 200 ⁇ m each deoxynucleotide triphosphate; and 50 picomoles of each primer, along with a concentration of MgCl 2 that is specific for each primer pair. All reactions can be performed in a Perkin Elmer-Cetus thermal cycler. The aforesaid components are denatured at 94 degrees C. for 30 seconds; annealed at 55 degrees C. for 45 seconds; and extended at 72 degrees C.
- the amplification products are analyzed by electrophoresis in a 1% Seakem/3% Nusieve agarose gel, a product of FMC Bioproducts of Rockland, Maine, followed by staining with ethidium bromide and U.V. transillumination. The products are then blotted onto Hybond nylon membranes (a product of Amersham, of Arlington Heights, Illinois). The membranes are crosslinked on a 300 nm U.V. transilluminator for five minutes and prehybridized in a mixture of 5X. Denhardt's solution; 0.5% SDS; and 100 ⁇ g/ml denatured salmon sperm DNA at 55 degrees C. for one hour.
- Filters are hybridized, in the above solution with labelled oligonucleotide probe for 4-6 hours. After hybridization, the hybridized filters are exposed to Kodak XAR-5 film between intensifying screens for times ranging from one hour, at room temperature, to as long as four days at -70 degrees C. Two positive controls consisting of 1 ng of genomic B. burgdorferi DNA derived from the cloned strain N40; and five negative controls, with no DNA added, may be utilized in confirmation of the observed results.
- the aforesaid procedure was performed on ticks that had recently fed on known positive and negative animals. The ticks were then processed as described above. Positive ticks were identified by the presence of a 146 base-pair OSP-A-specific amplification product which co-migrated with the positive controls. No such amplified species was observed among the known negatives.
- the primary target for PCR detection of Borrelia burgdorferi sequences is the gene encoding the OSP-A of the reference strain B31. The gene for this protein resides on a 49 kb linear plasmid present in multiple copies per cell and it serves as a highly conserved target for the sensitive detection of most North American isolates of B. burgdorferi.
- a second primer set directed against the flagellin gene of strain B31 is used. This primer pair produces a 200 base-pair genus-specific amplification product. Borrelial species of many types, including the Borrelia hermsii and recurrentis, are detected with these primers, but not Treponema, leptospira or several exo lagellum-bearing organisms.
- flagellin gene amplification products will be confirmed by slot-blot hybridization with an internal oligonucleotide probe (FLA-2) using the materials described herein.
- the method of this invention may greatly aid in diagnosing Lyme disease before symptoms appear, and thus can significantly limit the debilitating effects of the disease.
- the method can be performed with preserved tick specimens, which can be sent to remote laboratory for analysis. While the procedure for detecting the specific spirochete DNA sequences has been described specifically for use on tick samples, it will be understood that the same procedure can be used to test human and animal specimens.
- the latter specimens can be cerebrospinal fluid, blood, urine, skin and synovial fluid.
- DNA was extracted using standard procedures from synovial fluid specimens from patients suf-fering from arthritis. These samples all cultured negative for the Lyme spirochete using the prior art culture procedures; however, a number of the samples were found to contain spirochetal DNA by amplification OSP-A sequences, suggesting ongoing infection in these patients.
Abstract
Ticks, or other samples, are tested for the presence or absence of Lyme disease spirochete infestation by subjecting the sample to a polymerase chain reaction amplification for the detection of Borrelia burgdorferi spirochete nucleic acid sequences. Spirochete-specific primers and probes are used to perform the test.
Description
TESTING FOR SPIROCHETAL NUCLEIC ACID SEQUENCES
IN SAMPLES
In the specification
This invention relates to a method of testing arthropod vectors, such as ticks, or other samples, for the presence or absence of Borrelia burgdorferi-related genetic material to determine whether the ticks are spreaders of Lyme disease.
Lyme disease has only recently been recognized by the medical profession as a distinct clinical entity. At present in the United States alone, more than six thousand new human incidences of Lyme disease occur each year. Lyme disease has been determined to be global in scope, and is not merely restricted to the Northeastern United States. Symptoms of Lyme disease may be slow to manifest themselves, and may not be diagnosed by even a skilled physician until 'after the infection has been present for many years.
At the present time, Lyme disease may only be diagnosed after telltale symptoms have manifested themselves in an infected patient. As noted above, this can entail a considerable period of time after initial infection, and thus can expose the patient to complications from the infection which are painful and even life threatening, in extreme cases.
1 -
Lyme disease is initiated in humans by bites from infected ticks. The most common source of Lyme disease, in humans, is the deer tick. Deer ticks which carry the Lyme disease-causing parasite spirochete, Borrelia burgdorferi, will infect humans with Lyme disease when bitten. As noted above, the presence of the Lyme spirochetes in a locale will not be realized until after one has been bitten by an infected tick; thus prevention of the disease, until an effective vaccine is developed, is not known to be an option.
This invention is directed to a procedure for detecting the presence or absence of Borrelia burgdorferi in the tick population. Following the precepts of this invention, ticks found in the locale can be tested on a regular basis so that, if infected ticks are noted, the local populace can take extra precautions against exposure to ticks. Ticks from bitten patients can also obviously be tested for the spirochete. It is a relatively simple procedure to gather ticks without exposing oneself to the danger of being bitten. Since ticks are attracted to changes in surrounding color and temperature, one merely needs to drag a white sheet or the like about one's yard and the sheet will pick up ticks, if there are any in the area. Additionally, if one, or one's pet is bitten by a tick, the tick can be tested after removal from the skin of the bitten subject.
The procedure of this invention can be used on dessicated tick bodies, whereby the ticks to be tested can be killed by placing them in a vial containing alcohol, wherein the ticks will be preserved, for subsequent testing.
In general, the procedure of this invention involves grinding or otherwise rupturing the tick body to release its midgut contents. The midgut contents are then subjected to a polymerase chain reaction amplification for the detection of the presence -of Borrelia-specific nucleic acid sequences in the tick midgut contents. A positive result indicates that the subject tick is a carrier of the Lyme spirochete, while a negative result suggests the opposite.
Polymerase chain reaction (PCR) amplification of particular nucleic acid sequences in a sample is a procedure deriving from Cetus Corporation of Emeryville, California. U.S. Patents Nos. 4,683,195 to K. Mullis et al; and 4,683,202 to . Mullis, both granted July 28, 1987, describe the PCR process, and are both incorporated herein in their entireties. PCR amplification is preferred to the use of conventional direct fluorescent antibody staining followed by microscopic examination of the tick material, because the PCR procedure can be used on alcohol-preserved, dessicated ticks. The fluorescent antibody procedure cannot be used on alcohol-preserved ticks because of high levels of background fluorescence.
I have developed certain new reagents for use in utilizing the PCR procedure to detect the Borrelia nucleic acid sequences in suspect ticks. My reagents for PCR amplification consist of oligonucleotide primers and probes encoding the OSP-A (outer surface protein) and flagellin proteins.
The oligonucleotide primers can be prepared on an Applied Biosystems 320A oligonucleotide synthesizer, desalted in a oligonucleotide purification cartridge, and used in the PCR reactions without further purification.
The sequences of the primers used for the OSP PCR amplification procedure (along with their optimum magnesium concentrations) are as follows:
the OSP-A gene target (1.75 mM MgCl2) :
OSP-A2: 5'GTT TTG TAA TTT CAA CTG CTG ACC 3' ; and
OSP-A4: 5'CTG CAG CTT GGA ATT CAG GCA CTT C 3'; and the probe
OSP-A3: 5*GCC ATT TGA GTC GTA TTG TTG TAC TG 3' ; and the Flagellin gene target (1.75 mM MgCl2):
FLA-l: 5'GAT GAT GCT GCT GGC ATG GGA GTT TCT 663'; and
FLA-3: 5'CTG TCT GCA TCT GAA TAT GTG CCG TTA CCT G 3' ; and the probe
FLA-2: 5'ATT CAG ACA ACA GAA GGG AAT TTA GAA GTA G 3' .
For PCR analysis, tick specimens are air dried on filter paper disks for five minutes. Two hundred μl
(dry volume) of 0.5 mm glass beads are pre-treated in 1.0 ml of 1% bovine serum albumin in distilled water at 37 degrees C. for 30 minutes and then washed twice in 1.0 ml distilled water. The pre-treatment of the glass beads is necessary to prevent the beads from adsorbing the B. burgdorferi nucleic acid sequences during the PCR procedure. The ticks are placed whole into 0.6 ml microcentrifuge tubes containing 20 μl of a slurry of the pre-treated glass beads. The tick bodies are then crushed into the beads with a disposable plastic dowel for a time sufficient to liberate the midgut contents, and 25 μl of PCR buffer (lOmM Tris HCL, pH 8.3; 50 mM KCL; 1.75 mM MgCl2; 0.01% gelatin; 0.5% NP40 detergent; and 0.5% Tween 20 brand detergent) is added. The samples are then boiled for 5 minutes, and quenched immediately on ice, whereafter 5 μl aliquots of the treated samples are removed for amplification by the PCR procedure.
For amplification of Borrelia-specific sequences, 5 μl of the tick supernatant prepared as described above, is added to a 50 μl PCR reaction composition containing: 10 mM Tris HCL, pH 8.3; 50 mM KCL; 0.01% gelatin; 200 μm each deoxynucleotide triphosphate; and 50 picomoles of each primer, along with a concentration of MgCl2 that is specific for each primer pair. All reactions can be performed in a Perkin Elmer-Cetus thermal cycler. The aforesaid components are denatured at 94 degrees C. for 30 seconds; annealed at 55 degrees C. for 45 seconds; and extended at 72 degrees C. for l minute, for a total of 45 cycles.
The amplification products are analyzed by electrophoresis in a 1% Seakem/3% Nusieve agarose gel, a product of FMC Bioproducts of Rockland, Maine, followed by staining with ethidium bromide and U.V. transillumination. The products are then blotted onto Hybond nylon membranes (a product of Amersham, of Arlington Heights, Illinois). The membranes are crosslinked on a 300 nm U.V. transilluminator for five minutes and prehybridized in a mixture of 5X. Denhardt's solution; 0.5% SDS; and 100 μg/ml denatured salmon sperm DNA at 55 degrees C. for one hour. Filters are hybridized, in the above solution with labelled oligonucleotide probe for 4-6 hours. After hybridization, the hybridized filters are exposed to Kodak XAR-5 film between intensifying screens for times ranging from one hour, at room temperature, to as long as four days at -70 degrees C. Two positive controls consisting of 1 ng of genomic B. burgdorferi DNA derived from the cloned strain N40; and five negative controls, with no DNA added, may be utilized in confirmation of the observed results.
The aforesaid procedure was performed on ticks that had recently fed on known positive and negative animals. The ticks were then processed as described above. Positive ticks were identified by the presence of a 146 base-pair OSP-A-specific amplification product which co-migrated with the positive controls. No such amplified species was observed among the known negatives.
The primary target for PCR detection of Borrelia burgdorferi sequences is the gene encoding the OSP-A of the reference strain B31. The gene for this protein resides on a 49 kb linear plasmid present in multiple copies per cell and it serves as a highly conserved target for the sensitive detection of most North American isolates of B. burgdorferi.
To verify the presence of Borrelia-specific DNA in infected specimens, a second primer set directed against the flagellin gene of strain B31 is used. This primer pair produces a 200 base-pair genus-specific amplification product. Borrelial species of many types, including the Borrelia hermsii and recurrentis, are detected with these primers, but not Treponema, leptospira or several exo lagellum-bearing organisms.
The specificity of the flagellin gene amplification products will be confirmed by slot-blot hybridization with an internal oligonucleotide probe (FLA-2) using the materials described herein.
It will be readily appreciated that the method of this invention, if applied to human or veterinary specimens, may greatly aid in diagnosing Lyme disease before symptoms appear, and thus can significantly limit the debilitating effects of the disease. The method can be performed with preserved tick specimens, which can be sent to remote laboratory for analysis.
While the procedure for detecting the specific spirochete DNA sequences has been described specifically for use on tick samples, it will be understood that the same procedure can be used to test human and animal specimens. The latter specimens can be cerebrospinal fluid, blood, urine, skin and synovial fluid.
As an example of the latter, DNA was extracted using standard procedures from synovial fluid specimens from patients suf-fering from arthritis. These samples all cultured negative for the Lyme spirochete using the prior art culture procedures; however, a number of the samples were found to contain spirochetal DNA by amplification OSP-A sequences, suggesting ongoing infection in these patients.
Since many changes and variations of the disclosed embodiment of the invention may be made without departing from the inventive concept, it is not intended to limit the invention otherwise than as required by the appended claims.
What is claimed is:
Claims
1. A method of testing ticks for the presence or absence of Borrelia burgdorferi genetic material, comprising the steps of: a) extracting the midgut contents of the ticks; and b) performing a polymerase chain reaction amplification for the detection of Borrelia-specific nucleic acid sequences -in said tick midgut contents.
2. The method of Claim 1 wherein said ticks are desiccated prior to being tested.
3. The method of Claim 2 wherein said ticks are preserved in alcohol prior to being tested.
4. A method of testing ticks for the presence or absence of Borrelia burgdorferi genetic material, said method comprising the steps of: a) treating the ticks in such a manner so as to release the tick midgut contents for analysis; and b) performing a polymerase chain reaction amplification for the detection of Borrelia-specific nucleic acid sequences in said midgut contents.
5. The method of Claim 4 comprising the further step of preserving the ticks in alcohol prior to performing the test.
6. A primer mixture for use in a nucleic acid amplification procedure for detecting the presence of Borrelia DNA material in a sample, said mixture comprising the OSP A gene primers with the following sequences:
0SP-A2: 5'GTT TTG TAA TTT CAA CTG CTG ACC 3* ; and 0SP-A4: 5'CTG CAG CTT GGA ATT CAG GCA CTT C 3' ; and the Flagellin gene primer sequences:
FLA-1: 5'GAT GAT GCT GCT GGC ATG GGA GTT TCT 663'; and FLA-3: 5'CTG TCT GGA TCT GAA TAT GTG CCG TTA CCT G 3' .
7. A probe for use in a nucleic acid amplification procedure for detecting the presence of Borrelia DNA material in a sample, said probe being:
0SP-A3: 5'GCC ATT TGA GTC GTA TTG TTG TAC T63' .
8. A probe for use in a nucleic acid amplification procedure for detecting the presence of Borrelia DNA material in a sample, said probe being:
FLA-2: 5l TT CAG ACA ACA 6AA GGG AAT TTA GAA GTA G 3 ' .
9. A method which can be performed individually, or as a step in an integrated reaction, for detecting Borrelia-specific nucleic acids in a sample, said method comprising the steps of: adding to the specimen a primer mixture for a first Borrelia gene; adding to the specimen a primer mixture for a second Borrelia gene; performing a polymerase chain reaction amplification on the sample; adding to the specimen a first probe for detecting any amplified segments of said first Borrelia gene; and adding to the specimen a second probe for detecting any amplified segments of said second Borrelia gene.
10. The method of Claim 9 wherein said gene primer is an OSP-A gene primer.
11. The method of Claim 10 wherein said second gene primer is a Flagellin gene primer.
12. A method which can be performed individually, or as a step in an integrated reaction, for detecting Borrelia-specific nucleic acids in a sample, said method comprising the steps of: adding to the specimen a primer mixture for a Borrelia gene; performing a polymerase chain reaction amplification on the sample; and adding to the specimen a probe for detecting any amplified segments of said Borrelia gene.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US61768890A | 1990-11-26 | 1990-11-26 | |
| US617,688 | 1990-11-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1992009703A1 true WO1992009703A1 (en) | 1992-06-11 |
Family
ID=24474629
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1991/008737 WO1992009703A1 (en) | 1990-11-26 | 1991-11-21 | Testing for spirochetal nucleic acid sequences in samples |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO1992009703A1 (en) |
Cited By (41)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994019488A1 (en) * | 1993-02-19 | 1994-09-01 | La Region Wallonne | Borrelia burgdorferi spirochaete-specific nucleic acid probes |
| DE19605475C1 (en) * | 1996-02-14 | 1997-11-20 | Deutsches Krebsforsch | Method of recognizing the risk of disease after arthropod sting |
| WO2009155103A2 (en) * | 2008-05-30 | 2009-12-23 | Ibis Biosciences, Inc. | Compositions for use in identification of tick-borne pathogens |
| US7956175B2 (en) | 2003-09-11 | 2011-06-07 | Ibis Biosciences, Inc. | Compositions for use in identification of bacteria |
| US7964343B2 (en) | 2003-05-13 | 2011-06-21 | Ibis Biosciences, Inc. | Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
| US8017358B2 (en) | 2001-03-02 | 2011-09-13 | Ibis Biosciences, Inc. | Method for rapid detection and identification of bioagents |
| US8026084B2 (en) | 2005-07-21 | 2011-09-27 | Ibis Biosciences, Inc. | Methods for rapid identification and quantitation of nucleic acid variants |
| US8057993B2 (en) | 2003-04-26 | 2011-11-15 | Ibis Biosciences, Inc. | Methods for identification of coronaviruses |
| US8073627B2 (en) | 2001-06-26 | 2011-12-06 | Ibis Biosciences, Inc. | System for indentification of pathogens |
| US8084207B2 (en) | 2005-03-03 | 2011-12-27 | Ibis Bioscience, Inc. | Compositions for use in identification of papillomavirus |
| US8088582B2 (en) | 2006-04-06 | 2012-01-03 | Ibis Biosciences, Inc. | Compositions for the use in identification of fungi |
| US8097416B2 (en) | 2003-09-11 | 2012-01-17 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
| US8148163B2 (en) | 2008-09-16 | 2012-04-03 | Ibis Biosciences, Inc. | Sample processing units, systems, and related methods |
| US8158936B2 (en) | 2009-02-12 | 2012-04-17 | Ibis Biosciences, Inc. | Ionization probe assemblies |
| US8158354B2 (en) | 2003-05-13 | 2012-04-17 | Ibis Biosciences, Inc. | Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
| US8163895B2 (en) | 2003-12-05 | 2012-04-24 | Ibis Biosciences, Inc. | Compositions for use in identification of orthopoxviruses |
| US8173957B2 (en) | 2004-05-24 | 2012-05-08 | Ibis Biosciences, Inc. | Mass spectrometry with selective ion filtration by digital thresholding |
| US8182992B2 (en) | 2005-03-03 | 2012-05-22 | Ibis Biosciences, Inc. | Compositions for use in identification of adventitious viruses |
| US8187814B2 (en) | 2004-02-18 | 2012-05-29 | Ibis Biosciences, Inc. | Methods for concurrent identification and quantification of an unknown bioagent |
| US8214154B2 (en) | 2001-03-02 | 2012-07-03 | Ibis Biosciences, Inc. | Systems for rapid identification of pathogens in humans and animals |
| US8268565B2 (en) | 2001-03-02 | 2012-09-18 | Ibis Biosciences, Inc. | Methods for identifying bioagents |
| US8298760B2 (en) | 2001-06-26 | 2012-10-30 | Ibis Bioscience, Inc. | Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby |
| US8407010B2 (en) | 2004-05-25 | 2013-03-26 | Ibis Biosciences, Inc. | Methods for rapid forensic analysis of mitochondrial DNA |
| US8534447B2 (en) | 2008-09-16 | 2013-09-17 | Ibis Biosciences, Inc. | Microplate handling systems and related computer program products and methods |
| US8546082B2 (en) | 2003-09-11 | 2013-10-01 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
| US8550694B2 (en) | 2008-09-16 | 2013-10-08 | Ibis Biosciences, Inc. | Mixing cartridges, mixing stations, and related kits, systems, and methods |
| US8563250B2 (en) | 2001-03-02 | 2013-10-22 | Ibis Biosciences, Inc. | Methods for identifying bioagents |
| US8822156B2 (en) | 2002-12-06 | 2014-09-02 | Ibis Biosciences, Inc. | Methods for rapid identification of pathogens in humans and animals |
| US8871471B2 (en) | 2007-02-23 | 2014-10-28 | Ibis Biosciences, Inc. | Methods for rapid forensic DNA analysis |
| US8950604B2 (en) | 2009-07-17 | 2015-02-10 | Ibis Biosciences, Inc. | Lift and mount apparatus |
| US9080209B2 (en) | 2009-08-06 | 2015-07-14 | Ibis Biosciences, Inc. | Non-mass determined base compositions for nucleic acid detection |
| US9149473B2 (en) | 2006-09-14 | 2015-10-06 | Ibis Biosciences, Inc. | Targeted whole genome amplification method for identification of pathogens |
| US9194877B2 (en) | 2009-07-17 | 2015-11-24 | Ibis Biosciences, Inc. | Systems for bioagent indentification |
| US9393564B2 (en) | 2009-03-30 | 2016-07-19 | Ibis Biosciences, Inc. | Bioagent detection systems, devices, and methods |
| US9416409B2 (en) | 2009-07-31 | 2016-08-16 | Ibis Biosciences, Inc. | Capture primers and capture sequence linked solid supports for molecular diagnostic tests |
| US9598724B2 (en) | 2007-06-01 | 2017-03-21 | Ibis Biosciences, Inc. | Methods and compositions for multiple displacement amplification of nucleic acids |
| US9719083B2 (en) | 2009-03-08 | 2017-08-01 | Ibis Biosciences, Inc. | Bioagent detection methods |
| US9758840B2 (en) | 2010-03-14 | 2017-09-12 | Ibis Biosciences, Inc. | Parasite detection via endosymbiont detection |
| CN107475411A (en) * | 2017-09-19 | 2017-12-15 | 重庆出入境检验检疫局检验检疫技术中心 | A kind of Taqman real-time fluorescence PCR detection methods for detecting Borrelia burgdoyferi nucleic acid |
| US9873906B2 (en) | 2004-07-14 | 2018-01-23 | Ibis Biosciences, Inc. | Methods for repairing degraded DNA |
| US9890408B2 (en) | 2009-10-15 | 2018-02-13 | Ibis Biosciences, Inc. | Multiple displacement amplification |
-
1991
- 1991-11-21 WO PCT/US1991/008737 patent/WO1992009703A1/en unknown
Non-Patent Citations (1)
| Title |
|---|
| JOURNAL OF CLINICAL MICROBIOLOGY, Volume 28, No. 3, issued March 1990, PERSING et al., "Detection of Borrelia burgdorferi Infection in Ixodus dammini Ticks with the Polymerse Chain Reaction", pages 566-572. * |
Cited By (68)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994019488A1 (en) * | 1993-02-19 | 1994-09-01 | La Region Wallonne | Borrelia burgdorferi spirochaete-specific nucleic acid probes |
| BE1006728A3 (en) * | 1993-02-19 | 1994-11-29 | Wallone Region | PROBES Nucleic Acids spirochete Borrelia burgdorferi SPECIFIC. |
| US5817460A (en) * | 1993-02-19 | 1998-10-06 | La Region Wallonne | Nucleic acid probes specific to the spirochete Borrelia burgdorferi |
| DE19605475C1 (en) * | 1996-02-14 | 1997-11-20 | Deutsches Krebsforsch | Method of recognizing the risk of disease after arthropod sting |
| US9752184B2 (en) | 2001-03-02 | 2017-09-05 | Ibis Biosciences, Inc. | Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy |
| US8214154B2 (en) | 2001-03-02 | 2012-07-03 | Ibis Biosciences, Inc. | Systems for rapid identification of pathogens in humans and animals |
| US8563250B2 (en) | 2001-03-02 | 2013-10-22 | Ibis Biosciences, Inc. | Methods for identifying bioagents |
| US8265878B2 (en) | 2001-03-02 | 2012-09-11 | Ibis Bioscience, Inc. | Method for rapid detection and identification of bioagents |
| US8815513B2 (en) | 2001-03-02 | 2014-08-26 | Ibis Biosciences, Inc. | Method for rapid detection and identification of bioagents in epidemiological and forensic investigations |
| US8017358B2 (en) | 2001-03-02 | 2011-09-13 | Ibis Biosciences, Inc. | Method for rapid detection and identification of bioagents |
| US8802372B2 (en) | 2001-03-02 | 2014-08-12 | Ibis Biosciences, Inc. | Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy |
| US8268565B2 (en) | 2001-03-02 | 2012-09-18 | Ibis Biosciences, Inc. | Methods for identifying bioagents |
| US9416424B2 (en) | 2001-03-02 | 2016-08-16 | Ibis Biosciences, Inc. | Methods for rapid identification of pathogens in humans and animals |
| US8921047B2 (en) | 2001-06-26 | 2014-12-30 | Ibis Biosciences, Inc. | Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby |
| US8073627B2 (en) | 2001-06-26 | 2011-12-06 | Ibis Biosciences, Inc. | System for indentification of pathogens |
| US8380442B2 (en) | 2001-06-26 | 2013-02-19 | Ibis Bioscience, Inc. | Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby |
| US8298760B2 (en) | 2001-06-26 | 2012-10-30 | Ibis Bioscience, Inc. | Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby |
| US9725771B2 (en) | 2002-12-06 | 2017-08-08 | Ibis Biosciences, Inc. | Methods for rapid identification of pathogens in humans and animals |
| US8822156B2 (en) | 2002-12-06 | 2014-09-02 | Ibis Biosciences, Inc. | Methods for rapid identification of pathogens in humans and animals |
| US8057993B2 (en) | 2003-04-26 | 2011-11-15 | Ibis Biosciences, Inc. | Methods for identification of coronaviruses |
| US8476415B2 (en) | 2003-05-13 | 2013-07-02 | Ibis Biosciences, Inc. | Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
| US8158354B2 (en) | 2003-05-13 | 2012-04-17 | Ibis Biosciences, Inc. | Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
| US7964343B2 (en) | 2003-05-13 | 2011-06-21 | Ibis Biosciences, Inc. | Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
| US8242254B2 (en) | 2003-09-11 | 2012-08-14 | Ibis Biosciences, Inc. | Compositions for use in identification of bacteria |
| US8097416B2 (en) | 2003-09-11 | 2012-01-17 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
| US8546082B2 (en) | 2003-09-11 | 2013-10-01 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
| US8013142B2 (en) | 2003-09-11 | 2011-09-06 | Ibis Biosciences, Inc. | Compositions for use in identification of bacteria |
| US8394945B2 (en) | 2003-09-11 | 2013-03-12 | Ibis Biosciences, Inc. | Compositions for use in identification of bacteria |
| US8288523B2 (en) | 2003-09-11 | 2012-10-16 | Ibis Biosciences, Inc. | Compositions for use in identification of bacteria |
| US7956175B2 (en) | 2003-09-11 | 2011-06-07 | Ibis Biosciences, Inc. | Compositions for use in identification of bacteria |
| US8163895B2 (en) | 2003-12-05 | 2012-04-24 | Ibis Biosciences, Inc. | Compositions for use in identification of orthopoxviruses |
| US8187814B2 (en) | 2004-02-18 | 2012-05-29 | Ibis Biosciences, Inc. | Methods for concurrent identification and quantification of an unknown bioagent |
| US9447462B2 (en) | 2004-02-18 | 2016-09-20 | Ibis Biosciences, Inc. | Methods for concurrent identification and quantification of an unknown bioagent |
| US8173957B2 (en) | 2004-05-24 | 2012-05-08 | Ibis Biosciences, Inc. | Mass spectrometry with selective ion filtration by digital thresholding |
| US9449802B2 (en) | 2004-05-24 | 2016-09-20 | Ibis Biosciences, Inc. | Mass spectrometry with selective ion filtration by digital thresholding |
| US8987660B2 (en) | 2004-05-24 | 2015-03-24 | Ibis Biosciences, Inc. | Mass spectrometry with selective ion filtration by digital thresholding |
| US8407010B2 (en) | 2004-05-25 | 2013-03-26 | Ibis Biosciences, Inc. | Methods for rapid forensic analysis of mitochondrial DNA |
| US9873906B2 (en) | 2004-07-14 | 2018-01-23 | Ibis Biosciences, Inc. | Methods for repairing degraded DNA |
| US8084207B2 (en) | 2005-03-03 | 2011-12-27 | Ibis Bioscience, Inc. | Compositions for use in identification of papillomavirus |
| US8182992B2 (en) | 2005-03-03 | 2012-05-22 | Ibis Biosciences, Inc. | Compositions for use in identification of adventitious viruses |
| US8551738B2 (en) | 2005-07-21 | 2013-10-08 | Ibis Biosciences, Inc. | Systems and methods for rapid identification of nucleic acid variants |
| US8026084B2 (en) | 2005-07-21 | 2011-09-27 | Ibis Biosciences, Inc. | Methods for rapid identification and quantitation of nucleic acid variants |
| US8088582B2 (en) | 2006-04-06 | 2012-01-03 | Ibis Biosciences, Inc. | Compositions for the use in identification of fungi |
| US9149473B2 (en) | 2006-09-14 | 2015-10-06 | Ibis Biosciences, Inc. | Targeted whole genome amplification method for identification of pathogens |
| US8871471B2 (en) | 2007-02-23 | 2014-10-28 | Ibis Biosciences, Inc. | Methods for rapid forensic DNA analysis |
| US9598724B2 (en) | 2007-06-01 | 2017-03-21 | Ibis Biosciences, Inc. | Methods and compositions for multiple displacement amplification of nucleic acids |
| WO2009155103A3 (en) * | 2008-05-30 | 2010-03-18 | Ibis Biosciences, Inc. | Compositions for use in identification of tick-borne pathogens |
| WO2009155103A2 (en) * | 2008-05-30 | 2009-12-23 | Ibis Biosciences, Inc. | Compositions for use in identification of tick-borne pathogens |
| US8148163B2 (en) | 2008-09-16 | 2012-04-03 | Ibis Biosciences, Inc. | Sample processing units, systems, and related methods |
| US9027730B2 (en) | 2008-09-16 | 2015-05-12 | Ibis Biosciences, Inc. | Microplate handling systems and related computer program products and methods |
| US8252599B2 (en) | 2008-09-16 | 2012-08-28 | Ibis Biosciences, Inc. | Sample processing units, systems, and related methods |
| US9023655B2 (en) | 2008-09-16 | 2015-05-05 | Ibis Biosciences, Inc. | Sample processing units, systems, and related methods |
| US8534447B2 (en) | 2008-09-16 | 2013-09-17 | Ibis Biosciences, Inc. | Microplate handling systems and related computer program products and methods |
| US8550694B2 (en) | 2008-09-16 | 2013-10-08 | Ibis Biosciences, Inc. | Mixing cartridges, mixing stations, and related kits, systems, and methods |
| US8609430B2 (en) | 2008-09-16 | 2013-12-17 | Ibis Biosciences, Inc. | Sample processing units, systems, and related methods |
| US8796617B2 (en) | 2009-02-12 | 2014-08-05 | Ibis Biosciences, Inc. | Ionization probe assemblies |
| US9165740B2 (en) | 2009-02-12 | 2015-10-20 | Ibis Biosciences, Inc. | Ionization probe assemblies |
| US8158936B2 (en) | 2009-02-12 | 2012-04-17 | Ibis Biosciences, Inc. | Ionization probe assemblies |
| US9719083B2 (en) | 2009-03-08 | 2017-08-01 | Ibis Biosciences, Inc. | Bioagent detection methods |
| US9393564B2 (en) | 2009-03-30 | 2016-07-19 | Ibis Biosciences, Inc. | Bioagent detection systems, devices, and methods |
| US8950604B2 (en) | 2009-07-17 | 2015-02-10 | Ibis Biosciences, Inc. | Lift and mount apparatus |
| US9194877B2 (en) | 2009-07-17 | 2015-11-24 | Ibis Biosciences, Inc. | Systems for bioagent indentification |
| US9416409B2 (en) | 2009-07-31 | 2016-08-16 | Ibis Biosciences, Inc. | Capture primers and capture sequence linked solid supports for molecular diagnostic tests |
| US10119164B2 (en) | 2009-07-31 | 2018-11-06 | Ibis Biosciences, Inc. | Capture primers and capture sequence linked solid supports for molecular diagnostic tests |
| US9080209B2 (en) | 2009-08-06 | 2015-07-14 | Ibis Biosciences, Inc. | Non-mass determined base compositions for nucleic acid detection |
| US9890408B2 (en) | 2009-10-15 | 2018-02-13 | Ibis Biosciences, Inc. | Multiple displacement amplification |
| US9758840B2 (en) | 2010-03-14 | 2017-09-12 | Ibis Biosciences, Inc. | Parasite detection via endosymbiont detection |
| CN107475411A (en) * | 2017-09-19 | 2017-12-15 | 重庆出入境检验检疫局检验检疫技术中心 | A kind of Taqman real-time fluorescence PCR detection methods for detecting Borrelia burgdoyferi nucleic acid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO1992009703A1 (en) | Testing for spirochetal nucleic acid sequences in samples | |
| US20220251618A1 (en) | Amplicon rescue multiplex polymerase chain reaction for amplification of multiple targets | |
| Malloy et al. | Detection of Borrelia burgdorferi using the polymerase chain reaction | |
| Gomes et al. | Trypanosoma cruzi: optimization of polymerase chain reaction for detection in human blood | |
| Sritharan et al. | A simple method for diagnosing M. tuberculosis infection in clinical samples using PCR | |
| Schmidt | PCR in laboratory diagnosis of human Borrelia burgdorferi infections | |
| Navarro et al. | Diagnosis of human brucellosis using PCR | |
| Leyla et al. | Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples | |
| Hofmeister et al. | Cosegregation of a novel Bartonella species with Borrelia burgdorferi and Babesia microti in Peromyscus leucopus | |
| Andresen et al. | Diagnosis of visceral leishmaniasis by the polymerase chain reaction using blood, bone marrow and lymph node samples from patients from the Sudan | |
| KR101798211B1 (en) | Method for the detection and characterization of a toxinogenic Clostridium difficile strain | |
| Gwaltney et al. | Detection of Eperythrozoon suis using the polymerase chain reaction | |
| EP0119209A1 (en) | Identification of microorganisms | |
| Vojdani et al. | Multiplex PCR for the detection of mycoplasma fermentans, m. hominis, and m. penetrans in patients with chronic fatigue syndrome, fibromyalgia, rheumatoid arthritis, and Gulf War syndrome | |
| RU2360972C1 (en) | Method detection and evaluation of biotype, serogroup and toxigenicity of cholera agent and related kit | |
| Chu | Rapid and sensitive PCR-based detection and differentiation of aetiologic agents of human granulocytotropic and monocytotropic ehrlichiosis | |
| Khosravi et al. | ISOLATION OF BRUCELLA MELITENSISAND BRUCELLA ABORTUS FROM BRUCELLOSIS PATIENTS BY CONVENTIONAL CULTURE METHOD AND POLYMERASE CHAIN REACTION TECHNIQUE | |
| US6087097A (en) | PCR detection of Borrelia burgdorferi | |
| JPH03206899A (en) | Nucleic acid probe and method for use in detection of cryptocox neoholmance | |
| Schmitt et al. | Evaluation of PCR for diagnosis of bovine viral diarrhea virus in tissue homogenates | |
| RU2756924C2 (en) | Method for early pcr diagnostics of the aleutian mink disease virus carnivore amdoparvovirus | |
| JP2012528571A (en) | Advanced pathogen detection and screening | |
| RU2732626C1 (en) | System of oligonucleotide primers and probe for detecting dna mycoplasma bovigenitalium | |
| El-Zaatari et al. | Uses and cautions for use of polymerase chain reaction for detection of Helicobacter pylori | |
| Simpson et al. | The use of RAPDs for the analysis of parasites |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A1 Designated state(s): JP |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE |