CA1313111C - Method of identifying unknown organisms - Google Patents
Method of identifying unknown organismsInfo
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- CA1313111C CA1313111C CA000553239A CA553239A CA1313111C CA 1313111 C CA1313111 C CA 1313111C CA 000553239 A CA000553239 A CA 000553239A CA 553239 A CA553239 A CA 553239A CA 1313111 C CA1313111 C CA 1313111C
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- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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Abstract
ABSTRACT OF THE DISCLOSURE:
A direct test for bacterial organisms uses organism specific bacteriophage to combine with the target organisms.
Labels on the bacteriophage provide positive identification for the bacterial species or genus. Radioactive, fluorescent and enzymatic labels may be used.
A direct test for bacterial organisms uses organism specific bacteriophage to combine with the target organisms.
Labels on the bacteriophage provide positive identification for the bacterial species or genus. Radioactive, fluorescent and enzymatic labels may be used.
Description
~3~t ~ ~
METHOD OF IDENTIFYING UNKNOWN ORGANISMS
Backaround and Summary o the Invention:
Various tests procedures are known for detecting the presence of infectious organisms. Indirect methods typically test for the presence of antibodies produced by a host in response to the presence of infectious organisms. Direct testing methods may also be used, for example, specimens may be cultured to grow an organism and the organism can then be identified. Other test procedures have also been used in the art. Applicants are aware of the following U.S. patents which disclose a variety of microbiological test procedures.
U.S. Patent No. 3,654,090 U.S. Patent No. 4,~56,834 U.S. Patent No. 3,791,g32 U.S. Patent No. 4,281,061 U.S. Patent No. 3,826,613 U.S. Patent No. 4,287,300 U.S. Patent No. 3,867,517 U.S. Patent No. 4,308,348 U.S. Patent No. 4,016,043 U.S. Patent No. 4,347,311 U.S. Patent No. 4,098,876 U.S. Patent No. 4,374,925 U.S. Patent No. 4,104,126 U.S. Patent No. 4,376,110 U.S. Patent No. 4,244,940 U.S. Patent No. 4,469,786 The disclosures of the above patents may be referred to for further details of such procedures.
In the main, both indirect and direct testing procedures have been cumbersome and time consuming. Direct testing methods are often slow. For example, it may take several days, for an organism to be cultured successively, before a certain identification can be made. Other test procedures have other short comings, in particular, many test procedures have a high incidence of false positives and false negatives.
Applicants have discovered a new test method for directly testing for the presence of unknown organisms. Applicants method is fast acting and produces a test result which has a high degree of certainty. Applicants test procedure provides qualitative and quantitative evaluation of unknown organisms.
The test may be used to identify a particular species, type or ''' ~
13t31 1 1 genus.
Applicants method uses a specific agent which rapidly attaches to a target test organism. The agent is labeled, for positive identification. Applicant's method may use a variety 5 of conventional labeling or reporting techniques. For example, the agent may be marked or tagged for identification using radioactive or chemical marking, for example, peroxidase labeling, fluorescent labeling, biotin labeling or other identification for the agent. The marked or tagged agent is 10 then brought into contact with a test unknown organism. If the agent attaches to the organism its presence can be determined by detecting the tag or marker to provide a positive identification for that particular organism. Typically, applicants' agent is a bacteriophage. It is generally accepted that bacteriophages are 15 narrowly restricted in their range of host bacteria. Some bacteriophages are specific for a particular species of bacteria. However, there are also some bacteriophages which are specific to particular genuses.
Applicants have discovered that bacteriophages may be 20 labeled or tagged, as described herein, and can then be used to identify a bacterium, based on the binding or non-binding to that particular bacteria species, type, or genus. Since a bacteriophage is smaller than the organism to which it binds, free bacteriophage can be separated from bacteria by filtration through an appropriately sized medium, for example, inert filter paper. The filter traps the bacteria and permits the unbound smaller bacteriophage to pass through the filter. However, if the bacteria is the one to which the bacteriophage is specific, the bacteriophage will bind to the bacteria within a very short time. If the test specimen is filtered after this time has elapsed, the bacteria will be trapped on the filter with the bacteriophage attached. If the bacteriophage is labeled its presence can be determined by developing the tag or marker and the presence of that specific bacteria to which the particular bacteriophage attaches can then be determined. Of `` 1 31 3 î ~ 1 course, the greater the number of the sp~cific bacterial organisms present the greater the number of bacteriophage which will be found attached to the specimen on the filter, yielding a greater signal from the reporter groups.
Consequently, applicants' invention seeks to provide a specific test method for determining the presence of unknown organisms.
Further, applicants' invention seeks to provide a test method by which a labeled agent rapidly attaches to a specific microorganism to identify that organism.
Still further, applicants' invention seeks to provide a test method for unknown microorganisms which acts rapidly and with a high degree of reliability.
Further still, applicants invention seeks to provide a test reagent for determining the presence of organisms by labeling an organism specific agent.
Thus, the invention in one broadly claimed aspect provides a method of identifying specific organisms comprising selecting a binding agent for the specific organism, labeling the binding agent, contacting the labeled binding agent with a specimen of an unknown organism, binding the binding agent to the specific organism present in the specimen, separating the bound organism and binding agent from the specimen and testing to determine the presence of the label on the bound organisms.
Another broad aspect provides a specific test reagent for directly identifying salmonella type bacterial organisms comprising a specific bacteriophage capable of binding to the salmonella type bacteria, the bacteriophage having an identifiable label thereon.
Applicants invention may be further understood by reference to the following Description of the Preferred Embodiments.
i ~3~
r .
~3~31 1 1 DESCRIPTION OF THE PREFERRED EMBODIMENT~:
Bacteriophages are easy to acquire. Suitable wild bacteriophage may be harvested from nature, isolated very readily by conventional means and grown to substantial quantity on the specific host bacterium. Applicants have harvested and isolated bacteriopha~e specific for Salmonella, E. coli, 5taphylococcus aureus and Pseudomonas, among others. In addition, common bacteriophages, for example, T1, T2, T3, T4 and lambda, which are specific for E. coli, are readily available, as are other known bacteriophages. ATCC 15693-81 is specific for Pseudomonas aerugenosa, ATCC 6538P is specific for -3a-Staphylococcus aureus, ATCC 6051-B1 is specifia for Bacillus subtilus and ATCC 2307~-Bl i~ speci~ic for Listeria monooytogenes. Naturally available and collected baateriophage are specific for Camphylobacter, Mycobacterium tuberculosis and others.
The ready availability and rapid growth of cultures o~
bacteriophage is of particular advantage in using baaterlophage as a test reagent. In comparison, monoclonal and polyclonal antibodies are difficult to obtain in high yield and high degree of purity.
Bacteriophages rapidly bond to the outside of host bacteria and, though infecting the bacteria, leave the shell of the bacteriophage present on the surface of the host. The properties of bacteriophages in binding to a host are noted in Re combinant DNA A Short Course, Watson et al. pages 14, 15, 23 and 24, W.H. Freeman & Company, New York 1983 and have been noted in "The Mechanism of Virus Attachment to Host Cells. IV.
Physiochemical Studies on Virus and Cell Surface Groups," Arch.
~ioçhem. Biophys. Vol. 51 (1954), Puck et al., pages 229 through 245.
Bacteriophage is particularly advantageous as a test reagent in that relative to its host bacterium the bacteriophage is relatively large, enabling the tester to bind sizeable quantities of tags or marker elements, such asj enzyme markers, fluorescent markers, or radioactive markers, to the individual bacteriophage. The protein shell of the bacteriophage remains outside and attached to the bacterium after infecting the host bacterium so that the presence of the bacteriophage can be readily identified. Though being relatively large, the bacteriophage is also sufficiently smaller than the host bacteria to permit the unattached bacteriophage to be readily separated from the bacteria in a test specimen by filtration.
Labeling bacteriophages to provide a marker that identifies the presence of the bacteriophage or the bacteriophage bound to a host can be readily accomplished. The shell of bacteriophage is proteinaceous and a variety of markers can be bound to the bacteriophage surface by using a protein linker. For example, the enzyme horseradish peroxidase may be linked to a bacteriophage with a bifunctional cross linking reagent, dimethyl suberimidate 2HCl, an imidoester. The general reaction scheme is as follows:
, Cl /~ ~2 C
--c ` ~ ~-N~ _C
\O C ~ J ~ ~ ( R = PROTEIN ) generating a bacteriophage-linker-peroxidase complex. Other enzymes may also be used, for example, alkaline phosphatase and beta-galactosidase may also be used as well as the use of other bifunctional cross linkers such as other amino reacting cross linkers and thiol reacting cross linkers. Fluorescent labels may be attached by using a fluorescent label-fluorescene isothiocyanate through the epsilon-amino groups of lysine found in the bacteriophage capsomeres. Other fluorescent molecules such as rhodamine may also be used in a similar manner.
Labeling the bacteriophages with biotin type reporter groups using a succinimide ester group may also be used. Other conventional secondary labeling sub6tance~ may also be incorporated.
Radioactive labeling of the bacteriophages with various radioactive compounds could also be used, for example, radioactive iodine 125 may be linked to the protein of the bacteriophage shell by a conventional reaction.
Applicants have found that the Nakane method of binding horseradish peroxidase is particularly useful. ~he Nakane method is described in Standard Biochemical Methods. The procedure is as follows: Horseradish peroxidase is reacted with 0.032M formaldehyde in the presence of a 0.30M NaHC03 buffer, pH 8.1 to form HRP=CH2. ~he HRP=CH2 is reduced with 1 mg NaBH4/mg enzyme to form HRP-CH3 (methylated .
, .
, 1 3t ~ 1 1 1 horseradish peroxidase). ~h~ HRP-CH3 i5 bu~ered with 0.30M
NaHC03 and separated by chromotography on a 0.5m exclusion column. The HRP-CH3 is then oxidized with 0.04M NaI04 to form HRP-CH0 (horseradish peroxidase aldehyde). The HRP-CH0 is buffered to pH 9.0 with a Na2C03 buffer and separated on a O.5m exclusion column to produce the activated HRP enzyme. The bacteriophage is grown on a host bacterium culture, for example S. typhimurium. The lysed bacteriophage is separated from the culture through a 0.45 micron filter. The activated enzyme is added to the lysed bacteriophage and reacted for 4 to 6 hours at room temperature. The enzyme readily bonds to the protein coating of the bacteriophage, probably be attaching to lysine present in the protein coating. The tagged bacteriophage is buffered to pH 7.0 with a 50mM PO4 buffer and separated on an 0.5m exclusion column. The separated tagged bacteriophage is then assayed and adjusted for titer.
EXAMPLE I Salmonella S~ecies Type Test:
A Salmonella specific bacteriophage was harvested from nature and isolated on a host culture of Salmonella typhimurium. (A culture of this bacteriophage, designated ATCC
Number 40282, has been deposited with the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Maryland 20852 and is available to the public from the permanent callection.) After isolation and purification on a Salmonella typhimurium host culture, the bacteriophage was labeled with horseradish - peroxidase using the Nakane method. The labeled baateriophage was then tested against representative Salmonella species and the serologically closely related species of, Citrobacter, as shown in Table I. The Salmonella and Citrobacter organisms were graciously provided by Alma Murlin from her collection at the National Center for Disease Control, Atlanta, Georgia and by the USDA Veterinary Services Center, Amesj Iowa. The Citrobacter, Salmonella type, was provided by the Center for Disease Control, Atlanta, Georgia.
- ~ .. . . : .
Table I
oraanism Colorimetric Test Salmonella. Group ~
worthington Positive anatum Positive cholerae var. suis Positive newington Positive paratyphi B Positive montevideo Positive typhi Positive heidelberg Positive typhimurium Positive portsmouth Positive johannesburg Positive laardt Positive poona Positive berta Positive meleagridis Positive infantis Positive enteritidis Positive pomona Positive jauiana Positive paratyphi A Positive newport Positive agoha Positive cereo Positive brithday Positive luciana Positive london Positive 30 westerstead Positive tennessee Positive ' . ~, ' .
newbrunswick Positlve gaminara) Positive florida Positive alachua Positive krefold Positive paratyphi A Positive dublin Positive seftenberg Positive paratyphi C Positive drypool Positive inverness Positive barrilly Positive minnesota Positive newington Positive paratyphi A, japan Positive cereo Positive Salmonella. Grou~ 2 phoenix Positive neb-M23037 Positive Salmonella, Group 3 3a Positive 3b Positive Salmonella, Group 4 flint Positive 25 marina Positive Salmonella~ Group 5 brookfield Positive bongar Positive Citrobacter 30 Citrobacter spp., Salmonella type Negative freundii Negative amalonaticus Negative The test procedure for each organism listed in the Table I
was as follows: A culture of the organism was slurried to a concentration of 106 organisms per ml. A 100 ul sample of the organism slurry was combined with 50 ul of the labeled bacteriophage reagent. The reagent wa~ ~tandardized to a titer of lo8 p.f.u. The reagent and Salmonella sample (and Citrobacter~ were incubated for 20 minutes. After incubation the mixture was filtered through a 0.45 micron filter with vacuum. The filtered mixture was washed twice using a phosphate buffered saline solution containing o.05% TWEEN 20~TM). The last wash was vacuumed to dryness. A 100 ul saturated solution of para-para-biphenol, buffered to pH 7.0 by a 50m phosphate buffer, (substrate) was added to the filter and incubated for 15 minutes. A characteristic brown color indicated a positive test in all cases (presence of labeled bacteriophage on the filtered bacteria). All reactions were at room temperature.
Example II Reliability:
A test for reliability of the labeled bacteriophage was conducted on the array of bacteria shown in Table II and Table III. The bacteriophage used were obtained from the American Type Culture Collection, with the exception of applicants' Salumonella bacteriophage. The bacteriophage were specific for the particular organism, as noted herein. Each bacteriophage used was labeled with horseradish peroxidase using the Nakane method. The tests were conducted as described in Example I and developed using para-para-biphenol.
: .
~: , . , -: .
~Q~;
Labeled Test Number of Number of Num~er of Percent Bacteriophage Organism True False Tests False Positive Negative Negative ATCC 40282 Citrobacter, 0 0 16 0.00 Salmonella type T4 E. coli 162 15 177 8.47 ATCC 15692-Bl Pseudomonas 273 9 282 3.19 aeruginosa ATCC 40282 Salmonella 792 3 795 0.38 typhimurium ATCC 6538P Staph 181 17 198 8.59 aureus Total 44 1468 3.00 Table III
Labeled Test Number of Number of Number of Percent Bacteriophage Organism True False Tests False Negative Positive Positive -ATCC 40282 Citrobacter, 15 1 16 6.25 Salmonella type present T4 E. coli 2730 0 2730 0.00 not present ATCC 15692-B1 Pseudomonas 2616 9 2625 0.34 aeruginosa not present ATCC 40282 Salmonella 2313 3 2316 0.13 typhimurium not present ATCC 6538P Staph 2709 0 2709 0.00 aureus not present Total 13 10396 0.13 .
:
It will be appreciated by those s~illed in the art that various modifications may be made to the invention disclosed herein. The invention is not to be limited to the cpecific embodiments given herein for purposes of illustration, but is limited only by the scope of the appended claims and their equivalents.
METHOD OF IDENTIFYING UNKNOWN ORGANISMS
Backaround and Summary o the Invention:
Various tests procedures are known for detecting the presence of infectious organisms. Indirect methods typically test for the presence of antibodies produced by a host in response to the presence of infectious organisms. Direct testing methods may also be used, for example, specimens may be cultured to grow an organism and the organism can then be identified. Other test procedures have also been used in the art. Applicants are aware of the following U.S. patents which disclose a variety of microbiological test procedures.
U.S. Patent No. 3,654,090 U.S. Patent No. 4,~56,834 U.S. Patent No. 3,791,g32 U.S. Patent No. 4,281,061 U.S. Patent No. 3,826,613 U.S. Patent No. 4,287,300 U.S. Patent No. 3,867,517 U.S. Patent No. 4,308,348 U.S. Patent No. 4,016,043 U.S. Patent No. 4,347,311 U.S. Patent No. 4,098,876 U.S. Patent No. 4,374,925 U.S. Patent No. 4,104,126 U.S. Patent No. 4,376,110 U.S. Patent No. 4,244,940 U.S. Patent No. 4,469,786 The disclosures of the above patents may be referred to for further details of such procedures.
In the main, both indirect and direct testing procedures have been cumbersome and time consuming. Direct testing methods are often slow. For example, it may take several days, for an organism to be cultured successively, before a certain identification can be made. Other test procedures have other short comings, in particular, many test procedures have a high incidence of false positives and false negatives.
Applicants have discovered a new test method for directly testing for the presence of unknown organisms. Applicants method is fast acting and produces a test result which has a high degree of certainty. Applicants test procedure provides qualitative and quantitative evaluation of unknown organisms.
The test may be used to identify a particular species, type or ''' ~
13t31 1 1 genus.
Applicants method uses a specific agent which rapidly attaches to a target test organism. The agent is labeled, for positive identification. Applicant's method may use a variety 5 of conventional labeling or reporting techniques. For example, the agent may be marked or tagged for identification using radioactive or chemical marking, for example, peroxidase labeling, fluorescent labeling, biotin labeling or other identification for the agent. The marked or tagged agent is 10 then brought into contact with a test unknown organism. If the agent attaches to the organism its presence can be determined by detecting the tag or marker to provide a positive identification for that particular organism. Typically, applicants' agent is a bacteriophage. It is generally accepted that bacteriophages are 15 narrowly restricted in their range of host bacteria. Some bacteriophages are specific for a particular species of bacteria. However, there are also some bacteriophages which are specific to particular genuses.
Applicants have discovered that bacteriophages may be 20 labeled or tagged, as described herein, and can then be used to identify a bacterium, based on the binding or non-binding to that particular bacteria species, type, or genus. Since a bacteriophage is smaller than the organism to which it binds, free bacteriophage can be separated from bacteria by filtration through an appropriately sized medium, for example, inert filter paper. The filter traps the bacteria and permits the unbound smaller bacteriophage to pass through the filter. However, if the bacteria is the one to which the bacteriophage is specific, the bacteriophage will bind to the bacteria within a very short time. If the test specimen is filtered after this time has elapsed, the bacteria will be trapped on the filter with the bacteriophage attached. If the bacteriophage is labeled its presence can be determined by developing the tag or marker and the presence of that specific bacteria to which the particular bacteriophage attaches can then be determined. Of `` 1 31 3 î ~ 1 course, the greater the number of the sp~cific bacterial organisms present the greater the number of bacteriophage which will be found attached to the specimen on the filter, yielding a greater signal from the reporter groups.
Consequently, applicants' invention seeks to provide a specific test method for determining the presence of unknown organisms.
Further, applicants' invention seeks to provide a test method by which a labeled agent rapidly attaches to a specific microorganism to identify that organism.
Still further, applicants' invention seeks to provide a test method for unknown microorganisms which acts rapidly and with a high degree of reliability.
Further still, applicants invention seeks to provide a test reagent for determining the presence of organisms by labeling an organism specific agent.
Thus, the invention in one broadly claimed aspect provides a method of identifying specific organisms comprising selecting a binding agent for the specific organism, labeling the binding agent, contacting the labeled binding agent with a specimen of an unknown organism, binding the binding agent to the specific organism present in the specimen, separating the bound organism and binding agent from the specimen and testing to determine the presence of the label on the bound organisms.
Another broad aspect provides a specific test reagent for directly identifying salmonella type bacterial organisms comprising a specific bacteriophage capable of binding to the salmonella type bacteria, the bacteriophage having an identifiable label thereon.
Applicants invention may be further understood by reference to the following Description of the Preferred Embodiments.
i ~3~
r .
~3~31 1 1 DESCRIPTION OF THE PREFERRED EMBODIMENT~:
Bacteriophages are easy to acquire. Suitable wild bacteriophage may be harvested from nature, isolated very readily by conventional means and grown to substantial quantity on the specific host bacterium. Applicants have harvested and isolated bacteriopha~e specific for Salmonella, E. coli, 5taphylococcus aureus and Pseudomonas, among others. In addition, common bacteriophages, for example, T1, T2, T3, T4 and lambda, which are specific for E. coli, are readily available, as are other known bacteriophages. ATCC 15693-81 is specific for Pseudomonas aerugenosa, ATCC 6538P is specific for -3a-Staphylococcus aureus, ATCC 6051-B1 is specifia for Bacillus subtilus and ATCC 2307~-Bl i~ speci~ic for Listeria monooytogenes. Naturally available and collected baateriophage are specific for Camphylobacter, Mycobacterium tuberculosis and others.
The ready availability and rapid growth of cultures o~
bacteriophage is of particular advantage in using baaterlophage as a test reagent. In comparison, monoclonal and polyclonal antibodies are difficult to obtain in high yield and high degree of purity.
Bacteriophages rapidly bond to the outside of host bacteria and, though infecting the bacteria, leave the shell of the bacteriophage present on the surface of the host. The properties of bacteriophages in binding to a host are noted in Re combinant DNA A Short Course, Watson et al. pages 14, 15, 23 and 24, W.H. Freeman & Company, New York 1983 and have been noted in "The Mechanism of Virus Attachment to Host Cells. IV.
Physiochemical Studies on Virus and Cell Surface Groups," Arch.
~ioçhem. Biophys. Vol. 51 (1954), Puck et al., pages 229 through 245.
Bacteriophage is particularly advantageous as a test reagent in that relative to its host bacterium the bacteriophage is relatively large, enabling the tester to bind sizeable quantities of tags or marker elements, such asj enzyme markers, fluorescent markers, or radioactive markers, to the individual bacteriophage. The protein shell of the bacteriophage remains outside and attached to the bacterium after infecting the host bacterium so that the presence of the bacteriophage can be readily identified. Though being relatively large, the bacteriophage is also sufficiently smaller than the host bacteria to permit the unattached bacteriophage to be readily separated from the bacteria in a test specimen by filtration.
Labeling bacteriophages to provide a marker that identifies the presence of the bacteriophage or the bacteriophage bound to a host can be readily accomplished. The shell of bacteriophage is proteinaceous and a variety of markers can be bound to the bacteriophage surface by using a protein linker. For example, the enzyme horseradish peroxidase may be linked to a bacteriophage with a bifunctional cross linking reagent, dimethyl suberimidate 2HCl, an imidoester. The general reaction scheme is as follows:
, Cl /~ ~2 C
--c ` ~ ~-N~ _C
\O C ~ J ~ ~ ( R = PROTEIN ) generating a bacteriophage-linker-peroxidase complex. Other enzymes may also be used, for example, alkaline phosphatase and beta-galactosidase may also be used as well as the use of other bifunctional cross linkers such as other amino reacting cross linkers and thiol reacting cross linkers. Fluorescent labels may be attached by using a fluorescent label-fluorescene isothiocyanate through the epsilon-amino groups of lysine found in the bacteriophage capsomeres. Other fluorescent molecules such as rhodamine may also be used in a similar manner.
Labeling the bacteriophages with biotin type reporter groups using a succinimide ester group may also be used. Other conventional secondary labeling sub6tance~ may also be incorporated.
Radioactive labeling of the bacteriophages with various radioactive compounds could also be used, for example, radioactive iodine 125 may be linked to the protein of the bacteriophage shell by a conventional reaction.
Applicants have found that the Nakane method of binding horseradish peroxidase is particularly useful. ~he Nakane method is described in Standard Biochemical Methods. The procedure is as follows: Horseradish peroxidase is reacted with 0.032M formaldehyde in the presence of a 0.30M NaHC03 buffer, pH 8.1 to form HRP=CH2. ~he HRP=CH2 is reduced with 1 mg NaBH4/mg enzyme to form HRP-CH3 (methylated .
, .
, 1 3t ~ 1 1 1 horseradish peroxidase). ~h~ HRP-CH3 i5 bu~ered with 0.30M
NaHC03 and separated by chromotography on a 0.5m exclusion column. The HRP-CH3 is then oxidized with 0.04M NaI04 to form HRP-CH0 (horseradish peroxidase aldehyde). The HRP-CH0 is buffered to pH 9.0 with a Na2C03 buffer and separated on a O.5m exclusion column to produce the activated HRP enzyme. The bacteriophage is grown on a host bacterium culture, for example S. typhimurium. The lysed bacteriophage is separated from the culture through a 0.45 micron filter. The activated enzyme is added to the lysed bacteriophage and reacted for 4 to 6 hours at room temperature. The enzyme readily bonds to the protein coating of the bacteriophage, probably be attaching to lysine present in the protein coating. The tagged bacteriophage is buffered to pH 7.0 with a 50mM PO4 buffer and separated on an 0.5m exclusion column. The separated tagged bacteriophage is then assayed and adjusted for titer.
EXAMPLE I Salmonella S~ecies Type Test:
A Salmonella specific bacteriophage was harvested from nature and isolated on a host culture of Salmonella typhimurium. (A culture of this bacteriophage, designated ATCC
Number 40282, has been deposited with the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Maryland 20852 and is available to the public from the permanent callection.) After isolation and purification on a Salmonella typhimurium host culture, the bacteriophage was labeled with horseradish - peroxidase using the Nakane method. The labeled baateriophage was then tested against representative Salmonella species and the serologically closely related species of, Citrobacter, as shown in Table I. The Salmonella and Citrobacter organisms were graciously provided by Alma Murlin from her collection at the National Center for Disease Control, Atlanta, Georgia and by the USDA Veterinary Services Center, Amesj Iowa. The Citrobacter, Salmonella type, was provided by the Center for Disease Control, Atlanta, Georgia.
- ~ .. . . : .
Table I
oraanism Colorimetric Test Salmonella. Group ~
worthington Positive anatum Positive cholerae var. suis Positive newington Positive paratyphi B Positive montevideo Positive typhi Positive heidelberg Positive typhimurium Positive portsmouth Positive johannesburg Positive laardt Positive poona Positive berta Positive meleagridis Positive infantis Positive enteritidis Positive pomona Positive jauiana Positive paratyphi A Positive newport Positive agoha Positive cereo Positive brithday Positive luciana Positive london Positive 30 westerstead Positive tennessee Positive ' . ~, ' .
newbrunswick Positlve gaminara) Positive florida Positive alachua Positive krefold Positive paratyphi A Positive dublin Positive seftenberg Positive paratyphi C Positive drypool Positive inverness Positive barrilly Positive minnesota Positive newington Positive paratyphi A, japan Positive cereo Positive Salmonella. Grou~ 2 phoenix Positive neb-M23037 Positive Salmonella, Group 3 3a Positive 3b Positive Salmonella, Group 4 flint Positive 25 marina Positive Salmonella~ Group 5 brookfield Positive bongar Positive Citrobacter 30 Citrobacter spp., Salmonella type Negative freundii Negative amalonaticus Negative The test procedure for each organism listed in the Table I
was as follows: A culture of the organism was slurried to a concentration of 106 organisms per ml. A 100 ul sample of the organism slurry was combined with 50 ul of the labeled bacteriophage reagent. The reagent wa~ ~tandardized to a titer of lo8 p.f.u. The reagent and Salmonella sample (and Citrobacter~ were incubated for 20 minutes. After incubation the mixture was filtered through a 0.45 micron filter with vacuum. The filtered mixture was washed twice using a phosphate buffered saline solution containing o.05% TWEEN 20~TM). The last wash was vacuumed to dryness. A 100 ul saturated solution of para-para-biphenol, buffered to pH 7.0 by a 50m phosphate buffer, (substrate) was added to the filter and incubated for 15 minutes. A characteristic brown color indicated a positive test in all cases (presence of labeled bacteriophage on the filtered bacteria). All reactions were at room temperature.
Example II Reliability:
A test for reliability of the labeled bacteriophage was conducted on the array of bacteria shown in Table II and Table III. The bacteriophage used were obtained from the American Type Culture Collection, with the exception of applicants' Salumonella bacteriophage. The bacteriophage were specific for the particular organism, as noted herein. Each bacteriophage used was labeled with horseradish peroxidase using the Nakane method. The tests were conducted as described in Example I and developed using para-para-biphenol.
: .
~: , . , -: .
~Q~;
Labeled Test Number of Number of Num~er of Percent Bacteriophage Organism True False Tests False Positive Negative Negative ATCC 40282 Citrobacter, 0 0 16 0.00 Salmonella type T4 E. coli 162 15 177 8.47 ATCC 15692-Bl Pseudomonas 273 9 282 3.19 aeruginosa ATCC 40282 Salmonella 792 3 795 0.38 typhimurium ATCC 6538P Staph 181 17 198 8.59 aureus Total 44 1468 3.00 Table III
Labeled Test Number of Number of Number of Percent Bacteriophage Organism True False Tests False Negative Positive Positive -ATCC 40282 Citrobacter, 15 1 16 6.25 Salmonella type present T4 E. coli 2730 0 2730 0.00 not present ATCC 15692-B1 Pseudomonas 2616 9 2625 0.34 aeruginosa not present ATCC 40282 Salmonella 2313 3 2316 0.13 typhimurium not present ATCC 6538P Staph 2709 0 2709 0.00 aureus not present Total 13 10396 0.13 .
:
It will be appreciated by those s~illed in the art that various modifications may be made to the invention disclosed herein. The invention is not to be limited to the cpecific embodiments given herein for purposes of illustration, but is limited only by the scope of the appended claims and their equivalents.
Claims (14)
1. A method of identifying specific organisms comprising selecting a binding agent for the specific organism, labeling the binding agent, contacting the labeled binding agent with a specimen of an unknown organism, binding the binding agent to the specific organism present in the specimen, separating the bound organism and binding agent from the specimen and testing to determine the presence of the label on the bound organisms.
2. The method of claim 1 wherein the specific organism is a bacterium.
3. The method of claim 1 wherein the binding agent is a bacteriophage.
4. The method of claim 3 wherein the label is bound to the protein coating of the bacteriophage.
5. The method of claim 3 wherein the label is a peroxidase colorimetric indicator.
6. The method of claim 1 wherein the organism and bound binding agent are separated from the specimen by filtration.
7. The method of claim 6 wherein the binding agent is a bacteriophage and the label is a colorimetric indicator bound to the protein coating of the bacteriophage, the indicator being developed on the filter.
8. The method of claim 7 wherein the label is horseradish peroxidase.
9. The method of claim 1 wherein the binding agent is species specific.
10. The method of claim 1 wherein the binding agent is genus specific.
11. A specific test reagent for directly identifying salmonella type bacterial organisms comprising a specific bacteriophage capable of binding to the salmonella type bacteria, the bacteriophage having an identifiable label thereon.
12. The test reagent of claim 11 wherein the label is a peroxidase colorimetric indicator.
13. The test reagent of claim 11 wherein the label is attached to the protein shell of the bacteriophage.
14. The test reagent of claim 11 wherein the label is an activated peroxidase colorimetric indicator, the peroxidase being Nakane activated.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US93651586A | 1986-12-01 | 1986-12-01 | |
| US936,515 | 1986-12-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1313111C true CA1313111C (en) | 1993-01-26 |
Family
ID=25468751
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000553239A Expired - Fee Related CA1313111C (en) | 1986-12-01 | 1987-12-01 | Method of identifying unknown organisms |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0298094A1 (en) |
| AU (1) | AU1043788A (en) |
| CA (1) | CA1313111C (en) |
| WO (1) | WO1988004326A1 (en) |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0439354A3 (en) * | 1990-01-24 | 1992-06-17 | Amoco Corporation | Signal generating moiety and method for use |
| US5168037A (en) * | 1990-08-02 | 1992-12-01 | Phyllis Entis | Method for the preparation of a labelled virus without the inactivation of viral binding sites and method of assay utilizing said labelled virus |
| BE1006312A3 (en) * | 1991-11-29 | 1994-07-19 | Univ Catholique Louvain | Selection process for recombinant microorganisms with their area at least one molecule enzymatic activity. |
| AU3776493A (en) * | 1992-02-28 | 1993-09-13 | Paul W. Judkins | Method and diagnostic kit for determination of bacteria |
| DK63093D0 (en) * | 1993-06-02 | 1993-06-02 | Foss Electric As | IMPROVED METHOD |
| JP3615810B2 (en) * | 1994-12-05 | 2005-02-02 | 東北電子産業株式会社 | Bacteria detection method and detection apparatus |
| JP3270722B2 (en) * | 1996-09-27 | 2002-04-02 | オルガノ株式会社 | Bacteria detection method and detection device |
| WO1998047521A1 (en) * | 1997-04-24 | 1998-10-29 | Idaho Research Foundation, Inc. | Phages, methods for growing and detecting them and their use |
| US8216780B2 (en) | 2002-04-12 | 2012-07-10 | Microphage (Tm) Incorporated | Method for enhanced sensitivity in bacteriophage-based diagnostic assays |
| ATE469240T1 (en) | 2002-04-12 | 2010-06-15 | Colorado School Of Mines | METHOD FOR DETECTING LOW CONCENTRATIONS OF A TARGET BACTERIA USING PHAGES TO INFECT TARGET BACTERIAL CELLS |
| WO2006105504A1 (en) * | 2005-03-31 | 2006-10-05 | Microphage Incorporated | Apparatus and method for detecting microorganisms using flagged bacteriophage |
| US20080286757A1 (en) * | 2005-09-15 | 2008-11-20 | Microphage Incorporated | Method and Apparatus for Identification of Microorganisms Using Bacteriophage |
| WO2008064241A2 (en) * | 2006-11-20 | 2008-05-29 | Microphage Incorporated | Method and apparatus for enhanced bacteriophage-based diagnostic assay |
| JP5150856B2 (en) | 2007-06-15 | 2013-02-27 | マイクロファージ・インコーポレーテッド | Microbial detection method with improved bacteriophage amplification |
| US8697434B2 (en) | 2008-01-11 | 2014-04-15 | Colorado School Of Mines | Detection of phage amplification by SERS nanoparticles |
| US9441204B2 (en) | 2008-04-03 | 2016-09-13 | Colorado School Of Mines | Compositions and methods for detecting Yersinia pestis bacteria |
| CN105823876B (en) * | 2016-03-18 | 2018-01-16 | 南昌大学 | A kind of detection method for salmonella |
| US20190078133A1 (en) * | 2017-09-08 | 2019-03-14 | The Charles Stark Draper Laboratory, Inc. | Detection and identification of bacteria and determination of antibiotic susceptibility using bacteriophage and reporter molecules |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1303802A (en) * | 1969-01-16 | 1973-01-24 | ||
| US4104126A (en) * | 1976-01-29 | 1978-08-01 | Nichols Institute Of Endocrinology, Inc. | Non-isotopic substrate assay employing bacteriolysis products |
| US4282315A (en) * | 1979-09-13 | 1981-08-04 | Corning Glass Works | Preparation of enriched whole virus radioligand |
| AU2780284A (en) * | 1983-05-16 | 1985-11-21 | Packard Instrument Co. Inc. | Method of measuring atp and concentrating and measuring unicellular organisms |
| FR2560995B1 (en) * | 1984-03-07 | 1988-02-19 | Pasteur Institut | REAGENTS AND KITS FOR THE QUANTITATIVE DETERMINATION OF A VIRAL NUCLEIC ACID IN A BIOLOGICAL MEDIUM AND METHOD FOR DETERMINING SUCH A VIRAL NUCLEIC ACID |
| JPS61501489A (en) * | 1984-03-19 | 1986-07-24 | ユニバ−シテイ オブ イリノイ | Bacteriophages as recognition and identification agents |
| CA1277931C (en) * | 1984-06-05 | 1990-12-18 | Shimon Ulitzur | Detection and/or identification of microorganisms in a test sample usingbioluminescence or other exogenous genetically-introduced marker |
| FR2568588B1 (en) * | 1984-08-02 | 1987-02-13 | Inst Nat Sante Rech Med | PROBE AND METHOD FOR THE DETECTION OF DETERMINED MICRO-ORGANISMS, IN PARTICULAR LEGIONELLA IN THE MEDIA CONTAINING THE SAME |
-
1987
- 1987-12-01 EP EP19880900198 patent/EP0298094A1/en not_active Withdrawn
- 1987-12-01 WO PCT/US1987/003168 patent/WO1988004326A1/en not_active Application Discontinuation
- 1987-12-01 CA CA000553239A patent/CA1313111C/en not_active Expired - Fee Related
- 1987-12-01 AU AU10437/88A patent/AU1043788A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| EP0298094A1 (en) | 1989-01-11 |
| AU1043788A (en) | 1988-06-30 |
| WO1988004326A1 (en) | 1988-06-16 |
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