Method for the genetic detection of microorganisms
AssigneeThe Regents of the University of Michigan
InventorElliot Juni
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Abstract
A method for the genetic detection of specific microorganisms based on the phenomenon of transformation is disclosed. Variants such as auxotrophs which are distinguishable from their wild, parent strain are transformed by deoxyribonucleic acid (DNA) extracted from microorganism cells present in a biological test sample. The detection of transformed cells indicates the presence of the specific microorganism under determination in the sample. The transforming-DNA is in a sterile, crude form, thereby providing a simple, rapid, and highly specific test method.
Claims34
A method for detecting the presence of a specific microorganism in a biological sample comprising the steps o:
- a. contacting said biological sample with an aqueous solution containing a surfactant capable of lysing microorganism cells present in said biological sample to form a sterile, crude, aqueous extract comprising endogenous deoxyribonucleic acid;
- b. contacting a viable, transformable variant of said specific microorganism with said crude extract to form a mixture, said specific microorganism possessing a distinguishing characteristic relative to said variant, said variant being supported on a microbiological medium capable of diffusing aqueous solutions;
- and
- c. subjecting said mixture to an analysis for detecting the presence of said distinguishing characteristic;
- said surfactnat in step (a) being present in said aqueous solution at a concentration sufficient to lyse substantially all of the microorganism cells present in said biological sample while sufficient to destroy a substantial amount of said variant upon contacting said variant with said crude extract in step (b) and during the diffusion of said crude, aqueous extract into said microbiological medium.
A method for detecting the presence of a specific microorganism in a biological sample comprising the steps of:
- a. treating said biological sample with an aqueous solution of a surfactant which is in a sufficiently high concentration to lyse substantially all of the microorganism cells present in said sample and thereby release endogenous deoxyribonucleic acid in the form of a sterile, crude extract thereof, said surfactant being in a sufficiently low concentration so that upon contacting said crude extract with a number of microorganism cells growing on the surface of a microbiological medium capable of diffusing aqueous solutions which come into contact with a surface thereof, a substantial number of the cells are not destroyed by the solution of surfactant as it diffuses into the medium;
- b. contacting said crude extract with cells of an auxotroph of said specific microorganism, which cells are growing on the surface of a complete microbiological medium, said complete medium containing all of the nutrients required by said auxotroph for growth and being capable of diffusing aqueous solutions which come into contact with a surface thereof.
- c. incubating the resulting mixture for a predetermined period of time.
- d. transferring at least a portion of the resulting incubated mixture to a minimal microbiological medium containing all of the nutrients required by the prototroph of said auxotroph for growth but lacking at least one of the nutrients required by said auxotroph for growth, and
- e. detecting the development of any microbiological growth of the portion of said resulting incubated mixture transferred to said minimal microbiological medium, the development of microbiological growth indicating the presence of said specific microorganism in said biological sample.
Dependencies are inferred from the claim text.