Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-02-22
2003-04-29
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007320, C435S006120, C435S004000, C435S189000, C435S325000, C530S350000
Reexamination Certificate
active
06555312
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for detecting or identifying a bacterium in a specific and yet easy manner by employing a bacteriophage into which a light-emission gene is inserted. The method of the present invention can be utilized as a means for measuring or detecting a bacterium, and is particularly useful in fields of environmental, food, and medical applications.
2. Description of the Related Art
Numerous tests exist for determining the presence and identifying the type of bacterium. Most of these methods involve culturing a sample containing an unidentified bacterium in a medium (which may be of a variety of types) and thereafter observing the culturological characteristics, morphological characteristics, biological characteristics, sensitivity with respect to antibiotics, absorption of various colorants, serological characteristics, etc., of the unidentified bacterium. However, such conventional methods require selection of media and the like in order to effect the selective growth of a relevant bacterium while preventing the growth of irrelevant bacteria. Due to such requirements, conventional methods may take a considerable amount of time for identifying a relevant organic body (bacterium) after the isolation of the bacterium.
In order to solve the aforementioned problems, methods have been developed which detect the presence of a bacteria by introducing into a bacterium a foreign gene for producing a protein that expresses a detectable function, and allowing the introduced gene to be expressed so that the bacterium can be detected based on the expressed function.
Conventional bacterium detection methods which utilize biological light emission typically attain light emission by using an enzyme known as luciferase and its substrate, luciferin, to permit a reaction therebetween in the presence of adenosine triphosphate (ATP) of bacterial origin. According to such conventional methods, a reaction solution in which luciferase and luciferin are mixed is prepared; after a sample (e.g., meat) is crushed, the reaction solution is added to the crushed matter or an extract obtained after crushing. If the sample contains a bacterium, light emission can be observed because the adenosine triphosphate (ATP) which was contained in the bacterium will leak out of the bacterium during the crushing, so that the action of the leaking ATP allows a light emission reaction to occur in the reaction solution. The bacterium in question can be detected by detecting the intensity of light emission (see Japanese Laid-Open Publication No. 5-30997). However, this measurement system may also measure any ATP that is derived from cells other than the bacterium which is suspected to be present in the sample. As a result, there are problems of misdetection possibilities and substantial degradation in the sensitivity.
On the other hand, methods are also known which involve a step of introducing a gene which express luciferase into a vector, e.g., a bacteriophage, and a step of infecting a bacterium with this virus. According to this method, after the introduction of the gene, luciferin is introduced from outside of the bacterium in an invasive manner, and light emission is effected through an enzymatic reaction utilizing the ATP within the cell. The bacterium in question can be detected by detecting the intensity of light emission (see Japanese Publication for Opposition No. 6-34757). This technique will inevitably destroy all or some of the cells under examination because the introduction of luciferin is achieved by a method which is invasive to the bacterial cells. Hence, this technique is not exactly satisfactory in terms of misdetection possibilities and degradation in sensitivity.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a method for detecting a bacterium for measurement, including the steps of: allowing a bacteriophage to bind to the bacterium, the bacteriophage being capable of specifically binding to the bacterium and growing in the bacterium, whereby a gene within the bacteriophage which expresses a light-emission protein is introduced into the bacterium so that a protein is produced within the bacterium as a product of the gene; and providing an external factor in a non-invasive manner from outside of the bacterium, thereby causing only the actually-present bacterium to emit light in a specific manner.
In one embodiment of the invention, the bacterium is selected from a group including: Rhodospirillaceae, Chromatiaceae, Chlorobiaceae, Myxococcaceae, Archangiaceae, Cystobacteraceae, Polyangiaceae, Cytophagaceae, Beggiatoaceae, Simonsiellacea, Leucotrichaceae, Achromatiaceae, Pelonemataceae, Sprirochaetaceae, Spirillaceae, Pseudomonadaceae, Azotobacteraceae, Rhizobiaceae, Methylomonadaceae, Halobacteriaceae, Enterobacteriaceae, Vibrionaceae, Bacteroidacea, Neisseriaceae, Veillonellaceae, Organisms oxidizing ammonia or nitrite, Organisms metabolizing sulfur and sulfur compounds, Organisms depositing iron and/or manganese oxides, Siderocapsaceae, Methanobacteriaceae, Aerobic and/or facultatively anaerobic Micrococcaceae, Streptococcaceae, Anaerobic Peptococcaceae, Bacillaceae, Lactobacillaceae, Coryneform group of bacteria, Propionibacteriaceae, Actinomycetaceae, Mycobacteriaceae, Frankiaceae, Actinoplanaceae, Dermatophilaceae, Nocardiaceae, Streptomycetaceae, Micromonosporaceae, Rickettsiaceae, Bartonellaceae, Anaplasmataceae, Chlamydiaceae, Mycoplasmataceae, and Acholeplasmataceae
In another embodiment of the invention, the bacteriophage is selected from a group including T4, P2, T2, T7, &lgr;, MV-L2, PRD1, PM2, MV-L1, &PHgr;X174, fd, MS2, &PHgr;6, FELIX01, and G47.
In still another embodiment of the invention, the light-emission protein is green-fluorescent protein (GFP) or blue-fluorescent protein (BFP).
In still another embodiment of the invention, the external factor is light.
According to the present invention, there is also provided a method for identifying a bacterium present in a sample, including the steps of: allowing a bacteriophage to bind to the bacterium, the bacteriophage being capable of specifically binding to the bacterium and growing in the bacterium, whereby a gene within the bacteriophage which expresses a light-emission protein is introduced into the bacterium so that a protein is produced within the bacterium as a product of the gene; and providing an external factor in a non-invasive manner from outside of the bacterium, thereby causing only the actually-present bacterium to emit light in a specific manner.
According to the method of the present invention, a light-emission reaction can be effected without crushing or otherwise damaging a bacterium, such that the light-emission reaction occurs only within the bacterium. Accordingly, a bacterial measurement is achieved which is substantially free of misdetection possibilities and which provides high sensitivity. As a result, the method according to the present invention enables detection or identification of a bacterium in a quick and specific manner, without requiring complicated processes.
In particular, by adopting a protein which does not require any substrate for light emission (e.g., green-fluorescent protein) as a light-emission protein, it becomes possible to omit invasive introduction of an external factor from outside of the bacterium, a problematic step which is associated with conventional methods utilizing a luciferase-luciferin system.
Thus, the invention described herein makes possible the advantage of providing a method for detecting or identifying a bacterium species within a sample, in a specific, highly-sensitive, quick and secure manner, the method involving providing an external factor for the bacterial cell in a non-invasive manner so that the external factor induces a light-emission reaction.
This and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description with reference to the accompanying figures.
Matsushita Electric - Industrial Co., Ltd.
Portner Ginny Allen
Renner , Otto, Boisselle & Sklar, LLP
Smith Lynette R. F.
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