Method for detecting bacterial spores

1. Field of the Invention

This invention relates to a method for detecting bacterial spores using a combination of gas chromatography and mass spectrometry.

2. Description of Related Art

The detection of bacterial spores, or endospores, is useful for a wide variety of purposes. For example, potential biological agents used in warfare and by terrorists include spore-producing bacteria that cause diseases, such as anthrax, botulism, gas gangrene, and tetanus. The detection of spores of such bacteria can also be important in determining and implementing appropriate countermeasures against such agents. In agricultural technologies, spore-producing bacteria, such as Bacillus thuringiensis, are often used as insecticides. Spores in soil or plant samples can be monitored to ensure that the bacteria population is sufficiently high to be effective against insect pests. In the sanitation and hygiene fields, the detection of bacteria spores can be useful to monitor indoor environments, water quality, and food quality. In addition, the detection of nonviable bacterial spores can be useful to paleontologists.

Bacterial endospore concentrations are not easily determined with conventional techniques. Among the primary conventional techniques are microscopy and plate culture counting, both of which are extremely slow and tedious to perform.

U.S. Pat. No. 5,876,960 to Rosen discloses methods for detecting and quantifying bacterial spores in a sample. The method utilizes a lanthanide combined with a medium to be tested for endospore content. The lanthanide reacts with calcium dipicolinate, which is present in bacterial spores, to produce a lanthanide chelate, having distinctive absorbance and emission spectrums, which are detected using photoluminescence. This method is limited, however, as it is difficult to access dipicolinic acid because the endospore casing is difficult to penetrate.

Beverly et al., “Analysis of Dipicolinic acid in Bacterial Spores by Electron Monochromator-Mass Spectrometry,” 47^th ASMS Conference on Mass Spectrometry and Allied Topics, 1999 discloses a method of measuring dipicolinic acid in Bacillus cereus in which dried bacteria was directly introduced into a mass spectrometer and heated to 400° C. Bacteria spores typically contain from 7 to 14 percent by weight dipicolinic acid or salts thereof. Although the method was able to detect dipicolinic acid, the endospore casings were not reliably opened, making the results less than quantitative.

It would be desirable to develop a method for detecting bacterial spores that does not rely on time consuming and tedious microscopy and plate culture methods. Such a method should reliably and predictably open endospore casings so that the dipicolinic acid within can be accessed and measured as a surrogate for the quantity of bacterial spores present in a sample.

The present invention relates to a method of detecting the presence and quantity of bacterial spores. The method includes adding an electrophilic alcohol and an acid anhydride to a sample, admixing the sample with a gas chromatography solvent, and analyzing the sample. The sample may be analyzed using mass spectrometry, or other suitable methods of analysis.

Unless otherwise indicated, all numbers or expressions referring to quantities of ingredients, reaction conditions, etc. used herein are to be understood as modified in all instances by the term “about.”

The present invention is directed to a method of detecting the presence and quantity of bacterial spores. The method includes adding an electrophilic alcohol and an acid anhydride to a sample, optionally drying the sample, admixing the sample with a gas chromatography solvent, and injecting the mixture. The analysis may be performed using mass spectrometry, gas chromatography coupled with mass spectrometry, or any other suitable method of analysis.

Any suitable electrophilic alcohol may be used in the present invention, so long as it is able to form a diester with dipicolinic acid. In an embodiment of the present invention, the electrophilic alcohol is present in an amount sufficient to convert greater than 75 percent by weight, typically, 90 percent by weight, in many cases, greater than 95 percent by weight, and in other cases, 100 percent by weight of the dipicolinic acid to a corresponding dipicolinic diester of the electrophilic alcohol.

Suitable electrophilic alcohols include any short chained aliphatic alcohol, typically, C1 to C8 aliphatic alcohols, containing an electron withdrawing group. Examples of suitable electron withdrawing groups include, but are not limited to, fluoro, chloro, bromo, iodo, nitro, cyano, and the like.

In an embodiment of the present invention, the electrophilic alcohol is pentafluoropropanol.

The derivatization of dipicolinic acid, i.e., the reaction of the electrophilic alcohol with dipicolinic acid to form the dipicolinic acid diester of the electrophilic alcohol, is shown in Scheme 1.

Continue reading about Method for detecting bacterial spores...
Full patent description for Method for detecting bacterial spores

Brief Patent Description - Full Patent Description - Patent Application Claims

Click on the above for other options relating to this Method for detecting bacterial spores patent application.
###


How KEYWORD MONITOR works... a FREE service from FreshPatents
1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored.
3. Each week you receive an email with patent applications related to your keywords.  
Start now! - Receive info on patent apps like Method for detecting bacterial spores or other areas of interest.
###


Previous Patent Application:
Two-photon fluorescent probes for acidic vesicles in live cells and tissue and method of imaging acidic vesicles in live cells and tissue using the same
Next Patent Application:
Method and device for cell counting
Industry Class:
Chemistry: molecular biology and microbiology

###