Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-03-31
2001-08-14
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S007800, C435S091100, C435S091200, C435S283100, C536S023100, C536S024310, C536S024320
Reexamination Certificate
active
06274306
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a carrier for extracting bacteria, a method of extracting a microorganism and a method of assaying microorganisms.
2. Description of the Prior Art
In recent years, a garbage disposal for composting organic waste (the so-called kitchen garbage) discharged from a household kitchen or the like is now actively researched and developed. In the garbage disposal, microorganisms such as bacteria and protozoa degrade organic matter to form compost. During the composting process (organic degradation process) in such a garbage disposal, the degree of composting is evaluated by monitoring the temperature or the like. The state of the garbage disposal is adjusted to prepare high-quality compost on the basis of the evaluation.
In order to prepare high-quality compost, it is necessary to obtain information of the microorganisms (at least the types of the microorganisms) functioning in the garbage disposal. The information of the microorganisms is also necessary for excellently controlling degradation of the kitchen garbage with the microorganisms. In order to improve soil by adding the prepared compost, it is also important to obtain information of microorganisms contained in the soil.
In general, information of microorganisms is obtained by a method of sampling soil, wood chips or food containing bacteria, extracting the bacteria therefrom, isolating each bacterium included in the extracted bacteria and biochemically examining the same.
In order to examine bacteria in a garbage disposal, wood chips contained in the tank of the garbage disposal to serve as a treating carrier are sampled. The wood chips are introduced into a large quantity of sterilized physiological salt solution for liberating the bacteria from the wood chips by a suspension method. This solution is filtered through a filter for removing foreign matter such as the wood chips, large-sized protozoa and the like. The filtrate is further centrifuged for extracting the bacteria in the supernatant.
After extracting the bacteria in the aforementioned manner, the centrifuged supernatant is further diluted and inoculated on an agar medium. The bacteria are cultured in the agar medium, and each bacterium included in the bacteria is isolated. Each bacterium thus isolated is biochemically examined, for obtaining information of the microorganisms.
The wood chips employed as a treating carrier in the garbage disposal are composed of plant cell, and so they may have DNA. When analyzing DNA of the bacteria in the tank, therefore, the wood chips must be completely separated from the bacteria. Therefore, the treatment step by the suspension method and the filtration step with the filter are required as described above, to complicate the steps of extracting the bacteria.
Further, time is required for isolating each bacterium from the bacteria formed by a plurality of bacteria, and it is difficult to analyze bacteria which are hard to isolate by this method.
On the other hand, a PCR (polymerase chain reaction) method is employed for amplifying DNA. In the PCR method, a primer having a base sequence complementary to that at both ends of DNA (template DNA) to be amplified and heat-resistant DNA polymerase are employed for repeating a cycle formed by three stages of a thermal denaturation step, an annealing (heat treatment) step and an extension reaction step thereby enabling amplification of DNA fragments substantially identical to the template DNA. Employing this PCR method, a prescribed fragment in DNA of one of a small amount of bacteria can be amplified to hundred thousand to million times, for example.
In order to employ the PCR method, however, the base sequence of at least at both ends of a part of the template DNA must be known. If the types and base sequences of microorganisms functioning in the garbage disposal or existing in soil are unknown, therefore, DNA fragments of the microorganisms cannot be amplified in the conventional PCR method.
In this regard, there has been proposed a RAPD (random amplified polymorphic DNA) method or AP-PCR (arbitrarily primed-polymerase chain reaction) method of simultaneously amplifying a large amount of DNA fragments from a single type of DNA with a single primer, with no information of the base sequence. According to this method, the annealing temperature for the primer is reduced while the magnesium ion concentration in a reaction solution is increased during the PCR, thereby reducing sequence specificity of the primer in bonding. Thus, the primer is bonded to chromosome DNA of a microorganism with mismatching, to duplicate DNA fragments.
According to the RAPD method or AP-PCR method, some DNA fragments are amplified in a large amount with a single primer, with no information on the base sequence of the DNA to be amplified. A DNA fingerprint is obtained by separating the amplified DNA fragments by gel electrophoresis. The state of the microorganism can be analyzed by analyzing the DNA fingerprint.
When applying a conventional PCR method such as the RAPD (random amplified polymorphic DNA) method or the AP-PCR (arbitrarily primed-polymerase chain reaction) method to bacteria formed by a plurality of bacteria in analysis of chromosome DNA prepared from the bacteria, chromosome DNA of the bacteria can be readily associated with amplified DNA fragments and the efficiency of the analysis is improved by adjusting the primer and PCR conditions for setting the probability of amplification at a low value so that a single DNA fragment is amplified for 10
8
bp (base pairs), for example. When discriminating a group of bacteria formed by a plurality of bacteria, however, the number of types of amplified DNA fragments is so large that it is difficult to associate a bacterium which is a template with amplified DNA fragments, and hence it is difficult to discriminate an ecosystem formed by the group of bacteria.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a carrier for extracting bacteria which can simplify extraction of bacteria.
Another object of the present invention is to provide a method of extracting a microorganism which can readily extract a microorganism.
Still another object of the present invention is to provide a method of assaying microorganisms which can correctly discriminate a plurality of microorganisms.
A carrier for extracting bacteria according to an aspect of the present invention comprises a material body having a plurality of pores with no DNA, while the mean pore diameter of the pores is at least 5 &mgr;m and not more than 100 &mgr;m and the depth of each pore is at least 5 &mgr;m.
The carrier for extracting bacteria is arranged on a site for sampling bacteria for a prescribed time and thereafter recovered. After the recovered carrier for extracting bacteria is washed, the bacteria are liberated from the carrier for extracting bacteria.
The aforementioned carrier for extracting bacteria has a plurality pores suitable for inhabitation of bacteria, so that bacteria enter the pores and inhabit therein in a concentrated state. When recovering the carrier for extracting bacteria, therefore, the bacteria can be sampled from the sampling site in a concentrated state.
The bacteria, which enter the pores and inhabit therein, are hardly removed when washing the carrier for extracting bacteria. On the other hand, foreign matter etc. larger than the pores not entering the pores is readily removed by washing. Thus, a filtration step for separating the bacteria from the foreign matter can be shortened.
When liberating the bacteria from the carrier for extracting bacteria by crushing the carrier for extracting bacteria, DNA of the bacteria can be analyzed with no hindrance even if the extracted bacteria contain the carrier for extracting bacteria since the carrier for extracting bacteria is formed by the material body having no DNA. Therefore, the carrier for extracting bacteria may not be completely separated from the bacteria, and so the number of steps required for extracting microorganis
Akin Gump Strauss Hauer & Feld,L.L.P.
Horlick Kenneth R.
Sanyo Electric Co,. Ltd.
Siew Jeffrey
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