KR20040091462A - Quality Control Method for Microorganisms - Google Patents

Abstract

The present invention relates to a method for performing microorganism quality control by analyzing gene mutations by displaying the entire genome of a microorganism on a gel. Cutting into several fragments having a single strand of sticky ends, and, independently of this, preparing a hairpin ring structure adapter having a single strand of sticky ends that can complementarily complement both ends of the cut DNA fragment. step; Binding the genomic DNA fragments and hairpin ring structure adapters using DNA ligase; Removing the DNA fragment and the hairpin ring structure adapter not participating in the binding reaction by using an exonuclease; Amplifying the DNA fragment using a primer and a DNA polymerase that complementarily bind to a portion of the hairpin ring structure adapter; Separating the obtained amplification product on a gel; And it relates to a quality control method of the microorganism comprising the step of confirming the genetic variation of the microorganism.

Description

Quality Control Method for Microorganisms

The present invention relates to a method for performing quality control of a microorganism by displaying the entire genome of the microorganism on a gel and analyzing the genetic variation.

Currently, the Korea Food & Drug Administration (KFDA) has played a role in national health and health by producing excellent medicines according to KGMP (raw materials) and BGMP (raw materials) in accordance with the standards for manufacturing and quality control of excellent medicines. , Safety as food in the 1992 report, Safety evaluation of foods derived by modern biotechnology: concepts and principles, jointly prepared by the OECD Environment, Science, Technology and Industry Council. The concept of substantial equivalence, which is the basis of evaluation, was proposed. Accordingly, the Food and Drug Administration (FDA) has established food and feed products produced from new plant varieties created by newly developed technologies, including traditional methods (such as conventional cross-breeding) and genetic (DNA) recombination techniques. The FDA's 1992 Policy for the Supervision of Fruits, Vegetables, Grains and By-Products) was issued on 29 May 1992.

Substantial equivalence is a concept applied to assess whether a modified or new food or food ingredient is safe for human consumption by comparing existing organisms used as food or as a source of food.

Therefore, the relevant criteria for genetic modification, nutrition, and antibiotic resistance markers have been established in the Safety Criteria for Foods Produced from New Crop Species. <Review on the safety of genetically modified (mutated) foods>, Consumer Protection Agency)

Therefore, the standard method of evaluating the "substantial equivalence," etc. which is the basis of food safety evaluation considering the case where genetic mutations such as food microorganisms (ex. Lactic acid bacteria) affect the quality in the industry. It is required. On the other hand, the genome of a living organism is changed by many environmental factors, such as chemical mutagens, ultraviolet light, and naturally occurring mutations. Changes in physiological properties caused by mutations in these genomes are factors that determine the rate of reproduction of offspring depending on their adaptability to the environment in which the organism is located, and dominant species with high reproduction rates are evolutionarily selected. Many living things on Earth have evolved through this process and continue to evolve. Among these naturally evolved organisms, there are two main ways to determine the evolutionary flexibility of microorganisms.

First, biochemical methods are used to identify evolutionary flexibility. For example, cell morphology using electron microscopy, flagella observation using electron microscopy, lipid analysis of cells using gas chromatography, Diaminopimelic acid (DAP) structure analysis, analysis of Mycolic acid, HPLC (HPLC) Quinone analysis using High Performance Liquid Chromatography and sugar and protein analysis of whole cells.

Second, evolutionary flexibility is identified by molecular biological methods. For example, GC content (G + C%) analysis by HPLC, 16S or 18S rDNA assay, 28S rDNA D1 / D2 assay, Internal transcribed spacer (ITS) 5.8S rDNA assay and Restriction Fragment Length Polymorphism (RFLP) of rDNA region Analysis method and the like. Although the method can use several genes, these two parts are used as the basic unit because the globally accumulated database has been established for the internal transcribed spacer (ITS) of the rDNA gene and the D1-D2 portion of the large ribosomal DNA. Analyze the sequence. In addition, DNA fingerprinting using RFLP, AFLP, etc. may be used to identify species diversity or in cases where the two-sequence sequences do not vary enough to distinguish species levels. . These methods randomly target genes to identify specific types of genes. However, these methods have the disadvantage that all the strains to be compared must be tested together. In the D1 / D2 assay, the D1 / D2 portion is usually used for determining genus, and the ITS portion is used for identification of species level. However, in the case of mold, the range of taxa is different for each taxon, so even if the genus such as Penicillium is 100% identical to other species of ITS and D1 / D2, it is necessary to combine genes or morphological methods with faster evolutionary rate. In some cases. Conversely, in the case of Colletotrichum and Acremonium, up to 5-10% of nucleotide sequences may be different from one species.

It is also used to systematically analyze Mycibacterium kansasii with MK7 (GC content of 63%) using the GC content (G + C%) analysis through HPLC (US5,518,884). In the 16S rDNA assay, 16S rDNA is well conserved among groups of organisms that are very diverse or related among species or subspecies in the bacterial taxa and are used to identify them (Woese in Microbiol. Rev. 51: 221- 271 (1987) and Woese et al., In System, Appl. Microbiol. 6: 143-151 (1985)). Techniques for the use of oligonucleotides complementary to these 16S rDNAs for identifying specific organisms or groups of organisms have already been described (Gobel et al. In J. Gen. Microbiol 133: 1969-1974 (1987), Giovannoni et al. In J. Bacteriol. 170: 720-726 (198) and Stahl et al. In Appl. And Environ.Microbiol. 54: 1079-1084 (1988)). In addition, phylogenetic elucidation between each organism is helpful because the rate of change of the rDNA-decoding sequence usually occurs very slowly compared to the rate of change of the protein (reviewed in Fox et al., Sci. 209: 459-). 483 (1980)). Among the above-mentioned prior arts, the biochemical assay is mostly a method for analyzing physiological characteristics, and it is impossible to analyze all physiological variations in each cell. Because it is only a minute difference that can not be detected by the conventional biochemical analysis method is insufficient.

In addition, most biological assays use rDNA expression sites for analysis. RDNA is an analysis target as a sequence that is expected to be conserved even when evolution proceeds due to the slight variation in most cells. Although levels of mutations can occur on real genes and physiological variations are observable, the rDNA region remains virtually unchanged, and if the mutagen is strong enough to cause mutations in the rDNA region, most of the time it is fatal to the cell and the cell cannot even survive. Mutation detection is inadequate.

The other method is to analyze the sequence of the whole genome as the most reliable method of tracking the variation of the genome in accuracy, but it is an effective search method because it requires at least 6 months of time and huge cost. Can't.

Accordingly, an object of the present invention is to provide a method of performing microorganism quality control by analyzing the genetic variation by displaying the entire genome of the microorganism on a gel.

Another object of the present invention is to provide a method for identifying a low level of genetic mutation on the genome in a short time.

1 is a diagram showing whether or not mutations of strain (m) and wild strain (w) when the Lactobacillus spp . Treated with a chemical mutagen according to the present invention (here, arrows are different physiological characteristics M represents the detected mutant gene that is responsible for, M is the marker indicating the relative size of the deployed DNA, and numbers below indicate the size of the marker, respectively. Different sets of hairpin adapters found).

Figure 2 is a diagram showing whether the natural mutation occurs when passaged several times in a wild strain medium without a mutagen. Genomic DNA was extracted at each time after subculture, and only a specific set of 120 hairpin adapter sets was selected and repeatedly searched according to the present invention. As shown in the figure, it can be seen that there is no natural mutation incidence (strain quality change) through subculture in a medium without mutagen (wherein M is a marker indicating the relative size of the deployed DNA, The lower number indicates the size of the marker, respectively, and lanes 1 to 6 indicate the number of passages).

The present invention relates to a method for performing quality control of a microorganism by displaying the entire genome of the microorganism on a gel and analyzing the genetic variation.

In the present invention, quality control (QC) refers to management for maintaining and improving product quality by applying scientific principles, which are commonly used for interpretation and interpretation, and in the broadest sense, It is a concept that includes a series of systematic measures to produce the most marketable products most economically.

In the present invention, a genetic variation or genetic variation refers to a genetic variation occurring in an organism, and may be caused by a change in genetic composition such as a change in a gene, a combination change of a gene, a change in a chromosome, and a change in the number of chromosomes. It refers to the variation of the trait that occurs.

The present invention uses a restriction enzyme to cut the microbial whole genomic DNA into several fragments having a single strand of sticky ends and, independently of this, a single molecule capable of complementarily binding to the sticky ends of a single strand of such DNA segments. Preparing an adapter consisting of a sticky end of the strand and a hairpin ring portion; Binding the DNA fragment and the adapter having the hairpin ring structure using a DNA ligase; Removing an adapter having a DNA fragment and a hairpin ring structure not involved in the binding reaction by using an exonuclease; Optionally, removing the hairpin structure using alkaline solution, RNAase (RNase) or single stranded selective endonucleases; Amplifying a genomic DNA fragment using a primer complementarily binding to a site remaining after removal of only the hairpin ring portion of the adapter; Separating the amplified DNA product on a gel; And it relates to a quality control method of the microorganism comprising the step of confirming whether the genetic variation of the microorganism.

In the present invention, the arrangement of the hairpin adapter set is divided into rows and columns. For example, in a multi-well plate, if row 7 is CG and column 3 is AG, then the adapters with single-strand cohesive end sites having the sequence of CG and the adapters with single-strand cohesive end sites having the sequence of AG Mix into manna wells. If the number of bases constituting the single-stranded sticky end is two, the number of test tubes required at this time is 112. If the number of bases constituting the single-stranded end is N, the type of hairpin ring adapter is (4 ²ⁿ -2 x 4 ⁿ ) / 2. In addition, the 5 ', 3' end of the genomic DNA fragment and the 5 ', 3' end of the adapter must be complementary to each other. As described above, each adapter is dispensed onto a plate and then the restriction enzyme digested genomic DNA fragments are mixed in appropriate amounts for each plate. Then proceed with the ligation reaction. Once the ligation reaction is complete, genomic DNA fragments and adapters not involved in the binding reaction are removed using exonucleases. At this time, the DNA fragments ligated by the adapter having a hairpin ring structure at the end are not cleaved by the exonuclease.

Thereafter, treatment with alkaline solution, RNase, or single-strand-selective endonucleases results in the cleavage of only the hairpin ring portion into a double-stranded DNA fragment of general form. After the hairpin ring is removed, a polymerase chain reaction is performed using a primer capable of complementarily binding to a part of the adapter bound to the double-stranded DNA fragment.

And the polymerase chain reaction product is separated by a method such as polyacrylamide gel electrophoresis, and it is possible to determine whether genetic variation of the microorganism occurs by confirming each band pattern between the normal and the test subject.

In addition, in the case of mRNA, it is possible to determine whether the genetic variation of one microorganism using the method according to the invention.

Using the above method, according to the present invention, quality control of microorganisms can be performed by displaying the entire genome of the microorganisms on a gel and analyzing the genetic variation.

In addition, considering the case where genetic mutations such as food microorganisms, such as lactic acid bacteria, which are currently used industrially usefully through the method of the present invention affect the quality, the actual basis of the food safety evaluation is "substantial". Equivalence, substantial equivalence, etc.

In addition, the method of the present invention can be analyzed in a short time, low-level mutations made in a short time by the method of the present invention, it is possible to identify the causal gene of the scientifically useful microorganisms.

Hereinafter, the method of the present invention will be described in detail through examples. The following examples are only intended to illustrate and explain the contents of the present invention, and are not intended to limit the scope of the present invention.

Example 1 Identification of Mutations Between Mutant and Wild Strains That Cause Physiological Variation by NTG (N-methyl-N'-nitroso-N-Nitroguanidine)

1) mutant strain production

To obtain the mutant strains, Lactobacillus reuteri (ATCC 23272) was first used in MRS medium (1% beef extract, 1% peptone, 0.5% yeast extract, 0.1% Tween-80), 0.2% ammonium citrate. , 0.5% sodium acetate, 0.01% MgSO _4, 0.01% MnSO ₄ , 0.05% L-cysteine-HCl)) for 6 hours. The bacteria obtained by centrifugation were washed once with PBS (NaCl 8g / L, KCl 0.2g / L, Na ₂ HPO ₄ 1.44g / L, KH ₂ PO _4, pH 7.0,), and then NTG (N- methyl-N'-nitro-N-nitrosoguanidine) was added to the final 200 ㎍ / ml and incubated for 30 minutes at 37 ℃ to kill 99.9% of the strain. And in order to prevent the occurrence of back-mutation of this strain (MRS) was transferred three times using the liquid medium. The group of mutants obtained by the above method was properly diluted to form about 30 colonies on one plate, and the MRS medium (5% fructose, 1% beef extract, 1% peptone, 0.5% yeast containing high concentration of fructose) was formed. Extract, 0.1% Tween-80, 0.2% ammonium citrate, 0.5% sodium acetate, 0.01% MgSO _4, 0.01% MnSO ₄ , 0.05% L-cysteine-HCl) and incubate at 37 ° C. for 48 hours It was. More than 2,000 colonies formed in MRS solid medium were checked for viscosity using sterilized toothpicks, and one of the strains having the highest viscosity was selected.

2) Extraction of Genomic DNA

1.5 mL of each of the mutant and wild strains obtained in 1) was taken, and cultured for 48 hours at 37 ° C. in MRS medium, followed by centrifugation to extract only the cells. Diluted in 190 mL dilution solution I (50 mM glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA), 2 mL lysozyme (100 mg / ml) and 8 mL proteinase (50 mg / ml) After the addition was stored for 1 hour at 55 ℃. Thereafter, 10 mL of 10% SDS (Sodium dodecylsulfate) and 2 mL of RNase A (1 mg / ml) were added and stored at 37 ° C. for 30 minutes. Thereafter, about 88 mL of TE buffer (10 mM Tris-Cl pH8.0, 1 mM, EDTA) was added to the final volume of 300 mL, and then the same amount of phenol / chloroform / isoamylalcohol was added. 25: 24: 1 was added and vigorously stirred for 1 minute, followed by centrifugation. The aqueous solution layer was taken, and the same volume of chloroform was added and vigorously stirred, followed by centrifugation. Then transfer the aqueous layer to a new tube, adjust the volume to 300 mL with TE buffer, add 0.1 times the volume of sodium acetate (3M, pH5.2), mix well, add 2.5 times the volume of ethanol and mix well at 70 ° C. It was stored for 30 minutes. After centrifugation, the precipitate settled below was carefully washed with 70% ethanol, and then centrifuged to remove the ethanol layer, dried well in a vacuum dryer, and then dissolved in 50 mL of TE buffer.

3) Restriction fragmentation of genomic DNA using restriction enzyme Hpy118III of type IIs

30 units of Hpy118III restriction enzyme was added to 10 ug of each genomic DNA, and the restriction enzyme reaction was performed in 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM DTT, pH7.9 buffer solution.

4) Ligation of Genomic DNA Fragments and Hairpin Ring Adapters

The genomic DNA fragment obtained in 3) was purified by PCR purification kit (Bionia Co., Ltd.), and 1 µg of the obtained DNA fragment was buffered with 50 mM Tris-HCl, 10 mM MgCl ₂ , 5 mM DTT, 2 mM ATP, and 25 mg / ml BSA. After dilution in the solution, 2 μm of hairpin adapter was added thereto, and then 1 unit of T4 DNA ligase was added to selectively bind in a total volume of 50 μl.

5) Removal of unbound genomic DNA, adapters using exonuclease III

5 μL of the binding reactions obtained in 4) were added to a buffer solution containing 500 mM Tris-HCl (ph 8.0), 50 mM MgCl ₂ , and 100 mM 2-Mercaptoethanol, and 20 units of exonuclease III were added, followed by a total volume. The reaction mixture was adjusted to 30 μl and reacted at 37 ° C. for 45 minutes.

6) Amplification through polymerase chain reaction

3 μl of the reaction obtained in 5) was used as a template, and 1 unit of 1 uM, 100 mM Tris-HCl (pH 8.3), 400 mM KCl, 15 mM MgCl ₂ , 10 mM DTT, 5 ug / ml acetylated BSA, and 1 unit of Taq DNA polymerase were prepared. Volume 2 μl, then 5 minutes at 94 ° C, 35 seconds at 55 ° C, 2 minutes at 72 ° C, 30 times at 35 ° C at 94 ° C, 35 seconds at 55 ° C, 2 minutes at 72 ° C, 1times 94 ° C Amplified by 35 minutes at 55 ° C, 35 seconds at 72 ° C, and 5 minutes at 72 ° C.

7) Sample development in polyacrylamide gel using staining method

After 10 μl of the reaction product obtained in 6) was electrophoresed on a 4% polyacrylamide gel containing 8M urea, the gel was reacted for 30 minutes in a solution containing a fixed solution (10% acetic acid). Then, the gel was washed three times with distilled water from which ions were removed. This gel was stained for 30 minutes in a 1 L solution containing 1 g AgNO ₃ and 1.5 ml 37% formaldehyde. The stained gel was washed in deionized water for 5 seconds in deionized water, and then in a developer (40 g sodium carbonate, 1.5 ml 37% formaldehyde, 1 L solution containing 20 µl sodium thiosulfate (10 mg / ml)) adjusted to 4 ° C. After staining, the mixture was reacted with a fixed solution for 5 minutes, washed with distilled water for 10 minutes, and stored. The results are shown in FIGS. 1 and 2.

As shown in Figure 1, Lactobacillus spp. When the mutant strain (m) and the wild strain (w) treated with NTG (N-methyl-N'-nitroso-N-Nitroguanidine) as a chemical mutagen were analyzed according to the present invention, the mutant strain and the wild type strain Seven of the 120 hairpin adapter sets showed mutations in the genome. And although there is no change in the traditional 16s rDNA sequence, the method of the present invention was able to accurately detect the variation of the genomic genome. In FIG. 1, the arrow indicates the result of detecting the mutant genes causing different physiological characteristics, M is a marker indicating the relative size of the DNA, and lanes 1 to 7 are the mutant genes among the 120 sets of hairpin adapters. Represents a set of seven different hairpin adapters found.

Example 2. Confirmation of mutation of the strain through six passages

Wild strain Lactobacillus reuteri (ATCC 23272) was incubated in 37mL, 48 hours in 1.5mL MRS medium without a mutagen. 10 mL of this was taken and inoculated in 1.49 mL of fresh MRS medium, and the remaining 1.49 mL was centrifuged and stored at 70 ° C. And the same process was repeated five times. Six cells obtained at this time were taken and carried out according to the method given in Example 1 above. The results are shown in FIG.

As shown in FIG. 2, wild strains were passaged several times in a medium without a mutagen to analyze the developmental pattern of natural mutation, that is, quality control of the strain (Quality Control), in a medium without a mutagen. The genomic DNA extracted from each generation did not change during six passages with only a specific set of 120 hairpin adapter sets, and the development of the genome changed during six experiments of different orders. It is observed that there is no natural mutation pattern. Therefore, it can be seen that the quality of the strain is kept constant by the present invention. In Figure 2, M represents a marker indicating the relative size of the developed DNA, lanes 1 to 6 shows the number of passages 1-6 times.

As described above, according to the present invention, microorganism quality control can be performed by displaying the entire genome of the microorganisms on a gel and analyzing the genetic variation. In the long-term preservation of microorganisms according to the present invention can be appropriately used for quality control (Q.C) of microorganisms by checking the genetic variation of the microorganism. In addition, in the case of industrially useful food microorganisms (ex. Lactic acid bacteria) according to the present invention, "substantial equivalence," etc., which is the basis of safety evaluation of the food of these bacteria can be evaluated. In addition, according to the present invention, it is possible to efficiently analyze the low-level mutations made in a short time and efficiently for the whole genome in a short time, and thus, it is possible to identify the cause genes of the microorganisms which are useful academically.

Claims (4)

(i) cutting the microbial genomic DNA into several fragments having single-stranded coarse ends using restriction enzymes and, independently of this, a single strand capable of complementarily binding to both ends of the truncated genomic DNA fragment. Preparing a hairpin ring adapter having a tacky end; (ii) binding the genomic DNA fragment and the hairpin ring structure adapter using DNA ligase; (iii) using a exonuclease to remove genomic DNA fragments and hairpin ring structure adapters that did not participate in the binding reaction; (iv) amplifying the genomic DNA fragments using primers and DNA polymerases that complementarily bind to the hairpin structure adapter portion: (v) separating the amplification product on a gel: and (vi) microorganism quality control method comprising the step of identifying the genetic variation of the microorganism. 2. The method of claim 1, further comprising removing only the hairpin ring portion from the genomic DNA fragment bound with the hairpin ring adapter generated in step ii). The method of controlling quality of microorganisms according to claim 2, wherein only the hairpin ring portion is removed using a solution selected from the group consisting of alkaline solution, RNase, and single-strand-selective endonuclease. The method of claim 1, wherein the restriction enzyme is selected from the group consisting of Type IIs and Type IIIp restriction enzymes.