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CRISPR/Cas9 has potential for efficient molecular breeding. Recently, a foreign-DNA-free gene-targeting technology was established by introducing a preassembled Cas9 ribonucleoprotein (RNP) complex into the oyster mushroom Pleurotus ostreatus. However, the target gene was restricted to such a gene like pyrG, since screening of a genome-edited strain was indispensable and could be performed via examination of 5-fluoroorotic acid (5-FOA) resistance caused by the disruption of the target gene. In this study, we simultaneously introduced the Cas9 RNP complex targeting fcy1, a mutation that conferred P. ostreatus resistance to 5-fluorocytosine (5-FC), together with that targeting pyrG. A total of 76 5-FOA resistant strains were isolated during the first screening. Subsequently, a 5-FC resistance examination was conducted, and three strains exhibited resistance. Genomic PCR experiments followed by DNA sequencing revealed that mutations were successfully introduced into fcy1 and pyrG in the three strains. The results indicated that double gene-edited mutants could be obtained in one experiment employing 5-FOA resistance screening for strains with Cas9 RNP incorporation. This work may pave the way for safe CRISPR/Cas9 technology to isolate mutant strains in any gene of interest without an ectopic marker gene.

The evolution of mitochondria through variations in mitochondrial DNA (mtDNA) is one of the intriguing questions in eukaryotic cells. In order to assess the causes of the variations in mitochondria, the mtDNAs of the 21 strains of Lentinula edodes were assembled for this study, and analyzed together with four published mtDNA sequences. The mtDNAs were within the sizes of 117 kb ~ 122 kb. The gene number was observed consistent except for two mtDNAs, which carry a duplicated trnG1-trnG2 unit or a putative gene deletion. The size variation was largely attributed to the number of introns, repeated sequences, transposable elements (TEs), and plasmid-related sequences. Intron loss and gain were found from cox1, rnl, and rns of three mtDNAs. Loss of two introns in cox1 of KY217797.1 reduced its size by 2.7 kb, making it the smallest cox1 gene (8.4 kb) among the cox1s of the 25 mtDNAs, whereas gain of a Group II intron (2.65 kb) and loss of a Group I intron (1.7 kb) in cox1 of MF774813.1 resulted in the longest cox1 (12 kb). In rnl of L. edodes, we discovered four intron insertion consensus sequences which were unique to basidiomycetes but not ascomycetes. Differential incorporation of introns was the primary cause of the rnl size polymorphism. Homing endonucleases (HEGs) were suggestively involved in the mobilization of the introns because all of the introns have HEG genes of the LAGRIDADG or GIY-YIG families with the conserved HEG cleavage sites. TEs contributed to 11.04% of the mtDNA size in average, of which 7.08% was LTR-retrotransposon and 3.96% was DNA transposon, whereas the repeated sequences covered 4.6% of the mtDNA. The repeat numbers were variable in a strain-dependent manner. Both the TEs and repeated sequences were mostly found in the intronic and intergenic regions. Lastly, two major deletions were found in the plasmid-related sequence regions (pol2-pol3 and pol1-atp8) in the five mtDNAs. Particularly, the 6.8 kb-long deletion at pol2-pol3 region made MF774813.1 the shortest mtDNA of all. Our results demonstrate that mtDNA is a dynamic molecule that persistently evolves over a short period of time by insertion/deletion and repetition of DNA segments at the strain level.

Background The family Staphylinidae is the most speciose beetle group in the world. The outbreaks of two staphylinid species, Paederus fuscipes and Aleochara (Aleochara) curtula, were recently reported in South Korea. None of research about molecular markers and genetic diversity have been conducted in these two species. Objective To develop microsatellite markers and analyze the genetic diversity and population structures of two rove beetle species. Methods NGS was used to sequence whole genomes of two species, Paederus fuscipes and Aleochara (Aleochara) curtula. Microsatellite loci were selected with flanking primer sequences. Specimens of P. fuscipes and A. curtula were collected from three localities, respectively. Genetic diversity and population structure were analyzed using the newly developed microsatellite markers. Results The number of alleles ranged 5.727–6.636 (average 6.242) and 2.182–5.364 (average 4.091), expected heterozygosity ranged 0.560–0.582 (average 0.570) and 0.368–0.564 (average 0.498), observed heterozygosity ranged 0.458–0.497 (average 0.472) and 0.418–0.644 (average 0.537) in P. fuscipes and A. curtula, respectively. Population structure indicates that individuals of A. curtula are clustered to groups where they were collected, but those of P. fuscipes are not. Conclusion Population structures of P. fuscipes were shallow. In A. curtula, however, it was apparent that the genetic compositions of the populations are different significantly depending on collection localities.

The performance of photovoltaic devices can be improved and sustained when their surfaces are designed to increase light absorption of photoactive layers and minimize contaminant adsorption. Pyrami...

Background Exploring the microbiome in multiple body sites of a livestock species informs approaches to promote its health and performance through efficient and sustainable modulation of these microbial ecosystems. Here, we employed 16S rRNA gene sequencing to describe the microbiome in the oropharyngeal cavity, proximal colon, and vaginal tract of Jeju Black pigs (JBP), which are native to the Korean peninsula. Results We sampled nine 7-month-old JBP gilts raised under controlled conditions. The most abundant phyla that we found within the oropharyngeal microbiota were Proteobacteria, Bacteroidetes, Fusobacteria and Firmicutes, collectively providing core features from twenty-five of their genera. We also found a proximal colonic microbial core composed of features from twenty of the genera of the two predominant phyla, Firmicutes, and Bacteroidetes. Remarkably, within the JBP vaginal microbiota, Bacteroidetes dominated at phylum level, contrary to previous reports regarding other pig breeds. Features of the JBP core vaginal microbiota, came from seventeen genera of the major phyla Bacteroidetes, Firmicutes, Proteobacteria, and Fusobacteria. Although these communities were distinct, we found some commonalities amongst them. Features from the genera Streptococcus, Prevotella, Bacillus and an unclassified genus of the family Ruminococcaceae were ubiquitous across the three body sites. Comparing oropharyngeal and proximal colonic communities, we found additional shared features from the genus Anaerorhabdus. Between oropharyngeal and vaginal ecosystems, we found other shared features from the genus Campylobacter, as well as unclassified genera from the families Fusobacteriaceae and Flavobacteriaceae. Proximal colonic and vaginal microbiota also shared features from the genera Clostridium, Lactobacillus, and an unclassified genus of Clostridiales. Conclusions Our results delineate unique and ubiquitous features within and across the oropharyngeal, proximal colonic and vaginal microbial communities in this Korean native breed of pigs. These findings provide a reference for future microbiome-focused studies and suggest a potential for modulating these communities, utilizing ubiquitous features, to enhance health and performance of the JBP.

The interaction of mating pheromone and pheromone receptor from the B mating-type locus is the first step in the activation of the mushroom mating signal transduction pathway. The B mating-type locus of Lentinula edodes is composed of Bα and Bβ subloci, each of which contains genes for mating pheromone and pheromone receptor. Allelic variations in both subloci generate multiple B mating-types through which L. edodes maintains genetic diversity. In addition to the B mating-type locus, our genomic sequence analysis revealed the presence of a novel chromosomal locus 43.3 kb away from the B mating-type locus, containing genes for a pair of mating pheromones (PHBN1 and PHBN2) and a pheromone receptor (RCBN). The new locus (Bα-N) was homologous to the Bα sublocus, but unlike the multiallelic Bα sublocus, it was highly conserved across the wild and cultivated strains. The interactions of RcbN with various mating pheromones from the B and Bα-N mating-type loci were investigated using yeast model that replaced endogenous yeast mating pheromone receptor STE2 with RCBN. The yeast mating signal transduction pathway was only activated in the presence of PHBN1 or PHBN2 in the RcbN producing yeast, indicating that RcbN interacts with self-pheromones (PHBN1 and PHBN2), not with pheromones from the B mating-type locus. The biological function of the Bα-N locus was suggested to control the expression of A mating-type genes, as evidenced by the increased expression of two A-genes HD1 and HD2 upon the treatment of synthetic PHBN1 and PHBN2 peptides to the monokaryotic strain of L. edodes.

The New Zealand endemic genus Simkinion Park and Pearce, 1962 is redescribed and diagnosed to include six species, four of which are new: S. convexum sp. n., S. corniculum sp. n., S. schomannae sp....

Fermented total mixed ration (TMR) is a novel feed for ruminants in South Korea. The purpose of this study was to evaluate the effects of lactic acid bacteria (LAB) on the quality of TMR and in vitro ruminal fermentation. Strains of three LAB spp. (Lactobacillus plantarum, L. brevis, L. mucosae) were used in fermentation of TMR. Inoculations with the three LAB spp. lowered pH and increased concentrations of lactic acid, acetic acid, and total organic acid compared to non-LAB inoculated control (only addition of an equivalent amount of water) (p

Butyrate is known to play a significant role in energy metabolism and regulating genomic activities that influence rumen nutrition utilization and function. Thus, this study investigated the effects of an isolated butyrate-producing bacteria, Clostridium saccharobutylicum, in rumen butyrate production, fermentation parameters and microbial population in Holstein-Friesian cow. An isolated butyrate-producing bacterium from the ruminal fluid of a Holstein-Friesian cow was identified and characterized as Clostridium saccharobutylicum RNAL841125 using 16S rRNA gene sequencing and phylogenetic analyses. The bacterium was evaluated on its effects as supplement on in vitro rumen fermentation and microbial population. Supplementation with 106 CFU/ml Clostridium saccharobutylicum increased (p < 0.05) microbial crude protein, butyrate and total volatile fatty acids concentration but had no significant effect on NH3-N at 24 h incubation. Butyrate and total VFA concentrations were higher (p < 0.05) in supplementation with 106 CFU/ml Clostridium saccharobutylicum compared with control, with no differences observed for total gas production, NH3-N and propionate concentration. However, as the inclusion rate (CFU/ml) of C. saccharobutylicum was increased, reduction of rumen fermentation values was observed. Furthermore, butyrate-producing bacteria and Fibrobacter succinogenes population in the rumen increased in response with supplementation of C. saccharobutylicum, while no differences in the population in total bacteria, protozoa and fungi were observed among treatments. Overall, our study suggests that supplementation with 106 CFU/ml C. saccharobutylicum has the potential to improve ruminal fermentation through increased concentrations of butyrate and total volatile fatty acid, and enhanced population of butyrate-producing bacteria and cellulolytic bacteria F. succinogenes.

The interaction of mating pheromone and pheromone receptor from the B mating-type locus is the first step in the activation of the mushroom mating signal transduction pathway. The B mating-type locus of Lentinula edodes is composed of B�� and B�� subloci, each of which contains genes for mating pheromone and pheromone receptor. Allelic variations in both subloci generate multiple B mating-types through which L. edodes maintains genetic diversity. In addition to the B mating-type locus, our genomic sequence analysis revealed the presence of a novel chromosomal locus 43.3 kb away from the B mating-type locus, containing genes for a pair of mating pheromones (PHBN1 and PHBN2) and a pheromone receptor (RCBN). The new locus (B��-N) was homologous to the B�� sublocus, but unlike the multiallelic B�� sublocus, it was highly conserved across the wild and cultivated strains. The interactions of RcbN with various mating pheromones from the B and B��-N mating-type loci were investigated using yeast model that replaced endogenous yeast mating pheromone receptor STE2 with RCBN. The yeast mating signal transduction pathway was only activated in the presence of PHBN1 or PHBN2 in the RcbN producing yeast, indicating that RcbN interacts with self-pheromones (PHBN1 and PHBN2), not with pheromones from the B mating-type locus. The biological function of the B��-N locus was suggested to control the expression of A mating-type genes, as evidenced by the increased expression of two A-genes HD1 and HD2 upon the treatment of synthetic PHBN1 and PHBN2 peptides to the monokaryotic strain of L. edodes.

Fecal calprotectin is a noninvasive marker of gut inflammation and has been widely utilized in human gastrointestinal diagnostics. This marker, however, has not been extensively utilized in porcine samples. The aim of this study was to optimize a protocol for the extraction of porcine fecal calprotectin, and to the best of our knowledge this is the first study to be conducted in this regard. Freshly collected swine fecal samples were used in this study. We determined the variability of three commercial ELISA assays in the recovery of porcine fecal calprotectin. We further studied the effect of dilution factor and roller shaker homogenization on the yield of calprotectin from swine fecal samples. Calprotectin recovery was significantly different(p

Manipulation of both pore diameters and heights of two-dimensional periodic porous polymer films is important to extensively control their characteristics. However, except for using different sized colloid templates in replication methods, an effective method that tunes these factors has rarely been reported. We found that both parameters are controllable by adjusting the flow behaviors of polystyrene colloids and curing resin precursors during the preparation of phenolic resin and poly(dimethylsiloxane) periodic porous films by embedding their precursors into colloidal crystal monolayers. We adjust the flow behaviors by either varying film preparation temperatures (≥glass transition temperature of polystyrene) or using the precursors mixed with different amounts of solvents that renders the colloids viscous. Consequently, the pore diameters and film heights change by 36-56 and 56-84%, respectively. Such modulation results in the change in height to diameter ratios and the areal fractions of resins at air-film interfaces, thereby significantly changing the water contact angles on these surfaces and their photonic characteristics. This straightforward method does not require additional steps, differently sized colloids, or different amounts of precursors for these parameter controls.

Butyrate is known to play a significant role in energy metabolism and regulating genomic activities that influence rumen nutrition utilization and function. Thus, this study investigated the effects of an isolated butyrate-producing bacteria

The importance of probiotics in swine production is widely acknowledged as crucial. However, gaps still remain in the exact roles played by probiotics in modulation of gut microbiota and immune response. This study determined the roles of probiotic Lactobacillus plantarum strain JDFM LP11in gut microbiota modulation and immune response in weaned piglets. L. plantarum JDFM LP11 increased the population of lactic acid bacteria in feces and enhanced the development of villi in the small intestine. Metagenome analysis showed that microbial diversity and richness (Simpson, Shannon, ACE, Chao1) and the relative abundance of the Firmicutes were higher in weaned piglets fed probiotics. Five bacterial families were different in the relative abundance, especially; Prevotellaceae occupied the largest part of microbial community showed the most difference between two groups. Transcriptome analysis identified 25 differentially expressed genes using RNA-sequencing data of the ileum. Further gene ontology and immune DB analysis determined 8 genes associated with innate defense response and cytokine production. BPI, RSAD2, SLPI, LUM, OLFM4, DMBT1 and C6 genes were down-regulated by probiotic supplementation except PLA2G2A. PICRUSt analysis predicting functional profiling of microbial communities indicated branched amino acid biosynthesis and butyrate metabolism promoting gut development and health were increased by probiotics. Altogether, our data suggest that L. plantarum JDFM LP11 increases the diversity and richness in the microbial community, and attenuates the ileal immune gene expression towards gut inflammation, promoting intestinal development in weaned piglets.

Animal science nutrition studies are increasingly focusing on finding solutions to reduce methane (CH(4)) emissions. In the present study, we evaluated the effect of reductive acetogenic bacteria [acetogen probiotics (AP)] and lauric acid (LA) on in vivo rumen fermentation and microbial populations in Hanwoo steers. Four cannulated Hanwoo steers (392 ± 14 kg) were analyzed in a 4 × 4 Latin square design and were placed in hood-type chambers. They were fed similar amounts of concentrate and rice straw within and experimental design as follows: control (Con; 40 g DM basal feed, nonaddition of AP or LA), T1 = LA (40 g DM basal feed mixed with 40 g LA), T2 = AP (40 g DM basal feed, fermented with AP), and T3 = LA + AP (40 g DM basal feed, fermented with AP and mixed with 40 g LA). The animals were acclimatized to the diet for 15 d, followed by 6 d of the experimental period. Rumen fluid samples for metabolite and molecular analyses were collected 6 h after the morning feeding, with 2-h collection intervals. The enteric CH(4) production was monitored on the last 2 d of the experimental period. Concentrations of total volatile fatty acids increased with the increase in time after feeding. Acetate, propionate, and butyrate concentrations were observed to be higher in the treatments than in Con. The addition of LA and AP reduced CH(4) emission compared with that of Con (P < 0.01). Nuclear magnetic resonance spectroscopy results revealed no correlation between the LA and Con groups, but AP showed a correlation with LA and Con. Reduction in the number of protozoa which was accompanied by a decrease, because methanogens live symbiotically with protozoa. Supplementation of AP or LA alone and in combination decreased (P < 0.05) the methanogen population, whereas supplementation of LA alone significantly increased (P < 0.05) Ruminococcus flavefaciens and slightly increased total fungi. Thus, dietary supplementation of LA and AP has inhibitory effects on CH(4) production in Hanwoo cattle. If the effects of this method can be maintained, reductive acetogens could become an important part of strategies to lower ruminant CH(4) emissions.

KunKook University, Seoul 143-701, Republic of KoreaABSTRACTThis study was conducted to determine the quality of italian ryegrass(IRG) and whole- crop barley(WCB) silage combined with heterofermentative lactic acid bacteria(LAB) during fermentation. Six strains of LAB(L. plantarum IMAU 70164, L. acidophilus KACC 12419, L. casei KACC 12413, L. reuteri KCTC 3594, L. buchneri KACC 12416 and L. diolivorans KACC 12385) were used in this study. L. casei and L. reuteri had the highest propionic acid production and were therefore used for fermenting the forages. The forages were fermented using monoculture and co-culture of L. casei and L. reuteri for 30, 45 and 60 days of ensiling. Addition of LAB lowered the pH of the IRG silage(p

The effects of Lactobacillus mucosae (L. mucosae), a potential direct fed microbial previously isolated from the rumen of Korean native goat, on the rumen fermentation profile of brewers grain were evaluated. Fermentation was conducted in serum bottles each containing 1% dry matter (DM) of the test substrate and either no L. mucosae (control), 1% 24 h broth culture of L. mucosae (T1), or 1% inoculation with the cell-free culture supernatant (T2). Each serum bottle was filled anaerobically with 100 mL of buffered rumen fluid and sealed prior to incubation for 0, 6, 12, 24, and 48 h from which fermentation parameters were monitored and the microbial diversity was evaluated. The results revealed that T1 had higher total gas production (65.00 mL) than the control (61.33 mL) and T2 (62.00 mL) (p

The effects of Lactobacillus mucosae (L. mucosae), a potential direct fed microbial previously isolated from the rumen of Korean native goat, on the rumen fermentation profile of brewers grain were evaluated. Fermentation was conducted in serum bottles each containing 1% dry matter (DM) of the test substrate and either no L. mucosae (control), 1% 24 h broth culture of L. mucosae (T1), or 1% inoculation with the cell-free culture supernatant (T2). Each serum bottle was filled anaerobically with 100 mL of buffered rumen fluid and sealed prior to incubation for 0, 6, 12, 24, and 48 h from which fermentation parameters were monitored and the microbial diversity was evaluated. The results revealed that T1 had higher total gas production (65.00 mL) than the control (61.33 mL) and T2 (62.00 mL) (p

The present study aimed to evaluate the probiotic characteristics of certain microbial strains for potential use as feed additives. Three bacterial strains and a yeast previously isolated from different environments were investigated. The strains were subjected to molecular identification and established as Lactobacillus paracasei CP133, Lactobacillus plantarum CP134, Bacillus subtilis CP350 and Saccharomyces cerevisiae CP605. Lactobacillus sp. CP133 and CP134 exhibited antibiosis, antibiotic activity, and relative odor reduction ability. Bacillus subtilis CP350 was thermotolerant, reduced hydrogen sulfide gas and showed significant proteolytic activity, whereas Saccharomyces cerevisiae CP605 exhibited high acid and bile salt tolerance. In general, the isolates in this study demonstrated improved functional characteristics, particularly acid and bile tolerance and relative cell adhesion to HT-29 monolayer cell line. Results in this work provides multifunctional probiotic characteristics of the strains for potential development of probiotics and cleaning of the environment.

Sarpogrelate is a selective serotonin 5-HT2A-receptor antagonist used to treat patients with peripheral arterial disease. This drug is rapidly hydrolyzed to its main metabolite (R,S)-1-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]-3-(dimethylamino)-2-propanol (M-1), which is mainly excreted as a glucuronide conjugate. Sarpogrelate was also directly glucuronidated to an O-acyl glucuronide and a N-glucuronide by UDP-glucuronosyltransferases (UGTs) in human liver microsomes (HLMs). Since M-1 is pharmacologically more active than sarpogrelate, we examined glucuronidation of this metabolite in HLMs and characterized the UGTs responsible for M-1 glucuronidation. Diastereomers of O-glucuronide (SMG1 and SMG3) and a N-glucuronide (SMG2) were identified by incubation of M-1 with HLMs in the presence of uridine 5'-diphosphoglucuronic acid (UDPGA), and their structures were confirmed by nuclear magnetic resonance and mass spectrometry analyses. Two O-glucuronides were identified as chiral isomers: SMG1 as R-isomer and SMG3 as S-isomer. Using recombinant UGT enzymes, we determined that SMG1 and SMG3 were predominantly catalyzed by UGT1A9 and UGT2B4, respectively, whereas SMG2 was generated by UGT1A4. In addition, significant correlations were noted between the SMG1 formation rate and propofol glucuronidation (a marker reaction of UGT1A9; r = 0.6269, P < 0.0031), and between the SMG2 formation rate and trifluoperazine glucuronidation (a marker reaction of UGT1A4; r = 0.6623, P < 0.0015) in a panel of HLMs. Inhibition of SMG1, SMG2, and SMG3 formation by niflumic acid, hecogenin, and fluconazole further substantiated the involvement of UGT1A9, UGT1A4, and UGT2B4, respectively. These findings collectively indicate that UGT1A4, UGT1A9, and UGT2B4 are the major UGT isoforms responsible for glucuronidation of M-1, an active metabolite of sarpogrelate.

This study was conducted to evaluate the effects of feeding supplemental illite to Hanwoo steers on methane (CH4 ) emission and rumen fermentation parameters. An in vitro ruminal fermentation technique was conducted using a commercial concentrate as substrate and illite was added at different concentrations as treatments: 0%, 0.5%, 1.0%, and 2.0% illite. Total volatile fatty acids (VFA) were different (P < 0.05) at 24 h of incubation where the highest total VFA was observed at 1.0% of illite. Conversely, lowest CH4 production (P < 0.01) was found at 1.0% of illite. In the in vivo experiment, two diets were provided, without illite and with addition of 1% illite. An automated head chamber (GreenFeed) system was used to measure enteric CH4 production. Cattle received illite supplemented feed increased (P < 0.05) total VFA concentrations in the rumen compared with those fed control. Feeding illite numerically decreased CH4 production (g/day) and yield (g/kg dry matter intake). Rumen microbial population analysis indicated that the population of total bacteria, protozoa and methanogens were lower (P < 0.05) for illite compared with the control. Accordingly, overall results suggested that feeding a diet supplemented with 1% illite can have positive effects on feed fermentation in the rumen and enteric CH4 mitigation in beef cattle.

본 연구는 가축생균제용 유산균을 Real-time PCR정량분석법을 이용하여 분석하였다. SYBR Green1 방법과 Probe 방법을 이용하여 표준곡선을 제작한 결과, SYBR Green1 방법에서는 Slope 값이 -3.346이었고, Y절편은 33.18, $R^2$ 값은 0.993으로 나타났으며, Probe 방법에서는 Slope값이 -3.321이었고, Y절편은 39.10, $R^2$ 값은 0.995로 나타나, 이를 이용한 표준곡선 제작이 가능함을 알 수 있었다. SYBR Green1 방법을 이용한 생균제의 Lactobacilli 정성 정량 분석결과 Real-time PCR값은 4.46~6.56 log copies로 나타났고, 생균수 측정 결과 값은 5.63~7.59 log CFU/g로 나타났으며, Probe 방법을 이용한 생균제의 Lactobacilli 정성 정량 분석결과에서는 Real-time PCR 값은 5.51~7.00 log copies로 나타났으며, 생균수 측정 결과 값은 5.63~7.59 log CFU/g로 나타났다. 본 연구에서 실시한 RT PCR법은 3~4일이 소요되는 기존의 배지법과 비교하여 24시간 이내에 신속하게 검출이 가능하다고 여겨지며, 또한 RT PCR을 이용한 분석방법에서도 dye 사용과 primer 사용에 따라 결과값이 차이가 나타났음을 확인할 수 있었으며, Probe 방법을 이용하여 실험 한 결과가 민감한 결과를 나타내었음을 확인 할 수 있었다. 【This study was conducted using quantitative real-time PCR using Lactobacilli as probiotics. Quantitative real-time PCR (RT PCR) was conducted via a method involving SYBR Green 1 and a probe. Plasmid DNA was cloned using the 16S-23S rRNA intergenic species region. Gene clones were diluted from $10^2$ to $10^{10}$ . Standard curves were constructed via Ct values obtained from the results of Real-time PCR via the aforementioned SYBR Green 1 and probe method. Plasmid DNA was also cloned using the 16S-23S rRNA intergenic species region and the gene clones were diluted from $10^2$ to $10^{10}$ copy numbers via the probe method. Using RT PCR, a standard curve of plasmid DNA copy numbers was also determined. The slope value for the Y-axis intercept and $R^2$ value were measured as -3.346, 33.18, and 0.993, respectively, via the first method. For the second method, the slope value for the Y-axis intercept and $R^2$ were -3.321, 31.10 and 0.995, respectively. The PCR inhibitor could not express the detection curve at a copy number over $10^{10}$ via either method, owing to high DNA density. The DNA extract from probiotics was diluted without pre-culturing, and 16 products were amplified via both methods. The Ct value was 11.06~18.12 in the first method and 16.74~22.11 in the second method. Measured probiotics and log copy values were largely similar among the methods used. It was concluded that both methods are effective for analysis, but further research will be required to verify the optimal method.】