적조가 처음 시작되는 해역을 조기에 파악하기 위하여 Quantitative real-time PCR (qPCR)을 경남해역 적조현장에 활용하였다. 2019년 경남해역을 대상으로 Cochlodinium polykrikoides를 qPCR로 정량분석한 결과, 6월 초에 저밀도로(0.0015~0.0058 cells mL-1) 검출되기 시작하여 8월 중순에는 최대 0.163 cells mL-1 밀도로 증가하였고, 주로 남해도 주변에서 높게 검출되었다. 8월 말에는 현미경 검경으로 남해도 주변에서 높게 출현함이 확인되었고(최대 24 cells mL-1), 9월 2일에는 남해도에서 적조주의보가 발령되었고(최대 200 cells mL-1), 9월 11일에는 최대 12,000 cells mL-1까지 남해도 해역에서 발생하였다. 위 결과는 극미량의 C. polykrikoides이 적조발생 전에 남해도에서 검출 되었고 이후 같은 해역에서 적조가 발생되었음을 보여준다. 이는 qPCR이 극미량의 C. polykrikoides을 조기검출하는데 유용한 방법임을 보여준다.

Honey bee, Apis mellifera L., have been widely used as a model organism for biological science because of its highly developed sociality, specialized labor division and passive population management. In order to examine the expression patterns of genes putatively involved in social development in honey bee, quantitative real-time PCR (qRT-PCR) that has been widely used to investigate the expression level of target gene can be used in honey bee study. However, the selection and validation of optimal reference genes is a crucial step prior to running qRT-PCR. In the present study, therefore, the seasonal expression stability of five candidate reference genes in the abdomen of forager and nurse was investigated using three programs (geNorm, NormFinder and BestKeeper), and selected reference genes were validated by the normalization of expression level of vg encoding vitellogenin. Although three programs revealed slightly different gene stability values, overall the combination of two genes (rpS18 and gapdh encoding ribosomal protein S18 and glyceraldehyde-3-phosphate dehydrogenase, respectively) was resulted in the most suitable use for normalization of the target gene in forager. However, a single gene, either rpL32 or rpS18 in the nurse or either rpL32, rpS18, or gapdh in the comparison between foragers and nurses, were suggested to be applied for normalization of seasonal and labor-specific gene expression by qRT-PCR.

The fruit fly, Drosophila melanogaster, is a good model organism in various areas of biological science. Since D. melanogaster has been thought to be adapted to the chemical stress environment caused by the overripen, decay and fermented fruits, identification of the genes involved in chemical tolerance and investigation of their expression patterns are essential for better understanding of the physiological evolution in D. melanogaster. For investigation of the gene expression level, quantitative real-time PCR (qRT-PCR) can be applied to quantify gene expression level and selection of reliable reference gene(s) for normalization is an accurate step. In the present study, therefore, we validated the expression stabilities of ten candidate reference genes using three softwares (geNorm, NormFinder and BestKeeper) in D. melanogaster exposed to different concentrations of acetic acid, ethanol and 2-phenylethanol. Although three programs resulted in slightly different gene stability ranks, but overall tbp encoding TATA box binding protein was most stable gene in acetic acid and ethanol exposed fly, while nd encoding NADH dehydrogenase was the most suitable reference gene in the case of 2-phenylethanol treatment. In the comparison of three chemical treatment condition, nd was also suggested to be most optimal reference gene. In addition, optimal number of reference gene for accurate normalization was calculated by geNorm pairwise analysis, and selection of multiple reference genes was suggested to be better for target gene normalization method than use of a single reference gene.

과일이나 농작물의 부패 및 발효 환경에서는 Methanol, Ethanol, Acetic acid을 비롯한 다양한 화학물질들이 생산된다. Drosophila melanogaster는 이러한 발효·부패 환경에 서식하면서 일정 농도 이상의 다양한 화학물질에 지속적으로 노출되어 생존하도록 적응되어온 것으로 생각된다. 다양한 화학물질이 포함한 환경에 안정적으로 서식하기 위해서는 D. melanogaster는 화학물질에 능동적으로 반응하여 해독 유전자나 대사 관련 유전자의 발현량을 변화 시킴으로써 발효·부패 환경에서 생성되는 화학물질에 대한 높은 내성을 가지고 있을 것으로 판단된다. 현재까지 유전자의 발현량 측정을 위해 real-time PCR를 이용하여 reference gene의 발현량을 기준으로 정량화하는 방법이 가장 널리 사용되고 있다. 그러나 조직별, 환경별, 발달단계를 비롯한 다양한 조건에서 안정적으로 발현되는 reference 유전자 선정이 필수적으로 선행되어야 하므로 본 연구에서는 발효·부패 환경에서 생산되는 두 화학물질인 Methanol과 Ethyl Acetate에 노출된 D. melanogaster에서 안정적으로 발현되는 reference gene을 찾는 연구를 실시하였다. 본 연구에서는 다양한 농도의 Methanol과 Ethyl Acetate을 D. melanogaster에 노출시킨 후 RNA 추출과 cDNA 합성을 실시였고, 5가지 후보 reference gene (hsp22, nd, rpL18, tbp and ef-1b)의 안정적 발현 여부를 qRT-PCR을 통해 조사하였으며, 유전자 발현의 안정성을 측정하는 3가지 프로그램(geNorm, NormFinder, BestKeeper)을 이용해 비교·분석하였다. 본 학회에서는 연구의 과정과 그 결과를 발표하고자 한다.

본 연구는 국내에서 생산되거나 해외에서 수입되어 국내에서 유통되는 수산물 중에서 두족류를 문어류, 낙지류, 오징어류, 주꾸미류, 꼴뚜기류의 5개 그룹으로 구분하여 분석하였다. 두족류 5개 그룹을 판별을 하기 위해 미토콘드리아에 존재하는 유전자를 분석하였고, 그 중에서 COI (mitochondrial cytochrome C oxidase subunit I), 16s rRNA (16s ribosomal RNA), 12s rRNA (12s ribosomal RNA) 내에서 상당히 유사한 DNA 서열 부분과 일부 서열 변화 부분이 확인되었다. 명확하게 두족류 5개 그룹 판별을 하기 위해 COI, 16s rRNA, 12s rRNA 유전자의 일부 서열 변화 부분에서 그룹 특이적 프라이머 세트를 디자인하였다. 국내·외에서 확보한 두족류 시료(참문어, 낙지, 살오징어, 아메리카 대왕오징어, 갑오징어, 주꾸미, 모래주꾸미, 하이야주꾸미, 참꼴뚜기, 창꼴뚜기, 한치꼴뚜기)의 genomic DNA을 추출하여 각 그룹의 특이적 프라이머를 이용하여 SYBR 기반의 real-time PCR 시스템에 의해 분석되었고, threshold cycle (Ct) value와 같은 real-time PCR 결과 분석에 의해 두족류 내 그룹 판별이 가능하였다(Table 3).

Honey bee has been widely used as a model insect for biological sciences because of its sociality and specialized labor division. For the investigation of the seasonal and labor-dependent expression patterns of genes putatively involved in its sociality, quantitative real-time PCR (qRT-PCR) can be applied to quantify gene expression level and selection of reliable reference gene(s) for normalization is an accurate step. In this study, using three softwares (geNorm, NormFinder and BestKeeper), we evaluated seasonal expression stabilities of four reference genes that have been widely used for qRT-PCR in forager and nurse heads. Among four candidates, two genes, rpS18 and gapdh, were suggested to be the optimal reference genes for qRT-PCR.

The purpose of this study was to develop Streptococcus sobrinus-specific qPCR primers based on the nucleotide sequence of the RNA polymerase β-subunit gene (rpoB). The specificity of the primers was determined by conventional polymerase chain reaction (PCR) with 12 strains of S. sobrinus and 50 strains (50 species) of non-S. sobrinus bacteria. The sensitivity of the primers was determined by quantitative real-time PCR (qPCR) with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of S. sobrinus ATCC 33478T. The specificity data showed that the S. sobrinus-specific qPCR primers (RTSsob-F4/RTSsob-R4) detected only the genomic DNAs of S. sobrinus strains with a detection limit of up to 4 fg of S. sobrinus genomic DNA. Our results suggest that the RTSsob-F4/RTSsob-R4 primers are useful in detecting S. sobrinus with high sensitivity and specificity for epidemiological studies of dental caries..

Prevotella intermedia-specific quantitative real-time PCR (qPCR) primers were previously designed based on the nucleotide sequences of RNA polymerase β-subunit gene (rpoB). However, the several clinical strains isolated from Korean populations are not detectable by the qPCR primers. The purpose of this study was to develop new P. intermedia-specific qPCR primers based on the rpoB. The specificity of the primers was determined by conventional PCR with 12 strains of P. intermedia and 52 strains (52 species) of non-P. intermedia bacteria. The sensitivity of primers was determined by qPCR with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of P. intermedia ATCC 25611T. The data indicated that only P. intermedia strains were detected by the P intermedia-specific qPCR primers (RTPiF2/RTPiR2); in addition, as little as 40 fg of P. intermedia genomic DNA could be detected. These results suggest that these qPCR primers are useful in detecting P. intermedia from the bacterial infectious lesions including dental plaque and oral tissue lesions.

본 연구에서는 FCV 현탁액에 물리, 화학적 위생처리 후 복합효소처리라는 전처리과정을 적용한 뒤 real-time RTPCR법을 이용하여 살균효능을 분석하였다. RT-PCR 이전에 37oC에서 30분 동안 PK와 RNase A를 처리함으로써 UV, 열, 염소, 에탄올, 과초산계열 제품에 의해 불활성화 된 바이러스들은 음성 결과를 나타내었고, real-time RTPCR법을 통해 살균 효능을 정량분석한 결과, 복합효소처리를 했을 경우 무처리구보다 더 높은 살균 효능을 보이는 것을 확인할 수 있었다. 이로써 Nuanualsuwan S. 등11,18,29)의 선행연구에서와 같이 PK와 RNase A로 전처리하는 단계를 통하여 물리, 화학적 위생처리에 의해 손상되지 않은 바이러스가 RT-PCR 법에 의해 증폭되는 것을 방지함으로써 Real-time PCR법 에 대한 검출 감도를 높일 수 있음을 확인하였다. 또한, FCV를 검출하기 위해 사용된 RT-PCR과 real-time RT-PCR 두 방법 중에서도 real-time RT-PCR법이 가장 신속하면서도 민감도 높은 결과로 도출되었다. 따라서, 유전자 분석 이전에 복합효소처리는 물리, 화학적 위생처리에 의해 불활성화 된 바이러스의 RNA가 transcription 또는 증폭되는 것을 방지하기 위한 수단으로 real-time RT-PCR법과 결합 됨으로써 노로바이러스를 비롯한 식중독 바이러스를 검출 하는데 효과적으로 적용될 것으로 판단된다. 또한 식품현 장에서 전기영동 과정없이 신속하게 살아있는 바이러스만을 수치적으로 정량화함으로써 식품안전에도 기여할 것으 로 사료된다.

Norovirus causes acute gastroenteritis in all age groups and its food poisoning outbreaks are rapidly increasing in Korea. Reverse transcription-polymerase chain reaction (RT-PCR) is most widely used for the rapid detection of foodborne viruses due to high sensitivity. However, the false positive results of RT-PCR obtained against already inactivated viruses could be a serious drawbacks in food safety area. In this study, we investigated a method to yield true positive RT-PCR results only with alive viruses. To decompose the RNA genes from dead viruses, the enzymatic treatments composed of proteinse K and Ribonuclease A were applied to the sanitized and inactivated virus particles. Another aim of this study was to quantify the efficiencies of several major sanitizing treatments using realtime RT-PCR. Feline calicivirus (FCV) that belongs to the same Caliciviridae family with norovirus was used as a surrogate model for norovirus. The initial level of virus in control suspension was approximately 104 PFU/mL. Most of inactivated viruses treated with the enzymatic treatment for 30 min at 37oC were not detected in RT-PCR, Quantification results to verify the inactivation efficiencies of sanitizing treatments using real-time RT-PCR showed no false positive in most cases. We could successfully develope a numerical quantification process for the inactivated viruses after major sanitizing treatments using real-time RT-PCR. The results obtained in this study could provide a novel basis of rapid virus quantification in food safety area.

The chigger mite, Leptotrombidium pallidum, is widely distributed throughout South Korea and is a major vector for Orientia tsutsugamushi, the causative agent of scrub typhus. In this study, the genome size of the chigger mite was estimated to determine the necessary coverage level prior to whole genome sequencing. Cloning of EF1α and RpS3 as putative single copy reference genes were conducted and their partial sequences were determined. Using the serially diluted reference genes with known amount as standard templates, the weight of a single copy of the genome was predicted by a method based on quantitative real time PCR. The average genome length estimated from the weight using two methods was 191 ± 7 Mb. When the genome size of other arthropods (Drosophila melanogster, Apis mellifera and Tetranychus urticae), with their genome analysis completed, were estimated using the same method and compared with actual values, the estimation accuracy was 79.8-98.9%, suggesting our current estimation of L. pallidum genome size is reliable. The estimated L. pallidum genome size is in a similar range to other Acariform mites, such as the dust mite and scabie mite, but appoximately 10-fold smaller compared to the deer tick, which belongs to Parasitiform. Our finding provides key information for further genome sequencing and understanding of mite genome evolution.

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators since the outbreak of honeybee collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites, which affect the life span and fecundity of their host, have been discovered in B. terristris. In order to detect the microsporidian pathogen, Nosema Spp. in the field populations of B. terristris, we collected adults and isolated their genomic DNA for diagnostic PCR. The PCR primers specific for Nosema Spp. were newly designed and applied to gene amplification for cloning. Only small subunit ribosomal RNA(SSU rRNA) gene of N. ceranae was successfully amplified and sequenced among examined genes, which indicates that N. ceranae mainly infects the examined field population of B. terristris. To detect of SSU rRNA gene, two regions of SSU rRNA gene were selected by primary PCR analysis and further analyzed in quantitative real-time PCR(qRT-PCR). The qRT-PCR analysis demonstrated that SSU rRNA of N. ceranae was detected at concentrations as low as 0.85 ng/μl genomic DNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae infection in the field population as well as risk assessment of B. terristris.

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators. Recently, pathogens and parasites affect the life span and fecundity of their host and been isolated from B. terristris. In order to detect viral infection in the field populations of B. terristris, we collected adults and isolated total RNA for reverse transcriptase-polymerase chain reaction (PCR). The PCR primers specific for several viruses such as deformed wing virus, Israel acute paralysis virus, Kashmir bee virus and black queen cell virus (BQCV) were newly designed and applied to gene amplification for cloning. Only BQCV was successfully amplified and sequenced, which suggests that BQCV may mainly infects the examined field population of B. terristris. To detect of capsid protein gene of BQCV, 4 selected regions were analyzed by primary PCR and 1 region was successfully amplified, which was further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that BQCV was detected at concentrations as low as 0.1ng/μl total RNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of BQCV infection in the field population as well as risk assessment of B. terristris.

Bombus terrestris has played an important role in the pollination in agricultural fields for the alternatives in colony collapsing in the honeybee. Recently, some pathogens or parasites such as viruses, bacteria, mites have been discovered in B. terristris, which affects its life span and fecundity. In order to detect a microsporidian, Nosema apis. in the field population, we collected honeybees and isolated genomic DNA. PCR primers specific for 16S ribosomal RNA (16S rRNA) were synthesized and applied to gene amplification for cloning and quantitative real-time PCR (qRT-PCR). The amplified gene was cloned and sequenced to confirm the 16S rRNA gene. qRT-PCR analysis showed the detection limit of 16S rRNA of Nosema apis was approximately 0.5 ng/μl genomic DNA. This result suggests that detection via qRT-PCR can be applied for the diagnosis of pathogen infection.

We constructed a standard curve to quantify Listeria monocytogenes in ready-to-eat product, especially sausage samples, using real-time PCR. A standard curve was generated using serially diluted L. monocytogenes cells in distilled water. When cells were artificially inoculated in 10 g of sausage samples in 90㎖ buffered peptone water, the cell concentration of range was approximately 1.0×108 to 100 CFU/㎖. The standard curve of the serially diluted cells was linear for at least seven orders of magnitude from 103 to 109 CFU/㎖ of L. monocytogenes. When cells were diluted in sausages, the linearity range was from 104 to 108 CFU/㎖. The correlation coefficient (R2) of diluted cells was 0.9888 and the slope of the curve was —2.6621. The coefficient and slope of inoculated samples were 0.9916 and —2.747, respectively. The R2 value for serially diluted L. monocytogenes and artificially contaminated sausage samples were acceptable. The approach described in this study represents the potency of the quantification of L. monocytogenes in sausage samples by quantitative real-time PCR. It can be used in monitoring the presence and persistence of this pathogen in sausages.

Sperm chromatin integrity is essential for successful fertilization and development of an embryo. Reported here is a quantification of DNA fragments which is intimately associated with reproductive potential to provide one of criteria for sperm chromatin integrity. Three sperm populations were considered: CONTROL (no treatment), UV irradiation (48mW/, 1h) and (oxidative stress induced by hydrogen peroxide, 10 mM, 50 mM and 100 mM). DNA fragments in boar sperm were evaluated by using ligation-mediated quantitative real-time polymerase chain reaction (LM-qPCR) assay, which relies on real-time qPCR to provide a measure of blunt 5' phosphorylated double strand breaks in genomic DNA. The results in agarose gel electrophoresis showed no significant DNA fragmentation and no dose-dependent response to . However, the remarkable difference in shape and position was observed in melting curve of LM-qPCR. This result supported that the melting curve analysis of LM-qPCR presented here, could be more sensitive and accurate than previous DNA fragmentation assay method.

Sex-sorting of sperm is an assisted reproductive technology (ART) used by the livestock industry for the mass production of animals of a desired sex. The standard method for sorting sperm is the detection of DNA content differences between X and Y chromosome-bearing sperm by flow cytometry. However, this method has variable efficiency and therefore requires verification by a second method. We have developed a sex determination method based on quantitative real-time polymerase chain reaction (qPCR) of the porcine amelogenin (AMEL) gene. The AMEL gene is present on both the X and the Y chromosome, but the length and sequence of its noncoding regions differ between the X and Y chromosomes. By measuring the threshold cycle (Ct) of qPCR, we were able to calculate the relative frequency of X chromosome. Two sets of AMEL primers were used in these studies. One set (AME) targeted AMEL gene sequences present in both X and Y chromosome, but produced PCR products of different lengths for each chromosome. The other set (AXR) bound to AMEL gene sequences present on the X chromosome but absent esholthe Y-chromosome. Relative product levels were calculated by normalizing the AXR fluorescence to the AME fluorescence. The AMEL method accurately predicted the sex ratios of boar sperm, demonstrating that it has potential value as a sex determination method.