The ovary undergoes substantial physiological changes along with estrus phase to mediate negative/positive feedback to the upstream reproductive tissues and to play a role in producing a fertilizable oocyte in the developing follicles. However, the disorder of estrus cycle in female can lead to diseases, such as cystic ovary which is directly associated with decline of overall reproductive performance. In gene expression studies of ovaries, quantitative reverse transcription polymerase chain reaction (qPCR) assay has been widely applied. During this assay, although normalization of target genes against reference genes (RGs) has been indispensably conducted, the expression of RGs is also variable in each experimental condition which can result in false conclusion. Because the understanding for stable RG in porcine ovaries was still limited, we attempted to assess the stability of RGs from the pool of ten commonly used RGs (18S, B2M, PPIA, RPL4, SDHA, ACTB, GAPDH, HPRT1, YWHAZ, and TBP) in the porcine ovaries under different estrus phase (follicular and luteal phase) and cystic condition, using stable RG-finding programs (geNorm, Normfinder, and BestKeeper). The significant (p < 0.01) differences in Ct values of RGs in the porcine ovaries under different conditions were identified. In assessing the stability of RGs, three programs comprehensively agreed that TBP and YWHAZ were suitable RGs to study porcine ovaries under different conditions but ACTB and GAPDH were inappropriate RGs in this experimental condition. We hope that these results contribute to plan the experiment design in the field of reproductive physiology in pigs as reference data.

This study aimed to develop strain-specific polymerase chain reaction (PCR) primers to detect Fusobacterium hwasookii KCOM 1249T, F. hwasookii KCOM 1253, F. hwasookii KCOM 1256, F. hwasookii KCOM 1258, and F. hwasookii KCOM 1268 on the basis of nucleotide sequences of a gene specific to each strain. The unique genes for each F. hwasookii strain were determined on the basis of their genome sequences using Roary. The strain-specific PCR primers based on each strain-specific gene were designed using PrimerSelect. The specificity of each PCR primer was determined using the genomic DNA of the 5 F. hwasookii strains and 25 strains of oral bacterial species. The detection limit and sensitivity of each strain-specific PCR primer pair were determined using the genomic DNA of each target strain. The results showed that the strain-specific PCR primers correspond to F. hwasookii KCOM 1249T, F. hwasookii KCOM 1253, F. hwasookii KCOM 1258, F. hwasookii KCOM 1256/F. nucleatum subsp. polymorphum KCOM 1260, or F. hwasookii KCOM 1268/Fusobacterium sp. oral taxon 203 were developed. The detection limits of these strain-specific PCR primers ranged from 0.2 to 2 ng of genomic DNA for each target strain. The results suggest that these strain-specific PCR primers are valuable in quality control for detecting specific F. hwasookii strains.

This study aimed to develop Lautropia mirabilis -specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis . The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/ RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

사람 아이치바이러스 (Aichivirus A; AiV-A)는 positivesense single-strand RNA 비외피 바이러스로 지난 10년 동안 하수, 강, 지표 및 지상의 다양한 물환경에서 전 세계적으로 검출이 보고되고 있다. 지하수 등 물환경에서 AiV-A 진단을 위한 고감도 및 특이성이 우수한 방법의 개발이 요구됨에 따라, 본 연구에서는 기존 및 신규 설계된 프라이 머 세트를 기초로 역전사 (RT) 및 이중 중합효소연쇄반응이 가능한 조합을 개발하였다. 개발한 방법을 국내 음용 지하수 시료에 적용 및 평가하였으며, 그 결과 지하수 시료에서 AiV-A를 성공적으로 검출 및 동정할 수 있는 RTnested PCR primer sets가 선정되었고 후속적으로 동정할 수 있는 절차가 고안되었다. 본 연구 결과는 지하수 등 물 환경에서 AiV-A 오염을 탐지하기 위한 모니터링 시스템 마련에 기여할 것으로 기대된다.

Salmonella is one of the most important bacterial pathogens responsible for many zoonotic food-related infectious diseases. Quantitative detection of the foodborne Salmonella contamination in various food sources is therefore critical for preventing the related disease outbreaks. In this study, we developed and evaluated a reliable real-time polymerase chain reaction (RT-PCR) assay to detect the Salmonella contamination quantitatively. The experimental results showed that our invA gene-specific quantitative RT-PCR (qRT-PCR) assay provides a strong correlation between the Cq values and the direct plate counts of Salmonella species in the artificially formulated samples. Further study may be necessary to identify more accurate correlation and equation that can apply to Salmonella spp.

The purpose of this study was to develop Peptoniphilus mikwangii -specific quantitative real-time polymerase chain reaction (qPCR) primers based on the 16S ribosomal RNA (16S rDNA) gene. The specificity of the primers was determined by conventional PCR using 29 strains of 27 oral bacterial species including P. mikwangii. The sensitivity of the primers was determined by qPCR using the purified genomic DNA of P. mikwangii KCOM 1628T (40 ng to 4 fg). The data showed that the qPCR primers (RTB134-F4/RTB134-R4) could detect P. mikwangii strains exclusively and as little as 40 fg of the genomic DNA of P. mikwangii KCOM 1628T. These results suggest that the developed qPCR primer pair can be useful for detecting P. mikwangii in epidemiological studies of oral bacterial infectious diseases.

Recently, the importance of on-site detection of pathogens has drawn attention in the field of molecular diagnostics. Unlike in a laboratory environment, on-site detection of pathogens is performed under limited resources. In this study, we tried to optimize the experimental conditions for on-site detection of pathogens using a combination of ultra-fast convection polymerase chain reaction (cPCR), which does not require regular electricity, and nucleic acid lateral flow (NALF) immunoassay. Salmonella species was used as the model pathogen. DNA was amplified within 21 minutes (equivalent to 30 cycles of polymerase chain reaction) using ultra-fast cPCR, and the amplified DNA was detected within approximately 5 minutes using NALF immunoassay with nucleic acid detection (NAD) cassettes. In order to avoid false-positive results with NAD cassettes, we reduced the primer concentration or ultra-fast cPCR run time. For singleplex ultra-fast cPCR, the primer concentration needed to be lowered to 3 μM or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, 2 μM of each primer set needed to be used or the run time needed to be reduced to 14 minutes. Under the conditions optimized in this study, the combination of ultra-fast cPCR and NALF immunoassay can be applied to on-site detection of pathogens. The combination can be easily applied to the detection of oral pathogens.

This study is for the consideration of the existence tendency of Kudoa septempunctata in olive flounder. In general, muscle has shown a strong PCR positive reaction in spores containing tissues rather than non-containing tissues. However, blood PCR results showed opposed tendency. In various organs of the tested fish containing spores in muscle tissue, heart had shown positive reaction along with muscle at PCR analysis. Muscle fiber necrosis was observed at the histological observation, and this degeneration was common in both samples. The one sample was the PCR positive muscle containing spore and the other was the PCR positive muscle non-containing spore. Both of muscle tissues indicated a positive reaction at ISH (in-situ hybridization) against K. septempunctata.

본 연구에서는 오징어류에 해당하는 대왕오징어, 오징어, 문어, 한치 및 이를 이용한 가공식품에 대해서 분자생물 학적 기법을 활용한 시험법을 검토하였다. 시료 중 원료 성분 확인을 위하여 오징어류 4종에 대해 최적의 종 특이 프라이머를 디자인하였으며, 시료로부터 직접 genomic DNA를 추출하여 PCR을 실시하였다. PCR 수행과정에서 반응을 저해하는 염 성분을 제거하기 위하여 증류수를 이용하여 3~4회 세척 후 PCR을 실시한 결과, Single PCR의 경우 대왕오징어(552 bp), 오징어(463 bp), 문어(247 bp), 한치(354 bp)에 해당하는 종 특이적인 증폭산물을 확인하였으며, Multiplex PCR 의 경우 서로 다른 종 사이의 교차반응없이 동시다발적으로 증폭이 일어남을 확인할 수 있었다. 또한 이들 4종에 대해 PCR 민감도를 조사한 결과, 모두 약 0.1 ng/μl의 농도까지 검출이 가능함을 확인하였으며 multiplex PCR의 경우 약 0.25 ng/μl의 농도까지 검출이 가능함을 확인하였다. 이를 이용하여 오징어류가 함유된 수산물 가공식품 8건에 대해 적용성을 검토한 결과, 모든 시료에서 유효한 결과를 확인할 수 있었다. 따라서 본 연구에서 제작된 오징어류 4종에 대한 종 특이적 프라이머는 생물 상태뿐만 아니라 수산물 가공식품에 대해서도 이를 판별할 수 있어 식품안전관리에 활용할 수 있을 것으로 기대된다.

Salmonellosis is one of the most common food-borne diseases in both humans and animals. The recovery of Salmonella from fecal and environmental samples by bacteriological assays takes several days. Polymerase chain reaction (PCR) has become an important technique for the rapid detection of Salmonella in a variety of samples, including feces. For rapid identification of Salmonella by PCR, 1 mL of enrichment culture was harvested after overnight incubation and DNA was extracted by heat lysis. To investigate the optimal conditions for rapid PCR detection of Salmonella, three different primer sets and three different enrichment media were used on a panel of Salmonella strains and a panel of non-Salmonella strains. The results showed that selenite cysteine enrichment broth and a primer set designed for the invA gene provided the most specific and rapid detection of Salmonella by PCR after the enrichment step.

Mycoplasma (M.) hyopneumoniae is the causative agent of swine enzootic pneumonia, a disease that is prevalent in every country where pigs are raised. In this study, we aimed to develop a sensitive and specific PCR assay to detect M. hyopneumoniae in pigs. The suitability of this PCR assay for the detection of mycoplasmal infection was also tested using clinical lung samples from slaughtered pigs. We de- veloped a probe and M. hyopneumoniae-specific primer pairs, MhyoP-F and MhyoP-R, for the new PCR assay based on regions in the Mycoplasma protein P97 gene that are unique to M. hyopneumoniae. The developed PCR as- say was very specific and sensitive for the detection of M. hyopneumoniae. The assay was able to detect the equivalent of 10 pg of target template DNA, which indicates that the assay was very sensitive. In addition, the M. hyopneumoniae PCR assay detected only M. hyopneumoniae and no other Mycoplasma spp. or bacterial species of another genera. Further, the newly developed PCR assay effectively detected M. hyopneumoniae infection in pigs. We suggest that this PCR assay using M. hyopneumoniae-specific primer pairs, MhyoP-F and MhyoP-R, will be useful and effective for monitoring M. hyopneumoniae infection in pigs.

Currently, honeybee colonies are not stable and suffer from the infection of pathogens, affecting the pollination. For the alternatives to this difficulty, Bombus terrestris has been imported and used for pollination in agricultural fields. Although imported insects for pollination are very useful, the potential risk exposing to novel pathogens has been raised. To assess the risk primarily, we designed and synthesized PCR primers for detection of pathogens and parasites in B. terrestris. The samples were obtained from companies importing B. terrestris or field collections and genomic DNAs not showing physical shearing were purified. PCR for detection of pathogen- or parasite-specific gene revealed several DNA fragments were amplified in expected molecular size including Kashmir Bee Virus, Varroa jacobsoni, V. rindereri, Acarapis woodi and Aspergillus flavus. These amplified DNA fragments are in the process of cloning for DNA sequencing to confirm the target gene amplification. We also have plans to optimize the PCR conditions for each amplified target gene and try to develop biomarkers for diagnosis.

This study was aimed to develop a novel qualitative multiplex polymerase chain reaction (PCR) for simultaneous detection of genetically modified (GM) soy and maize within a single reaction. The specific primers designed to detect four respective GM events (A2704-12, MON88017, Bt11, and MON863) were included in the tetraplex PCR system. Each of PCR products for four GM events could be distinguished by agarose gel based on their different lengths. The specificity and reproducibility of this multiplex PCR were evaluated. This multiplex PCR consistently amplified only a fragment corresponding to a specific inserted gene in each of the four GM events and also amplified all four of the PCR products in the simulated GM mixture. These results indicate that this multiplex PCR method could be an effective qualitative detection method for screening GM soy and maize in a single reaction.

Guillain-Barré syndrome (GBS) is the most common cause of acute flaccid paralysis and autoimmune polyradiculoneuropathy. Campylobacter jejuni is the most commonly identified infectious trigger for GBS. A sialic-acid containing lipopolysaccharide (LPS) of Campylobacter is thought to be involved in the triggering of GBS. The galE (UDPgalactoset- epimerase) gene of Campylobacter spp. is involved in the synthesis of LPS. In this study, we detected the galE gene in Campylobacter spp. responsible for triggering the onset of GBS. The PCR assay detected the presence of the gene in 14 of the 25 (56%) Campylobacter isolates from domestic chicken, 20 of the 28 (71.4%) Campylobacter isolates from imported chicken and 50 of the 51 (98%) Campylobacter isolates from human clinical samples. Also, the specific 497-bp region of galE sequence in Campylobacters responsible for triggering the onset of GBS was amplified from GBS patient. These results could provide evidence of the first GBS-related C. jejuni infection in Korea.

이 연구의 목적은 고라니(Hydropotes inermis argyropus)의 위내용물을 대상으로 PCR-DGGE 방법을 이용하여 그 식이습성을 조사하는데 있다. 이 연구를 위해, 강원도 철원과 전라남도 동부지역 등에서 자연사 혹은 로드킬에 의해 죽은 고라니 사체의 위에서 식이물 샘플을 채취하였다. 총 44개체의 위내용물에서 각각 DNA를 추출하였고, 두 가지의 프라이머(rbcLZ1과 rbcL19bR)를 이용하여 ribulose-1,5-bisphosphate carboxylase large subunit(rbcL) gene을 PCR 증폭하였다. 44개의 샘플 중 29 샘플에서 성공적으로 PCR을 수행하였다. 이 29개 partial rbcL gene의 PCR product는 PCR-DGGE에 이용되었다. 식이물에 대한 분석결과 총 6과의 식물이 확인되었다. 강원도 철원의 경우, 5과가 나타난 반면, 전라남도 동부의 경우, 3과만이 확인되었다. 이 연구에서는 종수준의 먹이식물의 구별에는 실패하였 지만, 차후 이 PCR-DGGE 기법은 고라니를 포함한 초식동물의 식이습성을 분석하는데 하나의 가능성 있는 방법이 될 것으로 생각된다.

This research aimed to compare the detection methods of Anisakis simplex in Sea fish by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and macroscopic inspection. We examined 18 Trichiurus lepturus, 11 Scomber japonicus, and 65 Todarodes pacificus collected from the retail markets in the areas of Uljin, Kyuonggi province and Seoul. As the result of examinations, we found that detection rate of Anisakis simplex by macroscopic observation was 89% in Trichiurus lepturus, 90.9% in Scomber japonicus, 32.3% in Todarodes pacificus. The detection rate of Anisakis simplex by PCR-RFLP was 77.7% in Trichiurus lepturus, 81.8% in Scomber japonicus, 26.1% in Todarodes pacificus. We could conclude that PCR-RFLP method of Anisakis simplex was more specific rather than macroscopic observation.

Aging causes thymus involution, and genes in thymus play an important role in the development of the immune system. In this study, we compared genes expressed in thymus of neonatal and peripubertal rats using annealing control primers (ACPs)-based GeneFishing polymerase chain reaction (PCR) and semiquantitative reverse transcription (RT)-PCR. We identified 10 differentially expressed genes (DEGs) with 20 ACPs. Of 10 DEGs, bystin-like, collagen type V alpha 1 (COL5A1), and T-cell receptor beta-chain segment 2 (TCRB2) that are related to immune-function were detected in rat thymus. Bystin-like and TCRB2 were up-regulated, while COL5A1 was down-regulated in peripubertal thymus. Semiquantitative RT-PCR confirmed postnatal changes in expression of bystin-like, COL5A1, and TCRB2. These results suggest that bystin-like, COL5A1, and TCRB2 could regulate immune function controlled in thymus as age increases.