• The Plant Pathology Journal
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• v.26 no.4
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• pp.409-416
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• 2010

Fusarium species, a large group of plant pathogens, potentially pose quarantine concerns worldwide. Here, we focus on the development of a method for detecting four Fusarium species in quarantined plants in Korea: F. solani f. sp. cucurbitae, F. stilboides, F. redolens, and F. semitectum var. majus. Species-specific primers were designed from the nucleotide sequences of either the translation elongation factor-1 alpha (TEF1) gene or RNA polymerase II subunit (RPB2) gene. Two different primer sets derived from TEF1, all specific to F. solani f. sp. cucurbitae, were able to differentiate the two races (1 and 2) of this species. A set of nested primers for each race was designed to confirm the PCR results. Similarly, two primer sets derived from RPB2 successfully amplified specific fragments from five F. stilboides isolates grouped within a single phylogenetic clade. A specific TEF1 primer set amplified a DNA fragment from only four of the 12 F. redolens strains examined, which were grouped within a single phylogenetic clade. All of the F. semitectum var. majus isolates could be specifically detected with a single RPB2 primer set. The specificity of the primer sets developed here was confirmed using a total of 130 Fusarium isolates.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.292-292
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• 2004

배아의 성별 판별을 위해 할구를 biopsy하여 핵상에서 정밀 분석을 하였다. 이 실험에서는 8-에서 16-세포기 배아의 할구를 배아 성결정의 대표물로 사용하여 IVF소 배아를 분석 평가하였다. 55개의 배아를 PCT후 biopsy하여 분석하였다. PCR에 의한 성판별에서 biopsy한 single blastomere와 blastocyst의 성판별의 일치하는 비율은 80%인 것으로 나타났다. IVF 수정란을 염색체 상태에서 평가하기 위해 8- 16- 세포기의 할구를 Karyotyping 하였다. 할구의 Karyotyping을 위해 metaphase 상태에서 vinblastine sulfate에 계속적으로 노출시켜 metaphse Ⅱ 상태를 유도하였다. (중략)

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.210-217
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• 2018

A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

• Korean Journal of Agricultural Science
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• v.49 no.4
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• pp.795-807
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• 2022

The development and commercialization of industrial genetically modified (GM) organisms is actively progressing worldwide, highlighting an increased need for improved safety management protocols. We sought to establish an environmental monitoring method, using real-time polymerase chain reaction (PCR) and propidium monoazide (PMA) treatment to develop a quantitative detection protocol for living GM microorganisms. We developed a duplex TaqMan quantitative PCR (qPCR) assay to simultaneously detect the selectable antibiotic gene, ampicillin (AmpR), and the single-copy Escherichia coli taxon-specific gene, D-1-deoxyxylulose 5-phosphate synthase (dxs), using a direct cell suspension culture. We identified viable engineered E. coli cells by performing qPCR on PMA-treated cells. The theoretical cell density (true copy numbers) calculated from mean quantification cycle (Cq) values of PMA-qPCR showed a bias of 7.71% from the colony-forming unit (CFU), which was within ±25% of the acceptance criteria of the European Network of GMO Laboratories (ENGL). PMA-qPCR to detect AmpR and dxs was highly sensitive and was able to detect target genes from a 10,000-fold (10^-4) diluted cell suspension, with a limit of detection at 95% confidence (LOD_95%) of 134 viable E. coli cells. Compared to DNA-based qPCR methods, the cell suspension direct PMA-qPCR analysis provides reliable results and is a quick and accurate method to monitor living GM E. coli cells that can potentially be released into the environment.

• Korean Journal of Agricultural Science
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• v.38 no.2
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• pp.269-275
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• 2011

The objective of this study was to develop a rapid and reliable PCR method for sexing of morula or blastocyst stage bovine embryo. BOV97M and bovine 1.715 satellite DNA sequences were selected for amplification of male and bovine specific DNA, respectively. But the unbalanced number of copies of these two repetitive sequences required some modification of PCR method. Karyotyping of blastomeres were carried for the confirmation of sex determination in bovine embryos. The coincidence rate of sex between biopsied-single blastomere and matched blastocyst was 80.0%. When in vivo- and in vitro- derived embryos were compared, 61.8% and 56.7% were male in in vitro- and in vivo-derived embryos, respectively. In vivo-derived embryos showed better hatching rate than in vitro-derived embryos following biopsy of blastomeres. In conclusion, rapid and effective PCR could be applied to sexing of bovine preimplantation embryos using single blastomere. The sensitivity of this assay may eliminate the need for biopsy of more than one nucleated blastomere and reduce trauma to the embryos derived from biopsy procedure.

• Journal of Life Science
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• v.31 no.1
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• pp.66-72
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• 2021

This study developed a species identification method for the salted opossum shrimp of Acetes japonicus, A. chinensis (Korea, China), A. indicus (I, II), and Palaemon gravieri based on PCR-RFLP markers. Genomic DNA was extracted from the salted opossum shrimp. The COI gene was used to amplify 519 base pairs (bp) using specific primers. The amplified products were digested by Acc I and Hinf I, and the DNA fragments were separated by automated electrophoresis for RFLP analysis. When the amplified DNA product (519 bp) was digested with Acc I, A. japonicus, A. chinensis (Korea), and A. indius (II) showed two fragments, whereas a single band of 519 bp was detected in A. chinensis (China) and A. indius (I). Also, in the RFLP patterns digested by Hinf I, A. chinensis (Korea) and A. chinensis (China) showed a single band of 519 bp, while two fragments were observed in A. japonicus and A. indius (I) and four fragments in A. indius (II). The PCR amplicon of P. gravieri was digested by Acc I into 3 bands of 271, 202, and 46 bp and by Hinf I into a single band of 519 bp. Therefore, salted opossum shrimp-specific RFLP markers showing distinct differences between four species and two sub-species by PCR-RFLP analysis. Thus, the PCR-RFLP markers developed in this study are a good method for identifying the six types of salted opossum shrimp.

• Journal of Food Hygiene and Safety
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• v.30 no.1
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• pp.43-50
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• 2015

In this study, single PCR and multiplex PCR tests were examined for identification of four types of squid species (giant squid, cuttlefish, octopus, beka squid) purchased from fish market as well as aquatic processed products in Busan. To design the specific primers against each species, the nucleotide sequences of the mitochondrial 16s rRNA gene of Architeuthis dux, Todarodes pacificus, Enteroctopus dofleini, Enteroctopus megalocyathus, Uroteuthis chinensis, Uroteuthis duvauceli, Uroteuthis edulis groups were analyzed for the identification of each species registered in the GeneBank (www.ncbi.nlm.nih.gov) and have been used for comparative analysis. In order to obtain the size variation of amplified fragments on multiplex PCR, we designed KOJ-F, OJ-F, OCT-F, HAN-F, ALLR primers for each species. The optimal PCR conditions and primers were selected for four types of squid species to determine target base sequences in its PCR products. In the case of single PCR, giant squid was only amplified by KOJ-F/ALLR primer; cuttlefish was only amplified by OJ-F/ALLR primer; octopus was only amplified by OCT-F/ALLR primer; and beka squid was only amplified by HAN-F/ALLR primer. For multiplex PCR, the mixture of four kinds of genomic DNA (giant squid, cuttlefish, octopus, beka squid) been prepared as a template and used together with the mixture of KOJ-F/OJ-F/OCT-F/HAN-F/ALLR primers in the reaction. By the multiplex PCR, it is confirmed that four samples are correspond to multiple simultaneous amplicon. Finally, we validated the established methods of multiplex PCR in the aquatic processed products. Although the mitochondrial 16s rRNA primers used in this study was useful as a marker for detection of each species among them, the study indicated that the established multiplex PCR method can be more useful tool for monitoring the processed products.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.281-284
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• 1994

Sex determination in genomic DNA from human blood leucocytes was performed by amplification of human Y chromosome-specific DNA sequences using PCR technique. A clear DNA fragment(154 nucleotides long) was appeared only in the male genomic DNA, but no specific band was observed in the case of female genomic DNA and negative control. To know the sensitivity of this method, the amplification reaction was performed in genomic DNA diluted to 2pg equivalent to the amonut present in the single human cell, and clear band also observed. The PCR amplification was so succesfully performed in the single leucocyte separated from human blood using micromanipulator that this techniqe is assumed to be applied to single blstomere before embryo transfer.

• The Plant Pathology Journal
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• v.19 no.3
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• pp.159-161
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• 2003

The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.

• Korean Journal of Veterinary Service
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• v.32 no.2
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• pp.103-109
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• 2009

Influeza type A virus have been worldwide problematic in animals as well as in humans. In this study, the use of reverse-transcriptase polymerase chain reaction (RT-PCR) was described for detecting influenza virus type A. The primer of RT-PCR was designed from an nonstructural (NS) gene of Influenza A virus. By RT-PCR, a product with the size of 189 bp was detected only when influenza virus type A was used as template. No products could be detected with Influenza virus type B as well as other respiratory pathogens. The detection limit of the RT-PCR was up to $10^{0.3}TCID_{50}$ which is comparable to the sensitivity of cell culture method. The RT-PCR could detect the influenza A virus from nasal turbinates of the ferrets infected with influenza virus type A not type B.