• Korean Journal of Veterinary Research
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• v.41 no.3
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• pp.381-385
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• 2001

Neospora caninum infections have been associated with neonatal paresis as well as abortion around the world. Bovine abortion induced by N caninum was first reported in 1997 in Korea. Diagnosis of N caninum infection is usually based on histopathology and immunohistochemical detection of organism. However, often the tissues having lesion suggestive of N caninum infection were negative on immunohistochemistry. Here, we describe establishment of PCR-based diagnostic strategy for N caninum infection using DNA extracted from paraffin blocks containing the lesion. PCR was able to amplify N caninum-specific bands from the paraffin blocks containing at least moderate degree of inflammation. Compared to paraffin-blocks, DNA extracted from fresh tissues were less sensitive than that of paraffin blocks. This PCR-based method can be practically applicable for rapid diagnosis of bovine N caninum infection with high specificity and sensitivity. Based on this method, 17% of bovine abortion surveyed during a designated period was associated with N caninum infection.

• Korean Journal of Veterinary Research
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• v.40 no.1
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• pp.42-48
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• 2000

Akabane disease is transmitted through mosquitoes in cattle, sheep and goats. It shows congenital abnormalities including encephalomyetitis, hydranencephaly, neurogenic arthrogryposis, and deformed neonatal calves. Akabane viruses, 93FMX and K-9 strain, were isolated from fetal matrix of aborted cow and blood of healthy cow, respectively. S gene sequences of 93FMX and K-9 showed 100% homology with that of OBE-1 strain isolated in Japan. Based upon our sequencing data, we synthesized specific primers for PCR diagnosis. Using these primers, we were able to amplify the S gene of Akabane virus not only from the culture fluid of Vero cells but also from the brain tissue of suckling mouse inoculated with, Akabane virus. These PCR products were confirmed by Southern blot hybridization. Not only the sensitivity of PCR test was high enough to detect the viruses of $10^{1.0}TCID_{50}/ml$, but also the time for diagnosis was significantly shorter than that of the virus isolation by tissue culture method. This method was also effective for the detection of Akabane virus in the cerebrum of fetus. RT-PCR method may be used for a useful diagnostic test of the clinical cases of Akabane disease.

• Food Science and Biotechnology
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• v.16 no.1
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• pp.104-109
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• 2007

A multiplex polymerase chain reaction (PCR) method was developed to simultaneously detect three varieties of genetically modified (GM) canola. The construct-specific primers were used to distinguish the following three varieties of GM canola; GT73, MS8xRF3, and T45, using multiplex PCR. The FatA (fatty acyl-ACP thioesterase) gene was used as an endogenous canola reference gene in the PCR detection. The primer pair Canendo-FIR containing a 105 bp amplicon was used to amplify the FatA gene and no amplified product was observed in any of the 15 different plants used as templates. The GT73-KHUF1/R1 primer recognized the 3'-flanking region of GT73, resulting in an amplicon of 125 bp. The Barstar-F1/MS8xRF3-R primer recognized the junction region of bars tar and the NOS terminator introduced into MS8xRF3, resulting in a 162 bp amplicon, and the T45-F2/R2 primer recognized the junction region of PAT and the 35S terminator introduced into T45, resulting in an amplicon of 186 bp. This multiplex PCR allowed for the detection of construct-specific targets in a genomic DNA mixture of up to 1% GM canola containing GT73, MS8xRF3, and T45.

• Preventive Nutrition and Food Science
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• v.9 no.3
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• pp.276-282
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• 2004

Marginal Zn deficiency is prevalent through the world and yet human zinc status has not been properly assessed due to the lack of a reliable diagnostic indicator. One potential possibility for zinc status assessment using Zn-binding protein, metallothionein (MT)-mRNA, has been proposed. The purpose of the present study was aimed to show whether measurement of mononuclear cell (MNC) MT mRNA, using a competitive-reverse transcriptase-polymerase chain reaction (competitive-RT-PCR) assay, could indicate zinc status in human subjects. In this study, MNC MT-mRNA expression was measured using a competitive-RT-PCR to compare before and after 14 days of zinc supplementation (50 mg Zn/das zinc gluconate). RT-PCR oligonucleotide primers which were designed to amplify both a 278 bp segment of the human MT-2A cDNA and a 198 bp mutant competitor cDNA template from MNCs, were prepared. MT-2A mRNA was normalized by reference to the housekeeping gene, $\beta$-actin, mRNA for which was also measured by competitive-RT-PCR. There was considerable inter-individual variation in MT-mRNA concentration and yet, the mean MT-2A mRNA level increased 4.7-fold after Zn supplementation, as compared to before Zn supplementation. This MT-2A mRNA level was shown as the same pattern and, even more sensitive assay, compared to the conventional plasma and red blood cells (RBCs) Zn assessment in which plasma and RBCs zinc levels increased 2.3- and 1.2-fold, respectively (p<0.05). We suggest that MT competitive-RT-PCR can be a useful assessment tool for evaluating human zinc status.

• The Plant Pathology Journal
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• v.31 no.3
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• pp.212-218
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• 2015

In this study, we developed a species-specific PCR assay for rapid and accurate detection of three Xanthomonas species, X. axonopodis pv. poinsettiicola (XAP), X. hyacinthi (XH) and X. campestris pv. zantedeschiae (XCZ), based on their draft genome sequences. XAP, XH and XCZ genomes consist of single chromosomes that contain 5,221, 4,395 and 7,986 protein coding genes, respectively. Species-specific primers were designed from variable regions of the draft genome sequence data and assessed by a PCR-based detection method. These primers were also tested for specificity against 17 allied Xanthomonas species as well as against the host DNA and the microbial community of the host surface. Three primer sets were found to be very specific and no amplification product was obtained with the host DNA and the microbial community of the host surface. In addition, a detection limit of $1pg/{\mu}l$ per PCR reaction was detected when these primer sets were used to amplify corresponding bacterial DNAs. Therefore, these primer sets and the developed species-specific PCR assay represent a valuable, sensitive, and rapid diagnostic tool that can be used to detect three specific pathogens at early stages of infection and may help control diseases.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.9
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• pp.1229-1233
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• 2006

To find the candidate genes concerned with ovulation rate of sheep, Differential Display Reverse Transcription Polymerase Chain Reaction was employed to find the differently expressed cDNA controlling ovulation in the Small Tail Han sheep of polyembryony and in Tan sheep of single birth. Twenty-four primer pairs of three anchored primers and eight arbitrary primers were assembled to amplify the specialized bands from these sheep. Positive cross tests were applied to optimize the ascertainable PCR conditions in which different special bands can be identified by silver strain in one PCR tube. After eliminating the false positive PCR products by Northern hybridization, 24 differential display bands were acquired from the ovary in the Small Tail Han sheep. These EST bands were sequenced and 18 different ESTs were found in which five ESTs had several copies and 13 ESTs had only one copy. Comparing these ESTs with homologous sequences by BLAST in the GenBank, there were six ESTs with known open reading frame (ORF) and function, three ESTs with known ORF and no function, and 9 ESTs without homologous sequence. These ESTs partly represent several genes such as NOS2, tensin, TCRA, CDKN1A, ESR1 and ACTB which express especially in Small Tail Han sheep.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.645-650
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• 2019

Despite differences in virulence between strains of Toxoplasma gondii, rapid and accurate genotyping methods are lacking. In this study, a method was developed to detect and genotype T. gondii in food and environmental samples using PCR and a novel peptide nucleic acid (PNA) melting array. An alignment of genome sequences for T. gondii type I, II, and III obtained from NCBI was generated, and a single nucleotide polymorphism analysis was performed to identify targets for PCR amplification and a PNA melting array. Prior to the PNA melting array, conventional PCR was used to amplify GRA6 of T. gondii. After amplification, the PNA melting array was performed using two different PNA hybridization probes with fluorescent labels (FAM and HEX) and quenchers. Melting curves for each probe were used to determine genotypes and identify mutations. A 214-bp region of the GRA6 gene of T. gondii was successfully amplified by PCR. For all T. gondii strains (type I, II, and III) used to evaluate specificity, the correct genotypes were determined by the PNA melting array. Non-T. gondii strains, including 14 foodborne pathogens and 3 protozoan parasites, such as Giardia lamblia, Cryptosporidium parvum, and Entamoeba histolytica, showed no signal, suggesting that the assay has a high specificity. Although this is only a proof-of-concept study, the assay is promising for the fast and reliable genotyping of T. gondii from food and environmental samples.

• The Plant Pathology Journal
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• v.15 no.6
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• pp.335-339
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• 1999

Citrus tristeza virus(CTV), an aphid-borne closterovirus, is one of the most destructive pathogens of citrus. It has caused rapid decline in growth, stem pitting and death in citrus trees. A reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for detection of CTV and investigation of the CTV infection status of citrus and its related cultivars in Cheju island. For RT-PCR based CTV detection, primers were designed to amplify 670bp of coat protein gene. A screening test for CTV in citrus cultivars was conducted from March to July in 1999. Seventy individual citrus trees representing 9 species of 3 genera were tested. The infection rates of CTV for leaves from the years or older trees of late maturing citrus varieties such as Yuzu (C. junos Sieb. ex Tanaka), Navel orange (C.sinensis Osbeck), Kiyomitanger (C. unshiu x C. sinensis), and Shiranuhi ((C. unshiu x C. sinensis) x C. reticulata) were 100%, 80%, 60%, and 60% respectively. The CTV infection rates in Early satsuma mandarins such as 'Miyagawa Early' Satsuma mandarins (C. unshiu Marc. var. Miyagawa) and 'Okitsu Early' Satsuma mandarins (C. unshiu Marc. var. Okitsu) were 100%, and 60%, respectively. CTV was not detected in Cheju native Dangyooja (C. unshiu Marc. var. Osbeck), Trifoliate orange (Poncirus trifoliata) and Kumquat (Fortunella margarita Swingle). In conclusion, RT-PCR assay can be successfully applied to the detection of CTV in citrus trees.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.40 no.6
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• pp.356-361
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• 2007

Recently, the development of various culture-independent identification techniques for environmental microbes has greatly enhanced our knowledge of microbial diversity. In particular, denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments, generated using the polymerase chain reaction (PCR) is frequently used to examine the diversity of environmental bacterial populations. This method consists of direct extraction of the environmental DNA, amplification of the 200-600 bp 16S rDNA fragments with universal primers, and separation of the fragments according to their melting point on a denaturing gradient gel. In this study, we investigated the seaside microbial community in coastal areas of Busan, Korea, using culture-independent techniques. First, marine genomic DNA was extracted from seawater samples collected at Songjeong, Gwangahn, and Songdo Beaches. Then, PCR was used to amplify the bacterial 16S rDNA using universal primers, and DGGE was used to separate the amplified 500 bp 16S rDNA fragments. Finally, the tested 16S rDNA genes were further analyzed by sequencing. Based on these experiments, we found that DGGE analysis clearly showed variation among the regional groups. It can be used to monitor rapid changes in the bacterial diversity of various environments. In addition, the sequence analysis indicated the existence of many unculturable bacteria, in addition to Arcobacter, Pseudoaltermonas, and Vibrio species.

• Korean Journal of Microbiology
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• v.37 no.4
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• pp.294-298
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• 2001

Highly effective polymerase chain reaction (PCR) often brings about false positivity caused by contamination of the sample with target nucleic acids. To solve this problem, in situ PCR (ISPCR) has been developed and applied onto various tissue sections and suspension cultures. With combination of PCR and in situ hybridization, this method amplifies the nucleic acid targets in situ and detect the amplified products inside the cells over the background of various cell types. In order to amplify the nucleic acid targets inside the cells, permeabilisation of a sample is required for the entry of amplification reactants into a cell. Treatments of a sample for the purpose allow not only the entry of reactants into the cell but also the exit of amplification products out of the cell. As a means to reduce the leakage of the amplification products, two methods were applied to suspension cultures of HIV-infected Molt/LAV and U 1.1 cells, in which modified, tailed primers produced long linear amplificants whereas biotinylated dUTP instead of dTTP did bulky products.