• Korean Journal of Veterinary Research
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• v.38 no.3
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• pp.565-573
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• 1998

To study the specific tools for the diagnosis of Newcastle disease virus (NDV) in chicken, polymerase chain reaction (PCR) and its presumable conditions were evaluated for the detection of hemagglutinin-neuraminidase (HN) gene of NDV RNA. For these purposes, Kyojeongwon strain of the NDV was propagated in allantoic cavity of SPF embryonating chicken eggs, and viral RNA was extracted from fractionated virus after the allantoic fluids were ultracentrifuged with sucrose gradient. The first-strand cDNA was then made for the HN gene of NDV RNA by reverse transcription at $42^{\circ}C$ for 1 hour using specific primer complementary to the HN gene. The single-stranded cDNA was used as template in the PCR of the HN-DNA, and various conditions of the PCR were evaluated to set up method for the specific detection of the HN-DNA. The PCR conditions promising for the detection of HN gene consist of preheating at $94^{\circ}C$, 5 min, 30 cycles of denaturation at $94^{\circ}C$, 1 min, annealing at $55^{\circ}C$, 1 min and polymerization at $72^{\circ}C$, 2 min, and a cycle of extension at $72^{\circ}C$, 5 min. when NDVs of allantoic fluids without fractionation were applied to the above PCR condition, the HN genes were detected effectively not only from Kyojeongwon but from other velogenic strains such as Herts and a field isolate.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.4
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• pp.568-572
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• 2015

Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis' economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis' origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3'-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China.

• KSBB Journal
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• v.19 no.1
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• pp.72-77
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• 2004

The aim of this study was to isolate and identify the spoilage bacteria in the low salt cucumber brine. The PCR amplicons comprising a portion of the 16S rRNA gene of the isolated colonies were directly sequenced and the untrimmed whole sequencing results of the unknown strains were aligned with the type strains using BLAST of NCBI. Then Sequence Aligner and Sequence Match of RDP confirmed the outcome. The identified isolates were eight species and belong to three genuses: Clostridium, Lactobacillus, and Bacillus. The RFLP pattern of the 16S rRNA gene of isolates verified the identified species. From now on the complex spoiling process of law salt fermented cucumber could be analyzed using the isolated species individually or with certain combinations.

• Research in Plant Disease
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• v.23 no.1
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• pp.65-68
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• 2017

Barley yellow mosaic virus (BaYMV), which is transmitted by the root-inhabiting protist Polymyxa graminis, causes a soil-borne disease. In this study, we detected BaYMV from soil using two-step nested polymerase chain reaction (PCR). Specific primers based on a coat protein region of BaYMV segment RNA1 were used in the first round of amplification. Based on the sequenced amplicon, an inner primer was designed for the second round of amplification. A PCR product of 372 bp exhibited 98%-100% nucleotide sequence identity with the coat protein region of BaYMV segment RNA1. In this study, we propose an easy method for the detection of BaYMV from soil, may considerably assist in accurate fungus-transmitted virus diagnosis and subsequent disease forecasting. This is the first report on the detection of BaYMV from soil.

• The Journal of Korean Society of Virology
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• v.30 no.1
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• pp.29-37
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• 2000

The aim of this study was to investigate viral etiology in dilated cardiomyopathy (DCM) by polymerase chain reaction (PCR) or nested reverse transcription PCR (RT-PCR), and characterize the enteroviral RNA presented in the clinical specimens. Twenty-eight paraffin-embedded heart tissue samples were assayed to detect cytomegalovirus, herpes simplex virus type 1, type 2, parvovirus, adenovirus, and enterovirus (EV) with each specific primer. Of these 28 patients (mean age: 27, M: 24, F: 4), 26 were histologically diagnosed as DCM and 2 as myocardial infarction (MI). Nested RT-PCR detected enteroviral RNA in 7 (26.9%) of 26 patients with DCM, and none of patients with MI. And none of DNA viruses tested were detected from the samples. Amplified products were also genotyped by single-strand conformation polymorphism (SSCP). Three subtypes can be differentiated from 7 clinical specimens. Furthermore, direct sequence analysis was performed to determine whether genetic variation of EV is present in the explanted heart tissues from patients with DCM. Although most of the sequences among the wild isolates have the greatest similarity to those of coxsackievirus B3, there are specific regions of variable sequences (no 490 - no 510). The data suggest that enterovirus may be a major viral pathogen for the DCM in Korea and nucleotide sequence data indicate that coxsackievirus B3 may be a leading etiologic agent of DCM.

• Development and Reproduction
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• v.3 no.1
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• pp.69-74
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• 1999

Insulin-like growth factors (IGF-1 and IGF-2) play an important regulatory role in premplantation embryonic development. To study the role of IGF-1 during premplantation embryonic development in mouse, the presence of mRNA transcripts for IGF-1 and IGF-lR in the oocytes and preimplantation embryos was examined. In this study, the transcripts of IGF-1 was detected in oocytes using primers for IGF-1. The PCR products were identified by Msp I restriction enzyme digest. We revealed that the transcripts of IGF-1 and IGF-1R were presented in the oocytes and preimplantation embryos. The highest mRNA levels in GV stage oocytes were decreased at 4- or 8-cell stage and then reincreased upto blastocyst. The presence of IGF-1 and IGF-lR in GV-oocytes suggests that the transcripts in the early stage embryos were derived from maternal genome. Additionally, the presence of IGF-1 and IGF-lR in the oocytes and preimplantation embryos suggests that IGF-1 plays an autocrine role during preimplantation embryonic development through IGF-lR as a signalling pathway.

• Parasites, Hosts and Diseases
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• v.54 no.5
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• pp.631-636
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• 2016

This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler's diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in > $1{\times}10^3$ oocysts for C. parvum, > $1{\times}10^4$ cysts for G. lamblia, and > 1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.

• Journal of Preventive Medicine and Public Health
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• v.45 no.4
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• pp.235-243
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• 2012

Objectives: Nail has been a substitute DNA source for genotyping. To investigate the integrity and consistency of nail DNA amplification for biomarker study, nail clippings from 12 subjects were collected at monthly intervals. The possibility of longer amplification and existence of GAPDH RNA/protein, were also investigated with three nail samples. Methods: Three primer sets were designed for quantitative amplification of nuclear and mitochondrial genes and analysis of their consistency. The mean threshold cycles in amplification of the target genes were compared to test the consistency of polymerase chain reaction (PCR) performance among individual factors including age groups, sex, family, the nail source, and by the size of the amplification segments. Results: The amplification of the target genes from nail DNA showed similar integrity and consistency between the nail sources, and among the serial collections. However, nail DNA from those in their forties showed earlier threshold cycles in amplification than those in their teens or seventies. Mitochondrial DNA (mtDNA) showed better DNA integrity and consistency in amplification of all three targets than did nuclear DNA (nucDNA). Over 9 kb of mtDNA was successfully amplified, and nested quantitative PCR showed reliable copy numbers (%) between the two loci. Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts. Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study. Conclusions: Nail DNA might be a feasible intra-individual monitoring biomarker. Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.

• Research in Plant Disease
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• v.28 no.1
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• pp.54-56
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• 2022

A peanut plant showing wilt and browned symptom was found in the field of Gochang, Korea, in July 2021. The symptomatic peanut plant was collected from the field and isolation of the pathogen caused the wilt symptom was performed using the collected sample on TZC media. The dominated colony on media was isolated colony on media was isolated and subcultured of purification. The pure cultured bacteria was identified as Ralstonia solanacearum by sequencing of 16S rRNA gene. Multiplex polymerase chain reaction using phylotype-specific primer set identified isolate as phylotype I (R. pseudosolanacearum). Phylogenetic tree was constructed based on 16S rRNA sequence and it was closed with R. pseudosolanacearum. Pathogenicity of the isolates was assessed by soil drenching inoculation on 4-week-old peanut plant. The wilt symptom was successfully reproduced by inoculation of the isolates after 14 days. This is first report of bacterial wilt caused by R. pseudosolanacearum on peanut in Korea.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2004.11a
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• pp.23-25
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• 2004

This study was conducted to investigate the effects of dietary supplementation of Ecklonia cava kjellman(ECK) and crude lectin extracted from ECK (CLEEC) on performances and immune responses in broiler chicks. A total of two hundreds thirty four 1 day old male broiler chicks (Ross) were fed corn-soy based diets containing 0 % (with or without vaccination and Salmonella challenge), 1.0 % ECK, 0.05 %, 0.1 % and 0.3 % CLEEC for 38 days and vaccinated against inactivated ND-IB combined oil vaccine on the fourth day. After S. gallinarum challenge. mortality was measured daily. The spleens of birds were removed for RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) with primer sets for IFN-ν, IL-2, IL-6 and ${\beta}$-actin were performed with RNA samples. At the 28th day, pancreas weights were heavier 0.3 % CLEEC than 1.0 % ECK group. At the 21st day after ND-IB oil vaccine injection, dietary supplementation of ECK and CLEEC tended to increase or significantly (P<0.05) improved ND or IB titer compared to the positive control. Mortality was significantly (P<0.05) decreased by dietary CLEEC treatments. Chicken splenic IFN-ν, IL-2, and IL-6 cytokines mRNA expressions were enhanced by challenge with S, gallinarum. Dietary treatments did not affect mRNA expression of IFN-ν. However, IL-2 and IL-6 expressions in Salmonella challenged birds that fed the 1.0 % ECK or 0.05 % CLEEC groups were enhanced (P<0.05) compare to the positive control. The results demonstrated that dietary ECK and CLEEC enhanced humoral and cellular immunity and therefore. it can be concluded that dietary supplementation of ECK and CLEEC can be used as a feed additive for enhancement of immunocompetence without any adverse effects in broiler chicks.