• Clinical and Experimental Pediatrics
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• v.52 no.12
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• pp.1358-1363
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• 2009

Purpose:The aim of this study was to determine the prevalence of asymptomatic nasopharyngeal carriages in children using a multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) assay kit. Methods:We obtained nasopharyngeal swabs from 33 children without any underlying disease from July 25 to July 28, 2008. The children were free from the signs of respiratory tract infections at the time of sampling. DNA was extracted from the swabs and subjected to multiplex RT-PCR using a primer set for the detection of pneumococci ($Seeplex^{(R)}$ PneumoBacter ACE Detection Seegene, Seoul, Korea). The amplified PCR products were separated on 2% agarose gels and stained with either ethidium bromide or screen tape system (Lab901 Scotland, UK). Results:A total of 33 children (male, 15 female, 18) aged between 3.2 and 16.3 (median, 8.2) years were included in this study. The mRT-PCR detected colonized bacteria (Streptococcus pneumoniae, Hemophilus influenzae, Chlamydia pneumoniae, and Bordetella pertussis) in 30 children (90.9%). Of these, 13 children (39.4%) showed more than 2 bacteria: 12 children were positive for 2 bacteria (S. pneumoniae and H. influenzae) and 1 child was positive for 3 bacteria (S. pneumoniae, H. influenzae, and C. pneumoniae). Conclusion:mRT-PCR was found to be a sensitive tool for the detection of asymptomatic nasopharyngeal carriages. Clinical significances of the bacteria detected by mRT-PCR will have to be evaluated in the future.

• Korean Journal of Veterinary Research
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• v.41 no.4
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• pp.529-533
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• 2001

The infectivity of an isolate of avian pneumovirus (APV) from turkeys to Balb/c mice was investigated to examine the transmission possibility to mammals. Three different age groups (3, 5 and 7 weeks old) were exposed by oculonasal route with a cell cultured APV of turkey origin. No clinical signs were observed from both APV-inoculated and commingled mice. However, all the tissue samples including blood from mice in the APV-inoculated group were positive for APV by polymerase chain reaction (PCR) up to 6 days postinoculation. At 14 days postinoculation, APV was not detected from blood samples by PCR, but sera showed the presence of APV-specific antibodies. In commingled mice, APV was detected from lung and rectal swap samples by PCR. These results suggest that an APV isolate from turkey could be transmitted to mice by direct contact or other ways.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.184-190
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• 1999

The use of the polymerase chain reaction (PCR) method was described using two sets of primers based on the ceuN gene (JEJ 1 and JEJ 2) which encodes a protein involved in siderophore transport and 16S rRNA gene (pA and pB) for the sensitive and specific detection of enteropathogen Campylobacter jejuni. Six oligonucleotides were utilized in an amplification experiment and PCR products of predicted sizes were generated from whole cells and boiled cell lysates at the same intensity. Two sets of the primer pairs, JEJ and pAB, were specific enough for all C. jejuni strains tested for the direct use of whole cells without DNA extraction or lysis steps. In the PCR using the pAB primer pair, the detection limit, as determined by the ethidium bromide staining of the amplification products on agarose gels, was at the level of $10^1$ bacteria cells or less in both the pure culture and artificially inoculated milk and chicken enrichment samples, whereas the detection limit with the JEJ primer pair was relatively low, i.e. $10^3$ cells or more in the same PCR samples. The PCR method using either a primer JEJ or pAB was both repeatable and specific for the detection of C. jejuni in food. This method is simply completed within 4 h.

• Fisheries and Aquatic Sciences
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• v.26 no.11
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• pp.678-688
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• 2023

Flatfish are one of the largest families in the order Pleuronectiformes and are economically important edible marine fish species. However, they have similar morphological characteristics leading to challenges in classifying correctly, which may result in mislabeling and illegal sales, such as fraudulent labeling of processed food. Therefore, accurate identification is important to ensure the quality and safety of domestic markets in Korea. Species-specific primers were prepared from the mainly consumed eleven species of the order Pleuronectiformes. To rapidly identify the 11 flatfish species, a highly efficient, rapid, multiplex polymerase chain reaction (PCR) with species-specific primers was developed. Species-specific primer sets were designed for the mitochondrial DNA cytochrome c oxidase subunit I gene. Species-specific multiplex PCR (MSS-PCR) either specifically amplified a PCR product of a unique size or failed. This MSS-PCR analysis is easy to perform and yields reliable results in less time than the previous Sanger sequencing methods. This technique could be a powerful tool for the identification of the 11 species b the family Pleuronectidae and can contribute to the prevention of falsified labeling and protection of consumer rights.

• Journal of Life Science
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• v.19 no.9
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• pp.1328-1332
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• 2009

Anisakidosis is caused by anisakid nematodes (family Anisakidae) larvae which can cause not only direct tissue damage but also a severe allergic response related to excretory-secretion products. Lots of different species of anisakid larvae, including Anisakis simplex, Contracaecum, Goezia, Pseudoterranova, and Hysterothylacium, cause the anisakidosis. But it is difficult to diagnosis the species of larvae since the morphologies of larval anisakid nematodes are almost indistinguishable. In order to diagnosis the differential infections of larval anisakid nematodes, polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) of 18S rDNA - was conducted. Three major species of anisakid larvae including A. simplex, C.ontracaecum spp, and Goezia spp. were collected from mackerel (Scomber japonicus), mullet (Mugil cephalus), founder (Paralichthys olivaceus), eel (Astroconger myriaster) and red sea bream (Pagrus major). PCR amplified 18S rDNA from each species of anisakid larvae was digested with eight restriction enzymes including Taq I, Hinf I, Hha I, Alu I, Dde I, Hae III, Sau96 I, and Sau3A I. The original sizes of PCR amplified 18S rDNA were 2.0Kb in both anisakid larvaes and Goezia. Restrction enzymes including Hinf 1, Alu 1, Hha I, Dde 1 and Hae III cut differently and distinguished the A. simplex and Contracaecum type C'. However, Contracaecum type A showed two different restriction enzyme cutting patterns by Taq 1, Hinf I, Alu 1, and Dde 1. One of the patterns was the same as those of A. simplex, Contracaecum type C' and Goezia and the other was unique. These results suggest that PCR-RFLP pattern by Hinf 1, Alu 1, Hae I, Dde 1 and Hae III can be applied to differential diagnosis of human infection with A. simplex and Contracaecum type C'. Contracaecum type A needs further study of classification by morphological characteristics and genetic analysis.

• Journal of Food Hygiene and Safety
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• v.33 no.5
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• pp.325-329
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• 2018

This study developed a rapid detection method for Bacillus cereus in fresh-cut cabbages. Fresh-cut cabbage samples were inoculated at 1-, 2- and 3-Log CFU/g, and pathogens were enriched in tryptic soy broth containing 0.15% polymyxin B at $30^{\circ}C$, $37^{\circ}C$, and $42^{\circ}C$ to determine the detection limit and appropriate enrichment temperature for multiplex PCR detection. Enriched bacterial cells in enrichment broth were collected in a hydrophobic filter prior to DNA extraction for multiplex PCR. Filters were resuspended in distilled water, and DNA was extracted from the suspension. DNA samples were further analyzed by multiplex PCR. Detection limit of multiplex PCR was 5-Log CFU/mL. B. cereus cell counts were higher (P < 0.05) at $42^{\circ}C$ than other temperatures. Detection rate of 1-, 2-, and 3-Log CFU/g inoculated samples were 60%, 80%, and 100% after enrichment respectively. However, when enriched samples were filtered with hydrophobic membrane filter, detection rates became 100%, regardless of inoculation level. Results indicate a combination of enrichment with hydrophobic filtration improves rapid detection efficiency of B. cereus in fresh-cut cabbage by multiplex PCR.

• Clinical and Experimental Pediatrics
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• v.51 no.7
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• pp.729-735
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• 2008

Purpose : Chlamydia trachomatis is one of the most common sexually transmitted diseases and is also a cause of pneumonia in infants. Respiratory infections by respiratory viruses are also common for infants. The objectives of this study were to identify the clinical manifestations and to determine the prevalence of C. trachomatis respiratory infections and coinfections by respiratory viruses in infants younger than 6 months of age. Methods : For this study, we enrolled 6 months or younger infants who were admitted to the Dankook University Hospital between January 2002 and July 2007, with respiratory symptoms. Nasopharyngeal aspirates or throat swabs were collected within s d of hospitalization and C. trachomatis was detected using polymerase chain reaction (PCR). Patients who tested positive underwent multiplex PCR for respiratory viruses. Results : A total of 690 patients underwent chlamydial PCR testing and 36 (5.2%) had positive results. Of the 36, 28 (78%) were male; 30 were vaginally delivered. From the 36 patients positive for C. trachomatis, 26 underwent multiplex respiratory viral PCR; 12 were coinfected with viruses. Respiratory syncytial virus (RSV) was the most frequent pathogen that was detected in 6 patients. Increased C-reactive protein and fever were significant in patients coinfected with respiratory viruses. Conclusion : C. trachomatis can infected in infants delivered by cesarean section as well as in 6 months old or younger infants. Infant with C. trachomatis respiratory infections can also be coinfected with respiratory infection also coinfected with respiratory viruses. Further studies are needed to better understand the prevalence rates of the this infection and its coinfection rate with respiratory viruses.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.6
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• pp.892-897
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• 2005

Macrophage colony-stimulating factor (M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocyte lineage. Total and 16 poly (A) mRNA of bovine M-CSF were isolated from healthy bovine peripheral mononuclear cells stimulated by phobol 12-myristste 13-acetate (TPA). The more compatible cultured mononuclear cells were 5${\times}$10/ml for RNA isolation. TPA-activated mononuclear cells increased the level of M-CSF-mRNA more than concanavalin A (Con A) and lipopolysaccharide (LPS). The optimal analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) for14 Macrophage colonystimulating factor (M-CSF) as a growth factor required for bovine M-CSF was denaturation at 94$^{\circ}C$ for 1 minute, annealing at 57$^{\circ}C$ for 1 minute, extension at 72$^{\circ}C$ for 1 minute for 30 cycles. The size of cDNA of bovine M-CSF by RT-PCR was 774 base pairs. A 774 base pairs cDNA encoding bovine M-CSF was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). Ligated cDNA was transformed to competent cells and then plasmid isolation and digestion was performed. Molecular cloning and sequencing were performed for cDNA of bovine M-CSF. The size of cloned cDNA of bovine M-CSF was 774base pairs. The homology of base sequence and amino acid sequence was 88% and 86% compared with known human M-CSF, respectively. From a high degree of sequence similarity, the obtained cDNA of bovine M-CSF is thought be a specific gene of bovine M-CSF.

• Journal of Sericultural and Entomological Science
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• v.38 no.1
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• pp.7-12
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• 1996

Reproducible the random amplified polymorphic DNAs(RAPDs) patterns were obtained in the two silkworm strains(J111, Galwon) by adjusting concentration optimized of Taq DNA polymerase(one unit), dNTP(200$\mu$M), MgCl2(1.5mM) and template DNA(30ng). In addition, anealing temperature ranging 35$^{\circ}C$ to 42$^{\circ}C$ by the adjusted condition was investigated and fixed at 35$^{\circ}C$ in this study. Variation among individuals and between male and female of Jam 113 strain was not authorized. DNA polymorhpisms among silkworms were authorized by five RAPD markers using OPM04 random primer. Using the primer showing polymorhpims between parents(J111, Galwon) in thirty three individuals, RAPD-PCR for F2 analysis was performed and segregated 3 : 1 in the F2 population. Consequently, RAPDs detected in the parents were obtained as genetic markers, which can be used for construction of genetic map for this industrially particular insect, silkworm Bombyx mori

• Pediatric Infection and Vaccine
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• v.11 no.2
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• pp.153-157
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• 2004

Purpose : Ttransfusion transmitted virus(TTV) is a circular DNA and consists of diverse genotypes and variants. The pathogenecity of TTV is still unclear. Recently another circular single stranded DNA virus, distantly related to TTV was isolated from the sera of blood donors, designated as Transfusion transmitted virus like minivirus(TLMV). TTV and TLMV show greater sequence divergence from each other than between genotypes of TTV. We planned to know the prevalence of TLMV in children. Methods : TLMV DNA was detected by PCR primers from noncoding region of the genome in 88 children without hepatitis, aged 0~15 years. PCR products derived from 10 children were directly sequenced and phylogenetic analysis was undertaken. Results : TLMV DNA was detected in 49% of 88 children without hepatitis. The prevalence of TLMV varied with age : <1 y, 16%(4/25); 1~3 y, 62%(18/29); 4~6 y, 43%(7/16); 7~9 y. 16%(1/6); 10~15 y, 66%(8/12). Mixed infection with TTV was confirmed in 22% of 88 children. Pyhlogenetic analysis of 10 TLMV sequences showed much heterogeneity compared to sequences of GenBank. Conclusion : TLMV prevalence in children was 49% in Korean children. Our TLMV sequence did not cluster in any sequence of TLMV in the GenBank.