• Title/Summary/Keyword: multiplex polymerase chain reaction (PCR)

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Effects of the Genetic Polymorphisms on Urinary Excretion of 1-Hydroxypyrene and 2-Naphthol (일반인구에서 유전자 다형성이 요중 1-hydroxypyrene 및 2-naphthol의 배설량에 미치는 영향)

  • Hwang Moon-Young;Cho Byung-Mann;Moon Seong-Bae
    • Journal of Environmental Science International
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    • v.14 no.5
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    • pp.499-511
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    • 2005
  • This study was performed to determine the effects of genetic polymorphisms, such as glutathione S-transferase ${\mu}1(GSTM1)$, glutathione S-transferase ${\Theta}1\;(GSTM1)$, glutathione S-transferase ${\pi}l (GSTP1)$, aryl hydrocarbon N-acetyltransferase 2 (NAT2), cytochrome P450 2E1 (CYP2E1), cytochrome P450 1A1 (CYP1A1) on the concentrations of urinary 1-hydroxypyrene (1-OHP) and 2-naphthol in general population with no occupational exposure to polycyclic aromatic hydrocarbons (PAHs). Study subjects were 257 men who visited a health promotion center in Susan. A questionnaire was used to obtain detailed data about age, smoking, drinking, body fat mass, intake of fat etc. Urinary l-OHP and 2-naphthol concentration were analyzed by HPLC system with a fluorescence detector. A multiplex PCR method was used to identify the genotypes for GSTM1 and GSTT1. The polymorphisms of GSTP1, NAT2, CYP1A1 and CYP2E1 were determined by the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Urinary 1-OHP concentration was higher in deleted genotype of GSTM1, increased as smoking and alcohol drinking increased. Urinary 2-naphthol concentration was also rely on the age and smoking. Neither genetic polymorphism nor drinking-related factors were significantly related to urinary 2-naphthol concentration. No significant relation was found between physical characteristics and concentrations of urinary PAHs metabolites in the subjects, but the geometric mean of urinary 1-OHP and 2-naphthol was higher in the group with higher value compared to median value. These data suggest that in general population occupationally not exposed to PAHs, urinary concentration of PAHs metabolites is influenced by smoking, alcohol drinking and deleted genotype of GSTM1 in 1-OHP and smoking in 2-naphthol.

Molecular methods for diagnosis of microbial pathogens in muga silkworm, Antheraea assamensis Helfer (Lepidoptera: Saturniidae)

  • Gangavarapu Subrahmanyam;Kangayam M. Ponnuvel;Kallare P Arunkumar;Kamidi Rahul;S. Manthira Moorthy;Vankadara Sivaprasad
    • International Journal of Industrial Entomology and Biomaterials
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    • v.47 no.1
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    • pp.1-11
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    • 2023
  • The Indian golden muga silkworm, Antheraea assamensis Helfer is an economically important wild silkworm endemic to Northeastern part of India. In recent years, climate change has posed a threat to muga silk production due to the requirement that larvae be reared outdoors. Since the muga silkworm larvae are exposed to the vagaries of nature, the changing climate has increased the incidence of microbial diseases in the rearing fields. Accurate diagnosis of the disease causing pathogens and its associated epidemiology are prerequisites to manage the diseases in the rearing field. Although conventional microbial culturing methods are widely used to identify pathogenic bacteria, they would not provide meaningful information on a wide variety of silkworm pathogens. The information on use of molecular diagnostic tools in detection of microbial pathogens of wild silk moths is very limited. A wide range of molecular and immunodiagnostic techniques including denaturing gradient gel electrophoresis (DGGE), random amplified polymorphism (RAPD), 16S rRNA/ITSA gene sequencing, multiplex polymerase chain reaction (M-PCR), fluorescence in situ hybridization (FISH), immunofluorescence, and repetitive-element PCR (Rep-PCR), have been used for detecting and characterizing the pathogens of insects with economic significance. Nevertheless, the application of these molecular tools for detecting and typing entomopathogens in surveillance studies of muga silkworm rearing is very limited. Here, we discuss the possible application of these molecular techniques, their advantages and major limitations. These methods show promise in better management of diseases in muga ecosystem.

Clinical and laboratory profiles of hospitalized children with acute respiratory virus infection

  • Choi, Eunjin;Ha, Kee-Soo;Song, Dae Jin;Lee, Jung Hwa;Lee, Kwang Chul
    • Clinical and Experimental Pediatrics
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    • v.61 no.6
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    • pp.180-186
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    • 2018
  • Purpose: Despite the availability of molecular methods, identification of the causative virus in children with acute respiratory infections (ARIs) has proven difficult as the same viruses are often detected in asymptomatic children. Methods: Multiplex reverse transcription polymerase chain reaction assays were performed to detect 15 common respiratory viruses in children under 15 years of age who were hospitalized with ARI between January 2013 and December 2015. Viral epidemiology and clinical profiles of single virus infections were evaluated. Results: Of 3,505 patients, viruses were identified in 2,424 (69.1%), with the assay revealing a single virus in 1,747 cases (49.8%). While major pathogens in single virus-positive cases differed according to age, human rhinovirus (hRV) was common in patients of all ages. Respiratory syncytial virus (RSV), influenza virus (IF), and human metapneumovirus (hMPV) were found to be seasonal pathogens, appearing from fall through winter and spring, whereas hRV and adenovirus (AdV) were detected in every season. Patients with ARIs caused by RSV and hRV were frequently afebrile and more commonly had wheezing compared with patients with other viral ARIs. Neutrophil-dominant inflammation was observed in ARIs caused by IF, AdV, and hRV, whereas lymphocyte-dominant inflammation was observed with RSV A, parainfluenza virus, and hMPV. Monocytosis was common with RSV and AdV, whereas eosinophilia was observed with hRV. Conclusion: In combination with viral identification, recognition of virus-specific clinical and laboratory patterns will expand our understanding of the epidemiology of viral ARIs and help us to establish more efficient therapeutic and preventive strategies.

Origin of lactic acid bacteria in mulkimchi fermentation

  • Hwang, Chung Eun;Haque, Md. Azizul;Hong, Su Young;Kim, Su Cheol;Cho, Kye Man
    • Journal of Applied Biological Chemistry
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    • v.62 no.4
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    • pp.441-446
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    • 2019
  • The assortment of endophytic lactic acid bacteria (LAB) in kimchi derives from its raw vegetables, which include Chinese cabbage, radish, welsh onion, onion, garlic, red pepper, and ginger. These vegetables were examined during mulkimchi fermentation using gene-specific multiplex polymerase chain reaction and 16S ribosomal RNA sequence analysis. Sixteen species from five LAB genera (Leuconostoc, Lactobacillus, Lactococcus, Pediococcus, and Weissella) appeared in the raw kimchi materials. Interestingly, nine LAB species were identified in mulkimchi on fermentation day 0 as follows: Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc gelidum, Leuconostoc inhae, Leuconostoc mesenteroides, Lactobacillus plantarum, Lactobacillus sakei, Lactococcus lactis, and Weissella confusa. Seven additional LAB species were present in mulkimchi at fermentation day 9 as follows: Leuconostoc gasicomitatum, Leuconostoc kimchii, Lactobacillus brevis, Lactobacillus curvatus, Lactobacillus pentosus, Pediococcus pentosaceus, and Weissella koreensis. These species corresponded completely with the LAB in kimchi vegetables. Wei. confusa was the predominant LAB during early fermentation (pH 6.20 to 4.98 and acidity 0.20 to 0.64%), while Lac. sakei, Lac. plantarum, and Wei. koreensis became dominant later in fermentation (pH 4.98 to 3.88 and acidity 0.64 to 1.26%). These results collectively demonstrate that the LAB involved in mulkimchi fermentation originates from the raw vegetables examined.

Campylobacter Enteritis: Clinical Features and Laboratory Findings in Children Treated at a Single Hospital

  • Jang, Won Tae;Jo, Na Hyun;Song, Mi Ok;Eun, Byung Wook;Ahn, Young Min
    • Pediatric Infection and Vaccine
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    • v.26 no.1
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    • pp.22-31
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    • 2019
  • Purpose: Campylobacter species are common causes of bacterial enteritis. There is limited knowledge on its prevalence and clinical features because of its fastidious culture conditions. The purpose of this study was to identify the clinical features of Campylobacter enteritis in children. Methods: We obtained stool specimens from patients diagnosed with acute gastroenteritis in the Department of Pediatrics, Nowon Eulji Medical Center (NEMC) and identified the pathogens by performing cultures or polymerase chain reactions (PCR). We retrospectively reviewed the medical records of patients with Campylobacter enteritis at NEMC between January 2012 and December 2017. Results: Overall, 123 patients were diagnosed with Campylobacter enteritis (60 by culture and PCR in EnterNet and 110 by multiplex PCR). The median (interquartile range [IQR]) age of patients was 12 years (IQR, 8 to 16 years). The disease occurred all year round, but 69.9% from June to September. Symptoms included diarrhea (97.6%), fever (96.7%), abdominal pain (94.3%), vomiting (37.4%), and headache (34.1%). Compared with other treatments, treatment with azithromycin was associated with a shorter hospitalization period (P<0.05). Conclusions: Campylobacter enteritis is common during summer and mostly infects adolescent patients. It causes severe abdominal pain and fever preceding the onset of diarrhea. Prompt diagnosis and appropriate use of antibiotics reduces the duration of the disease.

Active Surveillance of Pertussis in Infants Under 6 Months of Age: A Single Center Experience from 2011 to 2013 (생후 6개월 미만의 영아에서 백일해에 대한 적극적인 감시활동: 2011-2013년 단일기관 연구)

  • Han, Young Ik;Choi, Ji Yeon;Lee, Hyewon;Lee, Teak-Jin
    • Pediatric Infection and Vaccine
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    • v.21 no.2
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    • pp.114-120
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    • 2014
  • Purpose: The objective of this study was to assess and investigate the epidemiology of pertussis in infants under 6 months of age. Methods: A prospective study was conducted between October 1, 2011 and April 30, 2013 in CHA Bundang Medical Center, Seongnam, South Korea. Polymerase chain reaction (PCR) or culture was used to detect Bordetella pertussis in nasopharyngeal aspirates from case patients who were hospitalized for acute lower respiratory tract infection (LRTI). In addition, multiplex real-time PCR assays were also performed to detect 6 etiologic viruses, including adenovirus, human metapeumo-virus, influenza virus, parainfluenza virus, respiratory syncytial virus and rhinovirus. Results: Of the 79 enrolled case patients, whose median age was 2 months of age, the most common diagnoses uncovered in this study were acute bronchiolitis (60%) and pneumonia (28%). B. pertussis infection was found in 13 cases (16%), in which 7 (53%) was coinfected with respiratory syncytial virus and 1 (7%) with influenza A virus. Of the 13 patients with B. pertussis infection, 6 (46%) were not vaccinated with the diphtheria, tetanus toxoid, and acellular pertussis vaccine, while 6 (46%) received 1 dose, and 1 (8%) received 2 doses. Conclusion: B. pertussis infection was present in 16% of under 6 month-old infants, who were hospitalized for acute LRTI. Therefore, a nationwide epidemiological surveillance of pertussis, including institutions that cater to infants under 6 months of age is necessary and needed.

Development of the Duplex PCR Method of Identifying Trachurus japonicus and Trachurus novaezelandiae (다중 PCR 분석법을 이용한 전갱이속 어종의 신속한 종판별 분석법 개발)

  • Park, Yeon Jung;Lee, Mi Nan;Kim, Eun Mi;Noh, Eun Soo;Noh, Jae Koo;Park, Jung Youn;Kang, Jung-Ha
    • Journal of Life Science
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    • v.28 no.9
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    • pp.1062-1067
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    • 2018
  • Reliable labeling of fish products can reassure consumers regarding the identity and quality of seafoods. Therefore, techniques that can identify adulteration or mislabeling are valuable. To rapidly identify two Trachurus species, Trachurus japonicus and Trachurus novaezelandiae, a highly efficient, rapid, duplex polymerase chain reaction (PCR) having two species-specific primers simultaneously was identified. This species-specific primer focused on a single nucleotide mismatch in the 3'-terminal base of a primer designed in the mitochondrial cytochrome c oxidase (COI) subunit I DNA. To optimize the duplex PCR condition, gradient PCR reactions were conducted to determine the primer annealing temperature and the primer concentration. The PCR's product was observed on the gel, suggesting that DNA molecules may be useful in differentiating the two species. The length of the amplification fragments were 103 bp for Trachurus japonicus and 214 bp for Trachurus novaezelandiae, which, along with the species-specific primer visualized by agarose gel electrophoresis, enabled accurate distinction of the species of the Trachurus genus. These results indicate that the duplex PCR, which has a species-specific primer based on single nucleotide polymorphism (SNP), can be useful for rapidly differentiating the two species of Trachurus. This duplex PCR analysis is simple, rapid, and reliable, and could be beneficial to protecting consumers' rights.

Diagnostic value of the Vesikari Scoring Sys­tem for predicting the viral or bacterial patho­gens in pediatric gastroenteritis

  • Shim, Dong Ho;Kim, Dong Yeon;Cho, Ky Young
    • Clinical and Experimental Pediatrics
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    • v.59 no.3
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    • pp.126-131
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    • 2016
  • Purpose: To evaluate the diagnostic value of the Vesikari Scoring System (VSS) as an early predictor of pathogens in children with acute gastroenteritis (AG). Methods: In this retrospective study, the VSS score, absolute neutrophil count (ANC), and C-reactive protein (CRP) levels were analyzed in 107 hospitalized children with AG, aged 6 months to 17 years. Patients were divided into nonspecific, viral, and bacterial groups according to the pathogens detected using a multiplex polymerase chain reaction (PCR) test. Results: Patients in the bacterial group had significantly higher CRP values and VSS scores compared to those in the viral group and significantly higher VSS scores compared to those in the nonspecific group (P<0.05). Patients in the viral group had significantly higher VSS scores than those in the nonspecific group (P<0.05). Logistic regression analysis revealed that VSS was the most effective diagnostic tool for predicting the type of pathogen (P<0.05). The area under the receiver operating characteristics curve of VSS was significantly greater than that for ANC and CRP (P<0.05). At a cutoff point of 10 in the VSS, an acceptable diagnostic accuracy could be achieved for distinguishing between bacterial and viral pathogens in AG. Conclusion: VSS can be considered a useful and reliable infectious marker for pediatric gastroenteritis. VSS may be a good early predictor of the type of pathogen, enabling development of a treatment plan before results from a stool culture or PCR test are available.

Detection of Foodborne Pathogens and Mycotoxins in Eggs and Chicken Feeds from Farms to Retail Markets

  • Lee, Minhwa;Seo, Dong Joo;Jeon, Su Been;Ok, Hyun Ee;Jung, Hyelee;Choi, Changsun;Chun, Hyang Sook
    • Food Science of Animal Resources
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    • v.36 no.4
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    • pp.463-468
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    • 2016
  • Contamination by foodborne pathogens and mycotoxins was examined in 475 eggs and 20 feed samples collected from three egg layer farms, three egg-processing units, and five retail markets in Korea. Microbial contamination with Salmonella species, Escherichia coli, and Arcobacter species was examined by bacterial culture and multiplex polymerase chain reaction (PCR). The contamination levels of aflatoxins, ochratoxins, and zearalenone in eggs and chicken feeds were simultaneously analyzed with high-performance liquid chromatography coupled with fluorescence detection after the post-derivatization. While E. coli was isolated from 9.1% of eggs, Salmonella species were not isolated. Arcobacter species were detected in 0.8% of eggs collected from egg layers by PCR only. While aflatoxins, ochratoxins, and zearalenone were found in 100%, 100%, and 85% of chicken feeds, their contamination levels were below the maximum acceptable levels (1.86, 2.24, and 147.53 μg/kg, respectively). However, no eggs were contaminated with aflatoxins, ochratoxins, or zearalenone. Therefore, the risk of contamination by mycotoxins and microbes in eggs and chicken feeds is considered negligible and unlikely to pose a threat to human health.

Pyramiding transgenes for potato tuber moth resistance in potato

  • Meiyalaghan, Sathiyamoorthy;Pringle, Julie M.;Barrell, Philippa J.;Jacobs, Jeanne M.E.;Conner, Anthony J.
    • Plant Biotechnology Reports
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    • v.4 no.4
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    • pp.293-301
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    • 2010
  • The feasibility of two strategies for transgene pyramiding using Agrobacterium-mediated transformation was investigated to develop a transgenic potato (Solanum tuberosum L. cv. Iwa) with resistance to potato tuber moth (PTM) (Phthorimaea operculella (Zeller)). In the first approach, cry1Ac9 and cry9Aa2 genes were introduced simultaneously using a kanamycin (nptII) selectable marker gene. The second approach involved the sequential introduction (re-transformation) of a cry1Ac9 gene, using a hygromycin resistance (hpt) selectable marker gene, into an existing line transgenic for a cry9Aa2 gene and a kanamycin resistance (nptII) selectable marker gene. Multiplex polymerase chain reaction (PCR) confirmed the presence of the specific selectable marker gene and both cry genes in all regenerated lines. The relative steady-state level of the cry gene transcripts in leaves was quantified in all regenerated lines by real-time PCR analysis. Re-transformation proved to be a flexible approach to effectively pyramid genes for PTM resistance in potato, since it allowed the second gene to be added to a line that was previously identified as having a high level of resistance. Larval growth of PTM was significantly inhibited on excised greenhouse-grown leaves in all transgenic lines, although no lines expressing both cry genes exhibited any greater resistance to PTM larvae over that previously observed for the individual genes. It is anticipated that these lines will permit more durable resistance by delaying the opportunities for PTM adaptation to the individual cry genes.