• Title/Summary/Keyword: Multiplex-polymerase chain reaction

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Impact of Tobacco on Glutathione S Transferase Gene Loci of Indian Ethnics

  • Senthilkumar, K.P.;Thirumurugan, Ramasamy
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.10
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    • pp.5037-5042
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    • 2012
  • Background: Tobacco contains agents which generate various potent DNA adducts that can cause gene mutations. Production of DNA adducts may be neutralized by glutathione S transferase (GST) along with other phase I and phase II enzyme systems. The existence of null type of GST among the population increases the susceptibility to various disorders and diseases. The present study focuses on the impact of high tobacco usage and possible null type mutation in GST loci. Methods: Genotypes of GST were detected by multiplex polymerase chain reaction in unrelated 504 volunteers of high tobacco using natives of Gujarat. Allelic frequencies were calculated using Statistical Package for Social Studies-16 software. Hardy Weinberg Equilibrium (HWE) was calculated using Chi square test. Two sided Fisher's significance test was used to compare allelic frequencies of different populations. Results: The frequency of homozygous null genotype of GSTM1 and GSTT1 were 20% (95% CI 16.7-23.9) and 35.5% (95% CI 31.4-39.9) respectively. The GSTM1 and GSTT1 null allele frequency distribution in the Gujarat population was significantly deviating from HWE. GSTT1 null frequency of Gujaratians was significantly higher and different to all reported low tobacco using Indian ethnics, while GSTM1 was not differing significantly. Conclusion: Tobacco usage significantly influences the rate of mutation and frequency of GSTT1 and M1 null types among the habituates. The rate of mutation in GSTT1 loci was an undeviating response to the dose of tobacco usage among the population. This mutational impact of tobacco on GSTT1 postulates the possible gene - environment interaction and selection of null genotype among the subjects to prone them under susceptible status for various cancers and even worst to cure the population with GSTT1 dependent drugs.

Prevalence of respiratory viral infection in children hospitalized for acute lower respiratory tract diseases, and association of rhinovirus and influenza virus with asthma exacerbations

  • Kwon, Jang-Mi;Shim, Jae Won;Kim, Deok Soo;Jung, Hye Lim;Park, Moon Soo;Shim, Jung Yeon
    • Clinical and Experimental Pediatrics
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    • v.57 no.1
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    • pp.29-34
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    • 2014
  • Purpose: In this study, we aimed to investigate the prevalence of year-round respiratory viral infection in children with lower respiratory tract infection (LRTI) and the relationship between respiratory viral infection and allergen sensitization in exacerbating asthma. Methods: We investigated the sources for acute LRTIs in children admitted to our hospital from May 2010 to April 2011. The 6 most common respiratory viruses were isolated from nasopharyngeal aspirate using multiplex reverse transcription-polymerase chain reaction in 309 children; respiratory syncytial virus (RSV), adenovirus (AV), parainfluenza virus (PIV), influenza virus (IFV), human metapneumovirus (hMPV), rhinovirus (RV). Atopic sensitization was defined if more than 1 serum specific Immunoglobulin E level measured using UniCAP (Pharmacia) was over 0.35 IU/mL. Results: RSV was the most common pathogen of bronchiolitis in hospitalized children through the year. RV or IFV infection was more prevalent in asthma exacerbations compared to other LRTIs. AV and hMPV were more likely to cause pneumonia. RV and IFV were associated with asthma exacerbations in children with atopic sensitization, but not in nonatopic children. Conclusion: RV and IFV are associated with hospitalization for asthma exacerbation in children with atopic sensitization.

Clinical manifestation of Campylobacter enteritis in children

  • Bae, Joon Yeol;Lee, Dong Hyuk;Ko, Kyung Ok;Lim, Jae Woo;Cheon, Eun Jeong;Song, Young Hwa;Yoon, Jung Min
    • Clinical and Experimental Pediatrics
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    • v.61 no.3
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    • pp.84-89
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    • 2018
  • Purpose: Timely antibiotic therapy in selected cases of diarrhea associated with bacterial infections can reduce the duration and severity of illness and prevent complications. The availability of a predictive index before identification of causative bacteria would aid in the choice of a therapeutic agent. Methods: The study included patients admitted to the pediatrics unit at Konyang University Hospital for acute inflammatory diarrhea from August 1, 2015 to July 31, 2016 who underwent multiplex polymerase chain reaction testing. Of 248 patients, 83 had positive results. The clinical symptoms and blood test results were examined in 61 patients with Campylobacter spp. (25 patients), Salmonella spp. (18 patients), and Clostridium perfringens (18 patients) infections. The mean age of the 61 patients (male:femal=31:30) was $84.0{\pm}54.8months$, and the mean hospital stay was $4.6{\pm}1.7days$. Results: There were no statistical differences in sex, age, clinical symptoms, or signs. Patients with Campylobacter infection were significantly older (P=0.00). C-reactive protein (CRP) levels in patients with Campylobacter infection were higher than those in the other 2 groups, at $9.6{\pm}6.1mg/dL$. The results of receiver-operating characteristic curve analysis showed that the cutoff age was ${\geq}103.5months$ (sensitivity, 72%; specificity, 86%) and the CRP cutoff level was ${\geq}4.55mg/dL$ (sensitivity, 80%; specificity, 69%). Conclusion: Age (${\geq}103.5months$) and higher CRP level (${\geq}4.55mg/dL$) were good predictors of Campylobacter enterocolitis. If neither criterion was met, Campylobacter enterocolitis was unlikely (negative predictive value 97.2%). When both criteria were met, Campylobacter enterocolitis was highly likely.

Laboratory Investigation of Trends in Bacterial Pneumonia in Cheonan, Korea, from January 2008 to September 2017

  • Yook, Young-Sam;Jeon, Jae-Sik;Park, Ji On;Kim, Jae Kyung
    • Journal of Microbiology and Biotechnology
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    • v.28 no.10
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    • pp.1730-1735
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    • 2018
  • Bacterial pneumonia is one of the most common causes of mortality in Korea. In 2016, the mortality rate from pneumonia was 16,476 deaths per 100,000, which was an 11% increase from the previous year. The aim of our study was to determine the distribution of the bacterial pathogens causing respiratory symptoms in different age groups over a 10-year period. Between January 2008 and September 2017, 1,861 specimens from 1,664 patients admitted to Dankook University Hospital with respiratory symptoms were examined. We used multiplex polymerase chain reaction (PCR) to detect six bacterial pneumonia pathogens: Bordetella pertussis, Chlamydophila pneumoniae, Haemophilus influenzae, Legionella pneumophila, Mycoplasma pneumoniae, and Streptococcus pneumoniae. We detected bacterial pneumonia pathogens in 1,281 (68.83%) specimens. Of the 1,709 pathogens detected, S. pneumoniae was the most common (48.57%; n = 830) followed by H. influenzae (40.08%; n = 685). Most infections were found among children younger than 10 years (92.69%; n = 1,584). Although S. pneumoniae was the most common pathogen detected in all age groups, M. pneumoniae infection increased in prevalence with age (p < 0.05). The rate of co-infection was also high among these patients (31.1%; n = 399), which peaked in 2015 (54.55%; n = 42/77). The prevalence of bacterial pneumonia in Cheonan, along with the proportion of co-infections among patients increased over the 10-year study period. The findings will aid the development of treatment and prevention guidelines.

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.

An outbreak of chronic fowl cholera in broiler breeder chickens in Korea

  • Kim, Jin-Hyun;Yoon, Mi-Young;Cho, Jae-Keun;Sung, Myung-Suk;Kim, Ki-Seuk
    • Korean Journal of Veterinary Service
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    • v.34 no.4
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    • pp.353-359
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    • 2011
  • Fowl cholera is a contagious acute and chronic disease caused by Pasteurella multocida in both domesticated and wild birds. Acute fowl cholera in both chickens and wild birds has recently been documented in Korea, but the chronic form has not been reported in Korea until now. This study describes the first outbreak of chronic fowl cholera in 13-week-old Arbor Acre broiler breeder chickens submitted to the College of Veterinary Medicine, Kyungpook National University in April 2006. The clinical signs of the affected flock of 9,621 chickens were lameness caused by swollen hock joints, diarrhea, ruffled feathers, and an average weekly mortality of 1.0%. At necropsy, purulent or caseous exudates were found in the hock and wing joints, humerus, and eyes, and severe pneumonia and pericarditis were discovered. Eleven bacterial isolates obtained from the liver, joint, infraorbital sinus and sternal bursa of the submitted chickens were all identified as Pasteurella multocida based on their physiological and biochemical characteristics. Five isolates were examined for antimicrobial susceptibility against 21 different antimicrobial agents including ampicillin. All were resistant to kanamycin, neomycin, and streptomycin, and some were resistant to gentamicin. The tested isolates were all susceptible to the other 17 antimicrobial agents. All 11 isolates were capsular serogroup A based on multiplex polymerase chain reaction. In addition, two of five isolates used in the antimicrobial susceptibility test were identified as somatic serotype 1 by an agar gel diffusion precipitin test, while the others were non-typable.

Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease

  • Lee, Hyoung-Song;Kim, Min Jee;Ko, Duck Sung;Jeon, Eun Jin;Kim, Jin Young;Kang, Inn Soo
    • Clinical and Experimental Reproductive Medicine
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    • v.40 no.4
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    • pp.163-168
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    • 2013
  • Objective: Preimplantation genetic diagnosis (PGD) is an assisted reproductive technique for couples carrying genetic risks. Charcot-Marie-Tooth (CMT) disease is the most common hereditary neuropathy, with a prevalence rate of 1/2,500. In this study, we report on our experience with PGD cycles performed for CMT types 1A and 2F. Methods: Before clinical PGD, we assessed the amplification rate and allele drop-out (ADO) rate of multiplex fluorescent polymerase chain reaction (PCR) followed by fragment analysis or sequencing using single lymphocytes. We performed six cycles of PGD for CMT1A and one cycle for CMT2F. Results: Two duplex and two triplex protocols were developed according to the available markers for each CMT1A couple. Depending on the PCR protocols, the amplification rates and ADO rates ranged from 90.0% to 98.3% and 0.0% to 11.1%, respectively. For CMT2F, the amplification rates and ADO rates were 93.3% and 4.8%, respectively. In case of CMT1A, 60 out of 63 embryos (95.2%) were diagnosed and 13 out of 21 unaffected embryos were transferred in five cycles. Two pregnancies were achieved and three babies were delivered without any complications. In the case of CMT2F, a total of eight embryos were analyzed and diagnosed. Seven embryos were diagnosed as unaffected and four embryos were transferred, resulting in a twin pregnancy. Two healthy babies were delivered. Conclusion: This is the first report of successful pregnancy and delivery after specific PGD for CMT disease in Korea. Our PGD procedure could provide healthy babies to couples with a high risk of transmitting genetic diseases.

Clinical Significance of Inflammatory Biomarkers in Acute Pediatric Diarrhea

  • Park, Yoonseon;Son, Minji;Jekarl, Dong Wook;Choi, Hyun Yoo;Kim, Sang Yong;Lee, Seungok
    • Pediatric Gastroenterology, Hepatology & Nutrition
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    • v.22 no.4
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    • pp.369-376
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    • 2019
  • Purpose: The aim of this study was to evaluate the clinical significance of inflammatory biomarkers in acute infectious diarrhea among children. Methods: Clinical parameters including fever, bacterial and viral etiology based on stool culture and multiplex polymerase chain reaction, and nine biomarkers including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and leukocytes in blood and calprotectin, lactoferrin, myeloperoxidase, polymorphonuclear elastase, leukocytes, and occult blood in feces were evaluated in children who were hospitalized due to acute diarrhea without underlying disease. Results: A total of 62 patients were included. Among these patients, 33 had fever, 18 showed bacterial infections, and 40 patients were infected with 43 viruses. Of all the biomarkers, CRP was significantly correlated with fever (p<0.001). CRP, ESR, calprotectin, lactoferrin, myeloperoxidase, fecal leukocytes, and occult blood were significantly associated with infection with bacterial pathogens (p<0.001, p=0.04, p=0.03, p=0.003, p=0.02, p=0.03, p=0.002, respectively). The combination of CRP and fecal lactoferrin at their best cut-off values (13.7 mg/L and $22.8{\mu}g/mL$, respectively) yielded a sensitivity of 72.2%, and a specificity of 95.5% for bacterial etiology compared with their individual use. Conclusion: Blood CRP is a useful diagnostic marker for both fever and bacterial etiology in acute pediatric diarrhea. The combination of CRP and fecal lactoferrin yields better diagnostic capability for bacterial etiology than their use alone for acute diarrhea in children without underlying gastrointestinal disease.

Genetic diversity of Indonesian cattle breeds based on microsatellite markers

  • Agung, Paskah Partogi;Saputra, Ferdy;Zein, Moch Syamsul Arifin;Wulandari, Ari Sulistyo;Putra, Widya Pintaka Bayu;Said, Syahruddin;Jakaria, Jakaria
    • Asian-Australasian Journal of Animal Sciences
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    • v.32 no.4
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    • pp.467-476
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    • 2019
  • Objective: This research was conducted to study the genetic diversity in several Indonesian cattle breeds using microsatellite markers to classify the Indonesian cattle breeds. Methods: A total of 229 DNA samples from of 10 cattle breeds were used in this study. The polymerase chain reaction process was conducted using 12 labeled primers. The size of allele was generated using the multiplex DNA fragment analysis. The POPGEN and CERVUS programs were used to obtain the observed number of alleles, effective number of alleles, observed heterozygosity value, expected heterozygosity value, allele frequency, genetic differentiation, the global heterozygote deficit among breeds, and the heterozygote deficit within the breed, gene flow, Hardy-Weinberg equilibrium, and polymorphism information content values. The MEGA program was used to generate a dendrogram that illustrates the relationship among cattle population. Bayesian clustering assignments were analyzed using STRUCTURE program. The GENETIX program was used to perform the correspondence factorial analysis (CFA). The GENALEX program was used to perform the principal coordinates analysis (PCoA) and analysis of molecular variance. The principal component analysis (PCA) was performed using adegenet package of R program. Results: A total of 862 alleles were detected in this study. The INRA23 allele 205 is a specific allele candidate for the Sumba Ongole cattle, while the allele 219 is a specific allele candidate for Ongole Grade. This study revealed a very close genetic relationship between the Ongole Grade and Sumba Ongole cattle and between the Madura and Pasundan cattle. The results from the CFA, PCoA, and PCA analysis in this study provide scientific evidence regarding the genetic relationship between Banteng and Bali cattle. According to the genetic relationship, the Pesisir cattle were classified as Bos indicus cattle. Conclusion: All identified alleles in this study were able to classify the cattle population into three clusters i.e. Bos taurus cluster (Simmental Purebred, Simmental Crossbred, and Holstein Friesian cattle); Bos indicus cluster (Sumba Ongole, Ongole Grade, Madura, Pasundan, and Pesisir cattle); and Bos javanicus cluster (Banteng and Bali cattle).

BRCA1/2 mutations, including large genomic rearrangements, among unselected ovarian cancer patients in Korea

  • Kim, Do-Hoon;Cho, Chi-Heum;Kwon, Sun Young;Ryoo, Nam-Hee;Jeon, Dong-Seok;Lee, Wonmok;Ha, Jung-Sook
    • Journal of Gynecologic Oncology
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    • v.29 no.6
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    • pp.90.1-90.12
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    • 2018
  • Objective: We performed small-scale mutation and large genomic rearrangement (LGR) analysis of BRCA1/2 in ovarian cancer patients to determine the prevalence and the characteristics of the mutations. Methods: All ovarian cancer patients who visited a single institution between September 2015 and April 2017 were included. Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and long-range polymerase chain reaction (PCR) were performed to comprehensively study BRCA1/2. The genetic risk models BRCAPRO, Myriad, and BOADICEA were used to evaluate the mutation analysis. Results: In total, 131 patients were enrolled. Of the 131 patients, Sanger sequencing identified 16 different BRCA1/2 small-scale mutations in 20 patients (15.3%). Two novel nonsense mutations were detected in 2 patients with a serous borderline tumor and a large-cell neuroendocrine carcinoma. MLPA analysis of BRCA1/2 in Sanger-negative patients revealed 2 LGRs. The LGRs accounted for 14.3% of all identified BRCA1 mutations, and the prevalence of LGRs identified in this study was 1.8% in 111 Sanger-negative patients. The genetic risk models showed statistically significant differences between mutation carriers and non-carriers. The 2 patients with LGRs had at least one blood relative with breast or ovarian cancer. Conclusion: Twenty-two (16.8%) of the unselected ovarian cancer patients had BRCA1/2 mutations that were detected through comprehensive BRCA1/2 genetic testing. Ovarian cancer patients with Sanger-negative results should be considered for LGR detection if they have one blood relative with breast or ovarian cancer. The detection of more BRCA1/2 mutations in patients is important for efforts to provide targeted therapy to ovarian cancer patients.