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

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Molecular genetic characterization of multiple antimicrobial resistant Salmonella spp. isolated from pigs and cattle (소와 돼지유래 다제내성 Salmonella속 균의 분자유전학적 특성)

  • Lee, Woo-Won;Jung, Byeong-Yeal;Lee, Gang-Rok;Lee, Dong-Soo;Kim, Yong-Hwan
    • Korean Journal of Veterinary Service
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    • v.32 no.1
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    • pp.61-76
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    • 2009
  • At the present study, it was aimed to explore the molecular genetic characterization of multiple antimicrobial resistant Salmonella spp. isolates from pigs and cattle. A total of 138 Salmonella Typhimurium (S. Typhimurium) isolates were typed with phage, among them, 83.3% of S. Typhimurium tested could divide into a 10 phage types. Definitive type 193 (DT193) (25.4%) and DT195 (24.6%) were exhibited as the dominant types. DT104 and U302 were found from pigs and cattle. On the other hand, S. Enteritidis had 6 phage types, of them, phage type 21 (PT21) and PT11b were the popular types. In the plasmid profiles, 135 of S. Typhimurium isolates were exhibited 1 to 6 plasmid bands which molecular weight ranged from 90 to 2kb. 35 isolates (25.4%) harbored a 90kb plasmid which is thought to be the serotype specific virulence plasmid. Two of twenty five S. Enteritidis had common plasmids at 2 and 1.5kb. With multiplex polymerase chain reaction, virulence genes (invA and spvC) were detected from all Salmonella spp. from 167 of S. Typhimurium, S. Enteritidis and chloramphenicol resistant S. Schwarzengrund, but some drug resistant genes, such as PSE-1, cml/tetR and flo were not determined but other drug resistant genes, for example TEM and int were found. The detection rates of spvC, TEM and int gene was 35.3%, 29.3% and 72.5%, respectively. The TEM gene was highly popular in S. Typhimurium, which was detected from ampicillin and amoxicillin resistant strains as 95.9%. int gene was able to detect from all the isolates identified as multidrug resistsnt (MDR), particularly DT193 was thought as the most prevalent virulence and multidrug resistance isolate. The major plasmid profile and drug resistance pattern of DT193 were 90, 40, 10.5, 6.3, 3.0kb and ACCbDNaPSSuT, respectively. MDR was commonly found in other phage types, particularly DT104, U302 and DT203.

Patterns of the Occurrence of TYLCV and ToCV with Whitefly on Summer-Cultivated Tomato in Greenhouse in Gwangju, Gyeonggi Province (경기도 광주 여름재배 시설토마토의 가루이 매개 바이러스 TYLCV, ToCV 발생현황)

  • Kwon, Yongnam;Cha, Byeongjin;Kim, Mikyeong
    • Research in Plant Disease
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    • v.28 no.1
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    • pp.39-45
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    • 2022
  • Patterns of occurrence of tomato yellow leaf curl virus (TYLCV) and tomato chlorosis virus (ToCV) with whitefly on summer-cultivated tomato in Gwangju-si, Gyeonggi Province were surveyed using multiplex reverse transcription-polymerase chain reaction in 2020. In addition, distribution of the whiteflies species and their viral transmission rates were investigated throughout the tomato growing season. The infection rates of TYLCV and ToCV increased sharply during harvest, and the single infection rates were 30.9% and 5.0%, respectively, with a mixed infection rate of the two viruses being the highest at 52.2%. Single infection with TYLCV and double infections with TYLCV and ToCV accounted for the majority with 83.1%. Bemisia tabaci were dominant over Trialeurodes vaporariorum in greenhouse grown plants, and all of the investigated B. tabaci biotypes were identified as Mediterranean (MED, formerly known as Q biotype). The transmission rate of TYLCV, detected in every sampled B. tabaci MED population, was 21.4%, and the mixed transmission rate with ToCV was 35.5%. Viruliferous MED whiteflies with ToCV showed a higher rate than that of T. vaporariorum. In the transplant stage, viruliferous rate of both TYLCV and ToCV of B. tabaci was 42.7%; this rate was highest in the harvest stage. In examination of tomato yield, the increase in the mixed infection rate of TYLCV and ToCV led to complete yield loss. When the mixed infection rate increased by 10%, the yield decreased by 405.4 kg/10a.

Association between Kawasaki disease and acute respiratory viral infections (가와사끼병과 급성 호흡기 바이러스 감염증의 연관성에 관한 연구)

  • Cho, Eun Young;Eun, Byung Wook;Kim, Nam Hee;Lee, Jina;Choi, Eun Hwa;Lee, Hoan Jong;Choi, Jung Yun
    • Clinical and Experimental Pediatrics
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    • v.52 no.11
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    • pp.1241-1248
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    • 2009
  • Purpose:The etiology of Kawasaki disease (KD) is still unknown. Recently, an association between human coronavirus NL63 (HCoV-NL63) and KD was implicated. Hence, we attempted to determine the association between KD and acute respiratory viral infections. Methods:Nasopharyngeal aspirate samples were obtained from 54 patients diagnosed with KD at the Seoul National University (SNU) Children's Hospital and SNU-Bundang Hospital between October 2003 and September 2006. Viral diagnoses of 11 respiratory viruses were made using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR): respiratory syncytial virus (RSV), adenovirus, rhinovirus (RV), parainfluenza viruses (PIVs) 1 and 3, influenza viruses (IFVs) A and B, human metapneumovirus (HMPV), human bocavirus (HBoV), HCoV OC43/229E, and HCoV-NL63. Clinical data were reviewed retrospectively. Results:The median age was 32 months (6 months-10.4 years). Respiratory symptoms were observed in 37 patients (69%). The following respiratory viruses were identified in 12 patients (22%): RV (n=4), PIV-3 (n=2), HBoV (n=2), and adenovirus, RSV, PIV-1, IFV-A, and HCoV-NL63 (n=1). Co-infection with PIV-3 and RV was observed in one patient. Respiratory symptoms were observed in 7 (58.3%) and 30 (71.4%) patients of the virus-positive and virus-negative groups (P>0.05). Response rate to intravenous immunoglobulin administration was 67% (n=8) and 86% (n=36) in the virus- positive and virus-negative groups (P>0.05). Conclusion:Respiratory symptoms were commonly observed in KD patients but the association between respiratory viruses and KD were not found. Large multicenter-based investigations are required to confirm the association between acute respiratory viral infections and KD.

Prevalence and Clinical Characteristics of Pertussis in Children, Cheonan, Korea (천안지역 소아 백일해 감염의 유병률과 임상적 고찰)

  • Lee, Kun Song;Son, Jae Sung;Chung, Eun Hee;Bae, Hong Ki;Lee, Mee Jeong;Yu, Jeesuk;Chang, Young Pyo;Park, Woo Sung;Kim, Jae Kyoung;Rheem, Insoo;Roh, Eui-Jung
    • Pediatric Infection and Vaccine
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    • v.16 no.2
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    • pp.175-182
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    • 2009
  • Purpose : A number of countries have experienced an increase in pertussis during the past decade. In particular, there has been an increase in the incidence rate among adolescents and adults. To learn more about the current epidemiology of pertussis, we studied the prevalence and clinical characteristics of pertussis in children in Cheonan, South Korea. Methods : We collected nasopharyngeal aspirates of 118 patients who were treated for respiratory symptoms at Dankook Univeristy Hospital between March 2008 and September 2009. We performed multiplex PCR for detection of Bordetella pertussis in those aspirates. Results : Of the 118 patients, 10 (8%) were positive by PCR for B. pertussis. Six episodes occurred during the period July to September 2009. Nine of the 10 patients were less than 3 months old. Seven of them had not received DTaP vaccine. The mean duration of coughing before diagnosis was 10.9${\pm}$5.2 days. Ten patients (100%) had paroxysmal cough and 8 (80%) had post-tussive vomiting. Only one patient had fever. One who had complications that include pneumonia, atelectasis and pneumomediastinum developed an absolute increase in leukocyte count (84,400/$mm^3$). There was a statistically significant relation between vaccine being received and development of complications (P =0.033). Conclusion : We suspect that there was an epidemic of pertussis between July and September 2009. Further investigation by a pediatric or nationwide surveillance system is needed to monitor the changing epidemiology for pertussis.

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The First Newborn Screening Study of T-Cell Receptor Excision Circle and κ-Deleting Recombination Excision Circle for Severe Combined Immunodeficiency in Korea: A Pilot Study (국내 최초 T-Cell Receptor Excision Circle과 κ-Deleting Recombination Excision Circle 신생아 선별검사에 관한 연구)

  • Son, Sohee;Kang, Ji-Man;Kim, Jong Min;Sung, Sein;Kim, Yi-Seoul;Lee, Haejeong;Kim, BitA Reum;Lee, Yeon Kyoung;Ko, Sun Young;Shin, Son Moon;Kim, Yae-Jean
    • Pediatric Infection and Vaccine
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    • v.24 no.3
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    • pp.134-140
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    • 2017
  • Purpose: Severe combined immunodeficiency (SCID) is the most serious form of primary immunodeficiency. Infants with SCID are susceptible to life-threatening infections. To establish newborn screening for SCID in Korea, we performed a screening test for T-cell receptor excision circle (TREC) and ${\kappa}$-deleting recombination excision circle (KREC) in neonates and investigated the awareness of SCID among their parents. Methods: Collections of dried blood spots from neonates and parent surveys were performed at the Samsung Medical Center and Cheil General Hospital & Women's Healthcare Center in Korea. The amplification crossing point (Cp) value <37.0 was defined as TREC/KRECpositive based on cutoff values from measuring multiplex real-time polymerase chain reaction. A Cp value >39.0 was defined as negative. Results: For TREC/KREC screening, 141 neonates were enrolled; 63 (44.7%) were male. One hundred forty neonates (99.3%) had positive TREC/KREC results at the time of the initial test; 82.3% and 75.9% were positive and 17.0% and 23.4% were weakly positive for TREC and KREC, respectively. In one neonate (0.7%), the initial TREC/KREC test result was negative. However, repeated tests obtained and confirmed a positive result. For an awareness survey, 168 parents were engaged. Only 2% of parents (3/168) knew that the newborn screening test for SCID had been introduced and performed in other countries. Eighty-four percent of parents (141/168) replied that nationwide newborn SCID screening should be performed in Korean newborns. Conclusions: In this study, newborn SCID screening was performed along with assessment of public awareness of the SCID test in Korea. The study results showed that newborn SCID screening can be readily applied for clinical use at a relatively low cost in Korea.

Natural Occurrence of Aflatoxigenic Aspergillus Species and Aflatoxins in Traditional Korean Fermentation Starters, Meju and Nuruk

  • Woo, So Young;Lee, Sang Yoo;Tian, Fei;Jeong, A-Yeong;Yoo, Cha Nee;Kang, Seung Yoon;Chun, Hyang Sook
    • Journal of Food Hygiene and Safety
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    • v.35 no.5
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    • pp.438-446
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    • 2020
  • Meju and nuruk (respectively soybean and malt) are traditional Korean fermentation starters that are vulnerable to contamination by harmful microorganisms such as aflatoxigenic fungi and their associated aflatoxins (AFs). In this study, Aspergillus spp. were isolated and identified from a total of 57 meju and 18 nuruk samples collected from Korean markets. Their potential aflatoxigenicity was investigated by examining the presence of three aflatoxin biosynthetic genes (aflO, aflP, and aflR) using multiplex polymerase chain reaction (mPCR) assays. Thereafter, aflatoxin production of isolates and the natural occurrence of AFs in meju and nuruk samples were analyzed by high-performance liquid chromatography (HPLC). A total of 177 Aspergillus isolates were identified and 130 isolates were obtained from meju samples. Of these, 25 isolates (19.2%) contained all three aflatoxin biosynthetic genes, and five (20%) of these isolates produced aflatoxins. Forty-seven of the Aspergillus isolates were obtained from nuruk samples, five of which (10.6%) expressed all three AF biosynthetic genes; however, none of these strains produced AFs. HPLC analysis showed that 88% (51/58) of the meju samples and 39% (7/18) of nuruk samples were not contaminated with AFs (below limit of detection). Among the isolates isolated from meju and nuruk, there were aflatoxigenic strains containing all three aflatoxin biosynthetic genes or producing aflatoxin in medium, but the frequency of aflatoxin contamination was low in the meju and nuruk samples.

Convenient Nucleic Acid Detection for Tomato spotted wilt virus: Virion Captured/RT-PCR (VC/RT-PCR) (Tomato spotted wilt virus를 위한 간편한 식물바이러스 핵산진단법: Virion Captured/RT-PCR (VC/RT-PCR))

  • Cho Jeom-Deog;Kim Jeong-Soo;Kim Hyun-Ran;Chung Bong-Nam;Ryu Ki-Hyun
    • Research in Plant Disease
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    • v.12 no.2
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    • pp.139-143
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    • 2006
  • Virion captured reverse transcription polymerase chain reaction (VC/RT-PCR) could detect plant virus quickly and accurately. In the VC/RT-PCR, no antibody is needed unlike immuno-captured RT-PCR (IC/RT-PCR) which had been improved method of RT-PCR for plant viruses, and virus nucleic acids can be obtained easily within 30minutes by property of polypropylene PCR tube which is hold and immobilized viral particles on its surface. For the virion capture of Tomato spotted wilt virus (TSWV), the extraction buffer was tested. The optimum macerating buffer for TSWV was 0.01M potassium phosphate buffer, pH 7.0, containing 0.5% sodium sulfite. The viral crude sap was incubated for 30 min at $4^{\circ}C$. The virions in the PCR tubes were washed two times with 0.01M PBS containing 0.05% Tween-20. The washed virions were treated at $95^{\circ}C$ immediately for 1 min containing RNase free water and chilled quickly in the ice. Disclosed virions' RNAs by heat treatment were used for RT-PCR. Dilution end point of $10^{-5}$ from plant's crude sap infected with TSWV showed relatively higher detection sensitivity for VC/RT-PCR. During multiple detection using two or more primers, interference was arisen by interactions between primer-primer and plant species. The result of multiplex RT-PCR was influenced by combinations of primers and the kind of plant, and the optimum extraction buffer for the multiplex detection by VC/RT-PCR should be developed.

Analysis of Palivizumab Prophylaxis in Patients with Acute Lower Respiratory Tract Infection Caused by Respiratory Syncytial Virus (Respiratory syncytial virus로 인한 급성 하기도 감염 입원 환자에서 Palivizumab 예방요법 유무에 따른 비교 분석)

  • Min, Sung Ju;Song, Jung Sook;Choi, Jang Hwan;Seon, Han Su;Kang, Eun Kyeong;Kim, Do Hyun;Kim, Hee Sup
    • Pediatric Infection and Vaccine
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    • v.18 no.2
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    • pp.154-162
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    • 2011
  • Purpose : The aim of this study was to identify the clinical characteristics of lower respiratory tract infection due to respiratory syncytial virus (RSV) in young children and to provide information for an effective guideline for palivizumab administration in Korea. Methods : We reviewed medical charts of 167 patients under 3 years of age who were hospitalized in Dongguk University Ilsan Hospital for lower respiratory tract infection between January 2007 and February 2011. Diagnosis of the virus was made based on the multiplex real time polymerase chain reaction. Results : There were 113 patients who were infected by respiratory syncytial virus. 90 patients were term infants and 23 patients were preterm infants. No difference was shown between term and preterm infants except the days of admission which was 9.0${\pm}$6.0 days and 12.6${\pm}$21.0 days respectively. In the preterm group their mean age at the time of admission was 5.21${\pm}$4.9 months and the mean gestational age was 33.1${\pm}$4.3 weeks, and the mean birth weight was 2,152${\pm}$950 g. Only 4 patients were born under 28 weeks gestational age and were candidates for palivizumab administration. Conclusion : Most of the patients with severe RSV lower respiratory tract infection were term or near term infants who were not candidates for palivizumab prophylaxis. A nationwide study is needed to make a new risk stratified guideline for RSV prophylaxis for our country.

Distribution of Disease Severity and Group A Rotavirus Genotypes (VP7 & VP4) in Children with Acute Rotavirus Gastroenteritis (급성 로타바이러스 장염 소아에서 질환의 중증도와 로타바이러스 VP7 & VP4 유전형의 분포에 대한 연구)

  • Oh, Hyun-Ju;Kang, Hyun-Sik;Kang, Ki-Soo;Kim, Youn-Woo;Hong, Jung-Yun;Shin, Kyung-Sue;Lee, Jin-Sook;Lee, Su-Hyun;Lee, Keun-Hwa;Cho, Moon-Je;Suh, Dong-In
    • Pediatric Gastroenterology, Hepatology & Nutrition
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    • v.14 no.2
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    • pp.148-154
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    • 2011
  • Purpose: We aimed to study the distribution of rotavirus genotypes (VP7 and VP4) and disease severity of rotavirus gastroenteritis prevalent in our community. Methods: Stool samples were collected from 156 children who were hospitalized with rotavirus gastroenteritis from December 2007 to June 2008. The disease severity of all patients was scored using the Vesikari scale. After extraction of ds-RNA of the rotavirus, cDNA synthesis using reverse transcription and polymerase chain reaction (RT-PCR) and multiplex PCR was performed. Following this, the final identification of genotypes was performed. Results: Of the 156 samples, VP7(G) and VP4(P) genotypes were identified in 147 (94.2%) and 140 (89.7%) samples, respectively. G1 (116 of 147 samples; 78.9%) and P[8] (137 of 140 samples; 97.9%) were the most prevalent, respectively. Of the 138 samples identified of combination types of VP7 and VP4, G1P[8] (111 samples; 80.4%) was the most prevalent. Other combination types varied with very low distribution rates. 9.4% of genotypes were not included in the new vaccines. The disease severity score was $11.8{\pm}3.3$ ($mean{\pm}2SD$). The distribution of disease severity was mild or moderate in 37.8% and severe in 62.2% of patients. Conclusion: The most prevalent genotype combination of rotavirus was G1P[8] and genotypes not included in the vaccines represented 9.4% in our community. Disease severity distribution of hospitalized children with rotavirus gastroenteritis was higher in the severe than in the mild and moderate categories.

Genetic Polymorphism of Epoxide Hydrolase and GSTM1 in Chronic Obstructive Pulmonary Disease (만성폐쇄성폐질환 발생에 Epoxide hydrolase와 GSTM1유전자 다형성의 의의)

  • Park, Sang Sun;Kim, Eun Joung;Son, Chang Young;Wi, Jeong Ook;Park, Kyung Hwa;Cho, Gye Jung;Ju, Jin Young;Kim, Kyu Sik;Kim, Yu II;Lim, Sung Chul;Kim, Young Chul;Park, Kyung Ok;Na, Kook Joo
    • Tuberculosis and Respiratory Diseases
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    • v.55 no.1
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    • pp.88-97
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    • 2003
  • Background : Although smoking is a major cause of chronic obstructive pulmonary disease (COPD), only 10-20% of cigarette smokers develop symptomatic COPD, which suggests the presence of genetic susceptibility. This genetic susceptibility to COPD might depend on variations in the activities of the enzyme that detoxify hazardous chemical products, such as microsomal epoxide hydrolase (mEPHX) and glutathione-S transferase M1 subunit (GSTM1) genes. Methods : The genotypes of 58 patients with COPD, and 79 age matched control subjects, were determined by a polymerase chain reaction, followed by restriction fragment length polymorphism (PCR-RFLP) for the mEPHX, and multiplex PCR for the GSTM1. Results : GSTM1 was deleted in 53.3% of the subjects. There was no difference in GSTM1 deletion rates between the COPD patients (32/58, 55.2%) and the control subjects (41/79, 51.9%). The combination patterns of two polymorphisms of mEPHX showed slow enzyme activity in 29(21.2%), normal in 73(53.3%) and fast in 32(23.4%). The COPD group (7/57, 12.3%) showed a significantly lower incidence of slow enzyme activity compared to the control subjects (22/77, 28.6%, p<0.05). However, when the COPD and control groups were compared with smokers only, there were no significant differences in the genotypes of GSTM1 and mEPHX. Conclusion : The genotypes of GSTM1 and mEPHX were not significant risk factors of COPD in this cohort of study.