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

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Regional differences in phylogenetic group of Escherichia coli strains isolated from children with urinary tract infection in Korea

  • Choi, Ui-Yoon;Han, Seung Beom;Lee, Soo Young;Kang, Jin Han;Kim, Sun Mi;Ma, Sang Hyuk
    • Clinical and Experimental Pediatrics
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    • v.55 no.11
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    • pp.420-423
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    • 2012
  • Purpose: We phylogenetically analyzed the Escherichia coli strains isolated from children with urinary tract infection (UTI) in 2 regions of Korea. Virulence factors (VFs) and antibiotic resistance of the strains were also determined to compare the possible differences. Methods: A total of 138 E. coli strains were collected from the 2 regions; Gyeongin (78 strains) and Gyeongnam (60 strains). The phylogenetic groups were determined using the triplex polymerase chain reaction (PCR) method and multiplex PCRs were used to detect 7 VFs genes (fimH, papC, iutA, hlyA, sfa/focDE, afa/draBC, and kpsMT II). We also tested for antibiotic resistance. Results: Phylogenetic groups, B2 (61.6%) and D (26.8%), comprised the majority of all isolated strains. Regional comparisons revealed that more B2 strains and fewer non-B2 (A+B1+D) strains were found in Gyeongnam, than in the Gyeongin region (P=0.033), and certain VFs were predominantly detected in Gyeongnam (P<0.05). Neither regional nor phylogenetic differences, in antibiotic resistance of the strains, were significant. Conclusion: We were able to confirm that the geographic location is an important determinant of the distribution of the phylogenetic groups and VFs among the E. coli strains that cause UTI in children.

Epidemiological Trends of Sexually Transmitted Infections Among Women in Cheonan, South Korea, 2006-2012

  • Kim, Jae Kyung
    • Journal of Microbiology and Biotechnology
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    • v.23 no.10
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    • pp.1484-1490
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    • 2013
  • A lack of investigation in specific regions has impeded the understanding of epidemiological trends in the prevalence of sexually transmitted infections (STIs) in South Korea. To help fill this research gap, this study used multiplex polymerase chain reaction (mPCR) to determine the prevalence of STIs detected in clinical specimens collected from women in Cheonan, South Korea between August 2006 and November 2012, and analyzed the prevalence of STIs according to age, bacterial pathogen, and time period. Of the 1,618 specimens collected from 1,523 patients, 536 (35.2%) tested positive for at least 1 pathogen, with 407 (25.2%) testing positive for 1 pathogen, 103 (6.4%) for 2 pathogens, 20 (1.2%) for 3 pathogens, and 6 (0.4%) for 4 pathogens (n = 697 pathogens total). The median ages of all patients and of STI-positive patients were 37.8 and 33.3 years, respectively, and both decreased annually over the study period. Mycoplasma hominis (MH) was detected in 62.1% of the positive specimens, Ureaplasma urealyticum (UU) in 28.4%, Chlamydia trachomatis (CT) in 23.1%, Trichomonas vaginalis (TV) in 7.8%, Mycoplasma genitalium (MG) in 6.5%, and Neisseria gonorrhoeae (NG) in 2.1%. Whereas the prevalence of MH, MG, and TV infection did not vary greatly over the study period, that of UU decreased by one-fifth and that of both CT and NG increased 4-fold. The results indicate great variability in the rates of infection with each pathogen and a decreasing trend in overall STI prevalence, age of patients seeking STI testing, and age of STI-positive patients.

Glutathione S-Transferase T1 and M1 Polymorphisms and Risk of Uterine Cervical Lesions in Women from Central Serbia

  • Stosic, Ivana;Grujicic, Darko;Arsenijevic, Slobodan;Brkic, Marija;Milosevic-Djordjevic, Olivera
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.7
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    • pp.3201-3205
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    • 2014
  • The aim of this study was to investigate the frequencies of GSTT1 and GSTM1 deletion polymorphisms in newly-diagnosed patients with uterine cervical lesions from central Serbia. Polymorphisms of GST genes were genotyped in 97 patients with cervical lesions and 50 healthy women using a multiplex polymerase chain reaction (PCR). The GSTM1 null genotype was significantly more prominent among the patients than in controls (74.2% vs 56.0%), the risk associated with lesions being almost 2.3-fold increased (OR=2.26, 95%CI=1.10-4.65, p=0.03) and 3.17-fold higher in patients above >45 years old (95%CI=1.02-9.79, p=0.04). The analysis of the two genotypes demonstrated that GSTM1 null genotype significantly increased risk only for low grade squamous intraepithelial lesion-LSIL (OR=2.81, 95%CI=1.03-7.68, p=0.04). GSTT1 null genotype or different genotype combinations were not found to be risk factors, irrespective to lesion stages, age or smoking. We found that the risk of cervical lesions might be significantly related to the GSTM1 null genotype, especially in women aged above 45 years. Furthermore, the GSTM1 polymorphism might have greater role in development of early stage lesions.

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.

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.

Application of Multiplex RT-PCR for Simultaneous Identification of Tomato Spotted Wilt Virus and Thrips Species in an Individual Thrips on Chrysanthemum (시설재배 국화에서 총채벌레의 종 동정 및 보독 바이러스 동시 검출을 위한 다중 진단법 적용)

  • Yoon, Ju-Yeon;Yoon, Jung-Beom;Seo, Mi-Hye;Choi, Seung-Kook;Cho, In-Sook;Chung, Bong-Nam;Yang, Chang Yeol;Gangireddygari, Venkata Subba Reddy
    • Research in Plant Disease
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    • v.26 no.4
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    • pp.264-271
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    • 2020
  • We have developed a simultaneous diagnostic method that can identify both the species of thrips and tomato spotted wilt virus (TSWV) that are problematic in chrysanthemum plants. This is a method of amplifying DNA by performing reverse transcription-polymerase chain reaction by simultaneously adding primers specific to TSWV coat protein (N) gene and primers specific to the internal transcribed spacer 2 region of Frankliniella occidentalis and F. intonsa using total nucleic acid extracted from one thrips. The sizes of DNA fragments for TSWV, F. occidentalis, and F. intonsa were 777, 287, and 367 bp, respectively. These results showed species identification of thrips and whether thrips carrying TSWV can be simultaneously confirmed. Further usefulness of the simultaneous diagnostic method was made from greenhouse survey at chrysanthemum greenhouses in Taean (Chungcheongnam-do) and Changwon (Gyeongsangnam-do) to investigate the identification of thrips species and the rate of thrips carrying TSWV. Of thrips collected from the greenhouses, 83.7% thrips was F. occidentalis and 72.9% F. occidentalis carried TSWV in Taean. Similarly, the diagnostic method showed that 92.2% thrips was F. occidentalis and 84.0% F. occidentalis carried TSWV in Changwon. These results confirm that F. occidentalis is a dominant thrips species and the thrips species plays a crucial role in the transmission of TSWV in chrysanthemum plants in the greenhouses. Taken together, this study showed a simple diagnostic method for thrips identification and epidemiological studies of the timing and spread of TSWV through thrips in chrysanthemum greenhouses in South Korea.

Clinical characteristics of acute lower respiratory tract infections due to 13 respiratory viruses detected by multiplex PCR in children (소아에서 13종 호흡기 바이러스에 의한 급성 하기도 감염의 임상 양상)

  • Lim, Jeong-Sook;Woo, Sung-Il;Baek, Yun-Hee;Kwon, Hyuk-Il;Choi, Young-Ki;Hahn, Youn-Soo
    • Clinical and Experimental Pediatrics
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    • v.53 no.3
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    • pp.373-379
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    • 2010
  • Purpose : This study was performed to investigate the epidemiologic and clinical features of 13 respiratory viruses in children with acute lower respiratory tract infections (ALRIs). Methods : Nasopharyngeal aspirates were prospectively obtained from 325 children aged 15 years or less from May 2008 to April 2009 and were tested for the presence of 13 respiratory viruses by multiplex real-time-polymerase chain reaction (RT-PCR). Results : Viruses were identified in 270 children (83.1%). Co-infections with ${\geq}2$ viruses were observed in 71 patients (26.3 %). Respiratory syncytial virus (RSV) was the most common virus detected (33.2%), followed by human rhinovirus (hRV) (19.1%), influenza virus (Flu A) (16.9%), human metapneumovirus (hMPV) (15.4%), parainfluenza viruses (PIVs) (8.3%), human bocavirus (hBoV) (8.0%), adenovirus (ADV) (5.8%), and human coronavirus (hCoV) (2.2%). Clinical diagnoses of viral ALRIs were bronchiolitis (37.5%), pneumonia (34.5%), asthma exacerbation (20.9%), and croup (7.1%). Clinical diagnoses of viral bronchiolitis and pneumonia were frequently demonstrated in patients who tested positive for RSV, hRV, hMPV, or Flu A. Flu A and hRV were most commonly identified in children older than 3 years and were the 2 leading causes of asthma exacerbation. hRV C was detected in 14 (4.3%) children, who were significantly older than those infected with hRV A ($mean{\pm}SD$, $4.1{\pm}3.5$ years vs. $1.7{\pm}2.3$ years; P =0.009). hBoV was usually detected in young children ($2.3{\pm}3.4$ years) with bronchiolitis and pneumonia. Conclusion : This study described the features of ALRI associated with 13 respiratory viruses in Korean children. Additional investigations are required to define the roles of newly identified viruses in children with ALRIs.

Analysis of Molecular Epidemiological Properties of Staphylococcus aureus Isolates from Domestic Animals and Human Patients by PCR (Polymerase Chain Reaction을 활용한 국내 동물과 사람환자에서 분리한 Staphylococcus aureus 분리주의 분자역학적 특성분석)

  • Woo Yong-Ku;Kim Shin
    • Korean Journal of Microbiology
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    • v.41 no.1
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    • pp.24-37
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    • 2005
  • This study was conducted to analyze the molecular epidemiological properties and to select the most efficient and reliable PCR method on 116 of Staphylococcus aureus (S. aureus) isolates from Korean cattle, black goat, pig, dog, chicken, mouse and also human clinical cases from hospital. The distribution patterns of SSG [species specific genes; coagulase (coa), protein A (spa), nuclease (nuc) and aroA (RsaI) gene] were analyzed by PCR method. Among the SSGs, the nuc-gene was found in all strains $(100\%)$ tested and followed by coa-gene $(87.9\%)$, spa-gene $(91.4\%)$ and aroA-gene $(26.7\%)$, in order. The genetic subtyping by RFLP method was performed on the coa [AluI] and aroA-gene [RsaI] PCR products. The mecA-gene PCR and PCR-RFLP techniques were chosen to detect and verify of MRSA strains. Only the human strains $(12.1\%)$ were detected the positive mecA-gene products (533 bp), which were divided into two specific bands [201 & 332 bp] by HhaI enzyme digestion. On coa-gene and spa-gene typing, coa-gene was typed with ten kinds of genotype and coa-3 type were determined as the most predominant genotype, while spa-gene was divided into eleven kinds of genotype and also spa-7 type were selected the most prevalent genotype based on their genetic variations. On the aroA and coa-gene subtyping by PCR-RFLP, aroA-gene products were discriminated with only seven types of genotype, while coa-gene products were further divided into an eleven genotype, respectively. In comparison of SID values of five PCR based typing methods, the coa-PCR-RFLP (SID0.894) was evaluated the most efficient and reliable tools and followed by coa-PCR (SID0.883) and aroA-PCR-RFLP (SID0.462), in order. In conclusion, we could determined that the coa-PCR-RFLP method was the most suitable genetic analysis tool for S. aureus and MRSA strains from domestic animals and humans.

Genetic Analysis of SCN5A in Korean Patients Associated with Atrioventricular Conduction Block

  • Park, Hyoung-Seob;Kim, Yoon-Nyun;Lee, Young-Soo;Jung, Byung-Chun;Lee, Sang-Hee;Shin, Dong-Gu;Cho, Yong-Keun;Bae, Myung-Hwan;Han, Sang-Mi;Lee, Myung-Hoon
    • Genomics & Informatics
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    • v.10 no.2
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    • pp.110-116
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    • 2012
  • Recent several studies have shown that the genetic variation of SCN5A is related with atrioventricular conduction block (AVB); no study has yet been published in Koreans. Therefore, to determine the AVB-associated genetic variation in Korean patients, we investigated the genetic variation of SCN5A in Korean patients with AVB and compared with normal control subjects. We enrolled 113 patients with AVB and 80 normal controls with no cardiac symptoms. DNA was isolated from the peripheral blood, and all exons (exon 2-exon 28) except the untranslated region and exon-intron boundaries of the SCN5A gene were amplified by multiplex PCR and directly sequenced using an ABI PRISM 3100 Genetic Analyzer. When a variation was discovered in genomic DNA from AVB patients, we confirmed whether the same variation existed in the control genomic DNA. In the present study, a total of 7 genetic variations were detected in 113 AVB patients. Of the 7 variations, 5 (G87A-A29A, intervening sequence 9-3C>A, A1673G-H558R, G3578A-R1193Q, and T5457C-D1819D) have been reported in previous studies, and 2 (C48G-F16L and G3048A-T1016T) were novel variations that have not been reported. The 2 newly discovered variations were not found in the 80 normal controls. In addition, G298S, G514C, P1008S, G1406R, and D1595N, identified in other ethnic populations, were not detected in this study. We found 2 novel genetic variations in the SCN5A gene in Korean patients with AVB. However, further functional study might be needed.

Glutathione S-transferase M1 and T1 Polymorphisms, Cigarette Smoking and HPV Infection in Precancerous and Cancerous Lesions of the Uterine Cervix

  • Sharma, Anita;Gupta, Sanjay;Sodhani, Pushpa;Singh, Veena;Sehgal, Ashok;Sardana, Sarita;Mehrotra, Ravi;Sharma, Joginder Kumar
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.15
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    • pp.6429-6438
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    • 2015
  • Glutathione S-transferases (GSTs) play an important role in detoxification of carcinogenic electrophiles. The null genotypes in GSTM1 and GSTT1 have been implicated in carcinogenesis. Present study was planned to evaluate the influence of genetic polymorphisms of GSTM1 and GSTT1 gene loci in cervical carcinogenesis. The study was conducted in Lok Nayak hospital, New Delhi. DNA from clinical scrapes of 482 women with minor gynaecologic complaints attending Gynaecology OPD and tumor biopsies of 135 cervical cancer cases attending the cancer clinic was extracted. HPV DNA was detected by standard polymerase chain reaction (PCR) using L1 consensus primer pair. Polymorphisms of GSTM1 and GSTT1 were analysed by multiplex PCR procedures. Differences in proportions were tested using Pearson's Chi-square test with Odds ratio (OR) and 95% confidence interval (CI). The risk of cervical cancer was almost three times in women with GSTM1 homozygous null genotype (OR-2.62, 95%CI, 1.77-3.88; p<0.0001). No association of GSTM1 or GSTT1 homozygous null genotypes was observed in women with normal, precancerous and cervical cancerous lesions among ${\leq}35$ or >35 years of age groups. Smokers with null GSTT1 genotype had a higher risk of cervical cancer as compared to non-smokers (OR-3.01, 95% CI, 1.10-8.23; p=0.03). The results further showed that a significant increased risk of cervical cancer was observed in HPV positive smoker women with GSTT1 (OR-4.36, 95% CI, 1.27-15.03; p=0.02) and GSTM1T1 (OR-3.87, 95% CI, 1.05-14.23; p=0.04) homozygous null genotypes as compared to HPV positive non smokers. The results demonstrate that the GST null genotypes were alone not associated with the development of cervical cancer, but interacted with smoking and HPV to exert effects in our Delhi population.