• Title/Summary/Keyword: 실시간 중합효소 연쇄반응

Search Result 69, Processing Time 0.032 seconds

Development of Real-time PCR Assays for Detection of Dirofilaria immitis from Infected Dog Blood (심장사상충에 감염된 개의 혈액에서 심장사상충 유전자를 검출할 수 있는 실시간 중합효소연쇄반응 기법 개발)

  • Oh, In Young;Kim, Kyung Tae;Jun, Jin Hyun;Shin, Jae-Ho;Sung, Ho Joong
    • Korean Journal of Clinical Laboratory Science
    • /
    • v.48 no.2
    • /
    • pp.88-93
    • /
    • 2016
  • Dirofilaria immitis is a filarial nematode parasite that causes cardiopulmonary dirofilariasis in dogs. The purpose of this study was the development of real-time PCR assays for efficient detection of D. immitis. The D. immitis-specific primers confirmed in our previous study and a newly designed TaqMan probe were used for quantitative diagnostics. First, SYBR Green real-time PCR was performed using the specific primers and serially diluted genomic DNA or plasmid DNA, and melting curve analyses were performed after amplification. The melting curve showed one specific peak in each of the genomic and plasmid DNA reactions, suggesting that the primers specifically amplify the D. immitis cytochrome c oxidase subunit I gene. Comparison of SYBR Green and TaqMan real-time PCR using serially diluted plasmid DNA showed higher efficiency and specificity with TaqMan real-time PCR. The real-time PCR assays developed in this study will provide improved diagnostic methods to overcome the limitations of conventional diagnostic tools and facilitate more rapid and accurate diagnoses.

Comparative Study of Target Genes and Protocols by Country for Detection of SARS-CoV-2 based on Polymerase Chain Reaction (PCR) (중합효소 연쇄반응 기반의 코로나-19 바이러스 검출법에 대한 국가별 목표 유전자 및 프로토콜 비교 연구)

  • Kim, Jin-Hee
    • The Journal of the Korea Contents Association
    • /
    • v.21 no.1
    • /
    • pp.465-474
    • /
    • 2021
  • Corona-19, a disease caused by 'Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)', was declared a global pandemic by the World Health Organization (WHO) in March 2020, and a real-time polymerase chain reaction test is performed as a diagnostic test for screening and confirmation in most countries. However, not only the target genes and protocols differ by countries, but also the procedures for reading the diagnosis results are diverse, so the criteria for confirmed patients differ by country. Therefore, in this review, we discussed the target genes, test techniques, and diagnostic criteria for each country notified by WHO. And the specificity and sensitivity, limits of detection, positive and negative controls, false positive bacteria candidates, and specimens, and the specifics of the control setting were also described. In addition, the characteristics of Korea's test were compared to each country's one. Finally, in order to obtain the same diagnosis result for SARS-CoV-2 in the future, standardized diagnosis methods and result interpretations for Corona-19 diagnosis were proposed.

Development of a Panel of Multiplex Real-Time Polymerase Chain Reaction Assays for Simultaneous Detection of Canine Enteric Bacterial Pathogens (개의 장내 병원균의 동시 검출을 위한 다중 실시간 중합효소연쇄반응분석 패널개발)

  • Jang, Hye-Jin;Han, Jae-Ik;Kang, Hyo-Min;Na, Ki-Jeong
    • Journal of Veterinary Clinics
    • /
    • v.32 no.2
    • /
    • pp.154-157
    • /
    • 2015
  • A major cause of diarrhea in a dog is an infection with bacteria which include Salmonella spp., Campylobacter (C.) spp., and Clostridium (Cl.) spp.. It is fastidious to identify these bacteria by the culture. The purpose of this experiment is to devise the method for detecting Cl. perfringens, C. jejuni, C. coli, and Salmonella spp. with rapid and high sensitivity. The fecal samples collected from 71 normal and 66 diarrheic dog feces were used to compare the prevalence of the enteric pathogens and to develop a multiplex real-time polymerase chain reaction (PCR) assay for clinical use. Detection of Cl. perfringens, C. coli, and C. jejuni in diarrhea feces was higher than normal feces. A developed multiplex real-time PCR is useful for determining the presence and quantity of pathogen-specific or other unique sequences with in a fecal sample.

Clinical review of Epstein-Barr virus viremia in Korean children by using real-time PCR (한국인 소아에서 실시간 중합효소연쇄반응으로 검출된 Epstein-Barr virus 바이러스혈증의 임상적 고찰)

  • Ko, Il Yong;Suh, Jin Suk;Kim, Hwang Min;Sohn, Joon Hyung;Yeh, Byung-Il;Lee, Taek Jin;Kim, Dong Soo
    • Pediatric Infection and Vaccine
    • /
    • v.14 no.2
    • /
    • pp.171-178
    • /
    • 2007
  • Purpose : We investigated clinical characteristics of real-time PCR proven EBV viremia patients who were not serologically diagnosed but clinically suspected, and compared it to serologically proven EBV infected patients. Methods : The study population consisted of 45 patients, who were suspected acute EBV infection at Wonju Christian Hospital Department of Pediatrics, Yonsei University Wonju College of Medicine from Jan. 2004 to Dec. 2006. real-time PCR of cell free serum was performed to prove EBV viremia. Then we chose $102.5copies/{\mu}g$ DNA as a suitable cutoff level for EBV associated diseases. Results : There are 4 patients diagnosed as EBV infection by serologically and 15 patients diagnosed as EBV viremia by real-time PCR quantitative measurement. The most common presenting symptoms and signs of EBV viremia was fever in 11 cases (73%). Atypical lymphocytosis was found in 4 cases (27%). Increased AST, ALT levels were observed in 13 cases (87%), 12 cases (80%), respectively. We could diagnose 5 cases of EBV viremia younger than one year of age. They revealed clinical symptoms which could be found in EBV infection. The serologically diagnosed patients had hepatomegaly and splenomegaly in 3 cases (75%). All serologically confirmed patients have leukocytosis above $20,000/mm^3$, among them 2 cases (50%) had higher percentage (>15%) of atypical lymphocytes. The AST/ALT level above 50 IU/L were demonstrated in all cases. Conclusion : Serologically unproven real-time PCR EBV viremia patients revealed similar clinical findings with that of serologically proven EBV infected patients. So, it is meaningful to perform EBV real-time PCR for the diagnosis of EBV infection especially for the cases younger than 1 year of age.

  • PDF

A Study on the Validation system of Detection for Biological Agents Using Real-Time PCR (실시간 중합효소 연쇄반응을 활용한 생물작용제 검증시스템 연구)

  • Cha, Younggil;Koo, Bonwoo;Kim, Seongjoo;Kim, Namil;Park, Hanoh
    • Journal of the Korea Institute of Military Science and Technology
    • /
    • v.20 no.5
    • /
    • pp.726-732
    • /
    • 2017
  • Bacillus anthracis, Vibrio cholerae, Variola virus and Shigella dysenteriae are classified as category A and B biological weapons. In this study suggest that 4 genes of Bacillus anthracis, 2 genes of Vibrio cholerae, 1 gene of Variola virus and 1 gene of Shigella dysenteriae were detective 50~500 fg of target DNA per reaction using real-time PCR based assay. Also analytical specificity did not show any cross-reactivity with other related bacteria. Reliable and one reaction could be effective early diagnostic and treatment for detection of unknown samples.

Multiplex Quantitative Real-time Polymerase Chain Reaction Assay for Rapid Detection of Mycobacterium avium subsp. paratuberculosis in Fecal Samples (분변 시료에서 Mycobacterium avium subsp. paratuberculosis 의 빠른 검출을 위한 다중 실시간 중합효소연쇄반응기법의 개발)

  • Han, Jae-Ik;Jung, Young-Hun;Choe, Changyong;Yoo, Jaegyu;Kang, Seog-Jin;Yoo, Hansang;Park, Hongtae;Kwon, Eung-Gi;Cho, Yong-Il
    • Journal of Veterinary Clinics
    • /
    • v.32 no.3
    • /
    • pp.219-223
    • /
    • 2015
  • Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis or Johne's disease, an intestinal granulomatous infection in domestic and wild animals. The study aimed to develop and evaluate a panel of multiplex quantitative real-time polymerase chain reaction (mqPCR) assay for simultaneous detection of three MAP-specific genes (IS900, F57 and ISMAP02 genes). The analytic sensitivity (i.e., limit of detection, expressed as cells per 1 ml) was 150 for IS900, 1500 for F57, and 50 for ISMAP02. The specificity of the method was determined by testing 152 bovine fecal samples. Based on the test, it showed that the assay simultaneously detected the target genes in short period of time and at lower cost compared to laboratory routine tests. The test agreement between the assay and routine test was 94%. The discrepancy in the results was due to samples that were tested positive by the panel but negative by the routine tests, suggesting that the assay has higher sensitivity than the routine tests. In conclusion, the mqPCR assay could be a rapid and accurate testing tool for investigating paratuberculosis or Johne's disease cases in domestic and wild animals.

Clinical Characteristics and Comparison of the Various Methods Used for the Diagnosis of the New Influenza A Pandemic in Korea (한국에서의 2009 신종 인플루엔자 A의 임상양상과 다양한 진단 방법들의 비교)

  • Kwon, Min Jung;Lee, Chang Kyu;Roh, Kyoung Ho;Nam, Myung Hyun;Yoon, Soo Young;Lim, Chae Seung;Cho, Yun Jung;Kim, Young Kee;Lee, Kap No
    • Laboratory Medicine Online
    • /
    • v.1 no.1
    • /
    • pp.26-34
    • /
    • 2011
  • Background: Laboratory diagnosis of new influenza A (H1N1) is crucial for managing patients and establishing control and prevention measures. We compared the diagnostic accuracies of the real time RT-PCR (rRT-PCR) test recommended for the confirmation of the new flu and the viral culture method used conventionally for viral disease with that of the rapid antigen test (RAT). Methods: We performed RAT, R-mix culture, and real-time PCR by using 861 respiratory samples collected from December 2009 to January 2010 and evaluated the abilities of these methods to detect new influenza A. The relationship among the positive rates of RAT, grades of culture, and the cycle threshold (Ct) values of rRT-PCR was also evaluated. Results: Of the 861 patients, 308 (35.8%) were diagnosed with new influenza A. The sensitivities, specificities, positive predictive values, and negative predictive values of the tests were respectively as follows: 59.7%, 99.5%, 98.4%, and 81.6% for RAT; 93.2%, 100%, 100%, and 96.3% for R-mix culture; and 95.8%, 100%, 100%, and 97.7% for rRT-PCR. Samples with weak positive grade in culture and those with Ct values of 30-37 in rRT-PCR showed positivities as low as 25.3% and 2.3% in RAT, respectively. The hospitalization rate and death rate of the confirmed patients were 3.2% and 0.3%, respectively, and gastrointestinal symptoms were observed in 7.2% of the patients. Conclusions: R-mix culture and rRT-PCR tests showed excellent reliability in the diagnosis of new influenza A and could be very useful, especially for samples with low viral load.

생쥐 초기 배아에서 Aquaporin 8과 9의 발현에 관한 연구

  • 신현상;계명찬;강수만;이성은;이지원;강한승;김문규
    • Proceedings of the Korean Society of Developmental Biology Conference
    • /
    • 2003.10a
    • /
    • pp.68-68
    • /
    • 2003
  • Aquaporin은 막관통 통로 단백질(transmembrane channel protein)로서, 삼투압의 농도구배에 따라 세포막을 가로질러 물분자를 이동시키는 기능을 하고 있다. 포유류 초기배아에서 포배강 형성은 영양외배엽세포에서 $Na^+ / K^+$ATPase에 의한 이온 농도 구배가 형성되면 auqaporin에 의해 물이 포배강으로 유입되면서 이루어진다. 본 연구에서는 생쥐 초기배아에서 반정량적인 역전사 중합효소 연쇄반응 방법(semi-quantitative RT-PCR)과 실시간 역전사 중합효소 연쇄반응 방법(real-time RT-PCR)을 통하여 AQP8과 9의 mRNA발현을 조사하고 다중 면역형광현미경 방법(confocal immunofluorescence microscopy)을 통해 단백질 발현양상을 분석하였다. AQP8 mRNA는 상실기까지 발현되지 않다가 포배기에 이르러 발현되었고 AQP9 mRNA는 수정란에서부터 발현되어 포배기에는 유의할 정도로 증가하였다. 따라서 AQP8 mRNA는 배아유전자가 활성화되어 나타나는 것이고 AQP9 mRNA는 모계유전자 기원임을 알 수 있었다. AQP8 단백질은 상실배 단계까지 발현되지 않다가 포배시기에 영양외배엽세포사이의 접합면에 발현되었고 AQP9 단백질은 상실배 시기에 할구 사이의 인접 부위에서 강하게 발현되었다가 포배시기에는 세포간의 접합면에 약하게 발현하는 경향을 나타내었다. 실시간 역전사 중합효소 연쇄반응 방법으로 조사한 결과 포배에서 물과 글리세롤을 통과시키는 AQP9는 mRNA의 발현양이 AQP8보다 약 4배 정도 많았다. 또한 포배기에 이르러서야 물만을 통과시키는 AQP8의 발현이 나타나는 것을 보아 포배강 형성시 외부에서 영양외배엽을 통해 포배강으로 유입되는 물의 이동(trans- trophectodermal water movements)에 AQP9보다 AQP8이 더 중요하게 관여할 것으로 사료된다., K, Pb, Cd, Cr, Co, Cu, Ni)을 측정하였다. 실험 조건1의 결과로서 각 국의 유아용 일회용 기저귀의 중금속 함량은 거의 유사한 경향을 나타내었으며 Cr, Zn, Pb, Ni, Mn, Mg, Li, K는 detection limit(2 ppm) 이하였고, Cd, Fe, Co, Cu, Ca, Al, Sr는 검출되었지만 기준치 이하였다. 실험 조건2의 결과로서 측정 항목(Cr, Sb, Cd, Pb, Ni, Co, Cu)중 Cr, Cd, Ni, Cu는 detection limit(0.1 ppm) 이하였고, Sb, Pb, Co는 검출되었지만 기준치 이하였다.았다. 4%의 경우에는 8$0^{\circ}C$이하로 온도를 낮추는 것이 좋은 상태를 나타내었다. 이와 같은 결과는 일반적으로 화학적 레팅을 4%, 7%에서한 선행결과와 상당히 다른 결과이다.염 농도가 증가할수록 감소 현상을 보였다.X>, 75BG30은 8.6$\mu\textrm{m}$, 75BG40은 7.02$\mu\textrm{m}$로 나타났다. 따라서 경화제 양에 관계없이 10$\mu\textrm{m}$ 이하로 나타나, 경화제 10$m\ell$만으로 미세한 크기를 얻을 수 있음을 알 수 있다. 젤리 강도 변화에 따른 차이는 300BF는 78.09$\mu\textrm{m}$ 300BG는 56.32$\mu\textrm{m}$로, 75BF나 75BG에 비하여 현저히 증가하여, 젤라틴의 젤리 강도는 캡슐 제조 조건의 주요한 변수임을 알 수 있다.추출물 투여시 혈당강하 및 혈중콜레스테롤 강하가 나타났으며, 상엽복합추출물 투여와 운동을 병행시 이러한 감소 효과가 더 뚜렷하게 나타났다.교육의 적임자로 보는 시각이 비교적 높았고 약 1/2정도는 영양교육에 참여하겠다는 의지를 가지고 있을 뿐만 아니라 실제로 영양지도를

  • PDF

Supraclavicular BCG Lymphadenitis Noted at 21 Months after BCG Vaccination Confirmed by a Molecular Method (분자생물학적 방법으로 확진한 접종 21개월 후에 발생한 BCG 쇄골상부 림프절염 1례)

  • Lee, Min Hyun;Chae, Moon-Hee;Park, Kyoung Un;Cho, Hye-Kyung
    • Pediatric Infection and Vaccine
    • /
    • v.21 no.2
    • /
    • pp.139-143
    • /
    • 2014
  • Bacille Calmette-$Gu\acute{e}rin$ (BCG) lymphadenitis is the most common complication of BCG vaccination. It commonly occurs in infants aged <6 months involving ipsilateral axillary lymph nodes. We described BCG lymphadenitis in a 22-month-old boy presenting swelling of left supraclavicular lymph node that was confirmed by real-time polymerase chain reaction (PCR) and the multiplex PCR targeting the region of difference (RD).