• Title, Summary, Keyword: real-time PCR

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Ultra Fast Real-Time PCR for Detection of Babesia gibsoni as Point of Care Test

  • Yang, Yong-Sung;Mun, Myung-Jun;Yun, Young-Min
    • Journal of Veterinary Clinics
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    • v.37 no.1
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    • pp.23-27
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    • 2020
  • Between May and November 2018, babesiosis was examined in 162 bloods samples obtained to an animal hospital in Jeju island for anemia and medical examination. Sixty-two of 162 (38.3%) were positive by PCR. The ultra fast real-time PCR test with blood directly analyzed without DNA extraction showed the same results. Accurate diagnosis, treatment and prognosis of babesiosis should be combined with clinical symptoms, blood tests, the babesia antibody test, and the PCR antigen test. Ultra fast real-time PCR, with these tests, is expected to be a point-of-care testing (POCT) for easy, fast and accurate diagnosis of babesiosis in the veterinary clinic.

Evaluation of Various Real-Time Reverse Transcription Quantitative PCR Assays for Norovirus Detection

  • Yoo, Ju Eun;Lee, Cheonghoon;Park, SungJun;Ko, GwangPyo
    • Journal of Microbiology and Biotechnology
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    • v.27 no.4
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    • pp.816-824
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    • 2017
  • Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.

SYBR Green I-based Real-time PCR Assay and Melting Curve Analysis for Rapid Detection of Staphylococcus aureus from Raw Milks Samples (Real-time PCR을 이용한 원유시료 유래 황색포도상구균의 신속 검출)

  • Jung, Jae-Hyuk;Jeong, Soon-Young;Lee, Sang-Jin;Choi, Sung-Sook
    • Journal of Food Hygiene and Safety
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    • v.23 no.2
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    • pp.121-128
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    • 2008
  • The aim of this study was to develop a LightCycler-based real time PCR (LC-PCR) assay and to evaluate its diagnostic use for the detection of Staphylococcus aureus in raw milk samples. Following amplification of 113 bp of coa gene encoding an coagulase precursor specific for Staphylococcus aureus, melting curve and DNA sequencing analysis was performed to verify the specificity of the PCR products. Amplification of 209 bp gene encoding an altered penicillin-binding protein, PBP2a (mecA), melting curve analysis and DNA sequencing analysis was performed to verify methicillin resistance Staphylococcus aureus (MRSA). According to this study, 6 of 647 raw milk samples showed S. aureus positive and 2 of them showed a mecA positive and the detection limit was 10 fg of DNA. And we also isolated Staphylococcus chromogenes a causative agent of exudative epidermitis in pigs and cattle from 3 samples.

Rapid Detection and Monitoring Therapeutic Efficacy of Mycobacterium tuberculosis Complex Using a Novel Real-Time Assay

  • Jiang, Li Juan;Wu, Wen Juan;Wu, Hai;Ryang, Son Sik;Zhou, Jian;Wu, Wei;Li, Tao;Guo, Jian;Wang, Hong Hai;Lu, Shui Hua;Li, Yao
    • Journal of Microbiology and Biotechnology
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    • v.22 no.9
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    • pp.1301-1306
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    • 2012
  • We combined real-time RT-PCR and real-time PCR (R/P) assays using a hydrolysis probe to detect Mycobacterium tuberculosis complex (MTBC)-specific 16S rRNA and its rRNA gene (rDNA). The assay was applied to 28 non-respiratory and 207 respiratory specimens from 218 patients. Total nucleic acids (including RNA and DNA) were extracted from samples, and results were considered positive if the repeat RT-PCR threshold cycle was ${\leq}35$ and the ratio of real-time RT-PCR and real-time PCR load was ${\geq}1.51$. The results were compared with those from existing methods, including smear, culture, and real-time PCR. Following resolution of the discrepant results between R/P assay and culture, the overall sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) of all samples (including non-respiratory and respiratory specimens) were 98.2%, 97.2%, 91.7%, and 99.4%, respectively, for R/P assay, and 83.9%, 89.9%, 72.3%, and 94.7%, respectively, for real-time PCR. Furthermore, the R/P assay of four patient samples showed a higher ratio before treatment than after several days of treatment. We conclude that the R/P assay is a rapid and accurate method for direct detection of MTBC, which can distinguish viable and nonviable MTBC, and thus may guide patient therapy and public health decisions.

Quantitative analysis of a myxosporean parasite, Parvicapsula sp. detected from emaciated olive flounder, Paralichthys olivaceus in Korea (국내 여윔 넙치에서 검출된 점액포자충 Parvicapsula sp.의 정량적 분석)

  • Kim, Seung Min;Jeong, Joon Bum
    • Journal of fish pathology
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    • v.31 no.2
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    • pp.101-107
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    • 2018
  • Quantitative analysis of a myxosporean parasite, Parvicapsula sp. in internal organs (kidney, intestine, spleen, brain and liver) from non-emaciated (farm-A) or emaciated (farm-B and farm-C) olive flounder Paralichthys olivaceus were performed by real-time PCR. The highest DNA copy number ($1.7{\times}10^7copies/mg$ tissue) was detected in kidney of the emaciated olive flounder from farm-C, while the DNA copy number was below detection limit in all the organs of the olive flounder from farm-B. There was not positive result in all of organs from olive flounder in farm-A. PCR and histopathological analysis were also performed using the same specimen and showed same results as those by real-time PCR.

Comparison of Standard Culture Method and Real-time PCR Assay for Detection of Staphylococcus aureus in Processed and Unprocessed Foods (가공식품과 비가공식품에서의 황색포도상구균 검출을 위한 배지법과 Real-time PCR법의 비교)

  • Lee, Jae-Hoon;Song, Kwang-Young;Hyeon, Ji-Yeon;Hwang, In-Gyun;Kwak, Hyo-Sun;Han, Jeong-A;Chung, Yun-Hee;Seo, Kun-Ho
    • Food Science of Animal Resources
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    • v.30 no.3
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    • pp.410-418
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    • 2010
  • Staphylococcus aureus is one of the major pathogens that can cause staphylococcal infection and food poisoning. In this study, we compared conventional culture methods and real-time PCR for detection of S. aureus in artificially inoculated milk, sausage, raw pork, and vegetable salad. The performance of a coagulase test for confirming S. aureus was also compared with a colony PCR test. Bulk food samples (500 g each) were artificially inoculated with S. aureus and divided into 20 samples (25 g or mL each). All samples were added to tryptic soy broth (225 mL/sample) with 10% NaCl and incubated at $37^{\circ}C$ for 24 h. After the enrichment, broth cultures were streaked onto Baird-Parker (BP) agar with egg yolk tellulite, and incubated at $37^{\circ}C$ for 24 h. In addition, 1 mL of broth cultures was collected to perform real-time PCR. Two suspicious colonies from the BP agar were picked up and plated on nutrient agar and incubated at $37^{\circ}C$ for 24 h followed, by a coagulase confirmation test and a colony PCR analysis. There were no statistical differences between culture methods and realtime PCR in food samples with low background microflora, such as milk and sausage. However, a significant statistical difference was found between the culture methods and real-time PCR for raw pork and vegetable salad. Furthermore, the colony PCR test of the presumptive colonies on BP agar for confirming S. aureus is more accurate and efficient than the coagulase test for unprocessed foods.

Real-Time PCR for Quantitative Detection of Bovine Parvovirus during Manufacture of Biologics (생물의약품 제조공정에서 Bovine Parvovirus 정량 검출을 위한 Real-Time PCR)

  • Lee, Dong-Hyuck;Lee, Jung-Hee;Kim, Chan-Kyong;Kim, Tae-Eun;Bae, Jung-Eun;Kim, In-Seop
    • Microbiology and Biotechnology Letters
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    • v.36 no.3
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    • pp.173-181
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    • 2008
  • Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biologics such as biopharmaceuticals, tissue-engineered products, and cell therapy. Manufacturing processes for the biologics have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine parvovirus (BPV) is one of the common bovine pathogens and has widely been known as a possible contaminant of biologics. In order to establish the validation system for the BPV safety of biologics, a real-time PCR method was developed for quantitative detection of BPV contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BPV DNA were selected, and BPV DNA was quantified by use of SYBR Green 1. The sensitivity of the assay was calculated to be $1.3{\times}10^{-1}\;TCID_{50}/mL$. The real-time PCR method was validated to be reproducible and very specific to BPV. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BPV. BPV DNA could be quantified in CHO cell as well as culture supernatant. Also the real-time PCR assay could detect $1.3{\times}10^0\;TCID_{50}/mL$ of BPV artificially contaminated in bovine collagen. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BPV contamination during manufacture of biologics.

PNA-mediated Real-Time PCR Clamping for Detection of EGFR Mutations

  • Choi, Jae-Jin;Cho, Min-Hey;Oh, Mi-Ae;Kim, Hyun-Sun;Kil, Min-Seock;Park, Hee-Kyung
    • Bulletin of the Korean Chemical Society
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    • v.31 no.12
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    • pp.3525-3529
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    • 2010
  • Tyrosine kinase inhibitors (TKIs) are currently used in the treatment of patients with advanced lung cancer. Recent studies on non-small cell lung cancer have shown that some patients carry somatic mutations in the epidermal growth factor receptor (EGFR) gene. Such mutations correlate with the effectiveness of certain TKIs. To detect a small amount of mutant EGFR among an abundance of wild-type EGFR, we have developed a highly sensitive and simple method using PNA-mediated real-time PCR clamping. The PNA-mediated real-time PCR clamping enables detection of EGFR mutants down to approximately 1% mutant -to- wild type. The total assay time was short as it required only 2.0 hr. Thus, PNA-mediated real-time PCR clamping can easily be applied to clinical samples for identification of DNA carrying EGFR mutations and also appear to be the best assay to detect somatic mutations.

DNA Heteropolymorphism of Chum Salmon Detected by Denaturing Gradient Gel Electrophoresis and Real Time PCR (Denaturing gradient gel electrophoresis와 real time PCR 방법을 이용한 연어 유전자들의 DNA 이형 다양성 검색)

  • Ham Seung Hub;Lee Suk Keun;Han Hyon Sob;Jin Deuk Hee
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.35 no.5
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    • pp.490-496
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    • 2002
  • In order to detect the DNA heteropolymorphism of chum salmon, selected essential genes were examined in different regional chum salmons, i.e., Korean, Japanese and American by denaturing gradient gel electrophoresis (DGGE) and real time PCR methods. From the promoter regions and introns of growth hormone, mtDNA NDI region, D-loop region, IGF-I, histone H3 and MCH2 several representative primer pairs were obtained and employed for the DGGE with the PCR products from the genomic DNAs of the different regional chum salmons. mtDNA NDI, D-loop region and IGE-I genes showed marked heteropolymorphism between Korean and American chum salmons. Intron C of growth hormone also showed a heteropolymorphism between Korean and Japanese chum salmons. Whereas heteropolnnorphism of histone liH and MCH2 genes was detected among in Korean, Japanese and Asnerican chum salmons in the examined region. The real time PCR disclosed the characteristic incremental production of target DNAs dependent on the heteropolymorphic conditions of genomic DNAa of chum salmons, thus the different regional chum salmons could be grouped by the variable incremental curies. Although the DGGE and real time PCR did not produce the identical results in this study, we suggest that the DGGE and real time PCR could be used for the primary screening of the DNA heteropolymorphism of different animal genome.

Detection of infectious canine hepatitis virus by TaqMan real-time PCR method (TaqMan 실시간 PCR법에 의한 개 전염성 간염 바이러스의 검출)

  • Wang, Hye-young;Choi, Jae-yong;Lee, Mi-jin;Park, Jin-ho;Cho, Mae-Rim;Han, Jae-cheol;Choi, Kyoung-seong;Chae, Joon-seok
    • Korean Journal of Veterinary Research
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    • v.44 no.4
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    • pp.655-662
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    • 2004
  • The aim of this work was the validation of a rapid real-time PCR assay based on TaqMan technology for the unequivocal identification of infectious canine hepatitis (ICH) virus, to be used directly on DNA purified from blood specimens. A real-time PCR system targeting at the E3 ORFA gene sequence of canine adenovirus type 1 was optimized and validated through comparative analysis of samples using conventional PCR system. The real-time PCR assay based on TaqMan technology could disclose 23 (37.7%) out of 61 samples as PCR positive. In contrast, 18 (29.5%) samples were found PCR positive when conventional PCR was applied on these samples. The use of the ABI Prism 7700 sequence detection system allowed the efficient determination of the amplified product accumulation through a fluorogenic probe. The entire real-time TaqMan PCR assay, including DNA extraction, amplification, and detection could be completed within 3 hours. The detection method of real-time TaqMan PCR assay was 1,000 times more sensitive than conventional PCR. Real-time TaqMan probe and primer set developed and optimized in this study is a sensitive, rapid and accurate method for detection of ICH virus and can be effective screening tool for the detection of ICH in a diagnostic laboratory routines.