• Title, Summary, Keyword: real-time PCR

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Rapid detection and quantification of porcine circovirus type 2 (PCV 2) DNA in Real-time PCR (Real-time PCR을 이용한 돼지써코바이러스 감염증 진단법 연구)

  • Kim, Eun-Gyeong;Hwang, Bo-Won;Lee, Jong-Min;Son, Byeong-Guk;Park, Ho-Jung;Kim, Tho-Kyoung
    • Korean Journal of Veterinary Service
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    • v.32 no.4
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    • pp.299-306
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    • 2009
  • Assay for the detection and quantification of porcine circovirus type 2 (PCV 2) with the real-time PCR were developed. TaqMan probe real-time using a set of primer/probe was developed for detection of PCV 2. In this study we applied real-time PCR assay to 320 samples, collected from pig farms. In 151 of 320 samples, PCV 2 DNA was detected by conventional PCR assay. All samples positive for PCV 2 DNA in conventional PCR assay were also positive in Real-time PCR assay, but 69 of 169 samples that tested negative for PCV 2 DNA in conventional assay were tested positive in TaqMan probe real-time PCR assay. The test of TaqMan probe real-time PCR resulted in detection and quantification limits of 101 copies per sample. TaqMan probe real-time PCR assay increased the number of samples in which PCV 2 was detected by 21%. TaqMan probe real-time PCR assay is very efficient method in contrast to the conventinal PCR, becoming increasingly important method for gene analysis.

Real-time Reverse Transcription Polymerase Chain Reaction Using Total RNA Extracted from Nasopharyngeal Aspirates for Detection of Pneumococcal Carriage in Children (소아에서 폐렴구균 집락률 측정을 위해 비인두 흡인 물의 총 RNA를 이용한 실시간 중합효소 연쇄반응법)

  • Kim, Young Kwang;Lee, Kyoung Hoon;Yun, Ki Wook;Lee, Mi Kyung;Lim, In Seok
    • Pediatric Infection and Vaccine
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    • v.23 no.3
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    • pp.194-201
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    • 2016
  • Purpose: Monitoring pneumococcal carriage rates is important. We developed and evaluated the accuracy of a real-time reverse transcription polymerase chain reaction (RT-PCR) protocol for the detection of Streptococcus pneumoniae. Methods: In October 2014, 157 nasopharyngeal aspirates were collected from patients aged <18 years admitted to Chung-Ang University Hospital. We developed and evaluated a real-time PCR method for detecting S. pneumoniae by comparing culture findings with the results of the real-time PCR using genomic DNA (gDNA). Of 157 samples, 20 specimens were analyzed in order to compare the results of cultures, real-time PCR, and real-time RT-PCR. Results: The concordance rate between culture findings and the results of real-time PCR was 0.922 (P<0.01, Fisher exact test). The 133 culture-negative samples were confirmed to be negative for S. pneumoniae using real-time PCR. Of the remaining 24 culture-positive samples, 21 were identified as S. pneumonia -positive using real-time PCR. The results of real-time RT-PCR and real-time PCR from 20 specimens were consistent with culture findings for all S. pneumoniae -positive samples except one. Culture and real-time RT-PCR required 26.5 and 4.5 hours to perform, respectively. Conclusions: This study established a real-time RT-PCR method for the detection of pneumococcal carriage in the nasopharynx. Real-time RT-PCR is an accurate, convenient, and time-saving method; therefore, it may be useful for collecting epidemiologic data regarding pneumococcal carriage in children.

Detection of Mycobacterium leprae by Real-time PCR Targeting Mycobacterium leprae-Specific Repetitive Element Sequence

  • Jin, Hyun-Woo;Wang, Hye-Young;Kim, Jong-Pill;Cho, Sang-Nae;Lee, Hye-Young
    • Biomedical Science Letters
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    • v.16 no.2
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    • pp.127-131
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    • 2010
  • Mycobacterium leprae detection is difficult even with molecular biological techniques due to the low sensitivity of current methodologies. In this report, real-time PCR targeting the M. leprae-specific repetitive element (RLEP) sequence was developed as a new diagnostic tool and evaluated using clinical specimens. For this, M. leprae DNAs were extracted from skin biopsy specimens from 80 patients and analyzed by real-time PCR using TaqMan probe. Then, the detection efficiency of the real-time PCR was compared with that of standard PCR. In brief, the rate of positive detection by the standard PCR and real-time PCR was 32.50% and 66.25%, respectively. The results seemed to clearly show that the TaqMan real-time PCR developed in this study may be a useful tool for sensitive detection of M. leprae from clinical specimens.

Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR for the Rapid Detection of Salmonella Typhimurium, Listeria monocytogenes and Cronobacter sakazakii Artificially Inoculated in Foods (식품에 인위접종된 Salmonella Typhimurium, Listeria monocytogenes, Cronobacter sakazakii의 신속검출을 위한 Real-time PCR과 Loop-mediated isothermal amplification 비교)

  • Kim, Jin-Hee;Oh, Se-Wook
    • Journal of Food Hygiene and Safety
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    • v.34 no.2
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    • pp.135-139
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    • 2019
  • The objective of this research was to compare loop-mediated isothermal amplification (LAMP) with real-time polymerase chain reaction (PCR) for the rapid detection of pathogens in foods. In this study, the limits of detection (LODs) for Salmonella Typhimurium, Listeria monocytogenes, and Cronobacter sakazakii were evaluated in various foods. Among 11 samples tested for S. Typhimurium, LAMP and real-time PCR had the same LODs in beef and duck meat whereas real-time PCR was more sensitive than the LAMP in 8 samples. However, S. Typhimurium in chocolate samples was not detected by real-time PCR. The sensitivity of real-time PCR was high in all samples inoculated with L. monocytogenes and C. sakazakii whereas LAMP was more sensitive than real-time PCR in oil-rich foods. Therefore, LAMP can be shown as an easrer, more convenient method, as well as effective analytical method for testing difficult samples when employed in PCR.

Comparison of Real-Time PCR and Conventional Culture Method for Detection of Cronobacter spp. in Powdered Foods (분말식품에서 Cronobacter spp. 검출을 위한 Real-Time PCR과 배지배양법의 비교검증)

  • Chon, Jung-Whan;Song, Kwang-Young;Kim, Sun-Young;Hyeon, Ji-Yeon;Kim, Yun-Gyeong;Hwang, In-Gyun;Kwak, Hyo-Sun;Seo, Kun-Ho
    • Korean Journal of Microbiology
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    • v.47 no.1
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    • pp.87-91
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    • 2011
  • The aim of this study was to compare the performance of conventional culture and real-time PCR for detection of Cronobacter spp. in powdered foods. Infant formula, baby food and Misugaru inoculated with Cronobacter were enriched in distilled water as first enrichment step, followed by incubating in Enterobacteriaceae enrichment (EE) broth as second enrichment step. A loopful of enriched sample was streaked onto Druggan-Forsythe-Iversen agar, followed by incubating at $37^{\circ}C$ for 24 h. One milliliter of the enriched distilled water and EE broth were used in real-time PCR assay. No statistical differences were observed in the number of positive samples between culture method and real-time PCR (p>0.05) in all types of food samples. The number of positives of real-time PCR was higher in the first enrichment media (distilled water) than the second enrichment media (EE broth), though there was no significant difference (p>0.05). It appears that some components of the second enrichment broth, EE broth, inhibit the reaction of real-time PCR. These results show that real-time PCR using a single enrichment with distilled water could be useful as an effective screening method for detection of Cronobacter while saving much time and labor compared to conventional culture method.

Rapid Identification of Vibrio vulnificus in Seawater by Real-Time Quantitative TaqMan PCR

  • Wang, Hye-Young;Lee, Geon-Hyoung
    • Journal of Microbiology
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    • v.41 no.4
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    • pp.320-326
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    • 2003
  • In order to identify Vibrio vulnificus in the Yellow Sea near Gunsan, Korea during the early and late summers, the efficiency of the real-time quantitative TaqMan PCR was compared to the efficiency of the conventional PCR and Biolog identification system^TM. Primers and a probe were designed from the hemolysin/cytolysin gene sequence of V. vulnificus strains. The number of positive detections by real-time quantitative TaqMan PCR, conventional PCR, and the Biolog identification system from seawater were 53 (36.8%), 36 (25%), and 10 strains (6.9%), respectively, among 144 samples collected from Yellow Sea near Gunsan, Korea. Thus, the detection method of the real-time quantitative TaqMan PCR assay was more effective in terms of accuracy than that of the conventional PCR and Biolog system. Therefore, our results showed that the real-time TaqMan probe and the primer set developed in this study can be applied successfully as a rapid screening tool for the detection of V. vulnificus.

Quantitative Analysis of Feline Calicivirus Inactivation using Real-time RT-PCR (Real-time RT-PCR을 이용한 Feline Calicivirus 불활성화의 정량적 분석)

  • Jeong, Hye Mi;Kim, Kwang Yup
    • Journal of Food Hygiene and Safety
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    • v.29 no.1
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    • pp.31-39
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    • 2014
  • Norovirus causes acute gastroenteritis in all age groups and its food poisoning outbreaks are rapidly increasing in Korea. Reverse transcription-polymerase chain reaction (RT-PCR) is most widely used for the rapid detection of foodborne viruses due to high sensitivity. However, the false positive results of RT-PCR obtained against already inactivated viruses could be a serious drawbacks in food safety area. In this study, we investigated a method to yield true positive RT-PCR results only with alive viruses. To decompose the RNA genes from dead viruses, the enzymatic treatments composed of proteinse K and Ribonuclease A were applied to the sanitized and inactivated virus particles. Another aim of this study was to quantify the efficiencies of several major sanitizing treatments using real-time RT-PCR. Feline calicivirus (FCV) that belongs to the same Caliciviridae family with norovirus was used as a surrogate model for norovirus. The initial level of virus in control suspension was approximately $10^4$ PFU/mL. Most of inactivated viruses treated with the enzymatic treatment for 30 min at $37^{\circ}C$ were not detected in RT-PCR, Quantification results to verify the inactivation efficiencies of sanitizing treatments using real-time RT-PCR showed no false positive in most cases. We could successfully develope a numerical quantification process for the inactivated viruses after major sanitizing treatments using real-time RT-PCR. The results obtained in this study could provide a novel basis of rapid virus quantification in food safety area.

Rapid detection and Quantification of Fish Killing Dinoflagellate Cochlodinium polykrikoides (Dinophyceae) in Environmental Samples Using Real-time PCR

  • Park, Tae-Gyu;Kang, Yang-Soon;Seo, Mi-Kyung;Kim, Chang-Hoon;Park, Young-Tae
    • Fisheries and aquatic sciences
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    • v.11 no.4
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    • pp.205-208
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    • 2008
  • The mixotrophic dinoflagellate Cochlodinium polykrikoides was reported to be linked to major fish kills in Korea and Japan since the 1990s. Rapid and sensitive detection of microalgae has been problematic because morphological identification of dinoflagellates requires light microscopic and scanning electron microscopic observations that are time consuming and laborious compared to real-time PCR. To address this issue, a real-time PCR probe targeting the ITS2 rRNA gene was used for rapid detection and quantification of C. polykrikoides. PCR inhibitors in water column samples were removed by dilution of template DNA for elimination of false-negative reactions. A strong association between cell quantification using real-time PCR and microscopic counts suggests that the real-time PCR assay is an alternative method for cell estimation of C. polykrikoides in environment samples.

Detection of Anthracnose Fungus Colletotrichum circinans by Conventional PCR and Real-time PCR (일반 PCR과 Real-time PCR을 이용한 탄저병균 Colletotrichum circinans 검출)

  • Kim, Jun Young
    • The Korean Journal of Mycology
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    • v.46 no.4
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    • pp.467-477
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    • 2018
  • Colletotrichum circinans, an anthracnose pathogen, causes serious damage to onions worldwide. In this study, specific molecular markers were developed to detect C. circinans accurately and quickly with both conventional and real-time PCR methods. The cirTef-F/cirTef-R and cirTu-F/cirTu-R primer sets, which are specific for C. circinans, were constructed by analyzing $tef-1{\alpha}$ and ${\beta}-tubulin$ genes in the fungus. Using the conventional PCR method, 100 pg and 1 ng of fungal DNA could be detected using the cirTef-F/cirTef-R and cirTu-F/cirTu-R sets, respectively. Using the real-time PCR method, 10 pg and 100 pg of fungal DNA could be detected more sensitively with the cirTef-F/cirTef-R and cirTu-F/cirTu-R sets, respectively. Detection of C. circinans from the artificially infected onion seeds was possible by using both conventional and real-time PCR methods and the developed cirTef-F/cirTef-R primer set. The PCR markers specific for C. circinans developed in this study may enhance the efficiency of fungal pathogen detection in imported vegetables and seeds.

Detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus using duplex real-time PCR assay with melting curve analysis on fresh lettuce

  • Lee, Na-Ri;Kwon, Kyung-Yoon;Choi, Sung-Wook;Koo, Min-Seon;Chun, Hyang-Sook
    • Journal of Food Hygiene and Safety
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    • v.26 no.2
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    • pp.114-119
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    • 2011
  • In this study, two duplex real-time PCR approach with melting curve analysis is presented for the detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus, which are important food-borne bacterial pathogens usually present in fresh and/or minimally processed vegetables. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the ${\beta}$-glucuronidase (uidA, E. coli), thermonuclease (nuc, S. aureus), hemolycin (hly, L. monocytogenes) and tetrathionate reductase (ttr, Salmonella spp.) genes. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic $T_m$ values demonstrating the specific and efficient amplification of the four pathogens; $80.6{\pm}0.9^{\circ}C$, $86.9{\pm}0.5^{\circ}C$, $80.4{\pm}0.6^{\circ}C$ and $88.1{\pm}0.11^{\circ}C$ for S. aureus, E. coli O157:H7, L. monocytogenes and Salmonella spp., respectively. For all the pathogens, the two duplex, real-time PCR was equally sensitive to uniplex real-time PCR, using same amounts of purified DNA, and allowed detection of 10 genome equivalents. When our established duplex real-time PCR assay was applied to artificially inoculated fresh lettuce, the detection limit was $10^3$ CFU/g for each of these pathogens without enrichment. The results from this study showed that the developed duplex real-time PCR with melting curve analysis is promising as a rapid and cost-effective test method for improving food safety.