• Title/Summary/Keyword: real-time TaqMan PCR

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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.

Development of Real-time Quantitative PCR Assay based on SYBR Green I and TaqMan Probe for Detection of Apple Viruses (사과 바이러스 검정을 위한 SYBR Green I 및 TaqMan probe 기반의 real-time PCR 검사법 개발)

  • Heo, Seong;Chung, Yong Suk
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.65 no.4
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    • pp.496-507
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    • 2020
  • Virus infections of apples result in lowered commercial qualities such as low sugar content, weakened tree vigor, and malformed fruits. An effective way to control viruses is to produce virus-free plants based on the development of an accurate and sensitive diagnostic method. In this study, real-time PCR assays based on SYBR Green I and TaqMan probes were developed for detecting ASGV, ASPV, and ApMV viruses. These methods can detect and quantify 103 to 1011 RNA copies/μL of each virus separately. Compared with methods with two different dyes, the SYBR Green I-based method was efficient for virus detection as well as for assay using the TaqMan probe. Field tests demonstrated that real-time PCR methods developed in this study were applicable to high-throughput diagnoses for virus research and plant quarantine.

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.

TaqMan Probe Real-Time PCR for Quantitative Detection of Mycoplasma during Manufacture of Biologics (생물의약품 제조공정에서 마이코플라스마 정량 검출을 위한 TaqMan Probe Real-Time PCR)

  • Lee, Jae Il;Kim, In Seop
    • KSBB Journal
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    • v.29 no.5
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    • pp.361-371
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    • 2014
  • Mycoplasma is well recognized as one of the most prevalent and serious microbial contaminants of biologic manufacturing processes. Conventional methods for mycoplasma testing, direct culture method and indirect indicator cell culture method, are lengthy, costly and less sensitive to noncultivable species. In this report, we describe a new TaqMan probe-based real-time PCR method for rapid and quantitative detection of mycoplasma contamination during manufacture of biologics. Universal mycoplasma primers were used for mycoplasma PCR and mycoplasma DNA was quantified by use of a specific TaqMan probe. Specificity, sensitivity, and robustness of the real-time PCR method was validated according to the European Pharmacopoeia. The validation results met required criteria to justify its use as a replacement for the culture method. The established real-time PCR assay was successfully applied to the detection of mycoplasma from human keratinocyte and mesenchymal stem cell as well as Vero cell lines artificially infected with mycoplasma. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of mycoplasma contamination during manufacture of biologics.

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.

Evaluation of a novel TaqMan probe-based real-time polymerase chain reaction (PCR) assay for detection and quantitation of red sea bream iridovirus

  • Kim, Guk Hyun;Kim, Min Jae;Choi, Hee Ju;Koo, Min Ji;Kim, Min Jeong;Min, Joon Gyu;Kim, Kwang Il
    • Fisheries and Aquatic Sciences
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    • v.24 no.11
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    • pp.351-359
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    • 2021
  • The red sea bream iridovirus (RSIV) belonging to genus Megalocytivirus is responsible for red sea bream iridoviral disease (RSIVD) in marine and freshwater fishes. Although several diagnostic assays for RSIV have been developed, diagnostic sensitivity (DSe) and specificity (DSp) of real-time polymerase chain reaction (PCR) assays are not yet evaluated. In this study, we developed a TaqMan probe-based real-time PCR method and evaluated its DSe and DSp. To detect RSIV, the probe and primers were designed based on consensus sequences of the major capsid protein (MCP) genes from megalocytiviruses including RSIV, infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primers were shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A 95% limit of detection (LOD95%) was determined to be 5.3 viral genome copies/µL of plasmid DNA containing the MCP gene from RSIV. The DSe and DSp of the developed real-time PCR assay for field samples (n = 112) were compared with those of conventional PCR assays and found to be 100% and 95.2%, respectively. The quantitative results for SYBR Green and TaqMan probe-based real-time PCR were not significantly different. The TaqMan probe-based real-time PCR assay for RSIV may be used as an appropriate diagnostic tool for qualitative and quantitative analysis.

Identification of Hanwoo and Holstein meat using MGB probe based real-time PCR associated with single nucleotide polymorphism (SNP) in Melanocortin 1 receptor (MC1R) gene (소 모색관련 MC1R 유전자의 SNP와 관련한 MGB probe에 기초한 real-time PCR을 이용한 한우육과 Holstein육의 판별)

  • Park, Sung-Do;Kim, Tae-Jung;Lee, Jae-Il
    • Korean Journal of Veterinary Research
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    • v.45 no.1
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    • pp.25-28
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    • 2005
  • The melanocortin 1 receptor (MC1R) plays an important role in regulation of melanin pigment synthesis within mammalian melanocytes. Mutations within the gene encoding MC1R have been shown to explain coat color variations within several mammalian species including cattle. To develope a rapid and accurate method for the identification of Hanwoo meat, we performed a single nucleotide polymorphism (SNP) analysis in Melanocortin 1 receptor (MC1R) gene using TaqMan$^{(R)}$ MGB probe-based real-time PCR. Two specific probes (one for Hanwoo and the other for Holstein and Black angus) were designed. At the 5' end of 2 TaqMan$^{(R)}$ MGB probes, 6-carboxyfluorescein (FAM) was labeled for Hanwoo, and VIC for Holstein and Black angus. As a result, Hanwoo samples showed FAM-positive signal only, whereas other samples showed VIC-positive. This result suggests that the TaqMan$^{(R)}$ MGB probe based real-time PCR technique would be very accurate, easy and reproducible method to discriminate between Hanwoo meat and Holstein/Black angus meat.

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
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    • v.48 no.2
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    • pp.88-93
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    • 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.

TaqMan probe real-time PCR for quantitative detection of bovine adenovirus type 1 during the manufacture of biologics and medical devices using bovine-derived raw materials (소유래 성분 원재료 사용 생물의약품과 의료기기 제조 공정에서 bovine adenovirus type 1 정량 검출을 위한 TaqMan probe real-time PCR)

  • Ko, Woon Young;Noh, Na Gyeong;Kim, In Seop
    • Korean Journal of Microbiology
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    • v.51 no.3
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    • pp.199-208
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    • 2015
  • Biologics and medical devices manufactured with bovine-derived raw materials have the risk of viral contamination. Therefore, viral validation study is essential to ensure the safety of the products. Bovine adenovirus type-1 (BAdV-1) is one of the common bovine viral pathogens. For quantitative detection of BAdV-1 during the manufacture of biologics and medical devices, a TaqMan probe real-time PCR method was developed. Specific primers and TaqMan probe for amplifying and detecting BAdV-1 DNA were designed. Specificity, limit of detection (LOD), and robustness of the method was validated according to international guideline on the validation of nucleic acid amplification tests for the pathogen detection. The sensitivity of the assay was found to be $7.44{\times}10^1\;TCID_{50}/ml$. The real-time PCR method was reproducible, very specific to BAdV-1, and robust. Moreover, the method was successfully applied to the validation of Chinese Hamster Ovary (CHO)-K1 cells artificially infected with BAdV-1, a commercial CHO master bank, and bovine type 1 collagen. The overall results indicate that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BAdV-1 contamination during the manufacture of biologics and medical devices using bovine-derived raw materials.

Development of TaqMan Probe Real-Time RT-PCR for Quantitative Detection of Porcine Transmissible Gastroenteritis Virus During the Manufacture of Biopharmaceuticals (생물의약품 제조 공정에서 Porcine transmissible gastroenteritis virus 정량 검출을 위한 TaqMan Probe Real-Time RT-PCR 개발)

  • Lee, Jae Il;Han, Sang Eun;Kim, In Seop
    • Microbiology and Biotechnology Letters
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    • v.43 no.3
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    • pp.267-274
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    • 2015
  • Biopharmaceuticals and the cell substrates used for their manufacture are currently tested for porcine adventitious viruses due to the widespread use of porcine trypsin in cell culture. Porcine transmissible gastroenteritis virus (PTGV) is one of the major adventitious porcine viruses causing contaminated during the manufacture of biopharmaceuticals. Therefore, rapid and sensitive detection of PTGV is essential in ensuring the safety of biopharmaceuticals. A TaqMan probe real-time RT-PCR method was developed for the quantitative detection of PTGV contamination in cell substrates, raw materials, manufacturing processes, and final products, as well as PTGV clearance validation. Specific primers for the amplification of PTGV RNA were selected, and PTGV RNA was quantified by use of a specific TaqMan probe. Specificity, limit of detection (LOD), and robustness of the method was validated according to international guidelines on the validation of nucleic acid amplification tests. The sensitivity of the assay was calculated to be 1.10 × 100 TCID50/ml. The real-time RT-PCR method was validated to be reproducible, very specific to PTGV, and robust. The established real-time RT-PCR assay was successfully applied to the validation of Chinese Hamster Ovary (CHO)-K1 cells artificially infected with PTGV.