• 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.

• The Plant Pathology Journal
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• v.31 no.1
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• pp.25-32
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• 2015

Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens.

• 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.

• Korean Journal of Veterinary Research
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• v.42 no.4
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• pp.513-522
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• 2002

Antigenic ompC genes of S. gallinarum, S. pullorum and S. dublin were characterized among Salmonella spp. isolated from chickens and other animals to identify genetic variation. Salmonella ompC gene fragment (1,027 bp) was amplified by PCR and the amplicons were cloned for comparison of nucleotide sequences. The identity of the sequences between S. gallinarum and S. pullorum, S. gallinarum and S. dublin, S. pullorum and S. dublin was 99.8%, 97.6% and 97.8%, respectively. Also, we found that ompC has some diversity between S. gallinarum and S. pullorum, and other Salmonella spp. which may be useful to type the organisms. Similar to diagnosis in other organisms, the TaqMan PCR method can be applied to rapid and accurate diagnosis of salmonellosis in chickens and other animals. We designed PCR primers and TaqMan probe for flagellin gene (fliC) for detection of Salmonella spp. by TaqMan PCR. The TaqMan PCR method was 10,000 times more sensitive than conventional PCR.

• 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.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.1004-1007
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• 2014

Culture is the gold standard for diagnosis of tuberculosis, but it takes 6 to 8 weeks to confirm the result. This issue is complemented by the detection method using polymerase chain reaction, which is now widely used in a routine microbiology laboratory. In this study, we evaluated the performance of the Seegene Anyplex TB PCR to assess its diagnostic sensitivity and specificity, and compared its results with the Roche Cobas TaqMan MTB PCR, one of the most widely used assays in the world. Five university hospitals located in the Chungcheong area in South Korea participated in the study. A total of 1,167 respiratory specimens ordered for acid-fast bacilli staining and culture were collected for four months, analyzed via the Seegene Anyplex TB PCR, and its results were compared with the Roche Cobas TaqMan MTB PCR. For detection of Mycobacterium tuberculosis, the diagnostic sensitivity and specificity of the Anyplex TB PCR were 87.5% and 98.2% respectively, whereas those of the Cobas TaqMan were 92.0% and 98.0% respectively (p value > 0.05). For smear-positive specimens, the sensitivity of the Anyplex TB PCR was 95.2%, which was exactly the same as that of the Cobas TaqMan. For smear-negative specimens, the sensitivity of the Anyplex TB PCR was 69.2%, whereas that of the Cobas TaqMan TB PCR was 84.6%. For detection of MTB, the Seegene Anyplex TB PCR showed excellent diagnostic performance, and high sensitivity and specificity, which were comparable to the Roche Cobas TaqMan MTB PCR. In conclusion, the Anyplex TB PCR can be a useful diagnostic tool for the early detection of tuberculosis in clinical laboratories.

• 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.

• Korean Journal of Clinical Laboratory Science
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• v.51 no.1
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• pp.64-70
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• 2019

Dirofilaria immitis (D. immitis) is a filarial nematode that causes cardiopulmonary dirofilariasis in dogs. In the late stages of infection, infected dogs show one or more symptoms and advanced heart disorder with perivascular inflammation. To detect D. immitis specifically and efficiently in the early stages of infection, a duplex TaqMan qPCR assay was developed based on previous studies using primers and probes specialized to detect D. immitis cytochrome c oxidase subunit I (COI) and dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH). As positive controls, plasmid DNAs were constructed from D. immitis COI or dog GAPDH and a TA-cloning vector. Simplex and duplex TaqMan qPCR assays were performed using the specific primers, probes, and genomic or plasmid DNA. The duplex reaction developed could detect D. immitis COI and dog GAPDH in the same sample simultaneously after optimization of the primer concentrations. The limit of detection was 25 copies for the simplex and duplex assays, and both showed good linearity, high sensitivity, and excellent PCR efficiency. The duplex assays for pathogen detection reduce the costs, labor, and time compared to simplex reactions. Therefore, the duplex TaqMan qPCR assay developed herein will allow efficient D. immitis detection and quantification from a large number of samples simultaneously.

• 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.

• 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 (LOD_95%) 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.