• Food Science and Biotechnology
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• 제18권5호
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• pp.1118-1123
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• 2009

Event-specific real-time polymerase chain reaction (PCR) detection method for genetically modified (GM) maize MIR604 was developed based on integration junction sequences between the host plant genome and the integrated transgene. In this study, 2 primer pairs and probes were designed for specific amplification of 100 and 111 bp DNA fragments from the zSSIIb gene (the maize endogenous reference gene) and MIR604. The quantitative method was validated using 3 certified reference materials (CRMs) with levels of 0.1, 1, and 10% MIR604. The method was also assayed with 14 different plants and other GM maize. No amplification signal was observed in real-time PCR assays with any of the species tested other than MIR604 maize. As a result, the bias from the true value and the relative deviation for MIR604 was within the range from 0 to 9%. Precision, expressed as relative standard deviation (RSD), varied from 2.7 to 10% for MIR604. Limits of detections (LODs) of qualitative and quantitative methods were all 0.1%. These results indicated that the event-specific quantitative PCR detection system for MIR604 is accurate and useful.

• Journal of Veterinary Science
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• 제22권3호
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• pp.40.1-40.12
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• 2021

Background: The microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen. Objectives: The present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees. Methods: A procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration. Results: UR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min. Conclusions: UR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.

• 한국미생물·생명공학회지
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• 제36권3호
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• pp.173-181
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• 2008

소의 혈액, 세포, 조직, 기관 등은 생물의약품과 조직공학제제, 세포치료제의 원료로 널리 사용되고 있다. 소유래 물질을 원료로 사용한 제제의 경우 소유래 원료 물질에 다양한 바이러스가 오염된 사례가 있기 때문에 바이러스 안전성 검증이 필수적이다. BPV는 소에게 가장 흔하게 감염되는 바이러스 중의 하나이다. 소유래 물질을 원료로 하는 생물의약품, 조직공학제제, 세포치료제 등에서 BPV 안전성을 확보하기 위해, 원료물질, 제조공정, 완제품에서 BPV를 정량적으로 검출하고, 제조공정에서 BPV 제거 검증을 위한 시험법으로 활용이 가능한 BPV real-time PCR 시험법을 확립하였다. BPV에 특이적인 primer를 선별하였으며, 형광염료 SYBR Green I을 사용하여 BPV DNA 정량 검출 시험법을 최적화하였다. 세포배양법에 의한 감염역가와 비교한 결과 real-time PCR 민감도는 $1.3{\times}10^{-1}\;TCID_{50}/mL$이었다. 확립된 시험법의 신뢰성(reliability)을 보증하기 위해 시험법 검증을 실시한 결과 특이성(specificity)과 재현성 (reproducibility)이 우수함을 확인하였다. 확립된 real-time PCR을 생물의약품 제조공정 검증에 적용할 수 있는지 확인하기 위하여 인위적으로 BPV를 오염시킨 CHO 세포주와 소유래 콜라겐에서 BPV 검출 시험을 실시하였다. BPV를 감염시킨 CHO 세포에서 세포변병효과를 관찰할 수 없었지만, 세포와 세포배양 상청액에서 BPV를 정량적으로 검출할 수 있었다. 소유래 콜라겐에서도 $1.3{\times}10^0\;TCID_{50}/mL$까지 정량적으로 검출할 수 있었다.

• 대한의생명과학회지
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• 제19권2호
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• pp.153-157
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• 2013

Although culture is the gold standard method to identify mycobacteria, its use in tuberculous lymphadenitis (TBL) is limited due to formalin fixation of the submitted specimens. We evaluated the performance of quantitative real-time PCR (q-PCR) for Mycobacterium Tuberculosis (MTB) in granulomatous lymphadenitis using formalin-fixed paraffin-embedded (FFPE) tissues. From 2000 to 2010, a total number of 117 cases of lymph node samples with granulomatous inflammation which were surgically removed and fixed in formalin were studied. Hematoxylin & Eosin (H&E) and Ziehl-Neelsen-stained (ZN) slides were reviewed. qPCR using Real TB-Taq$^{(R)}$ was performed for all cases to identify Mycobacterium tuberculosis. Thirteen non-tuberculous lymphadenopathy cases were used as negative control. Cervical lymph nodes were more frequently affected (60%, 70/117) than other sites. ZN stain for acid fast bacilli was positive in 19 (16.24%) cases. qPCR for tuberculosis was positive in 92 (78.63%) cases. Caseous necrosis was found in 103 (88.03%) cases. While the ZN stain and qPCR were both negative in all control cases, the qPCR showed a significantly higher positive rate (78.63% vs. 16.24%) compared to ZN stain in histologically diagnosed TBL. Quantitative real-time PCR proves to be more sensitive than ZN stain for diagnosis of tuberculous lymphadenitis.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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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.

• Fisheries and Aquatic Sciences
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• 제24권11호
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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.

• Parasites, Hosts and Diseases
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• 제50권2호
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• pp.103-111
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• 2012

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

• Genomics & Informatics
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• 제21권2호
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• pp.24.1-24.7
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• 2023

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.

• 미생물학회지
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• 제49권3호
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• pp.270-274
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• 2013

서양뒤영벌(Bombus terrestris)은 꿀벌의 봉군붕괴증후군(colony collapse disorder)에 대한 대체 화분매개곤충으로서 농업분야에서 중요한 역할을 하고 있다. 최근 서양뒤영벌에서 바이러스, 세균, 응애 등의 여러 병원체와 기생체가 발견되었고, 이들은 서양뒤영벌의 수명과 생식력 등에 영향을 주는 것이 알려져 있다. 서양뒤영벌 야외개체군에서 Nosema spp.를 탐지하기 위해, 서양뒤영벌 성충으로부터 genomic DNA를 추출하여 Nosema spp. 유전자들에 대해 polymerase chain reaction (PCR)을 수행하였다. 이들 유전자 중에서 small subunit ribosomal RNA (SSU rRNA) 유전자만이 증폭되었고, 염기서열분석을 통해 N. ceranae로 확인된 것은 조사된 야외개체군에서 N. ceranae가 서양뒤영벌의 주된 감염체임을 보여준다. Quantitative real-time PCR (qRT-PCR)을 이용하여 SSU rRNA 유전자를 탐지하기 위해, 먼저 PCR을 통해 SSU rRNA 유전자의 2개 영역에 대한 유전자 특이적 증폭을 확인하였다. qRT-PCR을 이용하여 각 개체에서 얻은 genomic DNA의 순차적인 농도희석를 통해 $0.85ng/{\mu}l$ 이하의 genomic DNA 농도에서도 SSU rRNA 유전자가 성공적으로 증폭되는 것이 확인되었다. 이러한 실험 결과, qRT-PCR를 이용한 N. ceranae 특이 유전자 증폭은 서양뒤영벌의 병원체 감염 진단 뿐만 아니라 생태계 위해성 평가에도 활용될 수 있을 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.569-573
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• 2010

A quantitative detection method for Salmonella in seafood was developed using a SYBR Green-based real-time PCR assay. The assay was developed using pure Salmonella DNA at different dilution levels [i.e., 1,000 to 2 genome equivalents (GE)]. The sensitivity of the real-time assay for Salmonella in seeded seafood samples was determined, and the minimum detection level was 20 CFU/g, whereas a detection level of 2 CFU/ml was obtained for pure culture in water with an efficiency of ${\geq}85%$. The real-time assay was evaluated in repeated experiments with seeded seafood samples and the regression coefficient ($R^2$) values were calculated. The performance of the real-time assay was further assessed with naturally contaminated seafood samples, where 4 out of 9 seafood samples tested positive for Salmonella and harbored cells <100 GE/g, which were not detected by direct plating on Salmonella Chromagar media. Thus, the method developed here will be useful for the rapid quantification of Salmonella in seafood, as the assay can be completed within 2-3 h. In addition, with the ability to detect a low number of Salmonella cells in seafood, this proposed method can be used to generate quantitative data on Salmonella in seafood, facilitating the implementation of control measures for Salmonella contamination in seafood at harvest and post-harvest levels.