• 대한의생명과학회지
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• 제14권2호
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• pp.115-118
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• 2008

Salmonella typhi is frequent causes of foodborne illness and its detection is important for monitoring disease progression. In this study, by using general PCR and novel LAMP (Loop Mediated Isothermal Amplification) assay, we evaluated the usefulness of LAMP assay for detection of Salmonella typhi. In this LAMP assay, forward inner primer (FIP) and back inner primer (BIP) was specially designed for recognizing target invA gene. Target DNA was amplified and visualized as ladder-like pattern of bands on agarose gel within 60 min under isothermal conditions at $65^{\circ}C$. When the sensitivity and reproducibility of LAMP were compared to general PCR, there was no difference of reproducibility but sensitivity of LAMP assay was more efficient than PCR (the detection limit of LAMP assay was 30 fg, while the PCR assay was 3 pg). These results indicate that the LAMP assay is a potential and valuable means for detection of Salmonella typhi, especially for its rapidity, simplicity and low cost.

• 한국식품위생안전성학회지
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• 제34권3호
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• pp.290-295
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• 2019

최근 한국에서 발생한 Salmonella로 인한 식중독 사고는 2018년 9월 학교급식에서 제공된 초콜릿 무스 케이크가 원인이 되었다. 이 연구의 목적은 Salmonella Typhimurium이 인위적으로 접종된 무스케이크와 티라미수에서 3M Molecular Detection Assay 2 - Salmonella와 식품공전에 등재된 방법인 분리배지와 real-time PCR을 비교하는 것이었다. 무스케이크 2종과 티라미수 2종 25 g에 225 mL BPW를 넣고 $37^{\circ}C$에서 24시간 동안 증균 배양하였다. 배양 후, 3M Molecular Detection Assay 2 - Salmonella, 분리배지 그리고 real-time PCR로 분석하였다. 초콜릿 무스 케이크를 제외하고 3가지 방법은 유사한 결과를 보였다. 초콜릿 무스 케이크에서 분리배지와 3M Molecular Detection Assay 2 - Salmonella는 모든 접종수준에서 동일한 결과를 나타낸 반면 real-time PCR은 $10^4CFU/25g$ 수준에서 1번의 양성결과를 제외하고 모두 검출되지 않았다. 초콜릿 무스에 S. Typhimurium을 $10^2CFU/25g$ 수준으로 접종하였을때, real-time PCR를 이용한 검출은 15%에서는 부분적인 음성을 나타냈고, 20-100% 함량의 초콜릿 무스에서는 모두 음성이었다. Real-time PCR로는 chocolate이 15% 이상 함유된 식품에서의 Salmonella균 검출이 불가능하였지만, LMAP 기반의 3M Molecular Detection Assay 2으로는 chocolate 농도에 관계없이 검출이 가능하였다.

• 한국동물위생학회지
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• 제32권4호
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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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• 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.

• Journal of Microbiology and Biotechnology
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• 제20권10호
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• pp.1463-1470
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• 2010

E. coli has long been widely used as a host system for the manufacture of recombinant proteins intended for human therapeutic use. When considering the impurities to be eliminated during the downstream process, residual host cell DNA is a major safety concern. The presence of residual E. coli host cell DNA in the final products is typically determined using a conventional slot blot hybridization assay or total DNA Threshold assay. However, both the former and latter methods are time consuming, expensive, and relatively insensitive. This study thus attempted to develop a more sensitive real-time PCR assay for the specific detection of residual E. coli DNA. This novel method was then compared with the slot blot hybridization assay and total DNA Threshold assay in order to determine its effectiveness and overall capabilities. The novel approach involved the selection of a specific primer pair for amplification of the E. coli 16S rRNA gene in an effort to improve sensitivity, whereas the E. coli host cell DNA quantification took place through the use of SYBR Green I. The detection limit of the real-time PCR assay, under these optimized conditions, was calculated to be 0.042 pg genomic DNA, which was much higher than those of both the slot blot hybridization assay and total DNA Threshold assay, where the detection limits were 2.42 and 3.73 pg genomic DNA, respectively. Hence, the real-time PCR assay can be said to be more reproducible, more accurate, and more precise than either the slot blot hybridization assay or total DNA Threshold assay. The real-time PCR assay may thus be a promising new tool for the quantitative detection and clearance validation of residual E. coli host cell DNA during the manufacturingprocess for recombinant therapeutics.

• Parasites, Hosts and Diseases
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• 제54권1호
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• pp.1-8
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• 2016

Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, $RIDA^{(R)}$ Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.

• The Plant Pathology Journal
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• 제38권4호
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• pp.417-422
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• 2022

Apple stem grooving virus (ASGV) is a destructive viral pathogen of pome fruit trees that causes significant losses to fruit production worldwide. Obtaining ASGV-free propagation materials is essential to reduce economic losses, and accurate and sensitive detection methods to screen ASGV-free plantlets during in vitro propagation are urgently necessary. In this study, ASGV was sensitively and accurately quantified from in vitro propagated apple plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The optimized RT-ddPCR assay was specific to other apple viruses, and was at least 10-times more sensitive than RT-real-time quantitative PCR assay. Furthermore, the optimized RT-ddPCR assay was validated for the detection and quantification of ASGV using micropropagated apple plantlet samples. This RT-ddPCR assay can be utilized for the accurate quantitative detection of ASGV infection in ASGV-free certification programs, and can thus contribute to the production of ASGV-free apple trees.

• 식물병연구
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• 제13권3호
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• pp.145-151
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• 2007

Xanthomonas axonopodis pv. glycines에 의해 발병되는 콩 불마름병은 한국에서 콩에 가장 많이 발생하는 중요한 세균성 병해 중 하나이다. 본 연구에서는 Xanthomonas axonopodis pv. glycines를 종자에서 검출하기 위해 PCR기법을 이용하였으며, 한국의 36개 주요 콩 품종의 종자 오염을 조사하였다. 그리고 병원균 검출과 동정을 위한 PCR assay와 dilution plating assay를 비교하였다. PCR assay를 이용하여 인공접종에 의한 이병종자와 자연감염된 이병종자로부터 병원균 검출을 확인하였다. PCR assay를 통한 이런 결과는 dilution plating assay와 비슷한 결과를 보여 주었으며 종자에서 병원균을 검출하는 다른 전통적인 방법보다 더 효과적인 방법으로 증명되었다. 36개 주요 콩 품종의 X. axonopodis pv. glycines에 의한 종자 전염을 확인한 결과 풍산나물콩, 만리콩, 태광콩, 대망콩, 아주까리콩에서 병원균이 검출되었다. 그러므로 PCR assay는 콩 종자에서 신속하고, 민감하게 X. axonopodis pv. glycines 특이적으로 검출할 수 있는 효과적인 방법으로 활용될 수 있을 것이다.

• 한국동물위생학회지
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• 제46권2호
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• pp.123-135
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• 2023

Feline calicivirus (FCV) is considered the main viral pathogen of feline upper respiratory tract disease (URTD). The frequent mutations of field FCV strains result in the poor diagnostic sensitivity of previously developed molecular diagnostic assays. In this study, a more sensitive real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for broad detection of currently circulating FCVs and comparatively evaluated the diagnostic performance with previously developed qRT-PCR assay using clinical samples collected from Korean cat populations. The developed qRT-PCR assay specifically amplified the FCV p30 gene with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 2%. Based on the clinical evaluation using 94 clinical samples obtained from URTD-suspected cats, the detection rate of FCV by the developed qRT-PCR assay was 47.9%, which was higher than that of the previous qRT-PCR assay (43.6%). The prevalence of FCV determined by the new qRT-PCR assay in this study was much higher than those of previous Korean studies determined by conventional RT-PCR assays. Due to the high sensitivity, specificity, and accuracy, the new qRT-PCR assay developed in this study will serve as a promising tool for etiological and epidemiological studies of FCV circulating in Korea. Furthermore, the prevalence data obtained in this study will contribute to expanding knowledge about the epidemiology of FCV in Korea.

• Journal of Applied Biological Chemistry
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• 제49권4호
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• pp.143-147
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• 2006

A rapid PCR-based assay for detecting hepatitis B viral DNA(HBV DNA) in serum and plasma was developed using a new PCR instrument named GenSpector(TMC-1000, Samsung electronics). PCR was carried out using a chip-based platform, which enabled 50 PCR cycles with internal controls, and melting-curve analysis in 30 minutes. Verification of the amplified HBV DNA product and the internal control was based on specific melting temperatures(Tm) analysis, executed by the GenSpector software. Primers were designed within the region conserved through HBV genotypes A to F. The lower limit of detection was 840 copies/ml serum, conducted with serial dilutions of a HBV DNA positive control(ACCURUN 325 series 700, Boston Biomedica Inc.). The assay was also compared to another assay for HBV DNA(Versant HBV DNA 3.0 assay, Bayer HealthCare) for 200 samples(each 100 clinical negative and positive samples). The sensitivity and specificity were 100% matched. This rapid PCR-based assay is specific, reproducible, and enables qualitative detection of HBV DNA.