• 핵의학기술
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• 제23권1호
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• pp.35-39
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• 2019

[목적] B형 간염바이러스(hepatitis B virus, HBV)감염은 전세계적으로 중요한 공중 보건 문제이며 만성간염, 간 경변, 간암의 주요 원인으로 알려져 있으며, 이러한 질환의 진단 및 치료에 B형 간염바이러스의 혈청학적 검사는 필수적이다. 항 바이러스 치료중인 B형 간염 환자를 대상으로 면역방사계수 측정법(IRMA; Immunoradiometric assay)과 화학발광 미세입자 면역분석법(CMIA; Chemiluminescent Micropartical Immunoassay)을 이용하여 HBe-Ag 검사를 시행하였고, 실시간 중합효소 연쇄반응(RT-PCR; Realtime-Polymerase Chain Reaction)법을 이용하여 혈청 내 HBV DNA 검출율 을 비교 분석 하였다. [대상 및 방법] 항 바이러스 치료가 시행중인 B형 간염 환자 270명을 대상으로 HBeAg 혈청 검사와 HBV DNA 정량 검사를 실시하였다. HBeAg 혈청 검사는 검출 원리가 다른 두 가지 혈청학적 검사법(IRMA, CMIA)을 적용 하였고, 혈청 내 HBV DNA는 Abbott m2000 System을 사용하여 실시간 중합효소 연쇄반응(RT-PCR; Realtime-Polymerase Chain Reaction)법으로 정량 측정 하였다. [결과] HBeAg 검출율은 면역방사계수법(IRMA)의 경우 24.1% (65/205), 화학발광 미세입자 면역 분석법(CMIA)에서는 82.2% (222/48)의 결과를 보였다. 혈청학적 검사방법(IRMA, CMIA)에 따른 HBeAg 검사결과의 일치율은 33% (89/270)이다. 실시간 중합효소 연쇄반응(RT-PCR)을 이용한 혈청 내 HBV DNA의 검출율은 29.3% (79/191)를 보였고, 혈청 내 HBV-DNA 농도는 $16IU/mL{\times}1.0{\times}10^9IU/mL$ 이며 검출한계는 <15IU/mL 이다. 면역방사계수법(IRMA)으로 HBeAg 검출결과가 양성일때 55.4%, 그리고 음성일때 20.9%의 HBV DNA 검출율과 $1.1{\times}10^8IU/mL$, $5.7{\times}10^5IU/mL$의 혈청내 HBV DNA농도를 나타냈다. 이에 반해 화학발광 미세입자 면역 분석법(CMIA)의 경우 HBeAg 검출결과가 양성일때 HBV DNA 검출율은 28.4%, 음성일때 33.3%의 결과를 나타냈으며 혈청 내 HBV DNA농도는 $6.0{\times}10^7IU/mL$, $2.4{\times}10^5IU/mL$ 이었다. 면역방사계수법과 화학발광 미세입자 면역 분석법에서 동일하게 HBeAg 검출 결과가 양성인 경우 HBV DNA 검출율은 62.3%의 결과를 보였으며, 혈청 내 HBV DNA 농도는 $1.1{\times}10^8IU/mL$ 이다. [결론] 혈청학적 검사법에 따른 HeAg 검출율은 많은 차이를 보였다. 이러한 차이는 검사kit에 사용된 Ab의 특성과 epitope, HBV의 genotype등 여러 가지 원인으로 생각된다. 혈청학적 검사 결과로 분류 된 그룹별 HBV DNA의 검출율과 농도를 비교한 결과, Group II(IRMA 양성, CMIA 양성, N=53)에서 높은 검출율과 농도를 확인할 수 있었다.

• 생명과학회지
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• 제18권3호
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• pp.374-380
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• 2008

본 연구는 무균성수막염 의심환자의 다양한 검체로부터 enterovirus의 진단을 위하여 real-time NASBA, 2 step RT-PCR 시험과 세포배양 시험을 각각 실시하여 각 시험법의 검출율, 특이도, 사용자 편리성, 시험소요 시간, 교차오염의 가능성 등을 비교 검토하였다. 비교시험 결과 전체 292건의 검체로부터 real-time NASBA에서 145건, 세포배양에서 101건, 2 step RT-PCR에서 86건이 양성으로 나타나 real-time NASBA가 가장 검출율이 높은 시험법임을 알 수 있었다. Enterovirus 외의 무균성수막염 원인바이러스에 대한 특이도 비교 시험결과 2 step RT-PCR 시험에서 rhinovirus 10건 중 1건이 위양성 반응을 나타내어 다른 시험법에 비해 특이도가 떨어지는 것으로 나타났다. Real-time NASBA는 하나의 튜브에서 증폭과 검출이 동시에 일어나 다른 시험과 비교하여 교차오염의 가능성이 낮으며 또한 시험 소요시간이 5시간 정도로 세포배양(5-14일 소요) 및 2 step RT-PCR(9시간소요) 에 비하여 신속하게 진단할 수 있어 일선병원이나 실험실에서 enterovirus를 검출을 위하여 적용할 수 있을 것으로 사료된다.

• 한국식품위생안전성학회지
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• 제29권1호
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• pp.31-39
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• 2014

본 연구에서는 FCV 현탁액에 물리, 화학적 위생처리 후 복합효소처리라는 전처리과정을 적용한 뒤 real-time RT-PCR법을 이용하여 살균효능을 분석하였다. RT-PCR 이전에 $37^{\circ}C$에서 30분 동안 PK와 RNase A를 처리함으로써 UV, 열, 염소, 에탄올, 과초산계열 제품에 의해 불활성화 된 바이러스들은 음성 결과를 나타내었고, real-time RTP-CR법을 통해 살균 효능을 정량분석한 결과, 복합효소처리를 했을 경우 무처리구보다 더 높은 살균 효능을 보이는 것을 확인할 수 있었다. 이로써 Nuanualsuwan S. 등의 선행연구에서와 같이 PK와 RNase A로 전처리하는 단계를 통하여 물리, 화학적 위생처리에 의해 손상되지 않은 바이러스가 RT-PCR법에 의해 증폭되는 것을 방지함으로써 Real-time PCR법에 대한 검출 감도를 높일 수 있음을 확인하였다. 또한, FCV를 검출하기 위해 사용된 RT-PCR과 real-time RT-PCR 두 방법 중에서도 real-time RT-PCR법이 가장 신속하면서도 민감도 높은 결과로 도출되었다. 따라서, 유전자 분석 이전에 복합효소처리는 물리, 화학적 위생처리에 의해 불활성화 된 바이러스의 RNA가 transcription 또는 증폭되는 것을 방지하기 위한 수단으로 real-time RT-PCR법과 결합됨으로써 노로바이러스를 비롯한 식중독 바이러스를 검출하는데 효과적으로 적용될 것으로 판단된다. 또한 식품현장에서 전기영동 과정없이 신속하게 살아있는 바이러스만을 수치적으로 정량화함으로써 식품안전에도 기여할 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제23권10호
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• pp.1291-1298
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• 2010

The aim of this research was to investigate the dynamic changes of the level of total prolactin receptor (PRLR) mRNA and the short form prolactin receptor (S-PRLR) mRNA in skin of cashmere goats from the initiation of cashmere fibre growth to active growth. Eighteen half-sib wethers were allocated randomly to two groups. Melatonin implants were used in order to initiate growth of cashmere fibre before the normal time and reduce blood plasma prolactin (PRL) concentration. Real-time reverse transcription quantitative polymerase chain reaction (real-time PCR) was used to determine PRLR mRNA expression levels of skin from June to November. The results showed that, in Chinese Inner Mongolia cashmere goats, there were seasonal variations in expression of total PRLR mRNA in skin with levels decreasing from June to October. Synchronously, the cashmere fibre growth rate gradually increased during this period, but the expression levels of S-PRLR mRNA did not decrease along with seasonal variation from initiation to active growth of cashmere fibre. These results suggest that expression levels of S- PRLR mRNA might be involved in the process of cashmere growth. It was also possible that the change of alternative splicing of PRLR occurred in the skin of cashmere goats from proanagen to anagen.

• The Plant Pathology Journal
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• 제39권4호
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• pp.309-318
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• 2023

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.

• 대한의생명과학회지
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• 제17권3호
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• pp.191-196
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• 2011

Noroviruses (noroV) are the major cause of nonbacterial gastroenteritis in humans worldwide. Since noroV cannot yet be cultured in vitro and their diagnosis by electron microscopy requires at least $10^6$ viral particles/g of stool a variety of molecular detection techniques represent an important step towards the detection of noroV. In the present study, we have applied real-time nucleic acid sequence-based amplification (real-time NASBA) for simultaneous detection of NoroV genogroup I (GI) and genogroup II (GII) using standard viral RNA. For real-time NASBA assay which can detected noroV GI and GII, a selective region of the genes encoding the capsid protein was used to design primers and genotype-specific molecular beacon probes. The specificity of the real-time NASBA using newly designed primers and probes were confirmed using standard viral RNA of noroV GI and GII. To determine the sensitivity of this assay, serial 10-fold dilutions of standard viral RNA of noroV GI and GII were used for reverse transcription polymerase chain reaction (RT-PCR) and real-time NASBA. The results showed that while agarose gel electrophoresis could detect RT-PCR products with 10 pg of standard viral RNA, the real-time NASBA assay could detect 100 fg of standard viral RNA. These results suggested that the real-time NASBA assay has much higher sensitivity than conventional RT-PCR assay. This assay was expected that might detect the viral RNA in the specimens which could have been false negative by RT-PCR. There were needed to perform real-time NASBA with clinical specimens for evaluating accurate sensitivity and specificity of this assay.

• 생명과학회지
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• 제20권8호
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• pp.1201-1206
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• 2010

현장의 환경 시료를 대상으로 Real-Time PCR법과 LC/MS법을 이용하여 마이크로시스틴 검출방법을 비교 연구하였다. 3종류의 primer쌍을 이용하여 3종의 Microcystis aeruginosa 표준균주를 대상으로 Real-Time PCR법을 실시한 결과 TOX2P/TOX2M primer를 이용한 균주에서만 마이크로시스틴이 검출되었다. 2009년 6~9월 사이에 남조류가 발생한 상수원수 시료를 정립된 Real-Time PCR법과 기존의 LC/MS법으로 실험한 결과 11개 시료 모두에서 마이크로시스틴이 검출되었고, 농도는 5.98~219.0 ${\mu}g/l$ 범위로 조사되었다. 정수처리 공정별 실험에서는 BAC 여과 단계에서 마이크로시스틴이 완전히 제거되는 것으로 나타났다. 실험결과 Real-Time PCR법은 기존의 표준시험방법인 LC/MS법 보다 분석시간을 많이 단축시키는 것으로 나타나 효과적인 마이크로시스틴 검출방법임을 알 수 있었다.

• Journal of Dairy Science and Biotechnology
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• 제33권4호
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• pp.253-262
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• 2015

The authentication and implications of misleading labeling in milk and dairy products is important to protect against cheating consumers from adulteration and to alert sensitive consumers to any undeclared potential allergens. This need to support milk and dairy products labeling has led to the development of specific analytical techniques for the analysis of milk and dairy products ingredients. Recently, several methods based on polymerase chain reaction (PCR), including restriction fragment length polymorphism (PCR-RFLP), multiplex PCR, species-specific PCR, and real-time PCR, have been proposed as useful means for identifying species of origin in milk and dairy products, as well as quantifying and detecting any adulteration. These methods have particular advantages owing to their high specificity and sensitivity, as well as rapid processing time. In this review, we provide an updated and extensive overview of the PCR-based methods used for milk and dairy products authentication with a particular focus on the application of PCR methods to detect adulteration.

• Journal of Gastric Cancer
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• 제19권3호
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• pp.235-253
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• 2019

Gastric cancer (GC) is one of the deadliest malignancies in the world. Currently, clinical treatment decisions are mostly made based on the extent of the tumor and its anatomy, such as tumor-node-metastasis staging. Recent advances in genome-wide molecular technology have enabled delineation of the molecular characteristics of GC. Based on this, efforts have been made to classify GC into molecular subtypes with distinct prognosis and therapeutic response. Simplified algorithms based on protein and RNA expressions have been proposed to reproduce the GC classification in the clinical field. Furthermore, a recent study established a single patient classifier (SPC) predicting the prognosis and chemotherapy response of resectable GC patients based on a 4-gene real-time polymerase chain reaction assay. GC patient stratification according to SPC will enable personalized therapeutic strategies in adjuvant settings. At the same time, patient-derived xenografts and patient-derived organoids are now emerging as novel preclinical models for the treatment of GC. These models recapitulate the complex features of the primary tumor, which is expected to facilitate both drug development and clinical therapeutic decision making. An integrated approach applying molecular patient stratification and patient-derived models in the clinical realm is considered a turning point in precision medicine in GC.

• Journal of Medicine and Life Science
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• 제20권3호
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• pp.107-114
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• 2023

Epidemiological control of coronavirus disease 2019 (COVID-19) is needed to estimate the infection period of confirmed cases and identify potential cases. The present study, targeting confirmed cases for which the time of COVID-19 symptom onset was disclosed, aimed to investigate the relationship between intervals (day) from symptom onset to testing the cycle threshold (CT) values of real-time reverse transcription-polymerase chain reaction. Of the COVID-19 confirmed cases, those for which the date of suspected symptom onset in the epidemiological investigation was specifically disclosed were included in this study. Interval was defined as the number of days from symptom onset (as disclosed by the patient) to specimen collection for testing. A locally weighted regression smoothing (LOWESS) curve was applied, with intervals as explanatory variables and CT values (CTR for RdRp gene and CTE for E gene) as outcome variables. After finding its non-linear relationship, a polynomial regression model was applied to estimate the 95% confidence interval values of CTR and CTE by interval. The application of LOWESS in 331 patients identified a U-shaped curve relationship between the CTR and CTE values according to the number of interval days, and both CTR and CTE satisfied the quadratic model for interval days. Active application of these results to epidemiological investigations would minimize the chance of failing to identify individuals who are in contact with COVID-19 confirmed cases, thereby reducing the potential transmission of the virus to local communities.