• Journal of Microbiology and Biotechnology
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• 제21권1호
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• pp.100-108
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• 2011

A method for the rapid detection and quantification of Newcastle disease virus (NDV) produced in an animal cell culture-based production system was developed to enhance the speed of the NDV vaccine manufacturing process. A SYBR Green I-based real-time RT-PCR was designed with a conventional, inexpensive RT-PCR kit targeting the F gene of the NDV LaSota strain. The method developed in this study was validated for specificity, accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The validation results satisfied the predetermined acceptance criteria. The validated method was used to quantify virus samples produced in an animal cell culture-based production system. The method was able to quantify the NDV samples from mid- or late-production phases, but not effective on samples from the early-production phase. For comparison with other quantifiable methods, immunoblotting, plaque assay, and tissue culture infectious dose 50 ($TCID_{50}$) assay were also performed with the NDV samples. The results demonstrated that the real-time RT-PCR method is suitable for the rapid quantification of virus particles produced in an animal cell-culture-based production system irrespective of viral infectivity.

• The Plant Pathology Journal
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• 제40권4호
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• pp.408-413
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• 2024

The emergence of rice black-streaked dwarf virus (RBSDV) poses a significant threat to global cereal crop cultivation, necessitating the urgent development of reliable detection and quantification techniques. This study introduces a reliable approach for the precise and sensitive quantification of the RBSDV in cereal crop samples, employing a reverse transcription digital polymerase chain reaction (RT-dPCR) assay. We assessed the specificity and sensitivity of the RT-dPCR assay proposed for precise RBSDV detection and quantification. Our findings demonstrate that RT-dPCR was specific for detection of RBSDV, with no cross-reactivity observed with other viruses infecting cereal crops. The RT-dPCR sensitivity was over 10 times that of RT-quantitative PCR (RT-qPCR). The detection limit of RT-dPCR was 0.096 copies/㎕. In addition, evaluation of RT-dPCR assay with field samples was conducted on 60 different cereal crop samples revealed that RT-dPCR (45/60) exhibited superior accuracy compared with RT-qPCR (23/60). In this study, we present a specific and accurate RT-dPCR assay for the detection and quantification of RBSDV.

• 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.

• 한국통신학회논문지
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• 제35권3B호
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• pp.440-452
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• 2010

소프트웨어 제품의 평가를 위해 국제표준 ISO/IEC 품질인증 시스템을 기반으로 국내 외 기관 및 연구소에서 품질에 대한 많은 방법론이 연구 및 적용되고 있으나, 복잡한 차원의 특수한 성질을 지닌 안티바이러스 소프트웨어를 평가하기에는 많은 문제를 동반한다. 따라서 본 논문에서는 적정 수준 이상의 요건을 갖춘 안티바이러스 소프트웨어의 품질평가 방법론을 마련하고자 평가항목 도출을 위한 프로세스와 정량화 방안을 정립하였으며 각 요인간의 상대적 중요도를 분석함으로써 가중치 정보를 객관화하였다. 정의된 정보(평가 항목, 가중치)를 기반으로 포털 사이트에서 수집한 공개용 안티바이러스 소프트웨어 70종에 대하여 리얼 테스트 환경에서 품질평가를 수행하였으며, 사용자들의 오랜 시간동안의 경험을 이용한 실증분석 결과 본 논문에서 정의한 평가항목과 가중치에 대한 정당성을 마련할 수 있었다.

• 대한미생물학회지
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• 제35권5_6호
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• pp.351-351
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• 2000

• 한국환경보건학회지
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• 제37권4호
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• pp.306-314
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• 2011

Objectives: Respiratory virus infections are the most common disease among all ages in all parts of the world and occur through airborne transmission. The purpose of this study was to detect and quantitate human respiratory viruses in residential environments. Methods: Air samples were collected from the residential space of apartments in the Seoul/Gyeonggi-do area. The samples were collected from indoor and outdoor air. Among respiratory viruses, influenza A virus, influenza B virus, parainfluenza virus, metapneumovirus, respiratory syncytial virus, and adenovirus were investigated by multiplex polymerase chain reaction. Among the virus-positive samples, we performed adenovirus quantification by real-time polymerase chain reaction. Results: Virus detection rates were 44.0%, 3.8%, 3.4%, and 17.3% in spring, summer, autumn, and winter, respectively. The virus detection rate was higher in winter and spring than in summer and autumn. Adenovirus was most commonly detected, followed by influenza A virus and parainfluenza virus. Virus distribution was not significantly different between indoor and outdoor environments. Conclusions: Although virus concentrations were not high in residential environments, residents in houses with detected viruses may have an increased risk of exposure to airborne respiratory viruses, especially in winter and spring.

• 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.

• KSBB Journal
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• 제16권6호
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• pp.579-591
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• 2001

The cell growth inhibitor effect of cervical cancer cells was investigated by liposome mediated transfection (pRcCMVp53/lipofectin) and by transfection using adenovirus (AdCMVp57). The papilloma virus cancer cell lines we used in this study were HPV16 positive, having inhibiter gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3. LacZ gene of E.coli was used as the marker gene for the transfection efficiency. The effect on the inhibition of tumor cell growth was measured by cell count and cell viability though ELISA analysis and MTT assay. The inhibition of tumor cell growth was confirmed by measuring each assay for six days, comparing with the normal control cell growth. The cell growth of cervical cancer calls by transfection was significantly reduced and showed tittle differences among the cell lines. To eliminate the potential problem of Ad(adenovirus) contamination during rAAV production, rAAV can be produced by a triple transfection of vector plasmic, packaging plasmid, and adenovirus helper plasmid. To examine the helper functions of Ad plasmids on the production of rAAV vector, we carried out cotransfection of three plasmids, AAV vector, packaging construct, and Ad helper plasmids. The optimized transfection condition for calcium phosphate method is 25ug of total DNA per 10-cm-diameter plate of 293 cell. We found that rAAV yields peaked at 48hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/ml based on the quantification of viral DNA. Recent1y, Kombucha(fungi) was identified as a very potent antileukefic agent. In the present study, effect of natural toxin(plankton) and Kombucha is PSP(GTXI-3, neoSTX), on various MTT assay cervical cancer cell line. Toxin(GTX 1-3, neoSTX) also inhibited the proliferation in primary cervical cancer calls in a dose-dependent toxin concentration. These results showed that toxin was very potent in inhibiting the proliferation of cervical cancer calls in vitro. Toxins and Kombuoha exhibited a dose dependent inhibition of cellular proliferation in cancer cell line.

• 미생물학회지
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• 제44권1호
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• pp.49-55
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• 2008

흰반점 바이러스(white spot syndrome virus, WSSV)는 양식산 새우에 감염하여 대량폐사를 일으키는 전염성이 매우 강한 병원성 바이러스이다. 본 연구에서는 강화도에 위치한 대하(Fenneropenaeus chinensis) 양식장의 양식수와 양식장으로 유입되는 해수에서 WSSV를 막여과법을 이용하여 농축하였으며, 새롭게 디자인한 primer와 Taqman probe를 사용하여 정량 실시간 PCR (quantitative real-time PCR, QRT-PCR)을 적용하여 WSSV를 정량하였다. 농도표준을 사용한 QRT-PCR 결과, 제작된 primer와 probe를 이용하여 WSSV가 정확하고 민감하게 검출됨을 확인하였다. 해수에 존재하는 WSSV와 물리화학적, 생물학적 환경요인간의 상관관계를 도출하기 위하여 양식수와 해수 유입수에서 대하 양식기간인 2007년 6월부터 9월까지 총 8회에 거쳐 다양한 환경요인을 분석하였다. 양식수 1L에 존재하는 WSSV의 양은 3,814-121,545 copy였으며, 이는 분원성 enterococci ($r^2=0.9$, p=0.02), 엽록소${\alpha}$ ($r^2=0.8$, p=0.03), 생화학적 산소요구량($r^2=0.8$, p=0.07)과 상관관계를 나타내었다. 결론적으로 본 연구에서 정립된 WSSV의 농축법 및 QRT-PCR 방법은 해수에 존재하는 WSSV를 정량하는데 효과적이었으며, 해수에 존재하는 WSSV의 양은 물리화학적 환경요인보다 생물학적 환경요인과 밀접한 관련을 보였다.

• Journal of Microbiology and Biotechnology
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• 제31권3호
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• pp.358-367
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• 2021

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.