• Journal of Microbiology and Biotechnology
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• v.21 no.1
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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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• v.40 no.4
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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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• v.38 no.4
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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.

• The Journal of Korean Institute of Communications and Information Sciences
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• v.35 no.3B
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• pp.440-452
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• 2010

For an evaluation of the software product, the national/international organizations and labs have been studying various methodologies for the quality on the basis of ISO/IEC Quality Assurance System, but they still have many issues in evaluation of the anti-virus software that has special characteristics of complexity. In this paper, to establish a methodology of the quality evaluation for the anti-virus software, fulfilling the requirements more than reasonable level, a process to draw the evaluation items and quantification was established. And the information of weight was objectified by analyzing the relative magnitude between each factors. Based on the defined information (evaluation item, weight), conducting the quality evaluations for 70 kinds of open anti-virus software collected from the portal sites in the real test environment, and as a result of the positive analysis with user's long-term experience, this paper justifies the evaluation item and the weight.

• The Journal of the Korean Society for Microbiology
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• v.35 no.5_6
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• pp.351-351
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• 2000

• Journal of Environmental Health Sciences
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• v.37 no.4
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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.

• Journal of Food Hygiene and Safety
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• v.29 no.1
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• pp.31-39
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• 2014

Norovirus causes acute gastroenteritis in all age groups and its food poisoning outbreaks are rapidly increasing in Korea. Reverse transcription-polymerase chain reaction (RT-PCR) is most widely used for the rapid detection of foodborne viruses due to high sensitivity. However, the false positive results of RT-PCR obtained against already inactivated viruses could be a serious drawbacks in food safety area. In this study, we investigated a method to yield true positive RT-PCR results only with alive viruses. To decompose the RNA genes from dead viruses, the enzymatic treatments composed of proteinse K and Ribonuclease A were applied to the sanitized and inactivated virus particles. Another aim of this study was to quantify the efficiencies of several major sanitizing treatments using real-time RT-PCR. Feline calicivirus (FCV) that belongs to the same Caliciviridae family with norovirus was used as a surrogate model for norovirus. The initial level of virus in control suspension was approximately $10^4$ PFU/mL. Most of inactivated viruses treated with the enzymatic treatment for 30 min at $37^{\circ}C$ were not detected in RT-PCR, Quantification results to verify the inactivation efficiencies of sanitizing treatments using real-time RT-PCR showed no false positive in most cases. We could successfully develope a numerical quantification process for the inactivated viruses after major sanitizing treatments using real-time RT-PCR. The results obtained in this study could provide a novel basis of rapid virus quantification in food safety area.

• KSBB Journal
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• v.16 no.6
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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.

• Korean Journal of Microbiology
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• v.44 no.1
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• pp.49-55
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• 2008

White Spot Syndrome Virus (WSSV) is one of the most virulent viral agents in the penaeid shrimp culture industry. In this study, WSSV in a Fenneropenaeus chinensis shrimp farm and an adjacent seawater were concentrated using a membrane filtration and quantified using the quantitative real-time PCR (QRT-PCR) method with newly designed primers and Taqman probe. Sensitivity of primers and probe was proven by WSSV standard curve assay in QRT-PCR. In order to demonstrate the relationship between WSSV and environmental parameters, physicochemical and biological parameters of the farm and influent seawaters were monitored from June to September, 2007. The abundance of WSSV ranged 3,814-121,546 copies per 1 liter of seawater, which was correlated with fecal enterococci ($r^2=0.9$, p=0.02), chlorophyll ${\alpha}$ ($r^2=0.8$, p=0.03) and $BOD_5$ ($r^2=0.8$, p=0.07). Subsequently, it is concluded that the QRT-PCR method using Taqman probe established in this study was efficient to clarify the quantification of WSSV in seawaters. Statistical analyses of environmental parameters obtained in this study also showed that the abundance of WSSV was correlated with several biological parameters rather than physicochemical parameters.

• Journal of Microbiology and Biotechnology
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• v.31 no.3
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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.