• Microbiology and Biotechnology Letters
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• v.36 no.1
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• pp.12-20
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• 2008

Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacture of the products. Mammalian cells are highly susceptible to minute virus of mice(MVM), and there are several reports of MVM contamination during the manufacture of biopharmaceuticals. In order to establish the validation system for the MVM safety, a real-time PCR method was developed for quantitative detection of MVM in cell lines, raw materials, manufacturing processes, and final products as well as MVM clearance validation. Specific primers for amplification of MVM DNA was selected, and MVM DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $6{\times}10^{-2}TCID_{50}/mL$. The real-time PCR method was proven to be reproducible and very specific to MVM. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with MVM. MVM DNA could be Quantified in CHO cell as well as culture supernatant. When the real-time PCR assay was applied to the validation of virus removal during a virus filtration process, the result was similar to that of virus infectivity assay. Therefore, it was concluded that this rapid, specific, sensitive, and robust assay could replace infectivity assay for detection and clearance validation of MVM.

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.610-620
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• 2021

There has been increasing interest in the head and neck squamous cell carcinoma (HNSCC) that is caused by high-risk human papillomavirus (HR-HPV) and has posed a significant challenge to Otolaryngologists. A rapid, sensitive, and reliable method is required for the detection of HR-HPV in clinical specimens to prevent and treat HPV-induced diseases. In this study, a multiple cross-linking spiral amplification (MCLSA) assay was developed for the visual detection of HPV-16. In the MCLSA assay, samples were incubated under optimized conditions at 62℃ for 45 min, and after mixing with the SYBR Green I (SGI) dye, the positive amplicons showed bright green fluorescence while the negative amplicons exhibited no obvious change. The specificity test revealed that the developed MCLSA technique had high specificity and could effectively distinguish all five HPV-16 strains from other pathogenic microorganisms. In terms of analytical sensitivity, the limit of detection (LoD) of MCLSA assay was approximately 5.4 × 101 copies/tube, which was 10-fold more sensitive than loop-mediated isothermal amplification (LAMP) and RT-PCR. The detection results of laryngeal cancer specimens collected from 46 patients with suspected HPV infection in the Liaoning region demonstrated that the positive detection rates of MCLSA and hybridized capture 2 kit were 32.61% (15/46). The true positive rate of the MCLSA assay was higher than that of RT-PCR (100% vs. 93.33%) and LAMP (100% vs. 86.67%). Therefore, the MCLSA assay developed in the present study could be a potentially useful tool for the point-of-care (PoC) diagnosis of HR-HPV, especially in resource-limited countries.

• Journal of Food Hygiene and Safety
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• v.34 no.3
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• pp.277-282
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• 2019

The object of this study is to compare the performance of the 3M Molecular Detection Assay 2 (3M MDA 2) and the Korea Standard Food Codex (KSFC) Method (i.e., isolation media and real-time PCR) in detecting Salmonella Typhimurium and Listeria monocytogenes in traditional Korean foods. Yuk-hwe and Yuk-sashimi (types of raw beef dishes) were artificially inoculated with $10^0-10^4CFU/25g$ of L. monocytogenes and S. Typhimurium. Citrobacter freundii and Listeria innocua were used as competitive microflora. After enrichment, the samples were analyzed using 3M MDA 2 and real-time PCR. All samples inoculated at concentrations of $10^0-10^4CFU/25g$ without competitive microflora were positive for S. Typhimurium and L. monocytogenes, as detected by 3M MDA 2 and Korea Standard Food Codex (KFSC) Method. In addition, part of the samples were positive for the presence of C. freundii and L. innocua. The 3M MDA 2 - Salmonella and Korea Standard Food Codex (KFSC) Method showed similar detection performances in Yuk-hwe and Yuk-sashimi. The 3M MDA 2 method for Salmonella and Listeria, which is a LAMP-based technology, can be used for rapid detection of S. Typhimurium and L. monocytogenes in raw beef. LAMP bioluminescence assays provide results on the subsequent day and are simple to use compared with the Korea Standard Food Codex (KFSC) Method, particularly in terms of DNA preparation.

• Journal of Food Hygiene and Safety
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• v.23 no.2
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• pp.121-128
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• 2008

The aim of this study was to develop a LightCycler-based real time PCR (LC-PCR) assay and to evaluate its diagnostic use for the detection of Staphylococcus aureus in raw milk samples. Following amplification of 113 bp of coa gene encoding an coagulase precursor specific for Staphylococcus aureus, melting curve and DNA sequencing analysis was performed to verify the specificity of the PCR products. Amplification of 209 bp gene encoding an altered penicillin-binding protein, PBP2a (mecA), melting curve analysis and DNA sequencing analysis was performed to verify methicillin resistance Staphylococcus aureus (MRSA). According to this study, 6 of 647 raw milk samples showed S. aureus positive and 2 of them showed a mecA positive and the detection limit was 10 fg of DNA. And we also isolated Staphylococcus chromogenes a causative agent of exudative epidermitis in pigs and cattle from 3 samples.

• Food Science and Preservation
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• v.21 no.2
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• pp.170-174
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• 2014

Hepatitis A virus (HAV) infection leads to acute liver failure and death through the intake of contaminated food. The polymerase chain reaction (PCR) has been used to detect HAV in food samples. HAV detection takes a long time, however, due to the virus concentration step required before PCR assay. In this study, a rapid method of detecting the HAVs present in lettuce using immunomagnetic separation combined with quantum dots (IMS-QDs) assay was developed. The detection limit of IMS-QDs for HAV was 10 $TCID_{50}/mL$, similar to the result that was obtained using RT-PCR combined with PEG or IMS. The application of IMS-QDs assay completed the viral detection within one hour, but this was not possible using PEG combined with RT-PCR. In conclusion, IMS-QDs assay is a rapid and efficient method for detecting HAV at a low concentration in agricultural products.

• BMB Reports
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• v.37 no.5
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• pp.546-551
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• 2004

The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.

• Korean Journal Plant Pathology
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• v.14 no.5
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• pp.382-385
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• 1998

Molecular identification and genetic relationships between a phytoplasma associated with chestnut little leaf (CLL) and phytoplasma isolates of other trees in Korea were amplified by polymerase chain reaction (PCR). These 16S rDNA sequences amplified from the various phytoplasmas were used in DNA heteroduplex mobility assays (HMA). In DNA HMA combined with PCR, the mobility shift was observed for a heteroduoplex formed in combined with CLL and jujube witches broom, but not for those formed in combined with CLL and each of sumac witches broom, paulownia witches broom, and mulberry dwarf. HMA combined with PCR has been shown to be a very useful method for detection and differentiation of phytoplasmas.

• Journal of Industrial Technology
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• v.28 no.A
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• pp.141-147
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• 2008

Several contaminated bacteria such as Lactobacillus brevis and Pediococcus damnosus in beer production cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. Recently, two contaminated strains were isolated from vessel of beer production and identified as Lactobacillus species by API kit identificaton as well as 16S-23S ITS sequencing analyses. Two isolated strains were named as Lactobacillus sp. HLA1 and Lactobacillus HLB2, respectively. A polymerase chain reaction (PCR) method was developed for the rapid and specific detection of Lactobacillus sp.. Two sets of primer pairs (HLA1-F/HLA1-R and HLB2-F/HLB2-R) were designed for the amplification of a 1576 base pair (bp) fragment of the HLA1 16S-23S rRNA gene and 1888 bp fragement of the HLB2 16S-23S rRNA. Amplified PCR products were highly specific to detect corresponding bacteria when other contaminated strains were used as PCR templates. However, detection of both strains were limited when $100{\mu}{\ell}$ of cultured samples were mixed with $100m{\ell}$ of beer sample in arbitrary manner. The sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

• 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.

• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.173-181
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• 2008

Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biologics such as biopharmaceuticals, tissue-engineered products, and cell therapy. Manufacturing processes for the biologics have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine parvovirus (BPV) is one of the common bovine pathogens and has widely been known as a possible contaminant of biologics. In order to establish the validation system for the BPV safety of biologics, a real-time PCR method was developed for quantitative detection of BPV contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BPV DNA were selected, and BPV DNA was quantified by use of SYBR Green 1. The sensitivity of the assay was calculated to be $1.3{\times}10^{-1}\;TCID_{50}/mL$. The real-time PCR method was validated to be reproducible and very specific to BPV. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BPV. BPV DNA could be quantified in CHO cell as well as culture supernatant. Also the real-time PCR assay could detect $1.3{\times}10^0\;TCID_{50}/mL$ of BPV artificially contaminated in bovine collagen. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BPV contamination during manufacture of biologics.