• 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.

• Korean Journal of Microbiology
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• v.41 no.3
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• pp.216-224
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• 2005

Chromatography has now been used successfully to provide the requisite purity for human plasma-derived biop-harmaceuticals such as coagulation factors and immunoglobulins. Recently, increasing attention has been focused on establishing efficient cleaning procedures to prevent potential contamination by microorganisms as well as carry-over contamination from batch to batch. The purpose of present study was to develop a cleaning validation system for the assurance of virus removal and/or inactivation during chromatography process. In order to establish an assay system for the validation of virus clearance during chromatography cleaning process, a quantitative real-time PCR method for porcine parvovirus(PPV) was developed, since PPV, a model virus for human parvovirus B19, has a high resistance to a range of physico-chemical treatment. Specific primers for amplification of PPV DNA was selected, and PPV DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 1.5 $TCID_{50}/ml$. The established real-time PCR assay was successfully applied to the validation of PPV removal and cleaning during SP-Sepharose cation chromatography for thrombin purification and Q-Sepharose anion chromatography for factor VIII purification. The comparative results obtained by real-time PCR assay and infectivity titrations suggested that the real-time PCR assay could be a useful method for chromatography cleaning validation and that it could have an additive effect on the interpretation and evaluation of virus clearance during the virus removal process.

• Journal of Microbiology and Biotechnology
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• v.19 no.4
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• pp.339-345
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• 2009

We evaluated the activity and abundance of the crude-oil-degrading bacterium Nocardia sp. H17-1 during bioremediation of oil-contaminated soil, using real-time PCR. The total petroleum hydrocarbon(TPH) degradation rate constants(k) of the soils treated with and without H17-1 were $0.103\;d^{-1}$ and $0.028\;d^{-1}$ respectively. The degradation rate constant was 3.6 times higher in the soil with H17-1 than in the soil without H17-1. In order to detect and quantify the Nocardia sp. H17-1 in soil samples, we quantified the genes encoding 16S ribosomal RNA(16S rRNA), alkane monooxygenase(alkB4), and catechol 2,3-dioxygenase(23CAT) with real-time PCR using SYBR green. The amounts of H17-1 16S rRNA and alkB4 detected increased rapidly up to 1,000-folds for the first 10 days, and then continued to increase only slightly or leveled off. However, the abundance of the 23CAT gene detected in H17-1-treated soil, where H17-1 had neither the 23CAT gene for the degradation of aromatic hydrocarbons nor the catechol 2,3-dioxygenase activity, did not differ significantly from that of the untreated soil($\alpha$=0.05,p>0.22). These results indicated that H17-1 is a potential candidate for the bioaugmentation of alkane-contaminated soil. Overall, we evaluated the abundance and metabolic activity of the bioremediation strain H17-1 using real-time PCR, independent of cultivation.

• 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.

• Molecular & Cellular Toxicology
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• v.3 no.4
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• pp.320-325
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• 2007

Promoter hypermethylation of the $p16^{INK4a}$ gene was investigated in 52 sets of samples of tumor tissue and adjacent normal tissue from Korean patients with colorectal cancer, using the proposed modified the Real-time PCR/SYBR Green detection method presented in this study. In normal tissue, 29 of 52 patients (56%) were methylated and in tumor tissue, 23 of 52 patients (44%) were methylated. The 34 cases (65.4%) showed a concordant DNA methylation pattern in both normal tissue and tumor tissue. Analyzing the association between the clinicopathologic features and DNA methylation status of the $p16^{INK4a}$ gene, the DNA methylation status according to by Duke's stage was different while other clinicopathological characteristics, including the age, sex, tumor stage, and histologic type of the patient were not found to be correlated with $p16^{INK4a}$ methylation. With multivariate logistic regression, it was observed that the DNA methylation status of $p16^{INK4a}$ gene in normal tissue was correlated with the DNA methylation status of the $p16^{INK4a}$ gene in tumor tissue (P=0.026). According to a Kaplan-Meier survival analysis, a difference in the survival rate by DNA methylation status was found, but it was not significant.

• The Plant Pathology Journal
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• v.33 no.2
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• pp.184-192
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• 2017

Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at $63^{\circ}C$ for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was $10^{-2}J2/0.5g$ of soil, which was 10 times more sensitive than conventional PCR ($10^{-1}J2/0.5g$ of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

• Molecular & Cellular Toxicology
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• v.4 no.2
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• pp.164-169
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• 2008

Methylmercury (MeHg) is known to have devastating effects on the mammalian nervous system. In order to characterize the mechanism of MeHg-induced neurotoxicity, we investigated the analysis of transcriptional profiles on human 8k cDNA microarray by treatment of $1.4{\mu}M$ MeHg at 3, 12, 24 and 48h in human neuroblastoma SH-SY5Y cell line. Some of the identified genes by MeHg treatment were significant at early time points (3h), while that of others was at late time points (48h). The early response genes that may represent those involved directly in the MeHg response included pantothenate kinase 3, a kinase (PRKA) anchor protein (yotiao) 9, neurotrophic tyrosine kinase, receptor, type 2 gene, associated with NMDA receptor activity regulation or perturbations of central nervous system homeostasis. Also, when SH-SY5Y cells were subjected to a longer exposure (48h), a relative increase was noted in a gene, glutamine-fructose-6-phosphate transaminase 1, reported that overexpression of this gene may lead to the increased resistance to MeHg. To confirm the alteration of these genes in cultured neurons, we then applied real time-RT PCR with SYBR green. Thus, this result suggests that a neurotoxic effect of the MeHg might be ascribed that MeHg alters neuronal receptor regulation or homeostasis of neuronal cells in the early phase. However, in the late phase, it protects cells from neurotoxic effects of MeHg.

• Korean Journal of Microbiology
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• v.47 no.2
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• pp.103-109
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• 2011

Pectobacterium carotovorum subsp. carotovorum is the causative agent of soft rot in crops such as potato and cabbages. Loop-mediated isothermal amplification (LAMP) is a simple DNA amplification method, as well as isothermal PCR technique. In this study, a new method for the rapid detection of Pectobacterium carotovorum subsp. carotovorum was developed using LAMP that named PCC-LAMP. Based on lytic murein transglycolase gene of Pectobacterium carotovorum subsp. carotovorum, a set of four primers for LAMP was designed. The optimal PCC-LAMP reaction temperature was established at $61^{\circ}C$. Under standard conditions, PCC-LAMP amplified $1{\times}10^3$ copies of clone PCC-pBX437 per reaction. Further, this method can also assay directly by SYBR Green I without electrophoresis. Amplification was not detected for five other bacterial species. In conclusion, PCC-LAMP may be a useful method for the detection Pectobacterium carotovorum subsp. carotovorum in the field.

• Journal of Food Hygiene and Safety
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• v.30 no.1
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• pp.103-108
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• 2015

Weissella spp. from traditional Korean foods of Kimchi were isolated and characterized against food-borne pathogenic Listeria monocytogens. The isolates were identified as W. cibaria 0D17 and W. confusa 0D23 from Kimchi by the biochemical characteristics and 16S DNA sequencing. The culture solutions of the isolates adjusted to pH 7.0 showed L. monocytogens inhibition. To analyze the quantitative detection of L. monocytogenes, real-time PCR was performed according to the SYBR Green I method. The isolates grew well and L. monocytogens did not grow during the co-culture with those strains at $37^{\circ}C$. Therefore, W. cibaria 0D17 and W. confusa 0D23 might be the candidates as the functional lactic acid bacteria for improving food safety.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.8
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• pp.987-992
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• 2010

This study was to detect the expression of heart fatty acid-binding protein (H-FABP) and adipocyte fatty acid-binding protein (A-FABP) gene mRNA in different tissues of Rugao and Luyuan chickens at 56 d and 120 d by real-time fluorescence quantitative reverse transcription polymerase-chain reaction (FQ-RT-PCR). The primers were designed according to the sequences of HFABP, A-FABP and GAPDH genes in Gallus gallus, which were used as target genes and internal reference gene, respectively. The levels of H-FABP and A-FABP gene expression were detected by SYBR Green I FQ-RT-PCR. The relative H-FABP and A-FABP gene mRNA expression level was calculated with 2-$^{{\Delta}Ct}$. Melting curve analysis showed a single peak of three genes. Intramuscular fat (IMF) content in breast muscle and leg muscle of the two chicken breeds at 120 d was higher than at 56 d. IMF content in breast muscle and leg muscle at 56 d and 120 d in Luyuan was significantly higher than in Rugao, however, abdominal fat of Luyuan was significantly lower than that of Rugao. The relative H-FABP gene mRNA expression level in cardiac muscle was the highest in both chicken breeds. The relative H-FABP and A-FABP gene expression of different tissues in Luyuan was higher than in Rugao. H-FABP gene mRNA expression had a negative effect on IMF of leg and breast muscles, and was significantly negatively correlated with IMF content. The relative A-FABP gene mRNA level in abdominal fat was higher than in liver. The A-FABP gene mRNA was not expressed in leg, breast and cardiac muscles. A-FABP gene mRNA expression level was significantly positively correlated with abdominal fat and had a significant effect on abdominal fat but not IMF content.