• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.555-559
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• 2013

Vibrio cholerae O1 and O139 are the major serotypes associated with illness, and some V. cholera non-O1 and non-O139 isolates produce cholera toxin. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the species-specific detection and quantitation of V. cholera using a primer pair based on an outer membrane lipoprotein lolB gene for the amplification of a 195 bp DNA fragment. The qPCR primer set for the accurate diagnosis of V. cholera was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.

• Genomics & Informatics
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• v.11 no.4
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• pp.277-281
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• 2013

RNA analysis has become a reliable method of body fluid identification for forensic use. Previously, we developed a combination of four multiplex quantitative PCR (qRT-PCR) probes to discriminate four different body fluids (blood, semen, saliva, and vaginal secretion). While those makers successfully identified most body fluid samples, there were some cases of false positive and negative identification. To improve the accuracy of the identification further, we tried to use multiple markers per body fluid and adopted the NanoString nCounter system instead of a multiplex qRT-PCR system. After measuring tens of RNA markers, we evaluated the accuracy of each marker for body fluid identification. For body fluids, such as blood and semen, each body fluid-specific marker was accurate enough for perfect identification. However, for saliva and vaginal secretion, no single marker was perfect. Thus, we designed a logistic regression model with multiple markers for saliva and vaginal secretion and achieved almost perfect identification. In conclusion, the NanoString nCounter is an efficient platform for measuring multiple RNA markers per body fluid and will be useful for forensic RNA analysis.

• Journal of fish pathology
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• v.24 no.2
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• pp.65-73
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• 2011

In analysis of DNA viruses from the contaminated shellfish using PCR, preparation method of template DNA is an important factor to get enough copy number of viruses. In this study, we evaluated the efficiency of PCR template of Megalocytivirus (sT50mg-D) DNA obtained from 50 mg digestive gland homogenate of oyster using commercial method, and compared with that obtained from 5 g of the same tissues (T5g-D) after PEG precipitation procedures of virus. Both templates DNA suspended in the same volume of distilled water showed positive results by primary PCR with 35 cycles, and the presence of Megalocytivirus was confirmed in oysters collected from cultured farms in Korea. Moreover, PCR with sT50mg-D allowed us to discriminate the contaminated oyster individually, that can not be done in PCR with T5g-D prepared from the mixture of three different individual oyster to get 5 g digestive gland homogenate. In quantitative analysis with real time PCR, Megalocytivirus concentrations in 50 ${\mu}l$ templates prepared using 0.5~50 mg of one positive sample were appeared in the range 6.14E+00~1.2E+02/${\mu}l$. We were not able to get positive result using template DNA contained less than 6.14E+00 copies. Consequently, 2-step PCR performed with DNA extracts from oyster homogenate of small amount (sT50mg-D) i) was enough to detect the contaminated Megalocytivirus in shellfish, ii) allowed us to do the analysis for individual shellfish rather than mixture of several shellfish and iii) showed the presence of Megalocytivirus in oyster from Korea.

• Communications for Statistical Applications and Methods
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• v.16 no.6
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• pp.1037-1046
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• 2009

Copy number variations(CNVs) are known as one of the most important factors in susceptibility to genetic disorders because they affect expression levels of genes. In previous studies, pyrosequencing, mini-sequencing real-time polymerase chain reaction(PCR), invader assays and other techniques have been used to detect CNVs. However, the higher the copy number in a genome, the more difficult it is to resolve the copies, so a more accurate method for measuring CNVs and assigning genotype is needed. PCR followed by a quantitative oligonucleotide ligation assay(qOLA) was developed for quantifying CNVs. The aim of this study was to compare the two methods for detecting and quantifying the CNVs of duplicated gene: the published pyrosequencing assay(pyro_CNV) and the newly developed qOLA_CNV. The accuracy and precision of the assay were evaluated for porcine KIT, which was selected as a model locus. Overall, the root mean squares(RMSs) of bias and standard deviation of qOLA_CNV were 2.09 and 0.45, respectively. These values are less than half of those of pyro CNV.

• Korean Journal of Microbiology
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• v.50 no.4
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• pp.296-301
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• 2014

Rotavirus is a major cause of acute gastroenteritis in young children in developed and developing countries. The use of probiotics for the treatment of gastrointestinal diseases is both safe and easily accessible. In this study, we evaluated the anti-rotaviral activities of probiotic mixtures in a Sprague-Dawley rat. 24 litters with their dams were randomly assigned to four groups; placebo, phosphate buffered saline (PBS), and two probiotic mixture (PRO-1 and PRO-2) groups. All rats were inoculated with rotavirus at dose of 8 log plaque forming units per rat at 5 days old. Animals in the PRO-1 and PRO-2 groups were orally administered probiotic mixtures 1 or 2, respectively, at a dose of 8 log colony forming units daily during 4 days. For control purposes, placebo and PBS groups were orally administered the same amount of placebo (containing maltose and polydextrose) or PBS once daily for 4 days, respectively. Antiviral analysis was performed by real-time quantitative PCR (RT-qPCR) and observing intestinal villi. As a result, weights of small intestines were greater in the PRO-1, PRO-2 groups than in control groups. Villi were short and villous epithelial necrosis was exhibited in control groups, but these morphological changes were not observed in PRO-1, PRO-2 treated rats. RT-qPCR analysis showed that VP7 gene level of rotavirus in fecal samples and small intestinal epithelial cells were lower in the PRO-1 and PRO-2 groups. These findings suggest that probiotic mixtures may be useful probiotics for the treatment of or as alternative therapies for rotaviral gastroenteritis.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.24 no.3
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• pp.422-428
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• 2019

Fish killing dinoflagellate Cochlodinium polykrikoides has been separated into four genetically differentiated subpopulations globally based on large-subunit (LSU) ribosomal RNA gene, and two subpopulations have been found in the South Sea, Korea. In this study, distributions of the East Asia and Philippines ribotypes were surveyed in the South Sea for 3 years (2014~2016) using quantitative real-time PCR (qPCR). The East Asia ribotype was detected in all sampling stations of the South Sea (Tongyeong~Wando) by 40~100% positives for 2014~2016, whereas the Philippines ribotype was detected in some areas of Tongyeong~Goheung by 1~2% positives for only 2016 when the Tsushima Warm Current (TWC) was particularly strengthened. These results indicate that the East Asia ribotype is the dominant subpopulation in the South Sea, also some of C. polykrikoides swimming cells might be transported from offshore to the South Sea via TWC.

• Korean Journal of Veterinary Service
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• v.45 no.1
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• pp.19-30
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• 2022

In this study, a simple loop-mediated isothermal amplification (LAMP) combined with visual detection method (vLAMP) assay was developed for the rapid and specific detection of African swine fever virus (ASFV), overcoming the shortcomings of previously described LAMP assays that require additional detection steps or pose a cross-contamination risk. The assay results can be directly detected by the naked eye using hydroxynaphthol blue after incubation for 40 min at 62℃. The assay specifically amplified ASFV DNA and no other viral nucleic acids. The limit of detection of the assay was <50 DNA copies/reaction, which was ten times more sensitive than conventional polymerase chain reaction (cPCR) and comparable to real-time PCR (qPCR). For clinical evaluation, the ASFV detection rate of vLAMP was higher than cPCR and comparable to OIE-recommended qPCR, showing 100% concordance, with a κ value (95% confidence interval) of 1 (1.00~1.00). Considering the advantages of high sensitivity and specificity, no possibility for cross-contamination, and being able to be used as low-cost equipment, the developed vLAMP assay will be a valuable tool for detecting ASFV from clinical samples, even in resource-limited laboratories.

• Journal of Veterinary Science
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• v.25 no.2
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• pp.28.1-28.11
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• 2024

Background: Porcine circovirus type 2 (PCV2) infection is ubiquitous around the world. Diagnosis of the porcine circovirus-associated disease requires clinic-pathological elements together with the quantification of viral loads. Furthermore, given pig farms in regions lacking access to sufficient laboratory equipment, developing diagnostic devices with high accuracy, accessibility, and affordability is a necessity. Objectives: This study aims to investigate two newly developed diagnostic tools that may satisfy these criteria. Methods: We collected 250 specimens, including 170 PCV2-positive and 80 PCV2-negative samples. The standard diagnosis and cycle threshold (Ct) values were determined by quantitative polymerase chain reaction (qPCR). Then, two point-of-care (POC) diagnostic platforms, convective polymerase chain reaction (cPCR, qualitative assay: positive or negative results are shown) and EZtargex (quantitative assay: Ct values are shown), were examined and analyzed. Results: The sensitivity and specificity of cPCR were 88.23% and 100%, respectively; the sensitivity and specificity of EZtargex were 87.65% and 100%, respectively. These assays also showed excellent concordance compared with the qPCR assay (κ = 0.828 for cPCR and κ = 0.820 for EZtargex). The statistical analysis showed a great diagnostic power of the EZtargex assay to discriminate between samples with different levels of positivity. Conclusions: The two point-of-care diagnostic platforms are accurate, rapid, convenient and require little training for PCV2 diagnosis. These POC platforms can discriminate viral loads to predict the clinical status of the animals. The current study provided evidence that these diagnostics were applicable with high sensitivity and specificity in the diagnosis of PCV2 infection in the field.

• Parasites, Hosts and Diseases
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• v.54 no.1
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• pp.39-46
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• 2016

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (${\beta}-actin$), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.

• Bulletin of the Korean Chemical Society
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• v.35 no.5
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• pp.1562-1564
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• 2014