• Korean Journal of Veterinary Service
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• v.45 no.2
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• pp.87-99
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• 2022

A novel porcine circovirus 4 (PCV4) was recently emerged in Chinese and Korean pig herds, which provided epidemiological situation where three pathogenic PCVs, PCV2, PCV3, and newly emerged PCV4, could co-infect pig herds in these countries. In this study, a new triplex quantitative real-time polymerase chain reaction (tqPCR) method was developed for the rapid and differential detection of these viruses. The assay specifically amplified each viral capsid gene, whereas no other porcine pathogenic genes were detected. The detection limit of the assay was below 10 copies/µL and the assay showed high repeatability and reproducibility. In the clinical evaluation using 1476 clinical samples from 198 Korean pig farms, the detection rates of PCV2, PCV3 and PCV4 by the tqPCR assay were 13.8%, 25.4%, and 3.8%, respectively, which were 100% agreement with those of previously reported monoplex qPCR assays for PCV2, PCV3, and PCV4, with a κ value (95% CI) of 1 (1.00~1.00). The prevalence of PCV2, PCV3, and PCV4 at the farm levels were 46.5%, 63.6%, and 19.7%, respectively. The co-infection analysis for tested pig farms showed that single infection rates for PCV2, PCV3, and PCV4 were 28.8%, 44.4%, and 9.6%, respectively, the dual infection rates of PCV2 and PCV3, PCV2 and PCV4, and PCV3 and PCV4 were 12.6%, 3.5%, and 5.1%, respectively, and the triple infection rate for PCV2, PCV3, and PCV4 was 1.5%. These results demonstrate that three pathogenic PCVs are widely spread, and their co-infections are common in Korean pig herds, and the newly developed tqPCR assay will be useful for etiological and epidemiological studies of these pathogenic PCVs.

• Food Science and Preservation
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• v.30 no.3
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• pp.383-394
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• 2023

The ultra-fast quantitative real-time polymerase chain reaction (qPCR) assay was developed and validated to differentiate the morphologically similar ones, Oncorhynchus keta and Oncorhynchus mykiss. Species-specific primers were designed for the COI genes of mtDNA. The species-specific primers designed for O. keta and O. mykiss were selectively amplified by O. keta and O. mykiss DNA, respectively. The sensitivity of O. keta and O. mykiss primers was 1 ng/μL. Quantitative testing showed that the results met the 'Guidelines on Standard Procedures for Preparing Analysis Method such as Food' proposed by the Ministry of Food and Drug Safety. The qPCR method developed and validated in this study for identifying O. keta and O. mykiss has advantages such as speed and field applicability. Therefore, this method is expected to help control forgery and alteration of raw materials in the seafood industry.

• Journal of Life Science
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• v.25 no.2
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• pp.136-150
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• 2015

Pseudomonas syringae pathovar tabaci is a plant pathogenic bacterium that causes wildfire disease in tobacco plants. In P. syringae pv. tabaci, PsyI, a LuxI-type protein, acts as an AHL synthase, while primary and secondary sequence analysis of PsyR has revealed that it is a homolog of the LuxR-type transcriptional regulator that responds to AHL molecules. In this study, using phenotypic and genetic analyses in P. syringae pv. tabaci, we show the effect of PsyR protein as a quorum-sensing (QS) transcriptional regulator. Regulatory effects of PsyR on swarming motility and production of siderophores, tabtoxin, and N-acyl homoserine lactones were examined via phenotypic assays, and confirmed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Further qRT-PCR showed that PsyR regulates expression of these virulence genes in response to environmental signals. However, an upstream region of the gene was not bound with purified MBP-PsyR protein; rather, PsyR was only able to shift the upstream region of psyI. These results suggested that PsyR may be indirectly controlled via intermediate-regulatory systems and that auto-regulation by PsyR does not occur.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1575-1579
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• 2012

Paenibacillus polymyxa is known to be a plant-growth-promoting rhizobacterium. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the specific detection and quantitation of P. polymyxa using a primer pair based on the sequence of a membrane-fusion protein for the amplification of a 268 bp DNA fragment. This study reports that the qPCR-based method is applicable for the rapid and sensitive detection of P. polymyxa and can be used as an alternative method for agricultural soil monitoring.

• Korean Journal of Veterinary Service
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• v.46 no.4
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• pp.269-281
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• 2023

In this study, a new triplex real-time quantitative reverse transcription polymerase chain reaction (tqRT-PCR) assay was developed for the rapid and differential detection of three feline viral pathogens including feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), and influenza A virus (IAV) in a single reaction. The assay specifically amplified three targeted viral genes with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with intra- and inter-assay coefficients of variation of less than 1%. Based on the diagnostic results of the assay using 120 clinical samples obtained from cats with feline respiratory disease complex (FRDC)-suspected signs, the prevalence of FCV, FHV-1, or IAV was 43.3%, 22.5%, or 0%, respectively, indicating that the diagnostic sensitivity was comparable or superior to those of previously reported monoplex qRT-PCR/qPCR assays. The dual infection rate for FCV and FHV-1 was 8.3%. These results indicate that FCV and FHV-1 are widespread and that co-infection with FCV and FHV-1 frequently occur in the Korean cat population. The developed tqRT-PCR assay will serve as a promising tool for etiological and epidemiological studies of these three bacterial pathogens, and the prevalence data for three feline viruses obtained in this study will contribute to expanding knowledge about the epidemiology of FRDC in the current Korean cat population.

• Genomics & Informatics
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• v.21 no.4
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• pp.52.1-52.10
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• 2023

Accurate and efficient microbial diagnosis is crucial for effective molecular diagnostics, especially in the field of human healthcare. The gold standard equipment widely employed for detecting specific microorganisms in molecular diagnosis is quantitative real-time polymerase chain reaction (qRT-PCR). However, its limitations in low metagenomic DNA yield samples necessitate exploring alternative approaches. Digital PCR, by quantifying the number of copies of the target sequence, provides absolute quantification results for the bacterial strain. In this study, we compared the diagnostic efficiency of qRT-PCR and digital PCR in detecting a particular bacterial strain (Staphylococcus aureus), focusing on skin-derived DNA samples. Experimentally, specific primer for S. aureus were designed at transcription elongation factor (greA) gene and the target amplicon were cloned and sequenced to validate efficiency of specificity to the greA gene of S. aureus. To quantify the absolute amount of microorganisms present on the skin, the variable region 5 (V5) of the 16S rRNA gene was used, and primers for S. aureus identification were used to relative their amount in the subject's skin. The findings demonstrate the absolute convenience and efficiency of digital PCR in microbial diagnostics. We suggest that the high sensitivity and precise quantification provided by digital PCR could be a promising tool for detecting specific microorganisms, especially in skin-derived DNA samples with low metagenomic DNA yields, and that further research and implementation is needed to improve medical practice and diagnosis.

• Genomics & Informatics
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• v.21 no.1
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• pp.13.1-13.8
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• 2023

Importance of accurate molecular diagnosis and quantification of particular disease-related pathogenic microorganisms is highlighted as an introductory step to prevent and care for diseases. In this study, we designed a primer/probe set for quantitative real-time polymerase chain reaction (qRT-PCR) targeting rgpA gene, known as the specific virulence factor of periodontitis-related pathogenic bacteria 'Porphyromonas gingivalis', and evaluated its diagnostic efficiency by detecting and quantifying relative bacterial load of P. gingivalis within saliva samples collected from clinical subjects. As a result of qRT-PCR, we confirmed that relative bacterial load of P. gingivalis was detected and quantified within all samples of positive control and periodontitis groups. On the contrary, negative results were confirmed in both negative control and healthy groups. Additionally, as a result of comparison with next-generation sequencing (NGS)-based 16S metagenome profiling data, we confirmed relative bacterial load of P. gingivalis, which was not identified on bacterial classification table created through 16S microbiome analysis, in qRT-PCR results. It showed that an approach to quantifying specific microorganisms by applying qRT-PCR method could solve microbial misclassification issues at species level of an NGS-based 16S microbiome study. In this respect, we suggest that P. gingivalis-specific primer/probe set introduced in present study has efficient applicability in various oral healthcare industries, including periodontitis-related microbial molecular diagnosis field.

• Korean Journal of Veterinary Service
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• v.41 no.1
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• pp.29-39
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• 2018

In this study, we developed a sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid visual detection of foot-and-mouth disease virus (FMDV) circulated in Korea. The RT-LAMP was completed in 40 min at $62^{\circ}C$ and the results of the assay were directly detected by naked eye without any detection process. The assay specifically amplified all 7 serotypes of FMDV RNAs but not amplified other viral and cellular nucleic acids. The sensitivity of the RT-LAMP was $10^2$, $10^3$ and $10^3TCID_{50}/mL$ for serotype O, A and Asia 1 FMDV, respectively, which was comparable to conventional reverse transcription polymerase chain reaction (RT-PCR) and relatively lower than that of real time quantitative RT-PCR (qRT-PCR). Clinical evaluation of the RT-LAMP using different serotypes of Korean and foreign FMDV strains showed a 100% (35/35) agreement with the results of the RT-PCR and qRT-PCR. These results indicated that RT-LAMP assay developed in this study could be a valuable diagnostic method for FMDV monitoring and surveillance.

• Biomedical Science Letters
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• v.28 no.1
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• pp.42-49
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• 2022

Tetrodotoxin (TTX) is a natural neurotoxin found in several species of puffer fish belonging to Tetraodon fugu genus and has been reported to affect processes such as proliferation, metastasis and invasion of various cancer cells. However, it was not revealed which genes were influenced by these reactions. In this experiment, it was examined in human SW620 colorectal cancer cells. The proliferation of SW620 cells was significantly reduced when treated with 0, 1, 10 and 100 μM TTX for 48 h. It was confirmed using Annexin V-propidium iodide staining that some apoptosis was induced. Differentially expressed genes (DEGs) affecting cell proliferation through RNA sequencing (RNA-seq) were selected. The expression change of DEGs was confirmed by conducting quantitative real-time polymerase chain reaction (qRT-PCR). As a result, the mRNA expression of FOS and WDR48 genes was found to be increased in the 100 μM TTX treatment group compared to the control group. On the other hand, the mRNA expression of ALKBH7, NDUFA13, RIPPLY3 and SELENOM genes was found to be reduced, and in the case of the ALKBH7 gene was identified to show significant differences. This experiment suggests that TTX can be used as an important fundamental data to elucidate the mechanism that inhibits the proliferation of SW620 cells.

• Journal of Veterinary Science
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• v.22 no.6
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• pp.87.1-87.12
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• 2021

Background: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. Objectives: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. Methods: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. Results: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 10⁰ copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. Conclusions: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.