• The Plant Pathology Journal
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• 제39권6호
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• pp.614-624
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• 2023

Botrytis cinerea is a major fungal plant pathogen that causes gray mold disease in strawberries, leading to a decrease in strawberry yield. While benzimidazole is widely used as a fungicide for controlling this disease, the increasing prevalence of resistant populations to this fungicide undermines its effectiveness. To investigate benzimidazole resistant B. cinerea in South Korea, 78 strains were isolated from strawberries grown in 78 different farms in 2022, and their EC₅₀ values for benzimidazole were examined. As a result, 64 strains exhibited resistance to benzimidazole, and experimental tests using detached strawberry leaves and the plants in a greenhouse confirmed the reduced efficacy of benzimidazole to control these strains. The benzimidazole resistant strains identified in this study possessed two types of mutations, E198A or E198V, in the TUB2 gene. To detect these mutations, TaqMan probes were designed, enabling rapid identification of benzimidazole resistant B. cinerea in strawberry and tomato farms. This study utilizes TaqMan real-time polymerase chain reaction analysis to swiftly identify benzimidazole resistant B. cinerea, thereby offering the possibility of effective disease management by identifying optimum locations and time of application.

• 대한화학회지
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• 제54권2호
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• pp.215-221
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• 2010

살모넬라는 음식과 식수에서 흔히 나오는 중요한 병원체로 세계 곳곳에서 급성 위장염과 같은 감염증을 일으키며, 일반적으로 인간의 혈청형 임상종으로는 Salmonella enterica의 혈청형인 S. Typhimurium과 S. Enteritidis가 있다. 일반적인 검출 방법으로 살모넬라를 기본으로 하여 선택적인 배양으로 샘플을 수집하였고 살모넬라를 일으키는 군체의 특징을 생화학과 혈청학상인 테스트를 하였으나 이러한 방법들은 일반적으로 시간이 걸리고 높은 감도를 보이지 않았다. 최근, 등온증폭반응법과 real-time PCR법을 이용하여 높은 감도, 특이성으로 현재 병원성 박테리아에 빠르게 수행할 수 있게 되었다. 본 연구에서는 등온증폭반응과 real-time PCR법을 사용하여 S. Typhimurium과 S. Enteritidis의 검출하였다. 선택적인 타겟 유전자로, invA를 살모넬라종의 염기서열에 특이적으로 임의복제 하였다. 등온증폭반응과 real-time PCR은 살모넬라종으로부터 임의의 염기서열을 증폭하여 검출하였고, invA는 S. Typhimurium과 S. Enteritidis의 두 가지 종을 모두 검출하였다. 이러한 등온증폭반응과 real-time PCR법으로 S.Typhimurium과 S. Enteritidis의 검출 가능성을 보였으며, 살모넬라 종에 대한 특이성, 민감성을 갖춘 유용한 검출방법을 제시하였다.

• Journal of Microbiology and Biotechnology
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• 제24권8호
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• pp.1026-1033
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• 2014

The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets.

• 한국축산식품학회지
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• 제31권6호
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• pp.827-833
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• 2011

The objective of this study was to develop a fluorogenic real-time PCR-based assay for detecting and quantifying amounts of cow milk in cow/goat milk mixtures or goat milk products. In order to quantify the exact amount of cow milk in cow/goat raw milk mixtures and commercial goat milk products, it was necessary to achieve quantitative extraction of total genomic DNA from the raw milk matrix. Both mammalian-specific PCR and cow-specific PCR were performed. A cow-specific 252 bp band obtained from the raw cow milk and raw goat milk mixtures, commercial goat milk, and two goat milk powders was identified, along with the relationship between the cow milk amount and band intensity of the electrophoresis image. The detection threshold was found to be 0.1%. The expression of cow's 12S rRNA in the cow/goat milk mixtures, commercial goat milk, and two goat milk powders was identified. The expression quantity of the milk 12S rRNA increased with increasing ratios of the cow/goat milk mixtures. Using these calibrated relative expression levels as a standard curve in the cow/goat raw milk mixtures, the contents of cow milk were 1.8% in the commercial goat milk, 9.6% in goat milk powder A, and 11.6% in goat milk powder C. However, cow milk was not detected in goat milk powder B.

• ALGAE
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• 제31권2호
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• pp.167-174
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• 2016

Biological response of cells to variable conditions should affect the expression level of certain genes. Quantification of these changes in target genes needs stable internal controls. Real-time quantitative polymerase chain reaction (PCR) has traditionally used reference or ‘housekeeping’ genes, that are considered to maintain equal expression in different conditions, to evaluate changes in target genes between samples and experimental conditions. Recent studies showed that some housekeeping genes may vary considerably in certain biological samples. This has not been evaluated in red algae. In order to identify the optimal internal controls for real-time PCR, we studied the expression of eleven commonly used housekeeping genes; elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, β-actin, polyubiquitin, 30S ribosomal gene, 60S ribosomal gene, beta-tubulin, alpha-tubulin, translation initiation factor, ubiquitin-conjugating enzyme, and isocitrate dehydrogenase in different life-history stages of Bostrychia moritziana. Our results suggest that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 30S ribosomal gene, have the most stable gene expression levels between the different life history stages (male, female, carposporophyte, and tetrasporophyte), while the other genes are not satisfactory as internal controls. These results suggest that the combinations of GAPDH and 30S would be useful as internal controls to assess expression level changes in genes that may control different physiological processes in this organism or that may change in different life history stages. These results may also be useful in other red algal systems.

• Journal of Veterinary Science
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• 제22권3호
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• pp.40.1-40.12
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• 2021

Background: The microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen. Objectives: The present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees. Methods: A procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration. Results: UR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min. Conclusions: UR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.

• 한국동물위생학회지
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• 제45권3호
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• pp.145-153
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• 2022

Pathogens such as feline herpesvirus, feline calicivirus, Bordetella bronchiseptica, Chlamydia felis, Mycoplasma felis and Pasteurella multocida usually cause feline upper respiratory tract disease (URTD). Real-time PCR was used to analyze the detection and prevalence of the most common respiratory pathogens in cats with (n=69) and without respiratory signs (n=31). Pathogens were detected in 53 cats, divided into 37 (69.8%) with a single pathogen, 15 (28.3%) with two pathogens, and 1 (1.9%) with three pathogens. M. felis had the highest detection rate in 29 (42.0%) cats, P. multocida was detected in 18 (26.1%), FHV in 10 (14.5%), FCV in 7 (10.1%), B. bronchiseptica in 3 (4.3%), and C. felis in 2 (2.9%). M. felis was the most frequently detected pathogen in cats living outdoors without vaccination. Of the 37 cats infected with single pathogen, nasal discharge was observed in 13 (35.1%), ocular signs in 6 (16.2%), drooling in 5 (13.5%), dyspnea in 3 (8.1%), and asymptomatic in 10 (27.0%). In 51 outdoor and 49 indoor cats, pathogens were detected in 35 (68.6%) and 18 (36.7%) cats, respectively. Of the 29 cats infected with M. felis, 22 (75.9%) showed respiratory signs, and 7 (24.1%) were healthy. In the age of the 53 positive cats, 10 (18.9%) were under the age of 1 year, 26 (49.1%) were aged 1~3 years, and 17 (32.1%) were aged 3 years or older. Although the number of cats in the study was small, the results can provide valuable data on the prevalence of URTD in Korean cats.

• The Plant Pathology Journal
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• 제38권3호
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• pp.229-238
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• 2022

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is the most serious soil-borne disease in the world and has become the main limiting factor of watermelon production. Reliable and quick detection and quantification of Fon are essential in the early stages of infection for control of watermelon Fusarium wilt. Traditional detection and identification tests are laborious and cannot efficiently quantify Fon isolates. In this work, a real-time polymerase chain reaction (PCR) assay has been described to accurately identify and quantify Fon in watermelon plants and soil. The FONRT-18 specific primer set which was designed based on identified specific sequence amplified a specific 172 bp band from Fon and no amplification from the other formae speciales of Fusarium oxysporum tested. The detection limits with primers were 1.26 pg/µl genomic DNA of Fon, 0.2 pg/ng total plant DNA in inoculated plant, and 50 conidia/g soil. The PCR assay could also evaluate the relationships between the disease index and Fon DNA quantity in watermelon plants and soil. The assay was further used to estimate the Fon content in soil after disinfection with CaCN₂. The real-time PCR method is rapid, accurate and reliable for monitoring and quantification analysis of Fon in watermelon plants and soil. It can be applied to the study of disease diagnosis, plant-pathogen interactions, and effective management.

• Journal of Animal Science and Technology
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• 제65권4호
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• pp.767-778
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• 2023

The aim of the research is to identify that porcine oocytes can function as recipients for interspecies cloning and have the ability to develop to blastocysts. Furthermore each mitochondrial DNA (mtDNA) in interspecises cloned embryos was analyzed. For the study, mouse-porcine and porcine-porcine cloned embryos were produced with mouse fetal fibroblasts (MFF) and porcine fetal fibroblasts (PFF), respectively, introduced as donor cells into enucleated porcine oocytes. The developmental rate and cell numbers of blastocysts between intraspecies porcine-porcine and interspecies mouse-porcine cloned embryos were compared and real-time polymerase chain reaction (PCR) was performed for the estimate of mouse and porcine mtDNA copy number in mouse-porcine cloned embryos at different stages.There was no significant difference in the developmental rate or total blastocyst number between mouse-porcine cloned embryos and porcine-porcine cloned embryos (11.1 ± 0.9%, 25 ± 3.5 vs. 10.1 ± 1.2%, 24 ± 6.3). In mouse-porcine reconstructed embryos, the copy numbers of mouse somatic cell-derived mtDNA decreased between the 1-cell and blastocyst stages, whereas the copy number of porcine oocyte-derived mtDNA significantly increased during this period, as assessed by real-time PCR analysis. In our real-time PCR analysis, we improved the standard curve construction-based method to analyze the level of mtDNA between mouse donor cells and porcine oocytes using the copy number of mouse beta-actin DNA as a standard. Our findings suggest that mouse-porcine cloned embryos have the ability to develop to blastocysts in vitro and exhibit mitochondrial heteroplasmy from the 1-cell to blastocyst stages and the mouse-derived mitochondria can be gradually replaced with those of the porcine oocyte in the early developmental stages of mouse-porcine cloned embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제30권9호
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• pp.1350-1357
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• 2017

Objective: The present study aimed to investigate whether the long-lasting, recurrent restricting of sows leads to the physiological and psychological reaction of discomfort. Methods: Sows (Large White) that had experienced restricting for about 0.5 or 3 years and agematched sows kept in a group housing system (loose sows) were compared. Pupillary light reflex parameters were measured at the weaning stage. Immediately after slaughter, blood samples were taken to measure serum cortisol levels, and the brain was dissected, gene expression in the hippo-campus, frontal cortex and hypothalamus was analyzed. Results: The serum cortisol levels were higher in the confined sows than in the loose sows. The full maturity, but not the young adolescent, confined sows had longer latency time in the onset of pupil constriction than their loose counterparts. Real-time polymerase chain reaction analyses revealed an increased expression of interleukin 6 mRNA in the hippocampus and decreased expression of brain derived neurotrophic factor mRNA in hippocampus and hypothalamus and to a lesser extent in the frontal cortex of the full maturity confined sows, compared with the full maturity loose sows. Conclusion: Taken together, these data indicated that recurrent restricting stress in full maturity sows leads to the physiological and psychological reaction of discomfort.