• 한국동물위생학회지
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• 제46권2호
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• pp.123-135
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• 2023

Feline calicivirus (FCV) is considered the main viral pathogen of feline upper respiratory tract disease (URTD). The frequent mutations of field FCV strains result in the poor diagnostic sensitivity of previously developed molecular diagnostic assays. In this study, a more sensitive real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for broad detection of currently circulating FCVs and comparatively evaluated the diagnostic performance with previously developed qRT-PCR assay using clinical samples collected from Korean cat populations. The developed qRT-PCR assay specifically amplified the FCV p30 gene with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 2%. Based on the clinical evaluation using 94 clinical samples obtained from URTD-suspected cats, the detection rate of FCV by the developed qRT-PCR assay was 47.9%, which was higher than that of the previous qRT-PCR assay (43.6%). The prevalence of FCV determined by the new qRT-PCR assay in this study was much higher than those of previous Korean studies determined by conventional RT-PCR assays. Due to the high sensitivity, specificity, and accuracy, the new qRT-PCR assay developed in this study will serve as a promising tool for etiological and epidemiological studies of FCV circulating in Korea. Furthermore, the prevalence data obtained in this study will contribute to expanding knowledge about the epidemiology of FCV in Korea.

• 식물병연구
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• 제30권1호
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• pp.50-59
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• 2024

Charcoal rot disease, caused by the fungus Macrophomina phaseolina, is one of the most important diseases of Sesame (Sesamum indicum) all over the world. However, the population biology of M. phaseolina is poorly understood. In this study, M. phaseolina isolates from five different regions of Iran (Khuzestan, Fars, Bushehr, Hormozgan, and Kohgiluyeh & Boyer-Ahmad provinces) (n=200) were analyzed for genetic variation using inter simple sequence repeats marker. In total, 152 unique haplotypes were identified among the 200 M. phaseolina isolates, and gene diversity (H=0.46-0.84) and genotypic diversity were high in each of the regions. The structure analysis clustered five Iranian populations into two distinct groups, the individuals from group 1 were assigned to the Bushehr population and the individuals from Khuzestan, Fars, Hormozgan and Kohgiluyeh & Boyer-Ahmad were aggregated and formed group 2. The results matched with genetic differentiation and gene flow among regions. Analyses of the distribution of gene diversity within and among five Iranian populations were 61% and 39%, respectively. Our results showed that infected seeds are thought to be the dominant mechanism responsible for the spreading of the pathogen in southern parts of Iran. In summary, it is essential to have local quarantine and prevent seed exchanges between geographical populations to restrict the dispersal of pathogen over long distances and provide certified seeds in Iran.

• 한국균학회지
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• 제46권2호
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• pp.186-192
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• 2018

PCR 기술을 이용하여 고추와 토마토의 탄저병을 일으키는 Colletotrichum coccodes 균을 빠르고 정확하게 검출하는 방법을 개발하였다. Colletotrichum 13종 22개 균주로 부터 translation elongation factor 1 alpha 유전자를 분석하여 C. coccodes에 특이적인 coccoTef-F/cocco Tef-R primer set를 제작하였다. 제작된 primer를 사용한 결과 일반 PCR 방법으로 10 ng, real-time PCR 방법으로는 10 pg 수준에서 C. coccodes가 특이적으로 검출이 가능하였다. 인공적으로 C. coccodes에 감염시킨 고추와 토마토 종자에서도 일반 PCR 방법과 real-time PCR 방법 모두 C. coccodes검출이 가능하였다. 본 연구에서 개발한 PCR 방법은 수출입되는 종자에서 신속하고 정확하게 탄저병균 C. coccodes를 특이적으로 검출하는데 활용될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1782-1786
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• 2015

A new improved PCR method has been developed for the rapid, reliable, and sensitive detection of Venturia nashicola, a destructive pathogen of scab disease in Japanese pear. The translation elongation factor-1 alpha gene-derived PCR primers specifically amplified a 257-bp-sized DNA band of the target gene from the genomic DNA of V. nashicola. No amplicon was produced from the genomic DNA of other Venturia spp. and reference fungal species tested. With the high detection limit of 10 fg DNA content, our real-time method could be used for the quarantine inspection and field monitoring of V. nashicola.

• 식물병연구
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• 제16권2호
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• pp.129-135
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• 2010

강낭콩 종자로부터 강낭콩(Phaseolus vulgaris L.)에서 halo blight(달무리마름병)을 일으키는 종자 전염 병원세균인 Pseudomonas savastanoi pv. phaseolicola를 검출하는 PCR 방법을 개발하였다. 프라이머 Psp-JH-F와 Psp-JH-R는 오직 Pseudomonas savastanoi pv. phaseolicola로 부터 513 bp 크기의 DNA를 증폭하였다. 1차 PCR 증폭 산물의 안쪽에서 디자인 한 nested PCR 용 프라이머인 psp-JH-F-ne and psp-JH-R-ne는 오직 Pseudomonas savastanoi pv. phaseolicola로부터 169 bp 크기의 DNA를 증폭하였다. 이들 프라이머들은 강낭콩, 완두, 대두 등을 포함 콩과 종자 추출액으로부터 어떤 비특이적 DNA도 증폭하지 않았다. 인공적으로 병원균을 접종한 강낭콩 종자를 이용하여 병원균 검출 민감도를 비교하였을 때, 본 연구에서 개발한 nested PCR 방법이 ELISA나 선택배지 보다 훨씬 높은 민감도를 보여주었다. 본 연구에서 개발한 PCR방법들은 강낭콩 종자로부터 Pseudomonas savastanoi pv. phaseolicola를 검출하는 매우 유용한 방법으로 생각된다.

• 식물병연구
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• 제22권4호
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• pp.269-275
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• 2016

E. pyrifoliae에 의한 과수 가지검은마름병은 국내에서 1995년 최초 발생이래 2016년까지 꾸준히 발생하여 과수농가에 피해를 주고 있는 세균병해이다. 가지검은마름병 발생 농가의 폐원조치 및 공적방제로 인한 경제적 피해 감소를 위하여, 병징이 관찰되는 이병조직 및 건전조직 내의 병원 세균을 검출하여 효율적 관리방안을 마련하고자 본 연구를 수행하였다. 가지검은마름병원세균의 검출은 genomic DNA 추출과정을 생략한 순수 균총만을 이용하는 colony-PCR을 이용하였으며, 이를 위해 ERIC 지역에서 제작된 가지검은마름병원세균 특이 프라이머 EpSPF/EpSPR 프라이머쌍을 선발하였다. 특이 프라이머를 활용한 colony-PCR 방법으로 2014-2015년 4-10월까지 사과나무 생육기간 동안 가지검은마름병 발생상황을 모니터링한 결과, $25^{\circ}C$ 일 평균 온도 기간인 5월 중순부터 7월 초순까지 발병이 가장 빈번하였다. 발병가지 내 병원세균의 존재유무 검정 결과 병징 부위와 이로부터 20 cm 내 건전조직에서만 병원세균이 지속적으로 검출되었다. 따라서 이미 발생한 가지검은마름병을 효율적으로 관리하기 위해 이병조직과 건전조직 경계 부위로부터 20 cm 이상에서 가지치기를 하는 것이 매우 적절할 것으로 판단된다.

• 생명과학회지
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• 제19권10호
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• pp.1451-1455
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• 2009

Porcine cytomegalovirus (PCMV) is a betaherpesvirus which causes reproductive failure in breeding sows and generalized infection in newborn piglets. It has worldwide distribution including Korea. Serological survey on this virus has been reported in 76.3% of pigs, but virological survey and epidemiological analysis on PCMV distribution have been reported in only a few papers in Korea. In this study, we investigated the virological prevalence and infection status of PCMV on a farm level in selected swine farms with respiratory diseases. A total of 1,938 blood samples taken from groups of pigs of different ages were collected from 31 farms distributed nationwide in 2006 and 2007 and tested by PCR to detect the presence of PCMV. Virological prevalence at farm level and pig level were 96.8% and 17.5%, respectively, suggesting that PCMV has endemically infected Korean pig herds. The prevalence at farm level in gilts, sows and suckling piglet groups were 16.7%, 36.7% and 56.7%, indicating that vertical infections frequently occurred in conception or newborn stage. Thereafter, detection rates of PCMV were slightly increased in pig groups aged 40 and 70 days (70.0% and 73.3%), and then gradually decreased as they aged - 33.3% in 100, 26.7% in 130 and 16.7% in 160 day old pig groups. The prevalence at pig level has similar patterns to that at farm level. With the passage of time, the variation of infection patterns of PCMV was investigated in four PCMV-positive farms. Three blood samples were collected at intervals of 6 months in each farm, and examined for presence of PCMV using PCR. The results revealed that once PCMV was introduced to the pig farms, it continuously circulated between and within groups of sows and piglets in those farms. Taken together, it can be concluded that PCMV has endemically infected Korean pig farms and has the potential risk for emerging pathogen in combination with the known endemic pathogens including porcine reproductive, respiratory syndrome virus and porcine circovirus type 2. Therefore, more research is needed on diagnosis, epidemiology and control strategy for PCMV on the field.

• 농업과학연구
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• 제42권2호
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• pp.99-103
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• 2015

Potato virus T (PVT) is a plant pathogen in the family Betaflexiviridae, group IV single-stranded positive sense RNA viruses. The major host of PVT is potato, and it has been reported in Ullucus tuberosus, Oxalis tuberosa and Tropaeolum tuberosum. This study aimed at developing reverse transcription (RT)-polymerase chain reaction (PCR) and nested PCR techniques for specific detection of PVT. Finally, Two RT-PCR primer sets were developed and verified. The RT-PCR products were amplified to 734 (PVT RT-PCR primer set 6) and 828 bp (PVT RT-PCR primer set 29) long to detect PVT. The nested PCR primer sets [PVT-N70/C20 ($734{\rightarrow}315bp$) and PVT-N75/C30 ($828{\rightarrow}529bp$)] were developed which are high sensitivity and verification for detection of PVT. Furthermore, a modified-positive control plasmid is use to verify contamination of laboratory in PVT detection. This study supported the diagnose PVT in potato or PVT related hosts.

• The Plant Pathology Journal
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• 제31권4호
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• pp.343-349
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• 2015

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most important bacterial diseases of citrus. Because citrus canker is not found in many countries including European Union and Australia, Xcc is strictly regulated in order to prevent its spread. In this study, the effects of X-irradiation on Xcc growth either in the suspension or on the surface of citrus fruits were investigated. The suspension containing $1{\times}10^7cfu/ml$ of Xcc was irradiated with different absorbed doses of X-irradiation ranging from 50 to 400 Gy. The results showed that Xcc was fully dead at 400 Gy of X-irradiation. To determine the effect of X-irradiation on quarantine, the Xcc-inoculated citrus fruits were irradiated with different X-ray doses at which Xcc was completely inhibited by an irradiation dose of 250 Gy. The $D_{10}$ value for Xcc on citrus fruits was found to be 97 Gy, indicating the possibility of direct application on citrus quarantine without any side sterilizer. Beside, presence of Xcc on the surface of asymptomatic citrus fruits obtained from citrus canker-infected orchards was noted. It indicated that the exporting citrus fruits need any treatment so that Xcc on the citrus fruits should be completely eliminated. Based on these results, ionizing radiation can be considered as an alternative method of eradicating Xcc for export of citrus fruits.

• 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.