• Journal of Veterinary Science
• /
• v.21 no.2
• /
• pp.22.1-22.9
• /
• 2020

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID₅₀/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

• Research in Plant Disease
• /
• v.17 no.1
• /
• pp.52-58
• /
• 2011

The aim of this research was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant pathogenic bacteria and seed borne virus in commercial Brassicaceae crop seeds, Xanthomonns campestris pv. campestris (Xcc) and Lettuce Mosaic Virus (LMV). Bacterial and virus diseases of Brassicaceae leaves are responsible for heavy losses. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcc and LMV in commercial Brassicaceae crop seeds (lettuce, kohlrabi, radish, chinese cabbage and cabbage), two pairs of specific primer (LMV-F/R, Xcc-F/R) were synthesized by using primer-blast program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The multiplex PCR for the two pathogens in Brassicaceae crop seeds could detect specifically without interference among primers and/or cDNA of other plant pathogens. The pathogen detection limit was determined at 1 ng of RNA extracted from pathogens. In the total PCR results for pathogen detection using commercial kohlrabi (10 varieties), lettuce (50 varieties), radish (20 varieties), chinese cabbage (20 varieties) and cabbage (20 varieties), LMV and Xcc were detected from 39 and 2 varieties, respectively. In the PCR result of lettuce, LMV and Xcc were simultaneously detected in 8 varieties.

• Research in Plant Disease
• /
• v.17 no.1
• /
• pp.44-51
• /
• 2011

The aim of this study was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant seed infection pathogenic bacteria and virus, Xanthomonns campestris pv. vesicatoria (Xcv), Clavibacter michiganensis subsp. michiganensis (Cmm), Erwinia carotovora subsp. carotovora (Ecc), Pepper mild mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV) in pepper and tomato seeds. Most of pepper and tomato bacterial and virus diseases are responsible for germination and growth obstruction. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcv, Cmm, Ecc, PMMoV and TMGMV in pepper and tomato seeds, five pairs (Cmm-F/R, Ecc-F/R, Xcv-F/R, PMMoV-F/R, TMGMV-F/R) of specific primer were synthesized by primer-blast program. The multiplex PCR for the five pathogens in pepper and tomato seeds could detect specially without interference among primers and/or cDNA of plant seeds and other plant pathogens. The PCR result for pathogen detection using 20 commercial pepper and 10 tomato seed samples, Ecc was detected from 4 pepper and 2 tomato seed samples, PMMoV was detected from 1 pepper seed sample, and PMMoV and TMGMV were simultaneously detected from 1 pepper seed sample.

• Pediatric Infection and Vaccine
• /
• v.27 no.2
• /
• pp.90-101
• /
• 2020

Purpose: The multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) test developed recently can help detect enteric pathogens of acute gastroenteritis (AGE). This study aimed to investigate the epidemiology of pathogens in children with AGE using the multiplex RT-PCR. Methods: From May 2015 to June 2018, multiplex RT-PCR tests were performed to identify pathogens in the feces of pediatric patients diagnosed with AGE at a secondary hospital in Seoul, Korea. Results: Of the 1,366 stool samples examined for viral pathogens, 483 (35.3%) tested positive for ≥1 pathogen. Group A rotavirus (RV) was detected in 106 cases (7.8%). The positivity rate increased annually from 3.0% (8/263) to 16.7% (48/288) and surged in 2018 (P<0.001). Norovirus (NoV) GII was the most common viral pathogen (263/1,366, 19.3%), and the positivity rate did not increase during the 3 years. Of the 304 stool samples tested for bacterial pathogens, Campylobacter spp. was the most common bacterial pathogen (32/304, 10.5%), followed by Clostridium difficile (22/304, 7.2%) and Salmonella spp. (17/304, 5.6%). The positivity rate of these bacterial pathogens did not change significantly during the study period. Conclusions: NoV GII is the main pathogen in childhood AGE since the introduction of RV vaccine, yet the number of rotavirus-infected patients increased during our study, especially in 2018. Therefore, further research is needed including the possibility of emergence of novel RV strains. Campylobacter spp. is the predominant cause of bacterial AGE in children. For proper treatment, the clinical characteristics of the bacteria should be taken into consideration, and continuous monitoring is necessary.

• Journal of Plant Biotechnology
• /
• v.42 no.2
• /
• pp.117-122
• /
• 2015

Canola is a crop globally used for production of oil and biofuel. Cultivation area and import volume of living modified (LM) canola have been increasing every year. As canola import dependence has reached 100% in Korea, efforts have been made for safety management of LM canola and ecological risk assessment. We developed a set of multiplex PCR method for simultaneous detection of 5 LM canola events (Topas 19/2, Rf3, Ms8, RT73 and T45) approved in Korea. The multiplex PCR assay developed allows amplification of estimated products of 5 LM canolas from event specific primer sets. Primer extension time was skipped for a time-consuming process and two annealing steps (20 cycles at $55^{\circ}C$ and 20 cycles at $60^{\circ}C$) were performed for yielding the best result which was sufficient to distinguish five LM canolas. Our results suggest that multiplex PCR method provides a cost and time-effective approach for LM canola detection.

• 한국생물공학회:학술대회논문집
• /
• 2003.10a
• /
• pp.232-234
• /
• 2003

• The Plant Pathology Journal
• /
• v.27 no.1
• /
• pp.44-52
• /
• 2011

Field surveys were conducted in 45 stone fruit orchards in seven districts of Isparta Province located in western Mediterranean region of Turkey important for stone fruit production. Leaf samples were collected from 175 trees showing virus-like symptoms. These samples were first tested by ELISA for five different RNA viruses including Apple mosaic ilarvirus (ApMV), Prunus necrotic ringspot ilarvirus (PNRSV), Prune dwarf ilarvirus (PDV), Plum pox potyvirus (PPV), Apple chlorotic leafspot trichovirus (ACLSV). While no ApMV and PPV infection was found, 46, 24 and 16 samples were tested positive for PDV, ACLSV and PNRSV, respectively, in ELISA showing about 45% of symptomatic trees in the region were infected with at least one of these viruses. In addition, it was found that nine sweet cherry trees were mixed infected with two or three of these viruses and PDV with an infection rate of 26.3% was the most widespread virus in symptomatic trees in western Mediterranean region. Thirty samples were selected and tested by a multiplex RT-PCR (mRT-PCR) for simultaneous detection of these viruses. While PPV was not detected, more than half of the tested 20 samples were individually or mixed infected with ApMV, ACLSV, PNRSV and PDV. The mRT-PCR results were confirmed by detection of these viruses individually in some of the field samples using RT-PCR with primes specific to each virus. Comparison of ELSA and mRT-PCR results of 30 samples showed that numbers of infected and mixed infected samples as well as infection and mixed infection rates were significantly higher in RT-PCR (20 and 66.7%) than in ELISA (14 and 46.7%). The results confirm that mRT-PCR is more sensitive than ELISA.

• Microbiology and Biotechnology Letters
• /
• v.47 no.2
• /
• pp.317-322
• /
• 2019

Gastroenteritis with diarrhea is one of the most infectious diseases in the world following respiratory infections. Notably, diarrhea-causing viruses (DVs) cause more than 70% of such cases. In this study, 3,065 stool specimens from patients with diarrhea (median age, 1.1 years; range, 0.0-91.1 years), who were admitted to the DanKook University Hospital, were examined using multiplex reverse transcription PCR (mRT-PCR). The target viruses were astrovirus (AstV), enteric adenovirus (EAdV), group A rotavirus (RotV), norovirus GI (NoV-GI), and norovirus GII (NoV-GII). The mRT-PCR results were analyzed based on various factors such as seasonality, age, presence of co-infection, and analyzed trends. The detection rate of the DVs during the study period was found to be 30.8% (n = 943/3,065). When the detection rate was analyzed monthly, the DV detection rate was found to be highest between December to January. Of the detected DVs, NoV-GII was the most common, accounting for 45.5% of the detected viruses (n = 446/980). Notably, 86.5% (n = 848/980) of the pathogens were detected in individuals who were less than 5 years of age. During the study period, NoV-GII and RotV showed alternating trends. In addition, both the number and rate of co-infections increased.

• The Plant Pathology Journal
• /
• v.40 no.2
• /
• pp.125-138
• /
• 2024

Citrus yellow vein clearing virus (CYVCV) is a member of the Alphaflexiviridae family that causes yellow vein clearing symptoms on citrus leaves. A total of 118 leaf samples from nine regions of six provinces in Korea were collected from various citrus species in 2020 and 2021. Viral diagnosis using next-generation sequencing and reverse transcription polymerase chain reaction (RT-PCR) identified four viruses: citrus tristeza virus, citrus leaf blotch virus, citrus vein enation virus, and CYVCV. A CYVCV incidence of 9.3% was observed in six host plants, including calamansi, kumquat, Persian lime, and Eureka lemon. Among the citrus infected by CYVCV, only three samples showed a single infection; the other showed a mixed infection with other viruses. Eureka lemon and Persian lime exhibited yellow vein clearing, leaf distortion, and water-soak symptom underside of the leaves, while the other hosts showed only yellowing symptoms on the leaves. The complete genome sequences were obtained from five CYVCV isolates. Comparison of the isolates reported from the different geographical regions and hosts revealed the high sequence identity (95.2% to 98.8%). Phylogenetic analysis indicated that all the five isolates from Korea were clustered into same clade but were not distinctly apart from isolates from China, Pakistan, India, and Türkiye. To develop an efficient diagnosis system for the four viruses, a simultaneous detection method was constructed using multiplex RT-PCR. Sensitivity evaluation, simplex RT-PCR, and stability testing were conducted to verify the multiplex RT-PCR system developed in this study. This information will be useful for developing effective disease management strategies for citrus growers in Korea.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 2003.10a
• /
• pp.150.1-150
• /
• 2003

A single-step multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for the simultaneous detection of five cucurbit-infecting viruses： cucumber mosaic virus (CMV), watermelon mosaic virus 2 (WMV2), zucchini yellow mosaic virus (ZYMV), cucumber green mottle mosaic virus (CGMMV), and kyuri green mottle mosaic virus (KGMMV). The multiplex RT-PCR provides a simple and rapid method for detecting various viruses in cucurbit plants, which will help diagnose many cucurbit plants at a time.