• Research in Plant Disease
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• v.20 no.3
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• pp.173-181
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• 2014

The early and accurate detection of plant viruses is an essential component to control those. Because the globalization of trade by free trade agreement (FTA) and the rapid climate change promote the country-to-country transfer of viruses and their hosts and vectors, diagnosis of viral diseases is getting more important. Because symptoms of viral diseases are not distinct with great variety and are confused with those of abiotic stresses, symptomatic diagnosis may not be appropriate. From the last three decades, enzyme-linked immunosorbent assays (ELISAs), developed based on serological principle, have been widely used. However, ELISAs to detect plant viruses decrease due to some limitations such as availability of antibody for target virus, cost to produce antibody, requirement of large volume of sample, and time to complete ELISAs. Many advanced techniques allow overcoming demerits of ELISAs. Since the polymerase chain reaction (PCR) developed as a technique to amplify target DNA, PCR evolved to many variants with greater sensitivity than ELISAs. Many systems of plant virus detection are reviewed here, which includes immunological-based detection system, PCR techniques, and hybridization-based methods such as microarray. Some of techniques have been used in practical, while some are still under developing to get the level of confidence for actual use.

• 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.

• Korean Journal of Veterinary Research
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• v.55 no.1
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• pp.9-12
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• 2015

Brachyspira (B.) hyodysenteriae is a causative agent of swine dysentery that is responsible for death and economic losses in the pig industry. It is imperative that clinical samples be delivered fresh for accurate diagnosis. The viability and DNA detection of B. hyodysenteriae using lab-made (phosphate buffered saline and modified tryptic soy broth) or commercial transport media (C, D, and E) were compared by culturing and real-time PCR at $4^{\circ}C$ or room temperature (RT), respectively. B. hyodysenteriae grown in D (Anaerobe Systems, USA) and E (Starplex Scientific, Canada) media was viable for 4 days at $4^{\circ}C$ and RT. However, B. hyodysenteriae in A, B, and C (culture swab; BD Biosciences, USA) media were not recovered after 2 days at RT. Ct values for real-time PCR at $4^{\circ}C$ and RT ranged from $27.2{\pm}2.1$ (C) to $29.6{\pm}0.5$ (B), and $28.0{\pm}0.9$ (E) to $30.2{\pm}1.5$ (B), respectively. Considering the field conditions, it is important that transport media is used for specimen isolation and PCR to obtain an accurate diagnosis of swine dysentery.

• Research in Plant Disease
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• v.24 no.4
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• pp.348-352
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• 2018

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay was developed for the detection of Clover yellow vein virus (ClYVV), Peanut mottle virus (PeMoV), and Tomato spotted wilt virus (TSWV), which were recently reported to infect soybean and azuki bean in Korea. Species-specific primer sets were designed for the detection of each virus, and their specificity and sensitivity were tested using mixed primer sets. From among the designed primer sets, two combinations were selected and further evaluated to estimate the detection limits of uniplex, duplex, and multiplex RT-PCR. The multiplex RT-PCR assay could be a useful tool for the field survey of plant viruses and the rapid detection of ClYVV, PeMoV, and TSWV in leguminous plants.

• The Plant Pathology Journal
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• v.36 no.6
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• pp.651-657
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• 2020

Lotus is one of the most important aquatic vegetables in China. Previously, we detected sweet potato latent virus from lotus (SPLV-lotus) and found that it has highly significant sequence diversity with SPLV-sweet potato isolates (SPLV-sp). Here, we developed serological methods for the detection of SPLV-lotus in Chinese lotus cultivation areas. Based on the high sensitivity of SPLV-lotus coat protein antiserum, rapid, sensitive and large-scale diagnosis methods of enzyme-linked immunosorbent assay (ELISA) and dot blot in lotus planting area were developed. The established ELISA and dot blot diagnostic methods can be used to detect SPLV-lotus from samples successfully. And our results also showed that the SPLV-lotus and sweet potato isolates appeared clearly distinction in serology. Our study provides a high-throughput, sensitive, and rapid diagnostic method based on serology that can detect SPLV on lotus, which is suggested to be included in viral disease management approach due to its good detection level.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2000.04a
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• pp.1-1
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• 2000

• Korean Journal of Veterinary Service
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• v.45 no.4
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• pp.305-316
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• 2022

Bordetella (B.) bronchiseptica, Mycoplasma (M.) felis, and Chlamydia (C.) felis are considered as main bacterial pathogens of feline upper respiratory tract disease (URTD). In this study, a new triplex quantitative real-time polymerase chain reaction (tqPCR) assay was developed for the rapid and differential detection of these bacteria in a single reaction. The assay specifically amplified three bacterial genes with the 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 1%. Based on the diagnostic results of the assay using 94 clinical samples obtained from cats with URTD signs, prevalence of B. bronchiseptica, M. felis, or C. felis was 10.6%, 36.2%, or 6.4%, respectively, indicating that the diagnostic sensitivity was comparable to those of previously reported monoplex qPCR assays. The dual infection rates for B. bronchiseptica and M. felis or M. felis and C. felis was 2.1% or 3.2%, respectively. These results indicated that M. felis has been widely spread, and its co-infection with B. bronchiseptica or M. felis has been frequently occurred in Korean cat population. The developed tqPCR assay will serve as a promising tool for etiological and epidemiological studies of these three bacterial pathogens and the prevalence data obtained in this study will contribute to expanding knowledge about the epidemiology of feline URTD in Korea.

• Research in Plant Disease
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• v.16 no.2
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• pp.125-128
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• 2010

Bacterial black spot disease of prune fruit (Prunus salicina cv. formosa) has outbroke around major prune production area, Gimcheon, Euiseong and Gunwi in Gyungbuk province and has caused severe economic loss. Integrons PCR primer was designed along with sample pre-incubation and nested PCR method to enhance detection sensitivity for early detection of bacteria in fields. Designed integrons PCR primer successfully detected Xanthomonas arboricola pv. pruni from field collected samples, fruit, leaf, branch and even in raindrop collected from prune orchard. Pre-incubation along with nested PCR enhanced sensitivity to detect X. arboricola pv. pruni from seemingly healthy looking, symptomless branches. Designed integrons PCR can be used in prune nursery fields and in plant quarantine practice for the detection of X. arboricola pv. pruni.

• Korean Journal of Veterinary Research
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• v.57 no.1
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• pp.37-42
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• 2017

Getah virus (GETV) infection causes sporadic outbreaks of mild febrile illness in horses and reproductive failure in pigs. In this study, we established a reverse transcription polymerase chain reaction (RT-PCR) method to detect GETV from suspected virus-infected samples. The reaction conditions were optimized and validated by using RNA extracted from GETV propagated in cell culture. A GETV-specific GED4 primer set was designed and used to amplify a 177 bp DNA fragment from a highly conserved region of the E1 glycoprotein gene in the GETV genome. RT-PCR performed with this primer set revealed high sensitivity and specificity. In the sensitivity test, the GED4 primer set detected GETV RNA at the level of $10^{2.0}\;TCID_{50}/mL$. In the specificity test, the GED4 primer set amplified only a single band of PCR product on the GETV RNA template, without non-specific amplification, and exhibited no cross-reactivity with other viral RNAs. These results suggest that this newly established RT-PCR method is useful for accurate identification of GETV infection in animals.

• Research in Plant Disease
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• v.27 no.1
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• pp.38-44
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• 2021

Xylella fastidiosa is the most damaging pathogen in many parts of the world. To increase diagnostic capability of X. fastidiosa in the field, the loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assay were developed to mqsA gene of citrate-synthase (XF 1535) X. fastidiosa and evaluated for specificity and sensitivity. Both assays were more robust than current published tests for detection of X. fastidiosa when screened against 16 isolates representing the four major subgroups of the bacterium from a range of host species. No cross reaction with DNA from healthy hosts or other species of bacteria has been observed. The LAMP and PCR assays could detect 10-4 pmol and 100 copies of the gene, respectively. Hydroxynaphthol blue was evaluated as an endpoint detection method for LAMP. There was a significant color shift that signaled the existence of the bacterium when at least 100 copies of the target template were present.