• Food Science of Animal Resources
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• v.29 no.5
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• pp.647-652
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• 2009

A duplex PCR technique was applied for specific identification of cow and goat milk in milk products by using primers targeting the mitochondrial 12S rRNA gene. Duplex PCR using primers specific for cow and goat generated specific fragments of 223bp and 326bp from cow and goat milk DNA, respectively. Duplex PCR was applied to 15 milk products purchased from the market to verify label statements. The labeling statements of four market milk products, three yoghurt products, and one whole milk powder product were confirmed in the duplex PCR. The labeling statements of five of seven infant milk powder products were also confirmed by duplex PCR but the other two products were shown to be contaminated with either cow or goat milk. The proposed duplex PCR provides a rapid and sensitive approach to detection of as little as 0.1% cow milk in goat milk and one-step detection of cow or goat milk in milk products.

• Journal of Plant Biotechnology
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• v.49 no.3
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• pp.193-206
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• 2022

Astringent persimmon (Diospyros kaki Thunb.) is an important fruit crop in Korea; it possesses significant medicinal potential. However, knowledge regarding the pathogens affecting this crop, particularly, viruses and viroids, is limited. In the present study, reverse transcription-polymerase chain reaction (RT-PCR) and high-throughput transcriptome sequencing (HTS) were used to investigate the viruses and viroids infecting astringent persimmons cultivated in Korea. A one-step multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of the pathogens was developed by designing species-specific primers and selecting the primer pairs via combination and detection limit testing. Seven of the sixteen cultivars tested were found to be infection-free. The RT-PCR and HTS analyses identified two viruses and one viroid in the infected samples (n = 51/100 samples collected from 16 cultivars). The incidence of single infections (n = 39/51) was higher than that of mixed infections (n = 12/51); the infection rate of the Persimmon cryptic virus was the highest (n = 31/39). Comparison of the monoplex and mRT-PCR results using randomly selected samples confirmed the efficiency of mRT-PCR for the identification of pathogens. Collectively, the present study provides useful resources for developing disease-free seedlings; further, the developed mRT-PCR method can be extended to investigate pathogens in other woody plants.

• Research in Plant Disease
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• v.26 no.3
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• pp.170-178
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• 2020

Most strawberry viruses exist relatively low titers in tissues, and strawberry tissues include high levels of contamination by polysaccharides and phenolic compounds. These traits make the efficiency of strawberry diagnosis difficult. In this study, we tested different commercially available kits and reagents to secure optimal RNA extraction methods to determine virus detection from strawberry leaves. Total RNA was isolated from leaves of strawberry mottle virus (SMoV)-infected strawberry cultivar 'Mihong'. The efficiency of total RNA for virus diagnosis was confirmed through SMoV detection by one-step or two-step reverse transcription and polymerase chain reaction (RT-PCR). Among those, the RNeasy plant RNA kit was best to isolate RNA and the isolated RNA was good enough for further applications. To ensure a reliable detection for strawberry viruses, synthetic diagnosis clones for major seven strawberry viruses such as strawberry mild yellow edge virus, SMoV, strawberry latent ring spot virus, strawberry crinkle virus, strawberry pallidosis associated virus, strawberry vein banding virus and strawberry necrotic spot virus have been constructed. Based on the synthetic genes in each clone, primer sets for seven strawberry viruses were designed and tested an RT-PCR condition through a simultaneous application of the same annealing temperature that allowed to achieve an efficient and convenient diagnosis.

• Journal of Microbiology
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• v.34 no.3
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• pp.229-235
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• 1996

Microgram quantities of DNA per gram soil were recovered with SDS- based and freeze-and thaw procedures. The average DNA fragment size was > 23 Kb. This method generated minimal shearing of extracted DNA. However, the DNA extracts still contained considerable amounts of humic impurities sufficient to inhibit PCR. Several approaches were used to reduce the interferences with the PCR (use of CTAF in extraction step, Elutip-d column purification, addition of BSA to PCR buffer) to accomplish PCR with DNA extract as a template. Most of the DNA extracts were not digested completely by restriction endonuclease, and CTAB-TREATED ane Elutip-d column purified DNA extracts were partially digested. Regarding as restriction enzyme digestion, all PCRs failed to amplify 16S rRNA gene fragments in the DNA extracts. In the case of DNA extracts only where BSA was added to PCR buffer, PCR was successfully conducted whether the DNA extracts were treated with CTAB or purified with columns. However, these two treatments were indispensable for humic impurity-rich DNA extracts to generate the PCR-compatible DNA samples. Direct extraction of DNA, coupled with these procedures to remove and relieve interferences by humic impurities and followed by the PCR, can be rapid and simple method for molecular microbiological study on soil microorganisms.

• Korean Journal of Plant Resources
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• v.16 no.1
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• pp.40-48
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• 2003

Cylindrocarpon destructans is the major pathogen inducing the root rot disease in ginseng. Up to now, there is no reliable and convenient method to analyze the spore density or population of this pathogen in ginseng-growing soil or any contaminated farmlands. Therefore, it will be very valuable to develop a new and reliable method in detecting the spore of this pathogen. In this study, a molecular biological technique using two step nested PCR method, was developed. Two universal ITS primers, ITS5F and ITS4R were used in the first round of PCR to amplify a fragment of ITS region from the genomic DNA of C. destructans. The specific prmers Nest 1 and Nest 2 were designed and used in the second round of PCR to amplify a inner fragment from the first round PCR product of C. destructans. C. destructans spore, only soil samples from the diseased ginseng farm produced the positive bands, suggesting its usefulness in detecting the C. destructans spores in soil samples. Thus it is recommended to first extract the whole genomic DNA from soil samples and use it for the PCR reaction, thereby eliminating the inhibitory activity of soil components.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.690-693
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• 2000

Efficient intron deletion with the correct splicing of the two exons of the human proinsulin gene was accomplished by a novel stepwise method using genomic DNA [5]. The two exons were separately amplified in two steps, using the second step primers that incorporated additional bases complementary to the other exon. The fragments were combined in a third PCR reaction. Cloning and sequencing of the PCR product demonstrated the correct splicing of the two exons. Expression studies, using the pET9a vector, revealed a protein band with the correct size with respect to human proinsulin as confirmed by SDS-PAGe and Western blot. Proinsulin concentration was estimated to be around 200 mg per liter culture, expressed as inclusion bodies. Protein secretion to the culture medium and periplasmic space was achieved by cloning in the pEZZ18 vector.

• The Plant Pathology Journal
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• v.32 no.5
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• pp.414-422
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• 2016

Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.91-98
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• 2016

The growth area of living modified (LM) cotton has steadily increased every year, since its first commercialization in 1996. Development of environmental risk assessment tools and techniques for LM cotton is required for ecosystem safety. We therefore developed multiplex PCR assays for simultaneous detection of two (MON15985, MON531) and four (GHB614, LLCOTTON25, MON88913 and MON1445) LM cotton events approved in Korea, with event specific primer pairs. The PCR reactions were optimized by using event specific primers of six LM cottons at various concentrations. The reactions allows amplification of estimated amplicons of MON15985 (214 bp), MON531 (270 bp), GHB614 (119 bp), LLCOTTON25 (164 bp), MON88913 (276 bp), and MON1445 (389 bp) from multiplex PCR reactions. The multiplex PCR assay developed allowed that two annealing steps (15 cycles at $55^{\circ}C$ and 25 cycles at $60^{\circ}C$) were performed for amplification of distinguished two LM cottons, and only one annealing step (50 cycles at $60^{\circ}C$) was necessary for tetraplex PCR. Primer extension step of all PCR reactions was skipped for time-effective amplification. Our methods suggest that two multiplex PCR assays can be cost-effective and a rapid diagnostic tool for environmental LMO monitoring of six LM cottons.

• Korean Journal of Microbiology
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• v.49 no.2
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• pp.112-117
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• 2013

Wheat streak mosaic virus (WSMV), a member of the genus Tritimovirus in Potyviridae, severely impacts wheat and corn seed worldwide, but has yet to be detected in Korea, and hence, every effort should be made to prevent its introduction. To prevent WSMV from entering the country, it is necessary to prepare a specific, sensitive, simple, and fast detection method for routine application to plant quarantine procedures. For this reason, a two-step diagnosis system consisting of RT-PCR and nested PCR is being used for WSMV detection. In addition, a novel positive control was developed for use with the system. WSMV has been detected in seed sweet corn from Japan and seed wheat from USA by a two-step diagnosis system, the details of which are described in this study. After sequence analysis, similarities of 80.6 and 100.0% with other isolates were determined by BLAST. They showed the same topology, which was classified as 4 genotypes by various phylogenetic trees, using a poly protein encoding sequence amplification. In this analysis, WSMV-JSweet-corn2868 (JX845574) is classified as clade B, while WSMV-Uwheat1944-1 (KC754959) and WSMV-Uwheat1944-2 (KC754960) belong to clade D.

• Journal of fish pathology
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• v.31 no.1
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• pp.9-13
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• 2018

We previously monitored nervous necrosis virus (NNV) in brain samples of artificially produced walleye Pollock (Gadus chalcogrammus) seedlings, with a low prevalence (1.8%, 1/55) but no clinical symptoms. Given that this virus is considered one of the most serious viral threats for almost all marine aquaculture fish species and characterized by both vertical and horizontal transmission, it would be interesting to monitor NNV in the fertilized eggs as well. We collected fertilized walleye pollock eggs from the farms located in Goseong during January to March, 2017. Approximately 50 mg of eggs were periodically taken from 4 each different batches, and 37 different pooled sample sets in total were made during sampling period. RNA was extracted from the eggs by using Trizol and cDNA was synthesized for RT-PCR for detecting NNV. Primers and PCR conditions are the same as previously described. As a result, NNV was not detected from any of the sample sets by one step PCR (0%, 0/37), suggesting NNV may not be a threat in walleye pollock aquaculture in Korea at present time. However, continuous monitoring for NNV should be conducted because introducing a new species into aquaculture industry involves potentials of disease outbreak and NNV is already known to cause outbreaks in gadoid fishes.