• Journal of Microbiology and Biotechnology
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• v.27 no.3
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• pp.471-479
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• 2017

White spot syndrome virus (WSSV) is a major threat to the shrimp farming industry and so far there is no effective therapy for it, and thus early diagnostic of WSSV is of great importance. However, at the early stage of infection, the extremely low-abundance of WSSV DNA challenges the detection sensitivity and accuracy of PCR. To effectively detect low-abundance WSSV, here we developed a pre-amplification PCR (pre-amp PCR) method to amplify trace amounts of WSSV DNA from massive background genomic DNA. Combining with normal specific PCR, 10 copies of target WSSV genes were detected from ${\sim}10^{10}$ magnitude of backgrounds. In particular, multiple target genes were able to be balanced amplified with similar efficiency due to the usage of the universal primer. The efficiency of the pre-amp PCR was validated by nested-PCR and quantitative PCR, and pre-amp PCR showed higher efficiency than nested-PCR when multiple targets were detected. The developed method is particularly suitable for the super early diagnosis of WSSV, and has potential to be applied in other low-abundance sample detection cases.

• Korean Journal of Plant Tissue Culture
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• v.28 no.6
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• pp.335-339
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• 2001

Grapevine leafroll-associated 3 closterovirus (GLRaV-3) is phloem limited virus, and one of the most severe viral diseases found in Korea. However, nonhomogenous distribution and low concentration and seasonal variations of GLRaV-3 in grapevines remain as main problems which prevent the introduction and molecular biology or serology experiments. Virus-infected plantlet in vitro was obtained from node tissue cuttings, which was GLRaV-3 infected 'kyoho' vines. The amount of purified virus was highest in vitro plantlet. Moreover, viruses seem to be relatively homogeneously distributed in all organs including leaf, stem and callus derived from in vitro plantlets. RT-PCR detection using in vitro plantlet tissue as template was most effective. When comparing ELISA to RT-PCR, RT-PCR detection was 1,000 times as effective as ELISA. These results can be explained by improved quality such as tenderness or less tannins in plantlet in vitro. In conclusion, until infected herbaceous host will be available, tissue culture can be usefully adopted as a technique for a good source of GLRaV-3 closterovirus for further studies.

• The Plant Pathology Journal
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• v.15 no.6
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• pp.335-339
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• 1999

Citrus tristeza virus(CTV), an aphid-borne closterovirus, is one of the most destructive pathogens of citrus. It has caused rapid decline in growth, stem pitting and death in citrus trees. A reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for detection of CTV and investigation of the CTV infection status of citrus and its related cultivars in Cheju island. For RT-PCR based CTV detection, primers were designed to amplify 670bp of coat protein gene. A screening test for CTV in citrus cultivars was conducted from March to July in 1999. Seventy individual citrus trees representing 9 species of 3 genera were tested. The infection rates of CTV for leaves from the years or older trees of late maturing citrus varieties such as Yuzu (C. junos Sieb. ex Tanaka), Navel orange (C.sinensis Osbeck), Kiyomitanger (C. unshiu x C. sinensis), and Shiranuhi ((C. unshiu x C. sinensis) x C. reticulata) were 100%, 80%, 60%, and 60% respectively. The CTV infection rates in Early satsuma mandarins such as 'Miyagawa Early' Satsuma mandarins (C. unshiu Marc. var. Miyagawa) and 'Okitsu Early' Satsuma mandarins (C. unshiu Marc. var. Okitsu) were 100%, and 60%, respectively. CTV was not detected in Cheju native Dangyooja (C. unshiu Marc. var. Osbeck), Trifoliate orange (Poncirus trifoliata) and Kumquat (Fortunella margarita Swingle). In conclusion, RT-PCR assay can be successfully applied to the detection of CTV in citrus trees.

• Research in Plant Disease
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• v.10 no.1
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• pp.53-57
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• 2004

Cucumber green mottle mosaic virus (CGMMV) was a major pathogen of watermelon and had affected seriously to watermelon production in Korea. Rapid and sensitive detection method of CGMMV associated with bottle gourd (Lagenafia siceraria) seeds was developed by using RT-PCR in this study. A pair of primeri Wmfl and Wmrl, specific for CGMMV was designed from coat protein gene sequences of CGMMV-W and used for amplifying 420 bp product in RT-PCR. To simplify the virus extraction procedure and reduce an inhibitor from the extract for the RT-PCR, some methods using ethanol precipitation, double filtration, polyethylene glycol precipitation and phenol/chloroform/isoamyl alcohol extraction procedure were compared and the phenol/chloroform/isoamyl alcohol extraction procedure was selected by its enhanced sensitivity. This detection method using the selected extraction step and the primers for RT-PCR could reliably detect an infected level of one CGMMV-infested seed in 1,000 seeds. This rapid and sensitive RT-PCR assay provides auseful tool for the specific detection of CGMMV in bottle gourd seed samples containing high levels of back-ground inhibitors.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.186-191
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• 2002

The usefulness of oligonucleotide DNA chip was evaluated for detection and primary level identification of major waterborne viruses in environmental samples. The enteric waterborne viruses included enterovirus, adenovirus, and rotavirus. Total intracellular RNA of 10 BGM cell plates showing virus-specific cytopathic effects was extracted at the third day after inoculation. The intracellular RNA was then subjected to either enterovirus-specific RT-PCR followed by sequencing analysis, or the DNA chip. Seven out of 10 positive samples in cell culture were positive but the other three sample were turned out to be negative by both RT-PCR and DNA chip analyses. Nucleotide sequencing results and the DNA chip hybridization results of the RT-PCR product were in complete agreement in the identification of the 7 positive samples as enteroviruses. Using the DNA chip, it took only 3∼4 hr to complete detection and primary level identification of target viruses and additional procedures such as gel electrophoresis or nucleotide sequencing were not necessary. We believe that the DNA chip system can be employed as a highly effective and new detection methodology for environmental viruses.

• Research in Plant Disease
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• v.21 no.4
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• pp.315-320
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• 2015

Soybean mosaic virus (SMV) is a prevalent pathogen that causes significant yield reduction in soybean production worldwide. SMV belongs to potyvirus and causes typical symptoms such as mild mosaic, mosaic and necrosis. SMV is seed-borne and also transmitted by aphid. Eleven SMV strains, G1 to G7, G5H, G6H, G7H, and G7a were reported in soybean varieties in Korea. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) method allowed one-step detection of gene amplification by simple procedure and needed only a simple incubator for isothermal template. This RT-LAMP method allowed direct detection of RNA from virus-infected plants without thermal cycling and gel electrophoresis. In this study, we designed RT-LAMP primers named SML-F3/B3/FIP/BIP from coat protein gene sequence of SMV. After the reaction of RT-LAMP, products were identified by electrophoresis and with the detective fluorescent dye, SYBR Green I under daylight and UV light. Optimal reaction condition was at $58^{\circ}C$ for 60 min and the primers of RT-LAMP showed the specificity for nine SMV strains tested in this study.

• Korean Journal of Food Science and Technology
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• v.44 no.5
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• pp.638-642
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• 2012

Human pathogenic viruses such as hepatitis A and E virus (HAV and HEV), which lead to acute liver failure and death, are foodborne pathogens associated with the consumption of virus-contaminated meats, filter-feeding bivalves, fruits, and salads. Two of the three swine farms examined in this study had HAV and HEV positive stool samples in a nested RT-PCR assay. The use of the immunomagnetic separation (IMS) facilitated the separation of HAV through interactions between the ligand on the virion surface and the antibody from the swine feces containing both HAV and HEV. The nested RT-PCR analysis was performed for the detection of HAV obtained from hepatocarcinoma cell line (PLC/PRF/5) contaminated with eluent fraction of IMS. This indicated that IMS has the potential to simultaneously isolate and concentrate target viruses by changing antibodies linked on the magnetic beads.

• Korean Journal of Veterinary Research
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• v.43 no.1
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• pp.129-137
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• 2003

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failures in sows and respiratory problems in piglets. The nucleocapsid(N) protein, encoded by the open reading frame 7 (ORF7) gene, is known to be the most abundant and antigenic protein in PRRS virus. Therefore, it was suggested that the N protein could be a suitable candidate for the detection of PRRS virus-specific antibodies and diagnosis of PRRS. In the present study, the ORF7 gene encoding the N protein was cloned and expressed as a fusion protein with the glutathione S-transferase (GST) in Escherichia coli. The resulting GST-N recombinant protein was used as an antigen for an indirect sandwich enzyme-linked immunosorbent assay (i-ELISA). Expressed GST-N recombinant protein was migrated at 41 kDa and reacted with ORF7-specific monoclonal antibody by Western blotting. In order to increase the specificity of the ELISA for the detection of PRRS virus-specific antibodes, an i-ELISA was developed using an anti-GST antibody as a capture antibody. The sensitivity and specificity of developed i-ELISA were 92% and 96%, respectively. Based on these results, it was suggested that the i-ELISA is a simple and rapid test for screening a large number of swine sera for the anti-PRRS virus antibodies.

• Korean journal of applied entomology
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• v.18 no.1 s.38
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• pp.29-33
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• 1979

An attempt was made to find out whether barley stripe mosaic virus (BSMV) occurs in barley and wheat cultivars in Korea. Modified Hamilton's technique for serological detection of barley stripe mosaic virus was used to assay for BSMV in mature seed embryos. Of the 51 barley and wheat seed lots assayed, BSMV was detected in 34 of them. BSMV was detected in covered, naked, and matting barley as well as in wheat. The level of BSMV infection in the infected cultivars varied from $2\~35\%$. This is the first reported occurrence of barley stripe mosaic virus in Korea.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.217-225
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• 1997

PCR amplication using the primers for gag, pol and env genes in BLV (bovine leukemia virus) proviral DNA and syncytium assay were carried out for the Korean native goats experimentally infected with bovine leukemia virus to investigate pathogenesis of BLV in the goats, and to establish a model animal for BLV infection. The oligonucleotide primers used in PCR revealed very high specificity. The minimal amount of FLK-BLV cellular chromosomal DNA to detect the integrated BLV proviral DNA was 10 ng. The peripheral blood lymphocytes from the goat infected with BLV were examined at regular intervals by PCR amplification and syncytium assay. Pol or gag genes were detected in none of three infected goats at the 1st week post-infection (p.i.). At the 4th week p.i., one of three goats showed the amplified gag gene. Thereafter detection rates for the genes were increased, indicating that the BLV proviral genes were integrated in all of the lymphocytes from three goats, at the 16th weeks p.i., when it was evident in syncytium assay that the lymphocytes from all of three goats were infested with infective BLV. Investigating the tissues from the necropsied goats at the 8th month p.i., the amplified BLV proviral genes and infective BLV were detected in all of the peripheral lymphocytes from three infected-goats. Among various tissues examined, the amplified BLV proviral genes were observed in spleen and superficial cervical, mandibular and retropharyngeal lymph nodes, and the infective BLV, in superficial cervical and mandibular lymph nodes. It was assumed that the Korean native goat was quite susceptible to BLV infection, indicating that the goat could be a good model animal for BLV.