• Korean Journal of Veterinary Research
• /
• v.43 no.3
• /
• pp.471-476
• /
• 2003

Malignant catarrhal fever (MCF) is a systemic disease of ruminants caused by ovine herpesvirus 2 (OvHV-2). OvHV-2 is a gamma herpesvirus, which induces frequent latent infection and often difficult to detect its antigens and even specific nucleic acids because of its low viral copies in the infected tissues. Histopathology, serology and polymerase chain reaction (PCR) were compared for the diagnosis of MCF using 10 bison infected with OvHV-2. Histopathological diagnosis was performed using the criteria which was based upon the pathognomic lesions. Serological diagnosis was conducted using its serum with competitive ELISA for the detection of antibodies of OvHV-2. Also, the nest PCR was performed with peripheral blood leukocytes for the detection of OvHV-2-specific DNAs. Primers 556 and 775 were used for the primary amplification, and primers 556 and 555 were used for the secondary amplification. As the results, positive cases were 6 by histopahology, 9 by serology and 10 by PCR. As comparing with other diagnostic methods, PCR was found to be more sensitive than histopathology and serology. The recent development of molecular diagnostic assays has provided powerful tools for investigating how viruses survive in nature. Development of PCR specific for viruses has dramatically improved the accuracy of diagnosis of viruses in clinically infected animals. Furthermore, amplification of viral genomic material by nest PCR represents the most sensitive method for the detection of viruses and might be detected successfully even though very low viral DNA copies. So, it could be used as the first choice for the detection of viral DNAs with low copies such as the status of latent infection. However, it has also some limitation of application like as false negative results by PCR inhibitors and false positive results by contamination. The results of this study suggest that the use of molecular biological methods like PCR may increase the accuracy for the diagnosis of infectious diseases. However, in diagnostic laboratory, it is recommended that PCR assay must be conducted with other diagnostic methods for more reliable diagnosis.

• Environmental Analysis Health and Toxicology
• /
• v.16 no.4
• /
• pp.197-204
• /
• 2001

Endocrine disruption in crucian carp (Carassius auratus) living in the branch of Han River were examined. Vitellogenin level in plasma was measured using ELISA system and aromatase mRNA level in brain was observed using RT-PCR technique. In all female fish, vitellogenin levels were in the range of 20∼40 $\mu\textrm{g}$/ml and aromatase mRNA expression could be detected on the agarose gel after RT-PCR. However, in case of males, vitellogenin level was elevated in only one fish, while vitellogenin was hardly detected in others. Aromatase was expressed in all males although the levels were relatively lower than the level in female fish. Testis-ova and any other histological changes of reproductive organ were not shown in both sexes.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.8
• /
• pp.1044-1050
• /
• 2014

Since there is no consensus about the most reliable assays to detect invasive aspergillosis from samples obtained by minimally invasive or noninvasive methods, we compared the efficacy of an enzyme-linked immunosorbent assay (ELISA) for galactomannan (GM) detection and quantitative real-time PCR assay (qRT-PCR) for the diagnosis of invasive pulmonary aspergillosis. Neutropenic, male Sprague-Dawley rats (specific pathogen free; 8 weeks old; weight, $200{\pm}20g$) were immunosuppressed with cyclophosphamide and infected with Aspergillus fumigatus intratracheally. Tissue and whole blood samples were harvested on days 1, 3, 5, and 7 post-infection and examined with GM ELISA and qRT-PCR. The A. fumigatus DNA detection sequence was detected in the following number of samples from 12 immunosuppressed, infected rats examined on the scheduled days: day 1 (0/12), day 3 (0/12), day 5 (6/12), and day 7 (8/12) post-infection. The sensitivity and specificity of the qRT-PCR assay was 29.2% and 100%, respectively. Receiver operating characteristic curve (ROC) analysis indicated a Ct (cycle threshold) cut-off value of 15.35, and the area under the curve (AUC) was 0.627. The GM assay detected antigen in sera obtained on day 1 (5/12), day 3 (9/12), day 5 (12/12), and day 7 (12/12) post-infection, and thus had a sensitivity of 79.2% and a specificity of 100%. The ROC of the GM assay indicated that the optimal Ct cut-off value was 1.40 (AUC, 0.919). The GM assay was more sensitive than the qRT-PCR assay in diagnosing invasive pulmonary aspergillosis in rats.

• Archives of Pharmacal Research
• /
• v.19 no.6
• /
• pp.486-489
• /
• 1996

A simple, rapid, specific and sensitive method for the detection of serum hepatitis C virus (HCV) RNA using the reverse transcription-polymerase chain reaction (RT-PCR) technique without conventional RNA extraction was developed. HCV template RNA from serum was obtained by boiling the serum at $95^{\circ}C$ for 2 min, cooling rapidly in ice and removing the proteins by cetrifugation. RT-PCR amplifications including the reverse transcription and first PCR amplification were performed in one vessel containing both of reverse transcriptase and Taq DNA polymerase. The detection of HCV RNA from $10^{-3}{\mu}l$. serum was possible with this method. The suitability of this method for clinical analysis was evaluated by assaying HCV RNA in 225 patient samples including anti-HCV antibody negatives (13 samples) and positives (212 samples) by enzyme-linked immunosorbent assay test (ELISA). Detections of HCV RNA with this method were in 4 of 13 anti-HCV antibody negative samples (30.8%) and 95 of 212 positive samples (44.8%). The present method can be completed in 1 hr and has a wide range of application for the clinical utilities to determine the viral RNAS.

• Journal of Plant Biotechnology
• /
• v.7 no.4
• /
• pp.259-265
• /
• 2005

Indian citrus ringspot virus (ICRSV)-free plants of Kinnow mandarin (Citrus nobilis Lour x C. deliciosa Tenora) were raised from virus-infected plants using unfertilised ovules as explants. Plants were tested by indirect ELISA and RT-PCR before using their explant. An amplified product of 539 bp was obtained by RT- PCR in ICRSV infected plants. Unfertilized ovules were excised from unopened flower buds of plants tested postive for virus and were cultured on Murashige and Skoog's (MS) basal medium supplemented with various concentrations of kinetin (KN) or malt extract (ME). Maximum induction (31.94%) of embryogenic callus was observed on MS medium supplemented with KN ($9.29\;{\mu}M$). Transfer of embryogenic calli to similar media composition resulted in somatic embryogenesis in all cultures, with an average number of 60.36 globular, 17.39 heart and 7.71 cotyledonary-shaped somatic embryos per culture. All cotyledonary shaped embryos developed into complete plantlets within 60 days on transfer to similar medium. Embryogenic callus induction, somatic embryo formation, maturation, germination and plantlet formation were achieved on MS medium supplemented with KN ($9.29\;{\mu}M$) alone. The plantlets derived from somatic embryos were transferred to sterilized soil, sand and vermiculite (3:1:1) mixture. After acclimatization, the plantlets were transferred to screen house and were indexed for ICRSV employing indirect ELISA and RT-PCR and found free of virus. A distinct feature of this study is the induction of somatic embryogenesis from unfertilised ovules to produce virus-free plants.

• Biomedical Science Letters
• /
• v.10 no.3
• /
• pp.219-230
• /
• 2004

This experiment was performed to examine the in vitro and in vivo affects of the two different haplotype strains of mice, BALB/c and C3H/HeN infected with Echinostoma hortense, and the manifestation of the profiles of cytokine in the splenocytes. In the in vitro experiment, the two mice's splenocytes were divided and stimulated with antigen of crude extracts and the antigen of excretory and secretory products of an adult warm and the manifestation of cytokine mRNA was verified with RT-PCR. As a result, the two different strains of mice both strongly manifested the Th2 cytokine rather than the Thl cytokine and in the case of the Th2 cytokine, the BALB/c mice manifested more strongly than the C3H/HeN mice. In the experiment using the ELISA method, the protem cytokine manifestation had the same result as the mRNA experiment. In the in vivo experiment, the mice was infected via oral route with the metacercaria of the Echinostoma hortense and the manifestation of cytokine was verified by RT-PCR and ELISA and the results were the same as the in vitro experiment. Therefore, in the two strains of BALB/c and C3H/HeN, the C3H/HeN showed a higher susceptivity to the Echinostoma hortense.

• Korean Journal of Veterinary Service
• /
• v.33 no.4
• /
• pp.345-351
• /
• 2010

Toxoplasma gondii is a species of parasitic protozoa in the genus Toxoplasma. The definitive host of T. gondii is the cat, but the parasite can be carried by the vast majority of warm-blooded animals, including humans. It is often found in the tissues of food animals including pigs and sheep. To determine the regional prevalence of infection with T. gondii, bloods (n=300) from domestic pigs and tissues (n=200) from slaughter pigs in Gyeongnam province were tested using an enzyme-linked immunosorbent assay (ELISA) and a polymerase chain reaction (PCR) for detection of antibody and antigen. A total of 115 sero-positive pigs were identified for a prevalence rate of 38.3%. Of the 50 herds from domestic pigs tested, 34 had at least one sero-positive pig for a herd prevalence rate of 68.0%. Sero-positive rates of pigs in fattening farm were higher than that of pigs in breeding company. Sero-positive rates of sows were higher than that of growing pigs. Seasonally, sero-positive rates of pigs were highest in winter (80.0%) and lowest in spring (23.8%). According to farm size, sero-positive rates of pigs were higher in small size farms (${\leq}$2,000) than that of big size farms (>2,000). However, none of the bloods (n=300) from domestic pigs and tissues (n=200) from slaughter pigs were positive for T. gondii specific DNA by PCR.

• Korean Journal of Ichthyology
• /
• v.9 no.1
• /
• pp.91-98
• /
• 1997

The transgene, pFV4CAT, containing CAT reporter gene regulated by carp $\beta$-actin promoter, was expressed in independent transgenic mud loach germ lines, determined by reverse transcriptase-PCR (RT-PCR) and enzyme-linked immunosorbant assay (ELISA). Expression of the transmitted transgene was found to be tissue-specific in F1 and F2 generations. Tissue specificity of the expression was dependent on each transgenic line with reproducible patterns. Liver and spleen did express the transgene more frequently than other tissues tested, and muscle and heart revealed the higher amount of CAT than other tissues, while testes showed the lowest expression level. The highest level of CAT expression in muscle from a transgenic F1 line was corresponding to 68-fold compared to the basal levels of controls.

• Korean Journal of Veterinary Service
• /
• v.37 no.1
• /
• pp.11-18
• /
• 2014

Porcine reproductive and respiratory syndrome (PRRS) causes a significant economic loss in the swine industry not only in Korea but also all over the world. Andong and Hapcheon region were selected for Area Regional Control (ARC) programme to reduce the shedding of PRRS virus (PRRSV) and decrease PRRS outbreaks. Before conducting the PRRS ARC, sera of pigs were tested for both antibody using ELISA and antigen using RT-PCR, then phylogenetic classifications was analysed. Pigs of 138/275 (50.2%) in Andong and 352/425 (82.8%) in Hapcheon were seropositive. Also, the RT-PCR results revealed that 27 heads (8.2%) in Andong, 112 heads (22.0%) in Hapcheon were positive for PRRSV antigen. PRRSVs were mainly detected between the ages of 40 to 60 days. PRRSV ORF5 regions were used to determine genetic clusters based on previous report. All PRRSV type I detected in both Andong and Hapcheon were classified as Cluster I. The PRRSV type II isolates in Andong were assorted to Cluster II, whereas the PRRSV type II isolates in Hapcheon were the viruses were unassembled into any cluster except one identified to Cluster III. Phylogenetic analysis indicated that new clusters of PRRSVs type II were prevalent in Hapcheon.

• Korean Journal of Microbiology
• /
• v.47 no.4
• /
• pp.323-327
• /
• 2011

Fecal specimens from acute gastroenteritis in Seoul from 2009 to 2010 were collected and then tested for the presence of Group A Rotavirus by ELISA. Among a total of 1,916 samples investigated, 354 samples (18.4%) were positive. The predominant genotypes of positive samples were confirmed as P6G[4] (35%), P8G[3] (28%), P8G[1] (24%), P4G[2] (10%), P8G[9] (3%), respectively. Among a total of 70 ground water samples investigated, 2 samples (2.8%) were positive. The genotypes of positive samples were confirmed as P8G[3] (100%). By this molecular investigation, genotypic distribution associated with rotavirus will be used for control and prevention of rotavirus related diseases.