• Journal of Yeungnam Medical Science
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• v.14 no.1
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• pp.155-167
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• 1997

The identification of serum HBV DNA is very important for the assessment of the disease activity in persistent infection, for the evaluation of the infectivity of an individuals blood. The dot blot, however, has limited sensitivity and sometimes inconsistent with other serological markers and clinical settings. Using the most important recent advance in molecular biology, the polymerase chain reaction(PCR), specific DNA sequences can be amplified more than a million-fold in a few hours and with this technique the detection of the extreme low level of DNA is possible. This study was to determine sensitivity of the PCR for the detection of serum HBV DNA in comparison with dot blot analysis and to investigate the serum HBV DNA status and clinical significance of PCR in patients with chronic HBsAg positive liver disease. The subjects of this study were 17 patients with asymptomatic HBsAg carriers(9 HBeAg positive patients, 8 anti-HBe positive patients), 91 chronic hepatitis B(50 HBeAg positive patients, 41 anti-HBe positive patients), 57 liver cirrhosis(21 HBeAg positive patients, 36 anti-HBe positive patients), 27 hepatocellular carcinoma(10 HBeAg positive patients, 17 anti-HBe positive patients). The results were summerized as following; The detection rates of HBV DNA by dot blot, PCR were 58.9%, 72.2% in HBeAg positive patients, 34.3%, 53.9% in anti-HBe positive patients. The detection rates of HBV DNA by PCR in HBeAg negative patients were 25.0% in asymptomatic HBsAg carriers, 61.0% in chronic hepatitis B, 52.8% in liver cirrhosis, 52.9% in hepatocellular carcinoma. The positive rate for HBV DNA is a significant difference between HBeAg positive and negative asymptomatic HBsAg carriers, but not significantly difference in other groups. In conclusions, this study confirmed that the PCR is much more sensitive than the dot blot analysis in detecting the HBV DNA in the sera of patients with chronic liver disease. The presence of HBV DNA in the serum was detected by PCR with higher sensitivity and it suggested that active viral replication is still going on in most patients with chronic HBsAg positive liver disease irrespective of HBeAg/anti-HBe status, and PCR may be used as a prognostic factor in asymptomatic HBsAg carriers.

• Korean Journal of Microbiology
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• v.47 no.2
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• pp.117-123
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• 2011

The purpose of this study was to evaluate and compare different elution and concentration methods for optimization of human rotavirus (HRV) detection method using real-time RT-PCR and cell culture techniques. The leafy vegetable samples (lettuce, Chinese cabbage) were artificially inoculated with HRV. Viruses were extracted from the vegetables by two different elution buffers, buffer A (100 mM Tris-HCl, 50 mM glycine, 3% beef extract, pH 9.5) and buffer B (250 mM Threonine, 300 mM NaCl, pH 9.5), and the extracted viruses were concentrated by filtration and PEG precipitation sequentially. To determine infectivity of the viruses, the viruses recovered from the samples were infected to the MA-104 cells, and integrated cell culture real-time RT-PCR was performed at 1, 48, 72, 96, 120, 144, 168 h post-infection (p.i.). The elution buffer A was more efficient in extracting the virus from the produce samples tested than the buffer B, 29.54% and 18.32% of recoveries, respectively. The sensitivity of real-time RT-PCR method was markedly improved when the virus was concentrated by the filtration method. When the viruses were eluted and concentrated by buffer A and filtration, respectively, the average recovery rate was approximately 51.89%. When the viruses recovered from samples were infected to MA-104 cell, infectious HRV was detected within 48 h p.i. by ICC/real-time RT-PCR, whereas cytopathic effects were not observed until 72 h p.i. The optimized detection method evaluated in this study could be useful for rapid and reliable detection of HRV in fresh produce products and applied for detection of other food-borne viruses.

• Journal of Preventive Medicine and Public Health
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• v.21 no.1 s.23
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• pp.89-98
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• 1988

While there have been not a few reports on the seroepidemiological characteristics of hepatitis B virus (HBV) infection in Korea, most of them, however, have had several limitations; operational definition of HBV infection, validity of detection methods of HBV serologic markers, size of the study population, and confirmation of the vaccination history against HBV, etc. In order to avoid such limitations, authors randomly selected 1,495 healthy adults among the 217,511 insured (target population) of Korean Medical Insurance Corporation, living in seoul, and tested HBV serologic markers by RIA method and conducted direct interview to them. Although HBV serologic markers (HBsAg, anti-HBs and anti-HBc) of all the subjects were tested, 392(26.2%) of interview failure cases and 361 vaccinee were excluded from the actual population. Finally, the serologic markers tested of 742 nonvaccinee (study population) only were analysed for the seroepidemiologic observation of the natural infection of HBV. The seroepidemiological characteristics of HBV infection in Korea were as follows ; 1. Point prevalence of HBs antigenemia was 11.7(9.1{\sim}14.3)% in male, which was slightly higher than that of female, 9.5($3.7{\sim}15.3$)%. This level was one of the highest among those of Asian-Pacific countries. Decreasing tendency of HBsAg prevalence alter the age of 50 was observed, which seems to be due to selective attrition of HBV chronic carriers among the healthy adults and/or to the limited-lasting duration of the HBs antigenemia, in part. 2. Point prevalence of anti-HBc(78.8% in male,50.9% in female) was higher than that of anti-HBs(65.2% in male,46.6% in female), respectively. And both of them were higher in male than in female. Increasing tendency of the prevalence of both antibodies was observed by age, which seems to be largely due to recurrent infection in adults and to some cumulative effect, in part, of their relatively longer-lasting duration. 3. The level of HBV infection defined by positive for at least one of the 3 serologic markers of HBV by RIA method was 84.7($81.8{\sim}87.6$)% in male and 61.2($51.9{\sim}70.5$)% in female, which was also one of the highest among those of Asian-Pacific countries. The proportion of susceptible population to HBV infection among healthy adults was 15.3% in male and 38.8% in female. 4. The relative frequency of current or past infection and chronic carrier among HBV infected person was estimated. The currently or past infected was estimated 75.7% in male and 71.8% in female, and chronic carrier state, 13.8% in male and 14.1% in female. The analysis of the geometric mean of the antibody titer in anti-HBs positive sera indicated also to be compatible with the above findings, suggesting that active, even though inapparent, infection of HBV occur so frequently among healthy adults in Korea.

• Journal of Veterinary Clinics
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• v.21 no.2
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• pp.102-108
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• 2004

Orf virus (ORFV), a member of genus Parapoxvirus (family-Poxviridae), a causative agent of contagious ecthyma in sheep and goat leading to a condition commonly known as vesicular dermatitis. Recently, twelve goats from Iksan in Jeonbuk province were observed with clinical signs like necrotic vesicular lesions around the mucosa of mouth, nasal cavity, eye, ear, teats, abdomen and groin. Based on these clinical symptoms, contagious ecthyma infection was suspected. The skin scrapping was collected from lesions for isolation of DNA and subsequent PCR amplification of ORFV specific 235 bp region of B2L gene. All of the samples were found positive by PCR analysis. Sequencing and further phylogenetic analysis of the PCR product revealed 100% identity to Japan isolate of ORFV (Okinawa, GenBank accession number AB080769), and showed 99.6% of similarity to New Zealand strain (NZ-2, GenBank accession number U06671). It was concluded that ORFV strain detected in the present study is homologous to Japan isolate and New Zealand strain. The PCR test based on amplification of B2L gene is a highly useful tools for rapid and specific diagnosis of contagious ecthyma.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10209-10215
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• 2015

To assess the contribution of tumor necrosis factor $(TNF){\beta}$ +252 polymorphisms to risk and prognosis of hepatocellular carcinoma (HCC), we enrolled 150 pairs of sex- and age-matched patients with HCC, patients with cirrhosis alone, and unrelated healthy controls. $TNF{\beta}$ +252 genotypes were determined by polymerase chain reaction with restriction fragment length polymorphism. Multivariate analysis indicated that $TNF{\beta}$ G/G genotype [odds ratio (OR), 3.64; 95%CI, 1.49-8.91], hepatitis B surface antigen (OR, 16.38; 95%CI, 8.30-32.33), and antibodies to hepatitis C virus (HCV) (OR, 39.11; 95%CI, 14.83-103.14) were independent risk factors for HCC. There was an additive interaction between $TNF{\beta}$ G/G genotype and chronic hepatitis B virus (HBV)/HCV infection (synergy index=1.15). Multivariate analysis indicated that factors associated with $TNF{\beta}$ G/G genotype included cirrhosis with Child-Pugh C (OR, 4.06; 95%CI, 1.34-12.29), thrombocytopenia (OR, 6.55; 95%CI, 1.46-29.43), and higher serum ${\alpha}$-fetoprotein concentration (OR, 2.53; 95%CI, 1.14-5.62). Patients with $TNF{\beta}$ G/G genotype had poor cumulative survival (p=0.005). Cox proportional hazard model indicated that $TNF{\beta}$ G/G genotype was a biomarker for poor HCC survival (hazard ratio, 1.70; 95%CI, 1.07-2.69). In conclusion, there are independent and additive effects between $TNF{\beta}$ G/G genotype and chronic HBV/HCV infection on risk for HCC. It is a biomarker for poor HCC survival. Carriage of this genotype correlates with disease severity and advanced hepatic fibrosis, which may contribute to a higher risk and poor survival of HCC. Chronic HBV/HCV infected subjects with this genotype should receive more intensive surveillance for early detection of HCC.

• Korean Journal of Veterinary Research
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• v.36 no.4
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• pp.845-858
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• 1996

The purpose of this study was to establish early diagnostic methods for the detection of Aujeszky's disease viral antigens and nucleic acid in nasal cells, and buffy coats from experimentally infected living pigs by a combination of immunocytochemistry, in situ hybridization with digoxigenin(DIG)-labled probe and electron microscopy. Forty days old piglets were inoculated intranasally with $10^{7.0}TCID_{50}$ of Aujeszky's disease virus (ADV, NYJ-1-87 strain). The viral antigens and nucleic acid of ADV were detected in nasal cells, and buffy coat for 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopical method. The results were compared with conventional methods such as a porcine Aujeszky's disease serodiagnostic(PAD) kit, neutralization test(NT) and virus isolation. 1. The viral antigens, nucleic acids and capsids of ADV were detected in nasal cells, buffy coats from 3 days to 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopy, respectively. 2. When viral antigens were detected by the immunocytochemical technique, a diffuse brown deposit was observed in the nucleus and cytoplasm of nasal cells, buffy coats and PK-15 cells under a microscope. 3. DIG-labeled DNA probe was prepared by amplification of conserved sequence of recombinant ADV-gp50 clone with polymerase chain reacction. When ADV-DNA was detected by ISH with DIG-labeled probe, purplish blue pigmentation were observed in the nuclei and cytoplasms of ADV-infected cells under a microscope. Positive signals were observed in nasal cells and in the buffy coat and PK-15 cells at the first day after inoculation. 4. Where ADV-capsids were detected by transmission electron microscopical method, aggregation of capsids was observed in the nuclei and cytoplasms of nasal cells, buffy coats and PK-15 cells. The results suggested that these methods were considered as the highly sensitive and reliable tools for rapid and confirmative diagnosis of Aujeszky's disease in living pigs.

• Journal of Food Hygiene and Safety
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• v.29 no.1
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• pp.31-39
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• 2014

Norovirus causes acute gastroenteritis in all age groups and its food poisoning outbreaks are rapidly increasing in Korea. Reverse transcription-polymerase chain reaction (RT-PCR) is most widely used for the rapid detection of foodborne viruses due to high sensitivity. However, the false positive results of RT-PCR obtained against already inactivated viruses could be a serious drawbacks in food safety area. In this study, we investigated a method to yield true positive RT-PCR results only with alive viruses. To decompose the RNA genes from dead viruses, the enzymatic treatments composed of proteinse K and Ribonuclease A were applied to the sanitized and inactivated virus particles. Another aim of this study was to quantify the efficiencies of several major sanitizing treatments using real-time RT-PCR. Feline calicivirus (FCV) that belongs to the same Caliciviridae family with norovirus was used as a surrogate model for norovirus. The initial level of virus in control suspension was approximately $10^4$ PFU/mL. Most of inactivated viruses treated with the enzymatic treatment for 30 min at $37^{\circ}C$ were not detected in RT-PCR, Quantification results to verify the inactivation efficiencies of sanitizing treatments using real-time RT-PCR showed no false positive in most cases. We could successfully develope a numerical quantification process for the inactivated viruses after major sanitizing treatments using real-time RT-PCR. The results obtained in this study could provide a novel basis of rapid virus quantification in food safety area.

• Korean Journal of Food Science and Technology
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• v.39 no.1
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• pp.71-76
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• 2007

Foodborne illness caused by Noroviruses (NVs) is increasing rapidly in Korea. This study developed an effective detection protocol for NVs found in contaminated oysters and lettuce through an investigation using the major steps of virus particle separation, concentration and RT-PCR. As a surrogate model for NVs, the cultivable feline calicivirus (FCV) that belongs to the same Caliciviridae family was used. Instead of using a time-consuming ultracentrifugation method, efficient methods based on solvent extraction and PEG precipitation procedure were applied. Direct homogenization of a 25g sample of whole oyster and lettuce in 175mL PBS provided the simplicity that would be needed in the actual field of food product examination. The overnight PEG precipitation step at $4^{\circ}C$ was reduced to 3 h by placing the reaction tube in ice and by adjusting the PEG concentrations. The application of the use of chloroform and 0.2 ${\mu}m$ syringe filtration together showed a better detection efficiency than the use of chloroform alone in removing PCR inhibitors for both oyster and lettuce samples. Also, dilution of the extracted RNA solution before PCR provided increased sensitivity. The improved detection protocol developed in this study could be efficiently applied to detect FCV and most likely NVs from oysters and lettuce.

• Journal of Yeungnam Medical Science
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• v.10 no.2
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• pp.306-312
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• 1993

To evaluate the meaning of anti-HCV detection in patients treated with IVIG, serum levels of aspartate aminotranstferase(AST), alanine aminotransterase(ALT), HCV Ab titer were measured after treatment with IVIG in 36 patients diagnised of Kawasaki disease or neonatal sepsis. Also polymerase chain reaction (PCR) for the detection of HCV was done in 8 patients with persistent HCV Ab positivity at 3 months after IVIG treatment. The results were as follows 1) HCV Ab was positive in all 36 patients at 1 week after IVIG treatment, but in only 8 cases it was positive at 3 months after IVIG treatment. 2) AST, ALT were elevated in 9 cases at 1 week after IVIG treatment, but they were normalized in all cases at 3 months after IVIG treatment. 3) PCR for the detection of HCV was done in 8 patients with persistent HCV Ab positivity at 3 months after IVIG treatment, but HCV was not isolated in any cases. These results suggested that detection of anti-HCV was merely transitory phenominon of HCV Ab transmission, did not show any evidence of HCV infection due to HCV transmission.

• Journal of Food Hygiene and Safety
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• v.30 no.2
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• pp.150-158
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• 2015

This article reports the development of an effective test procedure for detection of norovirus (NoV) in foods of diverse matrices. In this study, target foods included fermented milk, soybean paste, powders made from uncooked grains and vegetables, sesame leaves preserved in soy sauce, pickled mooli, and mooli. Viral recovery varied depending on the food matrices or elution buffers tested. Buffers were compared to determine effective elution buffers from artificially virus-contaminated foods. The conventional test procedure for concentrating viruses from food (elution-polyethylene glycol(PEG) precipitation-chloroform-PEG precipitation) was modified to save time by eliminating one PEG precipitation step. The modified procedure (elution-chloroform-PEG precipitation) was able to concentrate viruses more effectively than the conventional procedure. It also removed RT-PCR inhibitors effectively. The modified procedure was applied to target food for genogroup II NoV detection. NoV RNA was detected at the initial inoculum levels 3.125-12.5 RT-PCR units per 10-25 g tested food. The use of this newly established procedure should facilitate detection of low levels of norovirus in diverse foods.