• Korean Journal of Clinical Laboratory Science
• /
• v.48 no.2
• /
• pp.88-93
• /
• 2016

Dirofilaria immitis is a filarial nematode parasite that causes cardiopulmonary dirofilariasis in dogs. The purpose of this study was the development of real-time PCR assays for efficient detection of D. immitis. The D. immitis-specific primers confirmed in our previous study and a newly designed TaqMan probe were used for quantitative diagnostics. First, SYBR Green real-time PCR was performed using the specific primers and serially diluted genomic DNA or plasmid DNA, and melting curve analyses were performed after amplification. The melting curve showed one specific peak in each of the genomic and plasmid DNA reactions, suggesting that the primers specifically amplify the D. immitis cytochrome c oxidase subunit I gene. Comparison of SYBR Green and TaqMan real-time PCR using serially diluted plasmid DNA showed higher efficiency and specificity with TaqMan real-time PCR. The real-time PCR assays developed in this study will provide improved diagnostic methods to overcome the limitations of conventional diagnostic tools and facilitate more rapid and accurate diagnoses.

• Journal of the Korean Society for Precision Engineering
• /
• v.25 no.11
• /
• pp.37-44
• /
• 2008

• Pediatric Infection and Vaccine
• /
• v.14 no.2
• /
• pp.171-178
• /
• 2007

Purpose : We investigated clinical characteristics of real-time PCR proven EBV viremia patients who were not serologically diagnosed but clinically suspected, and compared it to serologically proven EBV infected patients. Methods : The study population consisted of 45 patients, who were suspected acute EBV infection at Wonju Christian Hospital Department of Pediatrics, Yonsei University Wonju College of Medicine from Jan. 2004 to Dec. 2006. real-time PCR of cell free serum was performed to prove EBV viremia. Then we chose $102.5copies/{\mu}g$ DNA as a suitable cutoff level for EBV associated diseases. Results : There are 4 patients diagnosed as EBV infection by serologically and 15 patients diagnosed as EBV viremia by real-time PCR quantitative measurement. The most common presenting symptoms and signs of EBV viremia was fever in 11 cases (73%). Atypical lymphocytosis was found in 4 cases (27%). Increased AST, ALT levels were observed in 13 cases (87%), 12 cases (80%), respectively. We could diagnose 5 cases of EBV viremia younger than one year of age. They revealed clinical symptoms which could be found in EBV infection. The serologically diagnosed patients had hepatomegaly and splenomegaly in 3 cases (75%). All serologically confirmed patients have leukocytosis above $20,000/mm^3$, among them 2 cases (50%) had higher percentage (>15%) of atypical lymphocytes. The AST/ALT level above 50 IU/L were demonstrated in all cases. Conclusion : Serologically unproven real-time PCR EBV viremia patients revealed similar clinical findings with that of serologically proven EBV infected patients. So, it is meaningful to perform EBV real-time PCR for the diagnosis of EBV infection especially for the cases younger than 1 year of age.

• Journal of Veterinary Clinics
• /
• v.32 no.2
• /
• pp.154-157
• /
• 2015

A major cause of diarrhea in a dog is an infection with bacteria which include Salmonella spp., Campylobacter (C.) spp., and Clostridium (Cl.) spp.. It is fastidious to identify these bacteria by the culture. The purpose of this experiment is to devise the method for detecting Cl. perfringens, C. jejuni, C. coli, and Salmonella spp. with rapid and high sensitivity. The fecal samples collected from 71 normal and 66 diarrheic dog feces were used to compare the prevalence of the enteric pathogens and to develop a multiplex real-time polymerase chain reaction (PCR) assay for clinical use. Detection of Cl. perfringens, C. coli, and C. jejuni in diarrhea feces was higher than normal feces. A developed multiplex real-time PCR is useful for determining the presence and quantity of pathogen-specific or other unique sequences with in a fecal sample.

• Journal of Veterinary Clinics
• /
• v.32 no.3
• /
• pp.219-223
• /
• 2015

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis or Johne's disease, an intestinal granulomatous infection in domestic and wild animals. The study aimed to develop and evaluate a panel of multiplex quantitative real-time polymerase chain reaction (mqPCR) assay for simultaneous detection of three MAP-specific genes (IS900, F57 and ISMAP02 genes). The analytic sensitivity (i.e., limit of detection, expressed as cells per 1 ml) was 150 for IS900, 1500 for F57, and 50 for ISMAP02. The specificity of the method was determined by testing 152 bovine fecal samples. Based on the test, it showed that the assay simultaneously detected the target genes in short period of time and at lower cost compared to laboratory routine tests. The test agreement between the assay and routine test was 94%. The discrepancy in the results was due to samples that were tested positive by the panel but negative by the routine tests, suggesting that the assay has higher sensitivity than the routine tests. In conclusion, the mqPCR assay could be a rapid and accurate testing tool for investigating paratuberculosis or Johne's disease cases in domestic and wild animals.

• Laboratory Medicine Online
• /
• v.1 no.1
• /
• pp.26-34
• /
• 2011

Background: Laboratory diagnosis of new influenza A (H1N1) is crucial for managing patients and establishing control and prevention measures. We compared the diagnostic accuracies of the real time RT-PCR (rRT-PCR) test recommended for the confirmation of the new flu and the viral culture method used conventionally for viral disease with that of the rapid antigen test (RAT). Methods: We performed RAT, R-mix culture, and real-time PCR by using 861 respiratory samples collected from December 2009 to January 2010 and evaluated the abilities of these methods to detect new influenza A. The relationship among the positive rates of RAT, grades of culture, and the cycle threshold (Ct) values of rRT-PCR was also evaluated. Results: Of the 861 patients, 308 (35.8%) were diagnosed with new influenza A. The sensitivities, specificities, positive predictive values, and negative predictive values of the tests were respectively as follows: 59.7%, 99.5%, 98.4%, and 81.6% for RAT; 93.2%, 100%, 100%, and 96.3% for R-mix culture; and 95.8%, 100%, 100%, and 97.7% for rRT-PCR. Samples with weak positive grade in culture and those with Ct values of 30-37 in rRT-PCR showed positivities as low as 25.3% and 2.3% in RAT, respectively. The hospitalization rate and death rate of the confirmed patients were 3.2% and 0.3%, respectively, and gastrointestinal symptoms were observed in 7.2% of the patients. Conclusions: R-mix culture and rRT-PCR tests showed excellent reliability in the diagnosis of new influenza A and could be very useful, especially for samples with low viral load.

• The Journal of the Korea Contents Association
• /
• v.21 no.1
• /
• pp.465-474
• /
• 2021

Corona-19, a disease caused by 'Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)', was declared a global pandemic by the World Health Organization (WHO) in March 2020, and a real-time polymerase chain reaction test is performed as a diagnostic test for screening and confirmation in most countries. However, not only the target genes and protocols differ by countries, but also the procedures for reading the diagnosis results are diverse, so the criteria for confirmed patients differ by country. Therefore, in this review, we discussed the target genes, test techniques, and diagnostic criteria for each country notified by WHO. And the specificity and sensitivity, limits of detection, positive and negative controls, false positive bacteria candidates, and specimens, and the specifics of the control setting were also described. In addition, the characteristics of Korea's test were compared to each country's one. Finally, in order to obtain the same diagnosis result for SARS-CoV-2 in the future, standardized diagnosis methods and result interpretations for Corona-19 diagnosis were proposed.

• Journal of the Korea Institute of Military Science and Technology
• /
• v.20 no.5
• /
• pp.726-732
• /
• 2017

Bacillus anthracis, Vibrio cholerae, Variola virus and Shigella dysenteriae are classified as category A and B biological weapons. In this study suggest that 4 genes of Bacillus anthracis, 2 genes of Vibrio cholerae, 1 gene of Variola virus and 1 gene of Shigella dysenteriae were detective 50~500 fg of target DNA per reaction using real-time PCR based assay. Also analytical specificity did not show any cross-reactivity with other related bacteria. Reliable and one reaction could be effective early diagnostic and treatment for detection of unknown samples.

• Journal of Korean Society of Water and Wastewater
• /
• v.23 no.2
• /
• pp.199-205
• /
• 2009

It is very important to differentiate of DNA derived from live or dead bacteria within mixed microbial communities in aquatic environments. Ethidium monoazide (EMA) is a DNA intercalating agent and the treatment of EMA with strong visible light cleaves the genomic DNA of bacteria. In dead bacterial cells, EMA intercalates into the genomic DNA, induces the cleavage of DNA, and inhibits the PCR amplification. In this study, we developed the EMA-PCR and EMA real-time PCR to detect the DNA derived from viable Escherichia coli (E.coli) in mixed cultures of live and dead E.coli. The treatment of EMA, $50{\mu}g/mL$, and 650 W visible halogen light exposure for 2 minutes cleaved the genomic DNA derived from heat killed E.coli but did not those of live E.coli. EMA-PCR could detect the DNA from live E.coli in mixed culture samples of live and dead E.coli at various ratio and there was no DNA amplification in only dead E.coli cultures. Similar results were observed in EMA real-time PCR. Further studies are needed to develop various EMA-PCR methods to detect viable waterborne pathogens such as Helicobacter pylori, Giardia lamblia, and so on.

• Journal of Veterinary Clinics
• /
• v.27 no.5
• /
• pp.508-513
• /
• 2010

Cytokines are important mediators of the immune response, and quantitating cytokine mRNA is a highly sensitive and attractive method for measuring cytokine production. The objective of the current study was to develop and validate a SYBR green quantitative real-time reverse transcriptase PCR (qRT-PCR) assay for measuring canine cytokine mRNA. The optimal annealing temperatures ($T_a$) of the designed primers were $62^{\circ}C$ for interleukin (IL)-$1{\beta}$, IL-6 and IL-10; $60^{\circ}C$ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tumor necrosis factor (TNF)-${\alpha}$; and $58^{\circ}C$ for high mobility group box 1 (HMGB1). Primer efficiencies of all primers calculated for standard curve samples were between 97.1% and 102.6%. No evidence of secondary structure or primer-dimer formation was seen via melt-curve analysis or gel electrophoresis. The developed qRT-PCR assays are highly specific and sensitive and can be used to quantify gene expression levels of canine cytokines.