• Biomedical Science Letters
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• v.16 no.2
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• pp.127-131
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• 2010

Mycobacterium leprae detection is difficult even with molecular biological techniques due to the low sensitivity of current methodologies. In this report, real-time PCR targeting the M. leprae-specific repetitive element (RLEP) sequence was developed as a new diagnostic tool and evaluated using clinical specimens. For this, M. leprae DNAs were extracted from skin biopsy specimens from 80 patients and analyzed by real-time PCR using TaqMan probe. Then, the detection efficiency of the real-time PCR was compared with that of standard PCR. In brief, the rate of positive detection by the standard PCR and real-time PCR was 32.50% and 66.25%, respectively. The results seemed to clearly show that the TaqMan real-time PCR developed in this study may be a useful tool for sensitive detection of M. leprae from clinical specimens.

• Journal of the Korean Chemical Society
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• v.51 no.1
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• pp.36-42
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• 2007

Hepatitis B is a serious public health problem leading to chronic infection and liver cancer. Quantitation of circulating hepatitis B virus (HBV) is important for monitoring disease progression and for assessing the response to antiviral therapy. In this study, by using Real-Time PCR and novel Micro-PCR assay method, we measured HBV concentration in the clinical sample. A total of 120 serum samples from patients with HBV infection collected was in Dankook university hospital to compare the detection limit, sensitivity, specificity and reproducibility of the two assay methods. These findings of this study suggest that Micro-PCR and Real-Time PCR assay methods are comparable to each other in there detection limit, sensitivity, and reproducibility for HBV DNA quantitation. However, Micro-PCR assay is more efficient than Real-Time PCR method, because Real-Time PCR is not so time - consuming, technically easy and need to reagent of a small quantity. It will be useful for rapid and reliable clinical diagnosis of HBV in many countries.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1453-1458
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• 2008

For quantitative PCR assay of Salmonella enterica serovar Typhimurium in food samples, a real-time PCR method was developed, based on DNA genome equivalent. Specific primers and probe designed based on the STM4497 gene of S. Typhimurium LT2 showed the specificity to S. Typhimurium. Threshold cycle (Ct) values of real-time PCR were obtained from a quantitative standard curve with genomic DNA of Salmonella Typhimurium. In addition, the recovery of S. Typhimurium inoculated artificially to chicken samples with $4.5{\times}10^5$ to 4.5 CFU/ml was evaluated by using real-time PCR and plate-count methods. Result showed that the number of cells calculated from the real-time PCR method had good correlation with that of the plate-count method. This real-time PCR method could be applicable to the detection and quantification of S. Typhimurium in food samples.

• Tuberculosis and Respiratory Diseases
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• v.69 no.4
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• pp.250-255
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• 2010

Background: The purpose of this study was to evaluate recently developed real-time polymerase chain reaction (PCR) assay kit to detect Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) in respiratory specimens. Methods: We assessed the positive rate of the real-time PCR assay to detect MTB and NTM in 87 culture-positive specimens (37 sputum, 50 bronchial washing), which were performed real-time PCR by using $Real-Q_{TM}$ MTB&NTM Kit from January 2009 to June 2009, at Gyeongsang University Hospital. To compare the efficacy with the TB-PCR assay, we evaluated 63 culture-positive specimens (19 sputum, 44 bronchial washing) for MTB or NTM, which were performed TB-PCR by using ABSOLUTETM MTB II PCR Kit from March 2008 to August 2008. Results: Among 87 specimens tested using real-time PCR, MTB and NTM were cultured in 58 and 29, respectively. The positive rate of real-time PCR assay to detect MTB was 71% (22/31) and 92.6% (25/27) in AFB stain-negative and stain-positive specimens. For NTM, the positive rate of real-time PCR was 11.1% (2/18) and 72.7% (8/11) in AFB stain-negative and stain-positive specimens. Among 63 specimens performed using TB-PCR, MTB and NTM were cultured in 46 and 17, respectively. The positive rate of TB-PCR was 61.7% (21/34) and 100% (12/12) in AFB stain-negative and stain-positive specimens. TB-PCR was negative in all NTM-cultured 17 specimens. Conclusion: TB/NTM real-time PCR assay is useful to differentiate MTB and NTM in AFB stain-positive respiratory specimens and it is as effective in detecting MTB with TB-PCR.

• Restorative Dentistry and Endodontics
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• v.33 no.6
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• pp.537-544
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• 2008

Polymerase chain reaction (PCR) can detect bacteria more rapidly than conventional plate counting. However DNA-based assays cannot distinguish between viable and dead cells due to persistence of DNA after cells have lost their vitality. Recently, propidium monoazide (PMA) treatment has been introduced. The purpose of this study is to evaluate the applicability of the PMA treatment and real-time PCR method for cell counting in comparison with plate counting and to evaluate the antibacterial efficacy of 2% CHX on E. faecalis using PMA treatment in combination with real-time PCR. Firstly, to elucidate the relationship between the proportion of viable cells and the real-time PCR signals after PMA treatment, mixtures with different ratios of viable and dead cells were used. Secondly, relative difference of viable cells using PMA treatment in combination with real-time PCR was compared with CFU by plate counting. Lastly, antibacterial efficacy of 2% CHX on E. faecalis was measured using PMA treatment in combination with real-time PCR. The results were as follows : 1. Ct value increased with decreasing proportion of viable E. faecalis. 2. There was correlation between viable cells measured by real-time PCR after PMA treatment and CFU by plate counting until Optical density (OD) value remains under 1.0. However, viable cells measured by real-time PCR after PMA treatment have decreased at 1.5 of OD value while CFU kept increasing. 3. Relative difference of viable E. faecalis decreased more after longer application of 2% CHX.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.4
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• pp.263-266
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• 2007

Vibrio vulnificusis a causative agent of serious diseases in humans resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately $84^{\circ}C$ for V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or $10^3$ V. vulnificus cells from pure cultured broth and $10^3$ cells in 1g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium using the rpoS gene in pure cultures and in infected oyster tissues.

• Food Science of Animal Resources
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• v.41 no.1
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• pp.85-94
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• 2021

A pig-specific real-time PCR assay based on the mitochondrial ND5 gene was developed to detect porcine material in food and other products. To optimize the performance of assay, seven commercial TaqMan master mixes and two real-time PCR platforms (Applied Biosystems StepOnePlus and Bio-rad CFX Connect) were used to evaluate the limit of detection (LOD) as well as the PCR efficiency and specificity. The LODs and PCR efficiencies for the seven master mixes on two platforms were 0.5-5 pg/reaction and 84.96%-108.80%, respectively. Additionally, non-specific amplifications of DNA from other animal samples (human, dog, cow, and chicken) were observed for four master mixes. These results imply that the sensitivity and specificity of a real-time PCR assay may vary depending on master mix and platform used. The best combination of master mix and real-time PCR platform can accurately detect 0.5 pg porcine DNA, with a PCR efficiency of 100.49%.

• Journal of Life Science
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• v.18 no.3
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• pp.374-380
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• 2008

Rapid detection of enterovirus (EVs) is important in the management of aseptic meningitis. We examined the relative efficiency and specificity of the real-time nucleic acid sequence-based amplification (NASBA) comparing with the established reverse transcription polymerase chain reaction (RT-PCR) and viral culture method which were used for the detection of enterovirus RNA in clinical specimens. Of the total 292 samples, 145 were found to be positive to enterovirus RNA by real-time NASBA, 101 were positive by viral culture, and 86 were positive by RT-PCR. 147 samples and 46 samples were determined to be negative and positive by all methods respectively, but 4 samples were positive only by real-time NASBA. To compare the specificity of each method, various clinical samples which were diagnosed for herpes simplex virus (HSV)-1, HSV-2, adenovirus, mumps, and rhinovirus were applied. Except one rhinovirus sample which was false positive to enterovirus RNA by RT-PCR, the other different samples were negative to all three methods. The real-time NASBA procedure can be completed within 5 hours in contrast with 9 hours for the RT-PCR and 3-14 days for the viral culture. From this study, it was suggested that the real-time NASBA assay could be a standardized, rapid, specific, and sensitive procedure for the detection of enterovirus RNA.

• Journal of Food Hygiene and Safety
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• v.34 no.3
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• pp.290-295
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• 2019

Salmonella spp. are frequently associated with food and are among the most important foodborne pathogens. The recent Salmonella out breaks in Korea was associated with chocolate mousse cakes served with school meals during September 2018. The objective of this research was to compare the 3M Molecular Detection Assay 2 - Salmonella and the Korean Standard Method of Salmonella in artificially inoculated mousse (chocolate and cheese) and tiramisu cakes. Mousse (chocolate and cheese) and tiramisu cakes were artificially inoculated with S. Typhimurium. Twenty five gram of sample was enriched with 225 mL buffered peptone water for incubation at $37^{\circ}C$ for 24 h. After enrichment, the cultures were analyzed by using the 3M Molecular Detection Assay 2 - Salmonella and the Korean Standard Method. Most of the inoculated samples showed similar results except the chocolate mousse cakes, in which real-time PCR was unable to detect S. Typhimurium even after $10^4CFU/25g$ of inoculation. However, S. Typhimurium inoculated at a concentration of $10^0CFU/25g$ was detected by using 3M Molecular Detection Assay 2 - Salmonella. In chocolate mousse, detection of S. Typhimurium using real-time PCR was partially successful when dark chocolate was added at less than 15%. Negative results in real-time PCR and 3M Molecular Detection Assay 2 - Salmonella were confirmed by gel electrophoresis. The data indicated that dark chocolate could inhibit amplification of the target gene in the PCR reactions. In conclusion, the 3M Molecular Detection Assay 2 - Salmonella was better than the Korean Standard Method (real-time PCR) for the detection of S. Typhimurium in chocolate mousse cakes and chocolate mousse.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.148-153
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• 2005

Malaria is a re-emerging infectious disease that is spreading to areas where it had been eradicated, such as Eastern Europe and Central Asia. To avoid the mortality from malaria, early detection of the parasite is a very important issue. The peripheral blood smear has been the gold standard method for the diagnosis of malaria infection. Recently, several other methods have been introduced for quantitative detection of malaria parasites. Real time PCR that employs fluorescent labels to enable the continuous monitoring of PCR product formation throughout the reaction has recently been used to detect several human malaria parasites. 18S rRNA sequences from malaria parasites have been amplified using Taqman real time PCR assay. Here, a SYBR Green-based real time quantitative PCR assay for the detection of malaria parasite-especially, Plasmodium vivax - was applied for the evaluation of 26 blood samples from Korean malaria patients. Even though SYBR Green-based real time PCR is easier and cheaper than Taqman-based assay, SYBR Green-based assay cannot be used because 18S rRNA cannot be specifically amplified using 1 primer set. Therefore, we used DBP gene sequences from Plasmodium vivax, which is specific for the SYBR Green based assays. We amplified the DBP gene from the 26 blood samples of malaria patients using SYBR Green based assay and obtained the copy numbers of DBP genes for each sample. Also, we selected optimal reference gene between ACTB and B2M using real time assay to get the stable genes regardless of Malaria titer. Using selected ACTB reference genes, we successfully converted the copy numbers from samples into titer, ${\sharp}$ of parasites per microliter. Using the resultant titer from DBP based SYBER Green assay with ACTB reference gene, we compared the results from our study with the titer from Taqman-based assay. We found that our results showed identical tendency with the results of 18S rRNA Taqman assay, especially in lower titer range. Thus, our DBP gene-utilized real time assay can detect Plasmodium vivax in Korean patient group semi-quantitatively and easily.