Despite recent economic prosperity, Korea still has high prevalence of tuberculosis. Molecular biologic characterization of Korean Mycobacterium tuberculosis strains might provide a deeper understanding of the forces contributing to the spread of tuberculosis in Korea. Therefore, we analyzed the cell lysate proteome of a representative Korean Mycobacterium tuberculosis isolate (K01) in comparison with laboratory reference strains H37Rv and H37Ra. Seven spots were strongly expressed only in K01 strain compared with M. tuberculosis H37Rv and H37Ra. Through continuous MALDI-MS analysis, these spots were identified as hypothetical protein Rv3849, secreted immunogenic protein Mpt64, Acetyl/propionyl-CoA Carbpxylase (AccD1), alkyl hydroperoxide reductase C (AhpC), N-acetylmuramyl-L-alanine amidase, a putative UDP glucose epimerase, and a transposase. A deeper study of these proteins may provide a clue in the development of effective new anti-tuberculosis vaccines against Korean M. tuberculosis isolates.
Korea ; Mycobacterium tuberculosis* ; Mycobacterium* ; Peroxiredoxins ; Prevalence ; Proteome* ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Transposases ; Tuberculosis ; UDPglucose 4-Epimerase ; Vaccines

BACKGROUND: The resurgence of tuberculosis and outbreaks of multidrug resistant (MDR) tuberculosis have increased the emphasis for the development of new susceptibility testing of the Mycobacterium tuberculosis for the effective treatment and control of the disease. Conventional drug susceptibility testings, such as those using egg-based or agar-based media have some limits, such as the time required and difficulties in determining critical inhibitory concentrations, but these are still being used in many diagnostic laboratories because of no better alternatives, considering cost and accuracy. To overcome these limits, a rapid and simple method for new susceptibility testing, using live and dead assays, was applied for a bacterial cell viability assay to distinguish dead from live bacterial cells based on two-color fluorescence. MATERIALS AND METHODS STRAINS: Forty strains were used in this study, 20 susceptible to all antituberculosis drugs and the other 20 resistant to the four first line antituberculosis drugs isoniazid, rifampicin, streptomycin and ethambutol. ANTIBIOTICS: The four antibiotics were dissolved in 7H9 broth to make the following solutions: 0.1micro gram isoniazid(INH)/ml, 0.4micro gram rifampicin(RMP)/ml, 4.0micro gram streptomycin(SM)/ml and 4.0micro gram ethambutol(EMB)/ml. RESULTS: Live and dead Mycobacterium tuberculosis cells fluoresced green and red with the acridin (Syto 9) and propidium treatments, respectively. These results are very well accorded with conventional drug susceptibility testing by proportional method on Lowensen-Jensen media (L-J) containing 4 drugs (INH, RMP, EMB and SM), showing a 93.7 % accordance rate in susceptible strains and 95% in resistant strains. CONCLUSION: The results of the drug susceptibility testing using the live and dead bacterial cell assay showed high accordance rates compared with the conventional proportion method on L-J. This finding suggests that the live and dead bacterial cell assay can be used as an alternative to conventional drug susceptibility testing for M. tuberculosis strains.
Anti-Bacterial Agents ; Cell Differentiation* ; Cell Survival ; Disease Outbreaks ; Ethambutol ; Fluorescence ; Isoniazid ; Mycobacterium tuberculosis* ; Mycobacterium* ; Propidium ; Rifampin ; Streptomycin ; Tuberculosis

No Abstract Available.
Korea* ; Mumps virus* ; Mumps*

The aim of this study was to establish a PCR for detecting of the hepatitis B virus (HBV) in blood and blood products. A primer pair set was designed to amplify a 513 bp fragment in the S-region of the HBV genome in the first PCR and a 233 bp fragment of first PCR amplicon in the second PCR with Rubisco (internal control). In order to assess the specificity of the PCR results, all the samples were tested cross-reactivity or interference in the assay. This method did not result in cross-reactivity with the non-HBV (HAV, HCV, HIV, CMV, HPV 18&6b, parvovirus B19/ or HSV 1&2) positive samples and was unaffected. In case of the HBV spiked blood products such as the immunogloubulin and coagulation factors, the lower detection limit of this method for the HBV DNA is 62.5 IU/ml. The PCR method is fully established in this study and will be a valuable method for the detection of the HBV in a variety of blood products, particularly, those derived from starting materials with a high titer of virus.
Blood Coagulation Factors ; DNA ; Genome ; Hepatitis B virus ; HIV ; Limit of Detection ; Parvovirus ; Polymerase Chain Reaction* ; Ribulose-Bisphosphate Carboxylase ; Sensitivity and Specificity

A number of recombinant proteins isolated from cell sources are being produced for biopharmaceuticals. Although most biopharmaceuticals are highly purified, there is a safety concern that such recombinant products could be contaminated with impurities including adventitious virus, mycoplasma, endotoxin and oncogenic DNA. Residual DNA in recombinant biopharmaceuticals is a potential risk factor and must be evaluated and removed to meet the regulatory guidelines. Recombinant HPV type 16 L1 VLPs, recombinant protein produced in Spodoptera frugiperda (Sf) 9 insect cells, is a HPV subunit vaccine candidate which has been studied as a preventive vaccine of cervical cancers. In this study, we performed detection and quantification of residual cellular DNA in the production of recombinant HPV type 16 L1 VLPs. HPV-16 L1 VLPs were purified by processes including detergent lysis, sonication treatment, sucrose cushion centrifugation, CsCl equilibrium density centrifugation, and DNase treatment which was added to inactivate residual cellular DNA after CsCl centrifugation step. We have developed a precise assay based on a dot-blot hybridization using digoxigenin random primed labeling DNA probes for the detection and quantification of residual cellular DNA during the purification process and final products. Detection limit of residual cellular DNA was 0.1 ng in this assay and the amount of residual cellular DNA in the final product was 0.5 ng~1 ng per 100 microgram of protein. This study describes safer and more sensitive methods alternative to radioactive techniques employed for residual cellular DNA quantification of biopharmaceuticals produced by recombinant protein technology and presents method validation data demonstrating precision and reproducibility.
Centrifugation ; Deoxyribonucleases ; Detergents ; Digoxigenin ; DNA Probes ; DNA* ; Human papillomavirus 16* ; Insects ; Limit of Detection ; Mycoplasma ; Recombinant Proteins ; Risk Factors ; Sonication ; Spodoptera ; Sucrose

BACKGROUND: Biomedical products such as viral vaccines can be contaminated with hazardous viruses during manufacturing processes and storage, thus causing harmful side effects. To assure the safety of biomedical products, highly effective and sensitive methods should be available to detect contaminating viruses. In this study, we performed recovery tests to determine the limit of detection of HIV-1. METHODS: An HIV-1 plasmid preparation was serially diluted and spiked into various culture media (DMEM, RPMI-1640, IMDM, GICM, and SDM) containing 10% fetal bovine serum (FBS). The HIV-1 plasmid was detected by PCR alone or a combination of PCR and ELISA (PCR/ELISA). RESULTS: When spiked into DMEM, RPMI, and IMDM, less than 4x10(-2) ng of HIV-1 plasmid was not detectable as HIV-1 PCR products in agarose gel. Intra- and inter-assays (n=6) showed that the PCR-ELISA system could detect PCR products diluted as much as 1, 875 times from HIV-1 plasmid serially spiked in various media. CONCLUSIONS: The PCR/ELISA system can be useful for the detection of trace amounts of hazardous viruses which may be present as contaminants in biological products.
Biological Products ; Culture Media ; Enzyme-Linked Immunosorbent Assay ; HIV-1* ; Limit of Detection ; Plasmids ; Polymerase Chain Reaction ; Sepharose ; Viral Vaccines

One-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using the MagNA Pure LC and LightCycler(TM) system was developed and validated for the detection and quantitation of hepatitis A virus (HAV) RNA. The assay was evaluated using in-house synthetic HAV RNA standard. The real-time RT-PCR assay could quantitate a dynamic range of HAV RNA standard between 10(2) and 10(8) copies per reaction. The regression coefficient of the standard curve was an 0.99. The detection limit of the assay was 31.3 RNA copies per reaction. The coefficient variations (CVs) of the assay in combination with automated RNA extraction were less than 1.91% in both intra- and inter-assay. The real-time RT-PCR assay for quantitative detection of HAV would serve a useful method for improving the safety of biological products.
Biological Products ; Hepatitis A virus* ; Hepatitis A* ; Hepatitis* ; Limit of Detection ; RNA*

Risk of viral contamination is one of major concerns common to all biologics derived from cultivated cells. Bovine viral diarrhoea virus (BVDV) has widely been known as a contaminant of cell culture-derived vaccines. The objective of the study was to assess the limit of detection and range of quantitation of the detection methods for BVDV using a reverse transcription-polymerase chain reaction (RT-PCR) assay, real-time RT-PCR assay, and RT-PCR-ELISA. One milliliter of cell culture supernatant containing 106.5+/-0.2 median tissue culture infectious dose (TCID50)/ml of BVDV NADL strain was subjected to RNA isolation. The isolated RNA was 10-fold serially diluted and each diluted sample (10-1 to 10-6) was subjected to RT-PCR on a GeneAmpR PCR System 9700 and/or LightCycler(TM). The amplified products were analyzedly (1) agarose gel electrophoresis for RT-PCR assay, (2) melting curve analysis for real-time RT-PCR assay (in this case a program is automatically linked to amplification step), and (3) ELISA using capture and detection probes for RT-PCR-ELISA. The limit of detection of the 3 assay methods was equally estimated to be 316 TCID50/ml of starting virus culture supernatant subjected to the assay. The quantitation range of real-time RT-PCR assay and RT-PCR-ELISA was estimated to be from 3.16x105 to 3.16x102 TCID50/ml of starting virus culture supernatant. The overall results suggested that the 3 assay methods for BVDV detection can be reliably applied to evaluate BVDV contamination in biologics derived from cell cultures.
Biological Products* ; Cell Culture Techniques* ; Electrophoresis, Agar Gel ; Enzyme-Linked Immunosorbent Assay ; Freezing ; Limit of Detection* ; Polymerase Chain Reaction ; Reverse Transcription ; RNA ; Vaccines

Commercial interests towards insect cell cultures have greatly been increased recently, in part due to the widespread use of insect virus-based vectors for efficient expressions of foreign proteins. Insect cell-derived biotechnology products should be free of adventitious agents such as arboviruses and mycoplasmas. The objective of this study was to establish techniques for the viral safety evaluation of insect cell-derived biotechnology products using Japanese encephalitis virus (JEV) as a model, since JEV is a member of arthropod-borne flaviviruses which has been known to be infectious to insect cells such as Sf9 cells. Quantitative assays for viral infectivity, concentrations of an antigen or a genome are a prerequisite for the studies of viral clearance validation. Here, we report the development of a quantitative assay for JEV RNA using real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay was performed using LightCycler and RNA amplification kit SYBR Green I. Viral RNA extracted from stock suspension of JEV with known infectivity titer was made to 10-fold serial dilution, and each dilution was subjected to the assay for the generation of a standard curve. A JEV specific primer was selected from 3' untranslated region with the expected band size of 323 base pairs. The real-time RT-PCR assay resulted in a successful amplification within 5 log dilution ranges of JEV RNA samples, and the sensitivity of the assay was calculated to be approximately 15 TCID50 per reaction. Highly reproducible standard curves were obtained from experiments performed on three different days. The real-time RT-PCR assay for the quantification of JEV will be an efficient alternative tool for viral clearance validation studies of insect cell-derived biotechnology products.
3' Untranslated Regions ; Arboviruses ; Asian Continental Ancestry Group* ; Base Pairing ; Biotechnology* ; Cell Culture Techniques ; Encephalitis Virus, Japanese* ; Encephalitis, Japanese* ; Flavivirus ; Genome ; Humans ; Insects* ; Mycoplasma ; RNA ; RNA, Viral ; Sf9 Cells

As viral vaccines sometimes induce the side effects which intimidate humans, it is urgently required to study about the side effects of viral vaccines. Most vaccines are derived from the biological forms. Hence it should be established the efficient and sensitive evaluation methods for the possibility of dangerous contaminants such as viruses and the stability of vaccines. A lots of vaccine products have been imported, or in sale or being developed in nation. Some of products may be mixed with the hazardous viruses derived from animal or human resources. Such hazardous viruses should be identified specifically and efficiently. Biological products are not permitted to distribute or obtain without presenting the result of absence experiment of hazardous viruses and prion and the validation data of inactivation experiment during processes. In order to detect viruses, there were TEM for viral particles, infectivity assays and detection methods for nucleic acids. However, these methods have defects such as insensitivity and inaccuracy. It should be considered to detect only the hazardous viruses with specificity, precision and accuracy during the processes of preparation, transportation and storage. It is noted to increase the detection limit in order to detect the minute hazardous factors during preparation processes for viral vaccines. This study is focused on the establishment of various sensitive PCR/ELISA methods for HBV virus which enhance the detection limit in order to detect the minute hazardous factors during preparation processes for viral vaccines.
Animals ; Biological Products ; Commerce ; Humans ; Limit of Detection ; Mass Screening* ; Nucleic Acids ; Sensitivity and Specificity ; Transportation ; Vaccines ; Viral Vaccines ; Virion