• Title/Summary/Keyword: DNA probe method

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Rapid Detection and Monitoring Therapeutic Efficacy of Mycobacterium tuberculosis Complex Using a Novel Real-Time Assay

  • Jiang, Li Juan;Wu, Wen Juan;Wu, Hai;Ryang, Son Sik;Zhou, Jian;Wu, Wei;Li, Tao;Guo, Jian;Wang, Hong Hai;Lu, Shui Hua;Li, Yao
    • Journal of Microbiology and Biotechnology
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    • v.22 no.9
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    • pp.1301-1306
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    • 2012
  • We combined real-time RT-PCR and real-time PCR (R/P) assays using a hydrolysis probe to detect Mycobacterium tuberculosis complex (MTBC)-specific 16S rRNA and its rRNA gene (rDNA). The assay was applied to 28 non-respiratory and 207 respiratory specimens from 218 patients. Total nucleic acids (including RNA and DNA) were extracted from samples, and results were considered positive if the repeat RT-PCR threshold cycle was ${\leq}35$ and the ratio of real-time RT-PCR and real-time PCR load was ${\geq}1.51$. The results were compared with those from existing methods, including smear, culture, and real-time PCR. Following resolution of the discrepant results between R/P assay and culture, the overall sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) of all samples (including non-respiratory and respiratory specimens) were 98.2%, 97.2%, 91.7%, and 99.4%, respectively, for R/P assay, and 83.9%, 89.9%, 72.3%, and 94.7%, respectively, for real-time PCR. Furthermore, the R/P assay of four patient samples showed a higher ratio before treatment than after several days of treatment. We conclude that the R/P assay is a rapid and accurate method for direct detection of MTBC, which can distinguish viable and nonviable MTBC, and thus may guide patient therapy and public health decisions.

Purification and Gene Analysis of Peptidyl Prolyl cia-trans Isomerase from Bacillus stearothermophilus (Bacillus stearothermophilus Peptidyl Prolyl cis-trans Isomerase의 정제 및 유전자 분석)

  • 김동주
    • The Korean Journal of Food And Nutrition
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    • v.15 no.2
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    • pp.104-111
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    • 2002
  • The peptidyl prolyl sis-trans isomerase (PPIase, EC 5.2.1.8) from bacillus stearothermophilus was extracted from the cells treated with by lysozyme. PPIase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPIase was estimated as 18kDa by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0∼8.0. The enzyme was considerably stable after heat treatment at 60$\^{C}$ for 30minutes, and the enzyme was quite stable up to 65$\^{C}$. The presence of the PPIase in the refolding solution accelerated the isomerization rate of the assay peptide. PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-l primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPI-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus.

Gene Cloning, Nucleotide Sequence and Efficent Expression of Peptidyl proryl cis-trans Isomerase from Bacillus stearothermophilus (Bacillus stearothermophilus의 Peptidyl Prolyl cis-trans Isomerase 유전자 분리 염기배열 및 발현)

  • 김동주
    • The Korean Journal of Food And Nutrition
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    • v.9 no.4
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    • pp.452-458
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    • 1996
  • A PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-1 primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI1-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPi-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus. The translated protein sequence of PPIase B. stearothermophilus was compared with sequence from periplasmic PPIase from Escherichina coil ; homogies of 16 and 58%, respectively, were found. The clond PPIase gene was over-expressed in E. coil cell using pUC19 as an expression vector. The enzyme was partially purified by heat treatment and colum chromatochraphy on DEAE-Sepharose CL-6B. The molecular weight of the enzyme was dermined to be about 18.0 kDal by SDS-PAGE.

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Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2000.05a
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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Detection of HER2 Status in Breast Cancer: Comparison of Current Methods with MLPA and Real-time RT-PCR

  • Pazhoomand, Reza;Keyhan, Elahe;Banan, Mehdi;Najmabad, Hossein;Karimlou, Masoud;Khodadad, Faranak;Iraniparast, Alireza;Feiz, Farnaz;Majidzadeh, Keivan;Bahman, Ideh;Moghadam, Fatemeh Aghakhani;Sobhani, Atoosa Madadkar;Abedin, Seyedeh Sedigheh;Muhammadnejad, Ahad;Behjat, Farkhondeh
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.12
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    • pp.7621-7628
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    • 2013
  • Human epidermal growth factor receptor (HER) status is an important prognostic factor in breast cancer. There is no globally accepted method for determining its status, and which method is most precise is still a matter of debate. We here analyzed HER2 mRNA expression by quantitative reverse transcription-PCR (qRT-PCR) and HER2 DNA amplification using multiplex ligation-dependent probe amplification (MLPA). In parallel, we performed a routine evaluation of HER2 protein by immunohistochemistry (IHC). To assess the accuracy of the RT-PCR and MLPA techniques, a combination of IHC and fluorescence in situ hybridization (FISH) was used, substituting FISH when the results of IHC were ambiguous (2+) and for those IHC results that disagreed with MLPA and qRT-PCR, this approach being termed IHC-FISH. The IHC results for four samples were not compatible with the MLPA and qRT-PCR results; the MLPA and qRT-PCR results for these samples were confirmed by FISH. The correlations between IHC-FISH and qRT-PCR or MLPA were 0.945 and 0.973, respectively. The ASCO/CAP guideline IHC/FISH correlation with MLPA was (0.827) and with RT-PCR was (0.854). The correlations between the IHC results (0, 1+ as negative, and 3+ as positive) and qRT-PCR and MLPA techniques were 0.743 and 0.831, respectively. Given the shortcomings of IHC analysis and greater correlations between MLPA, qRT-PCR, and FISH methods than IHC analysis alone with each of these three methods, we propose that MLPA and real-time PCR are good alternatives to IHC. However a suitable cut-off point for qRTPCR is a prerequisite for determining the exact status of HER2.

Cloning of a Glutathione S-Transferase Decreasing During Differentiation of HL60 Cell Line (HL6O 세포주의 분화 시 감소 특성을 보이는 Glutathione S-Transferase의 클로닝)

  • Kim Jae Chul;Park In Kyu;Lee Kyu Bo;Sohn Sang Kyun;Kim Moo Kyu;Kim Jung Chul
    • Radiation Oncology Journal
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    • v.17 no.2
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    • pp.151-157
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    • 1999
  • Purpose : By sequencing the Erpressed Sequence Tags of human 걸ermal papilla CDNA library, we identified a clone named K872 of which the expression decreased during differentiation of HL6O cell line. Materials and Methods : K872 plasmid DNA was isolated according to QIA plasmid extraction kit (Qiagen GmbH, Germany). The nucleotide sequencing was performed by Sanger's method with K872 plasmid DNA. The most updated GenBank EMBL necleic acid banks were searched through the internet by using BLAST (Basic Local Alignment Search Tools) program. Nothern bots were performed using RNA isolated from various human tissues and cancer cell lines. The gene expression of the fusion protein was achieved by His-Patch Thiofusicn expression system and the protein product was identified on SDS-PAGE. Results : K872 clone is 1006 nucleotides long, and has a coding region of 675 nucleotides and a 3' non-coding region of 280 nucleotides. The presumed open reading frame starting at the 5' terminus of K872 encodes 226 amino acids, including the initiation methionine residue. The amino acid sequence deduced from the open reading frame of K872 shares $70\%$, identity with that of rat glutathione 5-transferase kappa 1 (rGSTKl). The transcripts were expressed in a variety of human tissues and cancer cells. The levels of transcript were relatively high in those tissues such as heart, skeletal muscle, and peripheral blood leukocyte. It is noteworthy that K872 was found to be abundantly expressed in coloreetal cancer and melanoma cell lines. Conclusion : Homology search result suggests that K872 clone is the human homolog of the rGSTK1 which is known to be involved in the resistance of cytotoxic therapy. We propose that meticulous functional analysis should be followed to confirm that.

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Establishment and Application of a Multibead Serotyping Assay for Pneumococci in Korea (다중구슬 폐구균 혈청형 분석법의 국내 확립과 적용 연구)

  • Kim, Han Wool;Lee, Soyoung;Lee, Miae;Kim, Kyung-Hyo
    • Pediatric Infection and Vaccine
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    • v.22 no.2
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    • pp.97-105
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    • 2015
  • Purpose: Serotyping pneumococcal isolates is important to monitor efficacy of pneumococcal vaccines. Because of difficulties of typing pnueumocci, a multiplex bead-based (multibead) serotyping assay was recently introduced. The aim of this study is to establish a new multibead serotyping assay and to apply this method to analyze clinical isolates of pneumococci in Korea. Methods: To establish the multibead serotyping assay, six key reagents were transferred from University of Alabama at Birmingham (UAB) to Ewha Center for Vaccine Evaluation and Study (ECVES): bead set coated with polysaccharide and monoclonal antibody pool were used in one multiplex inhibition-type immunoassay and 2 bead sets coated DNA probe and 2 primer pools were used in two multiplex PCR-based assays. After multibead serotyping assay was set up, 75 test samples of pneumococci were analyzed whether ECVES is able to identify serotype correctly. After confirming the performance, serotyping assay was applied to identify serotypes of 528 clinical isolates of pneumococci collected from 3 different hospitals. Results: After establishment of the multibead pneumococcal serotyping assay system at ECVES, 75 test samples were analyzed. There was no discrepancy of serotypes of 75 test samples between the results assigned at UAB and those at ECVES. The serotypes of 528 pneumococci isolated from patients or healthy subjects were determined in 94.3% of isolates (498/528). Conclusions: The multibead pneumococcal serotyping assay can be successfully established in Korea. With this method, surveillance of serotypes of pneumococci isolated from patients as well as healthy subjects could be studied.

Glucose Transporter Gene Expression in Human Lung Cancer Cell Lines (사람 폐암 세포주에서 포도당 운반 단백 유전자의 발현)

  • Kim, Woo-Jin;Yim, Jae-Joon;Lee, Jae-Ho;Yoo, Chul-Gyu;Chung, Hee-Soon;Han, Sung-Koo;Chung, June-Key;Shim, Young-Soo;Kim, Young-Whan
    • Tuberculosis and Respiratory Diseases
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    • v.45 no.4
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    • pp.760-765
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    • 1998
  • Background: Glucose uptake has been found to be increased in cancer cells, and FDG-PET imaging is used for diagnosis of cancer using this phenomenon. However, the exact mechanism of increased glucose uptake in cancer cells has not been clarified. Recent studies demonstrated the presence of glucose transporter(GLUT) mRNA expression in gastrointestinal cancer and head and neck cancer, and suggested that GLUT may be associated with glucose uptake in cancer cells. In lung cancer cells, glucose metabolism is also known to be increased. We evaluated GLUT mRNA expression in human lung cancer cell lines in order to find out the mechanism of increased glucose uptake in lung cancer. Method: Total RNA was isolated from 15 human lung cancer cell lines and immortalized bronchial epithelial cell line(BEAS-2B). After electrophoresis of $20{\mu}g$ total RNA, Northern blot analysis was done using GLUT1 cDNA and GLUT3 cDNA as probes. Results: Thirteen of 14 human lung cancer cell lines expressed GLUT1 mRNA and 10 of 14 human lung cancer cell lines expressed GLUT3 mRNA. Eight human lung cancer cell lines expressed both GLUT mRNAs. BEAS-2B expressed GLUT1 mRNA and did not express detectable GLUT3 mRNA. Conclusion: The increase of glucose metabolism in lung cancer may be associated with GLUT1 and GLUT3 expression.

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Southern Analysis after Long-range PCR: Clinical Application in Korean Patients with Myotonic Dystrophy 1

  • Yum, Mi-Sun;Lee, Beom Hee;Kim, Gu-Hwan;Lee, Jin-Joo;Choi, Seung Hoon;Lee, Joo Yeon;Kim, Jae-Min;Kim, Yoo-Mi;Ko, Tae-Sung;Yoo, Han-Wook
    • Journal of Genetic Medicine
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    • v.10 no.1
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    • pp.33-37
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    • 2013
  • Purpose: Myotonic dystrophy 1 (DM1, OMIM 160900) is an autosomal-dominant muscular disorder caused by an expansion of CTG repeats in the 3' UTR of the DMPK gene. Variable expansions of CTG repeats preclude the accurate determination of repeat size. We tried to show the clinical and analytical validity of the application of Southern blotting after long-range PCR was demonstrated in Korean DM1 patients. Materials and Methods: The Southern blotting of long-range PCR was applied to 1,231 cases with clinical suspicion of DM1, between 2000 and 2011. PCR was performed using genomic DNA with forward 5'-CAGTTCACAACCGCTCCGAGC-3' and reverse 5'-CGTGGAGGATGGAACACGGAC-3' primers. Subsequently, the PCR fragments were subjected to gel electrophoresis, capillary transfer to a nylon membrane, hybridization with a labeled (CAG)10 probe. The correlation between clinical manifestations and the CTG repeat expansions were analyzed. Results: Among a total of 1,231 tested cases, 642 individuals were diagnosed with DM1 and the range of the detected expansion was 50 to 2,500 repeats; fourteen cases with mild DM1 ($75{\pm}14$ repeats), 602 cases with classical DM1 ($314{\pm}143$ repeats), and 26 cases with congenital DM1 ($1,219{\pm}402$ repeats). The positive and negative predictive values were 100%. The age at test requested and the CTG repeat numbers were inversely correlated (R=-0.444, P<0.01). Conclusion: This study indicates that Southern blotting after long-range PCR is a reliable diagnostic method DM1.

The Chemotoxonoic Relationship of Vibrio cholerae non-O1 by Fatty Acid Compositions (지방산 조성에 의한 vibrio cholerae non-O1의 화학분류학적 관계)

  • 성희경;이원재;장동성
    • Journal of Food Hygiene and Safety
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    • v.13 no.2
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    • pp.149-154
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    • 1998
  • The authors attempted utilization of fatty acid composition of vibrios as a tool for identification of the strains. Fatty acid of 49 strains of Vibrio cholerae non-O1, V. cholerae O1, V mimicus, V vulinificus and V parahaemolyticus was analyzed by gas-liquid chromatography column. According to the statistical analysis of the fatty acid data, the relationship between the Vibrio species and serotypes of the strains was discussed. Forty one kinds of fatty acid were detected from the tested strains and 35 kinds of fatty acids among the detected fatty acids were significant factors to identify the vibrios. The predominant fatty acids were 16:0, 16:1 cis 9, 18:1 trans 9/6/cis 11 and 15:0 iso 2OH/16:1 cis 9 as above about 20% in total. Fatty acid compositions of the Vibrio species were an important factor in identifying their subspecies either predominant fatty acids or minor ones. According to the analysed results by a conventional statistical processing method (UPGMA) and prepared dendrogram, V cholerae non-01 had more closer relationship with V. mimicus compared with V. cholerae 01. Moreover, the distribution of hydroxy acid was a significant factor for identifying V cholerae subspecies. Comprising all the 10 serotypes detected from V. cholerae non-01 examined such as O2, O5, O8, O10, O14, O27, O37, O39, O45 and O69, we could group them into seven subspecies by cluster analysis with the similarity value of fatty acid composition as above 92%. It means that there is a significant relationship between serotypes and fatty acid composition of V. cholerae. These results indicated that numerical analysis of fatty acid composition data of V cholerae non-01 could classifY them into subspecies, and also which may provide a useful epidemiologic information or a basis for further analysis such as PCR and DNA probe analysis.

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