• Title, Summary, Keyword: qPCR

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Quantitative analysis of oral disease-causing bacteria in saliva among bacterial culture, SYBRgreen qPCR and MRT-PCR method (타액내 구강질환 원인 균의 세균배양법, SYBR green qPCR법, MRT-PCR법 간의 정량분석)

  • Park, Yong-Duk;Oh, Hye-Young;Park, Bok-Ri;Cho, Ara;Kim, Dong-Kie;Jang, Jong-Hwa
    • Journal of Korean society of Dental Hygiene
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    • v.17 no.2
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    • pp.319-330
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    • 2017
  • Objectives: The purpose of this study was to compare SYBR Green qPCR, TaqMan, and bacterial selective medium cultures for accurate quantitative analysis of oral microorganisms. Methods: The SYBR Green method is widely used to analyze the total amount of oral microorganisms in oral saliva. However, in this study, MTR-PCR method based on TaqMan method was performed using newly developed primers and probes. In addition, it was designed to confirm the detection agreement of bacteria among bacteria detection method. Results: As a result of MRT-PCR and SYBR Green qPCR analysis, more than 40 times (0.9-362.9 times) bacterium was detected by MRT-PCR. In addition, more bacteria were detected in saliva in the order of MRT-PCR, SYBR Green qPCR, and bacterium culture, and the results of MRB-PCR and SYBR Green qPCR showed the highest agreement. The agreement between the three methods for detecting P. intermedia was similar between 71.4 and 88.6%, but the agreement between MRT-PCR and SYBR Green qPCR was 80% for S. mutans. Among them, the number of total bacteria, P. intermedia and S. mutans bacteria in saliva was higher than that of SYBR Green qPCR method, and bacterium culture method by MRT-PCR method. P. intermedia and S. mutans in saliva were detected by MRT-PCR and MRT-PCR in 88.6% of cases, followed by the SYBR Green qPCR method (80.0%). Conclusions: The SYBR Green qPCR method is the same molecular biology method, but it can not analyze the germs at the same time. Bacterial culturing takes a lot of time if there is no selective culture medium. Therefore, the MRT-PCR method using newly developed primers and probes is considered to be the best method.

Development of Quantitative Real-Time PCR Primers for Detection of Prevotella intermedia

  • Park, Soon-Nang;Kook, Joong-Ki
    • International Journal of Oral Biology
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    • v.40 no.4
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    • pp.205-210
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    • 2015
  • Prevotella intermedia-specific quantitative real-time PCR (qPCR) primers were previously designed based on the nucleotide sequences of RNA polymerase ${\beta}$-subunit gene (rpoB). However, the several clinical strains isolated from Korean populations are not detectable by the qPCR primers. The purpose of this study was to develop new P. intermedia-specific qPCR primers based on the rpoB. The specificity of the primers was determined by conventional PCR with 12 strains of P. intermedia and 52 strains (52 species) of non-P. intermedia bacteria. The sensitivity of primers was determined by qPCR with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of P. intermedia ATCC $25611^T$. The data indicated that only P. intermedia strains were detected by the P intermedia-specific qPCR primers (RTPiF2/RTPiR2); in addition, as little as 40 fg of P. intermedia genomic DNA could be detected. These results suggest that these qPCR primers are useful in detecting P. intermedia from the bacterial infectious lesions including dental plaque and oral tissue lesions.

Development of TaqMan Probe-Based Real-Time PCR Method for erm(A), erm(B), and erm(C), Rapid Detection of Macrolide-Lincosamide-Streptogramin B Resistance Genes, from Clinical Isolates

  • Jung, Jae-Hyuk;Yoon, Eun-Jeong;Choi, Eung-Chil;Choi, Sung-Sook
    • Journal of Microbiology and Biotechnology
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    • v.19 no.11
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    • pp.1464-1469
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    • 2009
  • To achieve more accurate and rapid detection of macrolide-lincosamide-streptogramin B resistance genes, erm(A), erm(B), and erm(C), we developed a TaqMan probe-based real-time PCR (Q-PCR) method and compared it with conventional PCR (C-PCR), which is the most widely using erm gene identification method. The detection limit of Q-PCR was 5 fg of genomic DNA or 5-8 CFU of bacterial cells of Staphylococcus aureus. The utilization of Q-PCR might shorten the time to erm detection from 3-4 h to about 50 min. These data indicated that Q-PCR assay appears to be not only highly sensitive and specific, but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay will permit rapid and accurate identification of erm genes from clinical and other samples.

Comparative Quantification of LacZ (β-galactosidase) Gene from a Pure Cultured Escherichia coli K-12

  • Han, Ji-Sun;Kim, Chang-Gyun
    • Environmental Engineering Research
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    • v.14 no.1
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    • pp.63-67
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    • 2009
  • Escherichia coli K-12 (E. coli K-12) is a representative indicator globally used for distinguishing and monitoring dynamic fates of pathogenic microorganisms in the environment. This study investigated how to most critically quantify lacZ ($\beta$-galactosidase) gene in E. coli K-12 by two different real-time polymerase chain reaction (real-time PCR) in association with three different DNA extraction practices. Three DNA extractions, i.e., sodium dodecyl sulfate (SDS)/proteinase K, magnetic beads and guanidium thiocyanate (GTC)/silica matrix were each compared for extracting total genomic DNA from E. coli K-12. Among them, GTC/silica matrix and magnetic beads beating similarly worked out to have the highest (22-23 ng/${\mu}L$) concentration of DNA extracted, but employing SDS/proteinase K had the lowest (10 ng/${\mu}L$) concentration of DNA retrieved. There were no significant differences in the quantification of the copy numbers of lacZ gene between SYBR Green I qPCR and QProbe-qPCR. However, SYBR Green I qPCR obtained somewhat higher copy number as $1{\times}10^8$ copies. It was decided that GTC/silica matrix extraction or magnetic beads beating in combination with SYBR Green I qPCR can be preferably applied for more effectively quantifying specific gene from a pure culture of microorganism.

Peptoniphilus mikwangii-specific quantitative real-time polymerase chain reaction primers

  • Park, Soon-Nang;Kook, Joong-Ki
    • International Journal of Oral Biology
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    • v.44 no.3
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    • pp.96-100
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    • 2019
  • The purpose of this study was to develop Peptoniphilus mikwangii-specific quantitative real-time polymerase chain reaction (qPCR) primers based on the 16S ribosomal RNA (16S rDNA) gene. The specificity of the primers was determined by conventional PCR using 29 strains of 27 oral bacterial species including P. mikwangii. The sensitivity of the primers was determined by qPCR using the purified genomic DNA of P. mikwangii KCOM $1628^T$ (40 ng to 4 fg). The data showed that the qPCR primers (RTB134-F4/RTB134-R4) could detect P. mikwangii strains exclusively and as little as 40 fg of the genomic DNA of P. mikwangii KCOM $1628^T$. These results suggest that the developed qPCR primer pair can be useful for detecting P. mikwangii in epidemiological studies of oral bacterial infectious diseases.

Assessment of Korean Paddy Soil Microbial Community Structure by Use of Quantitative Real-time PCR Assays (한국의 논 토양 미생물 다양성 분석을 위한 Quantitative Real-time PCR의 응용)

  • Choe, Myeong-Eun;Lee, In-Jung;Shin, Jae-Ho
    • Korean Journal of Environmental Agriculture
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    • v.30 no.4
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    • pp.367-376
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    • 2011
  • BACKGROUND: In order to develop effective assessment method for Korean paddy soil microbial community structure, reliable genomic DNA extraction method from paddy soil and quantitative real-time PCR (qRT-PCR) method are needed to establish METHODS AND RESULTS: Out of six conventional soil genomic DNA extraction methods, anion exchange resin purification method was turn to be the most reliable. Various PCR primers for distinguishing five bacterial phylum (${\alpha}$-Proteobacteria, ${\beta}$-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes), all bacteria, and all fungi were tested. Various qRT-PCR temperature conditions were also tested by repeating experiment. Finally, both genomic DNA extraction and qRT-PCR methods for paddy soil were well established. CONCLUSION: Quantitative real-time PCR (qRT-PCR) method to assess paddy soil microbial community was established.

Genotypic Identification of Cystoisospora in Immunocompromised Patients Using Tm-Variation Analysis

  • Basyoni, Maha M.A.;Elghobary, Hany Ahmed Fouad
    • The Korean Journal of Parasitology
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    • v.55 no.6
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    • pp.601-606
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    • 2017
  • Cystoisospora is responsible for morbidity in immunocompromised patients. PCR is sensitive for diagnosing Cystoisospora; however, it needs reevaluation for differential molecular diagnosis of cystoisosporiasis. We aimed at evaluating melting curve analysis (MCA) after real-time PCR (qPCR) in diagnosis and genotyping of Cystoisospora as an alternative to conventional PCR. We included 293 diarrheic stool samples of patients attending the Department of Clinical Oncology and Nuclear Medicine of Cairo University Hospitals, Egypt. Samples were subjected to microscopy, nested PCR (nPCR), and qPCR targeting the internal transcribed spacer 2 region (ITS2) of the ribosomal RNA (r RNA) gene followed by melting temperatures ($T_ms$) analysis and comparing the results to PCR-RFLP banding patterns. Using microscopy and ITS2-nPCR, 3.1% and 5.8% of cases were Cystoisospora positive, respectively, while 10.9% were positive using qPCR. Genotyping of Cystoisospora by qPCR-MCA revealed 2 genotypes. These genotypes matched with 2 distinct melting peaks with specified $T_ms$ at $85.8^{\circ}C$ and $88.6^{\circ}C$, which indicated genetic variation among Cystoisospora isolates in Egypt. Genotype II proved to be more prevalent (65.6%). HIV-related Kaposi sarcoma and leukemic patients harbored both genotypes with a tendency to genotype II. Genotype I was more prevalent in lymphomas and mammary gland tumors while colorectal and hepatocellular tumors harbored genotype II suggesting that this genotype might be responsible for the development of cystoisosporiasis in immunocompromised patients. Direct reliable identification and differentiation of Cystoisospora species could be established using $qPCR-T_ms$ analysis which is useful for rapid detection and screening of Cystoisospora genotypes principally in high risk groups.

Development of Quantitative Real-Time PCR Primers for Detection of Streptococcus sobrinus

  • Park, Soon-Nang;Kook, Joong-Ki
    • International Journal of Oral Biology
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    • v.41 no.3
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    • pp.149-154
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    • 2016
  • The purpose of this study was to develop Streptococcus sobrinus-specific qPCR primers based on the nucleotide sequence of the RNA polymerase ${\beta}-subunit$ gene (rpoB). The specificity of the primers was determined by conventional polymerase chain reaction (PCR) with 12 strains of S. sobrinus and 50 strains (50 species) of non-S. sobrinus bacteria. The sensitivity of the primers was determined by quantitative real-time PCR (qPCR) with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of S. sobrinus ATCC $33478^T$. The specificity data showed that the S. sobrinus-specific qPCR primers (RTSsob-F4/RTSsob-R4) detected only the genomic DNAs of S. sobrinus strains with a detection limit of up to 4 fg of S. sobrinus genomic DNA. Our results suggest that the RTSsob-F4/RTSsob-R4 primers are useful in detecting S. sobrinus with high sensitivity and specificity for epidemiological studies of dental caries..

Quantification of Her-2/Neu Gene in Breast Cancer Patients using Real Time-Polymerase Chain Reaction (Q-PCR) and Correlation with Immunohistochemistry Findings

  • Abdul Murad, Nor Azian;Razak, Zuraini Abdul;Hussain, Rosniza Muhammmad;Syed Hussain, Sharifah Noor Akmal;Ching Huat, Clarence Ko;Siti Aishah, Che Md. Ali;Abdullah, Norlia;Muhammad, Rohaizak;Ibrahim, Naqiyah;Jamal, Rahman
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.3
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    • pp.1655-1659
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    • 2013
  • Background: HER-2/neu is a proto-oncogene that encodes a transmembrane tyrosine kinase growth factor which is crucial for stimulating growth and cellular motility. Overexpression of HER-2/neu is observed in 10-35% of human breast cancers and is associated with pathogenesis, prognosis as well as response to therapy. Given the imperative role of HER-2/neu overexpression in breast cancer, it is important to determine the magnitude of amplification which may facilitate a better prognosis as well as personalized therapy in affected patients. In this study, we determined HER-2/neu protein expression by immunohistochemistry (IHC) concurrently with HER-2/neu DNA amplification by quantitative real time-polymerase chain reaction (Q-PCR). Materials and Methods: A total of 53 paired tissue samples from breast cancer patients were frozen-sectioned to characterize the tumour and normal tissues. Only tissues with 80% tumour cells were used in this study. For confirmation, Q-PCR was used to determine the HER-2/neu DNA amplification. Results: We found 20/53 (37.7%) of the tumour tissues to be positive for HER-2/neu protein overexpression using IHC. Out of these twenty, only 9/53 (17%) cases were in agreement with the Q-PCR results. The concordance rate between IHC and Q-PCR was 79.3%. Approximately 20.7% of positive IHC cases showed no HER-2/neu gene amplification using Q-PCR. Conclusion: In conclusion, IHC can be used as an initial screening method for detection of the HER-2/neu protein overexpression. Techniques such as Q-PCR should be employed to verify the IHC results for uncertain cases as well as determination of HER-2/neu gene amplification.

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.