scholarly journals Detection of Infectious Adenovirus in Cell Culture by mRNA Reverse Transcription-PCR

2003 ◽  
Vol 69 (12) ◽  
pp. 7377-7384 ◽  
Author(s):  
Gwangpyo Ko ◽  
Theresa L. Cromeans ◽  
Mark D. Sobsey
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Reverse Transcription ◽  
Radiation Detection ◽  
Virus Infectivity ◽  
Mrna Transcript ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Mrna Detection ◽  
Primer Sets

ABSTRACT We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models. Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture. Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41. The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay. The mRNA of Ad2 was detected as early as 6 h after infection at 106 infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture. The mRNA of Ad41 was detected as soon as 24 h after infection at 106 IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times. To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation. Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner. These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples.

scholarly journals Detection of Polioviruses in Sewage Using Cell Culture and Molecular Methods

2026 ◽  
Vol 65 (4) ◽  
pp. 479-483
Author(s):  
Agnieszka Figas ◽  
Magdalena Wieczorek ◽  
Bogumiła Litwińska ◽  
Włodzimierz Gut
Keyword(s):  
Cell Culture ◽  
Cell Lines ◽  
Cell Cultures ◽  
Environmental Samples ◽  
Genetic Material ◽  
Culture Technique ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Step Algorithm ◽  
Cell Culture Technique

The work presented here demonstrates the utility of a two-step algorithm for environmental poliovirus surveillance based on: preselection of sewage samples tested for the presence of enteroviral genetic material-RT-PCR assay and detection of infectious viruses by cell culture technique (L20B for polioviruses and RD for polio and other non-polio enteroviruses). RD and L20B cell lines were tested to determine their sensitivity for isolation of viruses from environmental samples (sewage). Finally, we wanted to determine if sewage concentration affects the results obtained for RT-PCR and cell cultures.

scholarly journals Detection and Identification of Human Parainfluenza Viruses 1, 2, 3, and 4 in Clinical Samples of Pediatric Patients by Multiplex Reverse Transcription-PCR

2000 ◽  
Vol 38 (3) ◽  
pp. 1191-1195 ◽  
Author(s):  
Jose C. Aguilar ◽  
María P. Pérez-Breña ◽  
María L. García ◽  
Nieves Cruz ◽  
Dean D. Erdman ◽  
...  
Keyword(s):  
Cell Culture ◽  
Reverse Transcription ◽  
Pediatric Patients ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Parainfluenza Viruses ◽  
Cell Culture Isolation ◽  
Human Parainfluenza Viruses

We describe a multiplex reverse transcription-PCR (m-RT-PCR) assay that is able to detect and differentiate all known human parainfluenza viruses (HPIVs). Serial dilution experiments with reference strains that compared cell culture isolation and m-RT-PCR showed sensitivities ranging from 0.0004 50% tissue culture infective dose (TCID50) for HPIV type 4B (HPIV-4B) to 32 TCID50s for HPIV-3. As few as 10 plasmids containing HPIV PCR products could be detected in all cases. When 201 nasopharyngeal aspirate specimens from pediatric patients hospitalized for lower respiratory illness were tested, m-RT-PCR assay detected 64 HPIVs (24 HPIV-3, 23 HPIV-1, 10 HPIV-4, and 7 HPIV-2), while only 42 of them (21 HPIV-1, 14 HPIV-3, 6 HPIV-2, and 1 HPIV-4 isolates) grew in cell culture. Our m-RT-PCR assay was more sensitive than either cell culture isolation or indirect immunofluorescence with monoclonal antibodies for the detection of HPIV infections. Also, HPIV-4 was more frequently detected than HPIV-2 in this study, suggesting that it may have been underestimated as a lower respiratory tract pathogen because of the insensitivity of cell culture.

Comet and cup genes in Drosophila spermatogenesis: the first demonstration of post-meiotic transcription

2008 ◽  
Vol 36 (3) ◽  
pp. 540-542 ◽  
Author(s):  
Carine Barreau ◽  
Elizabeth Benson ◽  
Helen White-Cooper
Keyword(s):  
In Situ Hybridization ◽  
Expression Pattern ◽  
Reverse Transcription ◽  
Protein Product ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Cyst ◽  
Reverse Transcription Pcr

Post-meiotic transcription is widespread in mammalian spermatogenesis, but is generally believed to be absent from Drosophila spermatogenesis. Genes required during meiosis, in early spermatids or later in spermiogenesis are typically transcribed in primary spermatocytes in Drosophila. Their mRNAs are then stored in the cytoplasm until the protein product is needed. Recently, using in situ hybridization, we identified 17 Drosophila genes, collectively named ‘comets’ and ‘cups’, whose mRNAs are most abundant in, and localize to the distal ends of, elongating spermatids. Using a single-cyst quantitative RT–PCR (reverse transcription–PCR) assay, we confirmed this unusual expression pattern and conclusively demonstrate the existence of post-meiotic transcription in Drosophila spermatids. We found that transcription of comets and cups occurs just before protamines can be detected in spermatid nuclei.

scholarly journals BAALC and ERG Expression in Egyptian Patients with Acute Myeloid Leukemia, Relation to Survival and Response to Treatment

2016 ◽  
Vol 4 (2) ◽  
pp. 264-270 ◽  
Author(s):  
Aml Soliman ◽  
Asmaa Abdel Aal ◽  
Reham Afify ◽  
Noha Ibrahim
Keyword(s):  
Polymerase Chain Reaction ◽  
Acute Myeloid Leukemia ◽  
Reverse Transcription ◽  
Myeloid Leukemia ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Age And Sex ◽  
Acute Myeloid

AIM: Aim was to detect Brain and Acute Leukemia, Cytoplasmic (BAALC) and ETS-related gene (ERG) expression in patients with acute myeloid leukemia (AML) as well as to study their biologic and prognostic impact on the disease outcome and survival.PATIENTS AND METHODS: The current study was carried out on 44 patients with denovo acute myeloid leukemia, as well as 44 age and sex matched controls. The quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was performed for estimation of BAALC and ERG expression.RESULTS: The current study was carried out on 44 patients with denovo acute myeloid leukemia, as well as 44 age and sex matched controls. The quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was performed for estimation of BAALC and ERG expression. BAALC was expressed in 36 (81.82%) of AML cases versus 10 (22.72%) of the control group which was highly statistically significant (P < 0.001). While ERG was positive in 39(88.64%) of cases and 8(18.18 %) of controls and that was also highly statistically significant (P < 0.001).CONCLUSION: Further researches still needed to clarify the role of BAALC and ERG in the pathogenesis of leukemia and their importance as targets for treatment of AML.

scholarly journals The Detection of Enteroviruses in Sewage Using Caco-2 Cells

2013 ◽  
Vol 62 (1) ◽  
pp. 97-100 ◽  
Author(s):  
MAGDALENA WIECZOREK ◽  
ŁUKASZ KURYK ◽  
AGNIESZKA WITEK ◽  
ANNA DIUWE ◽  
BOGUMIŁA LITWIŃSKA
Keyword(s):  
Cell Culture ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Beef Extract

The work presented here demonstrates the utility of Caco-2 cells to detect enteroviruses in sewage. Viruses were concentrated by beef extract elution and organic flocculation prior to analysis by cell culture assays and RT-PCR. Enteroviruses were detected in all sewage samples, but only one sample was positive solely in RT-PCR assay. We proved that Caco-2 cells were more effective than RD and L20B cells in enterovirus isolation, depending on procedures used in the inoculation process.

Multiplex Nested Reverse Transcription-Polymerase Chain Reaction for Respiratory Viruses in Acute Otitis Media

2003 ◽  
Vol 112 (3) ◽  
pp. 252-257 ◽  
Author(s):  
Toshio Ishibashi ◽  
Hiroko Monobe ◽  
Masanobu Shinogami ◽  
Yuka Nomura ◽  
Jun Yano
Keyword(s):  
Polymerase Chain Reaction ◽  
Otitis Media ◽  
Reverse Transcription ◽  
Acute Otitis Media ◽  
Respiratory Viruses ◽  
Molecular Technique ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

Because respiratory viruses play an important role in the causation and pathogenesis of acute otitis media (AOM), determining which virus has infected a child is important with respect to vaccines and antiviral drugs. In some instances, this information might be used to prevent the occurrence of AOM. We used a rapid, economical, and sensitive diagnostic system involving a multiplex nested reverse transcription–polymerase chain reaction (RT-PCR) assay to detect various respiratory viruses in clinical specimens of middle ear fluid (MEF) from children with AOM in our hospital. Multiplex RT-PCR was completed on 40 MEF samples from 28 infants and children less than 6 years old with AOM. Viral RNA was detected in 17 MEF samples (43%). Respiratory syncytial virus type A was present in 12 samples, adenovirus in 3, rhinovirus in 2, and influenza A (H3N2) in 1. The multiplex RT-PCR assay is recommended to clinical laboratories that are considering adoption of a molecular technique for viral diagnosis.

scholarly journals Evaluation of the PAX8/PPARG Translocation in Follicular Thyroid Cancer with a 4-Color Reverse-Transcription PCR Assay and Automated High-Resolution Fragment Analysis

2010 ◽  
Vol 56 (3) ◽  
pp. 391-398 ◽  
Author(s):  
Alicia Algeciras-Schimnich ◽  
Dragana Milosevic ◽  
Bryan McIver ◽  
Heather Flynn ◽  
Honey V Reddi ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
High Resolution ◽  
Reverse Transcription ◽  
Thyroid Tissue ◽  
Molecular Testing ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fragment Analysis ◽  
Reverse Transcription Pcr

Abstract Background: Molecular testing of thyroid malignancies, in combination with cytologic and histologic examination, is becoming increasingly attractive as a tool for refining traditional morphologic diagnosis. The molecular changes associated with follicular thyroid carcinoma (FTC) are point mutations in RAS oncogenes or the presence of PAX8/PPARG (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement. Methods: We developed and validated a clinical assay for the detection of PAX8/PPARG rearrangements that uses a 4-color reverse-transcription PCR (RT-PCR) assay and high-resolution fragment analysis. Results: The RT-PCR assay is applicable for detecting the various described fusion transcripts of PAX8/PPARG in formalin-fixed, paraffin-embedded thyroid tissue and in fine-needle aspirate biopsy washes from thyroid nodules. The analytical sensitivity of the assay is 1 abnormal cell in a background of 100–10 000 translocation-negative cells. A comparison of the RT-PCR assay with dual-fusion fluorescence in situ hybridization showed an overall concordance of 95%. With this assay, we obtained a prevalence for the PAX8/PPARG rearrangement in FTC of 62% (13 of 21 cases), compared with a 5% prevalence (3 of 55) for other follicular cell–derived neoplasms. Conclusions: The introduction of this assay into clinical practice could provide useful information for the diagnosis and possibly for the prognosis and treatment of thyroid cancer in the future.

scholarly journals Quantitative Reverse Transcription-PCR Assay with an Internal Standard for the Detection of Prostate-specific Antigen mRNA

1999 ◽  
Vol 45 (9) ◽  
pp. 1397-1407 ◽  
Author(s):  
Alice Ylikoski ◽  
Minna Sjöroos ◽  
Åke Lundwall ◽  
Matti Karp ◽  
Timo Lövgren ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Reverse Transcription ◽  
Specific Antigen ◽  
Internal Standard ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

Abstract Background: Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. Methods: The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates. Results: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 106 copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 ± 200 to 44 100 ± 4900 (mean ± SD) in the samples, corresponding to ∼1–100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 ± 100 and 2000 ± 900 (mean ± SD) PSA mRNA copies in 5 mL of blood for the 2 males]. Conclusions: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.

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