DETECTION OF RESPIRATORY SYNCITIAL VIRUS (RSV) IN LOWER ACUTE RESPIRATORY INFECTIONS BY NESTED REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION

2011 ◽  
pp. 11-15
Author(s):  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Respiratory Infections ◽  
Clinical Samples ◽  
Acute Respiratory Infections ◽  
Rt Pcr ◽  
Minimal Level ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

Objective: To develop and apply a nested reverse transcription- polymerase chain reaction (nested RT-PCR) for detection of RSV in lower acute respiratory infections. Materials and methods: A nested reverse transcription- polymerase chain reaction was used to amplify a sequence of the F gene in the RSV genomic RNA, optimized and compared the sensitivity and specificity of this assay with the control samples and then applied this procedure for diagnosing RSV from clinical samples. Results: This nested RT-PCR assay amplified the specific target fragment of RSV RNA and did not amplify any sequence of genomes of the tested common viruses and bacteria causing respiratory infections. The minimal level of detection of this procedure was 102 copies/ml. Results for detection of RSV on 109 samples of throat swabs or nasopharyngeal swabs from children with lower respiratory infections showed that twenty seven patients were positive with RSV ( 24.8%), among which six out of 30 (20%) were with bronchitis, seven out of 26 ( 27%) were with bronchiolitis and fourteen out of 53 (26.4%) were with pneumonia. Conclusion: This nested RT-PCR was found to be useful and reliable for detection of RSV in respiratory infections.

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.

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.

Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea

2004 ◽  
Vol 72 (3) ◽  
pp. 496-501 ◽  
Author(s):  
Xiaoli L. Pang ◽  
Bonita Lee ◽  
Nasim Boroumand ◽  
Barbara Leblanc ◽  
Jutta K. Preiksaitis ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Stool Specimens ◽  
Polymerase Chain

scholarly journals Systematic Review and Meta-analysis of Real-time Polymerase Chain Reaction assay for the detection of COVID-19 from clinical samples in low-and middle-income countries: Protocol

2021 ◽  
Author(s):  
Emmanuel Oladipo Babafemi
Keyword(s):  
Systematic Review ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meta Analysis ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Middle Income ◽  
Polymerase Chain

Abstract Background: COVID-19 has spread globally since its discovery in Hubei province, China in December 2019 and became pandemic in 2020. COVID-19 is a new betacoronavirus and a variant of severe acute respiratory syndrome coronavirus 2 (SARA- CoV-2). Rapid, accurate and reliable diagnosis of COVID-19 will prevent the spread and allow for appropriate management. The main objective of this systematic review is to identify, appraise and summarise the published evidence on the diagnostic performance and effectiveness of SARS-CoV-2 virus in the diagnosis of current or previous COVID-19 using real-time polymerase chain reaction (RT-PCR) assay in low-and middle-income countries (LMICs). Methods: We will search MEDLINE/PubMed, EMBASE, BIOSIS, LILACS, Cochrane Infectious Diseases Group Specialised Register (CIDG SR), Global Health, and CINAHL for published studies for the diagnosis of COVID-19 using real-time polymerase chain reaction assay in LMICs There will be no restriction regarding the language, date of publication, and publication status. We will include retrospective, cross-sectional and cohort observational studies will be included in the review. Selection of studies, data extraction and management, assessment of risk of bias, and quality of evidence will be performed by two independent reviewers (EB and BC). A third researcher (GM) will be consulted in case of discrepancies. Depending on the availability and quality of the data, a meta-analysis will be performed. Otherwise, findings will be qualitatively reported. Discussion: To our knowledge, this is the first systematic review and meta-analysis to assess the uptake of RT-PCR assay for SARS-CoV-2 detection from clinical samples in human in LMICs. This review will make available evidence on the uptake, accuracy, approach, and interpretation of results of this assay in the context of COVID-19 diagnosis which will meet an urgent need, considering the diagnostic challenges of RT-PCR assay for COVID-19 diagnosis in humans. Systematic review registration: PROSPERO CRD42021271894

scholarly journals Detection of minimal residual disease in acute promyelocytic leukemia by a reverse transcription polymerase chain reaction assay for the PML/RAR-alpha fusion mRNA

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1689-1694 ◽  
Author(s):  
WH Jr Miller ◽  
K Levine ◽  
A DeBlasio ◽  
SR Frankel ◽  
E Dmitrovsky ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Retinoic Acid ◽  
Complete Remission ◽  
Acute Promyelocytic Leukemia ◽  
Reverse Transcription ◽  
Promyelocytic Leukemia ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

The characteristic reciprocal translocation t(15;17) of acute promyelocytic leukemia (APL) disrupts the PML gene on chromosome 15 and the retinoic acid receptor-alpha (RAR-alpha) gene on chromosome 17. PML/RAR-alpha fusion mRNAs are then transcribed and can be detected by a newly described reverse transcription polymerase chain reaction (RT- PCR) assay. Using RT followed by nested PCR amplification for PML/RAR- alpha, we serially evaluated bone marrow aspirates from patients with APL who were treated with all-trans retinoic acid (RA) for induction, followed by all-trans RA as maintenance or cytotoxic drugs as consolidation. At diagnosis, PML/RAR-alpha mRNA was detected in all patients. After initial therapy with all-trans RA, the RT-PCR assay remained positive after induction of complete remission in 31 of 32 evaluable patients. Maintenance treatment by all-trans RA alone was associated with persistent assay positivity and subsequent clinical relapse in 13 of 13 patients. By contrast, the test became negative in 19 of 20 newly diagnosed patients who received consolidation chemotherapy; the 1 patient who remained positive relapsed at 12 months. Three of the 19 assay-negative patients later converted to positive and subsequently relapsed; the remaining 16 patients have remained RT-PCR negative in sustained first remission, with a median follow-up duration that exceeds 24 months (range, 12+ to 34+ months). Despite induction of complete remission in a high proportion of patients, all-trans RA rarely eradicates molecular evidence of disease in patients with APL; however, subsequent treatment with cytotoxic chemotherapy frequently converts the RT-PCR assay for PML/RAR-alpha to negative. Serial negative tests are associated with prolonged disease- free survival, whereas persistence of a positive test after treatment is highly correlated with subsequent relapse. This test identifies patients in remission at high risk for relapse who may benefit from additional antileukemic therapy.

scholarly journals An international, interlaboratory ring trial confirms the feasibility of an extraction-less “direct” RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0261853
Author(s):  
Margaret G. Mills ◽  
Emily Bruce ◽  
Meei-Li Huang ◽  
Jessica W. Crothers ◽  
Ollivier Hyrien ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Correct Identification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Ring Trial ◽  
Feasible Option ◽  
Polymerase Chain

Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). “Extraction-less” or “direct” real time–reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

scholarly journals Performance of Real-Time Reverse Transcription Polymerase Chain Reaction for the Detection of Foot-and-Mouth Disease Virus during Field Outbreaks in the United Kingdom in 2007

2009 ◽  
Vol 21 (3) ◽  
pp. 321-330 ◽  
Author(s):  
Scott M. Reid ◽  
Katja Ebert ◽  
Katarzyna Bachanek-Bankowska ◽  
Carrie Batten ◽  
Anna Sanders ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
United Kingdom ◽  
Real Time ◽  
Reverse Transcription ◽  
Foot And Mouth Disease ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The United Kingdom ◽  
Polymerase Chain

Rapid and accurate diagnosis is essential for effective control of foot-and-mouth disease (FMD). The present report describes the practical steps undertaken to deploy a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to process the samples received during the outbreaks of FMD in the United Kingdom in 2007. Two independent real-time RT-PCR assays targeting different regions (5′UTR and 3D) of the FMD virus (FMDV) genome were used to confirm the presence of FMDV in clinical samples collected from the first infected premises. Once the FMDV strain responsible had been sequenced, a single real-time RT-PCR assay (3D) was selected to test a total of 3,216 samples, including material from all 8 infected premises. Using a 96-well automated system to prepare nucleic acid template, up to 84 samples could be processed within 5 hr of submission, and up to 269 samples were tested per working day. A conservative cut-off was used to designate positive samples, giving rise to an assay specificity of 99.9% or 100% for negative control material or samples collected from negative premises, respectively. For the first time, real-time RT-PCR results were used to recognize preclinical FMD in a cattle herd. Furthermore, during the later stages of the outbreaks, the real-time RT-PCR assay supported an active surveillance program within high-risk cattle herds. To the authors' knowledge, this is the first documented use of real-time RT-PCR as a principal laboratory diagnostic tool following introduction of FMD into a country that was FMD-free (without vaccination) and highlights the advantages of this assay to support control decisions during disease outbreaks.

Real-time reverse transcription-polymerase chain reaction for detection of SYT-SSX translocation in synovial sarcoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 9553-9553
Author(s):  
J. Liu ◽  
K. Qu ◽  
C. Chai ◽  
H. Li ◽  
A. Sferruzza ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Synovial Sarcoma ◽  
Internal Control ◽  
Reverse Transcription ◽  
Gel Electrophoresis ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

9553 Background: Synovial sarcoma is the most common non-rhabdomyosarcomatous soft tissue sarcoma in children and adolescents. A specific translocation, t(X;18), induces fusion of the SYT gene on chromosome 18 to an SSX gene on chromosome X. The resulting fusion gene consists of at least 2 subtypes with different breakpoints: SYT-SSX1(X;18)(p11.23;q11.2) and SYT-SSX2 (X;18)(p11.21;q11.2). Because t(X;18) transcripts occur in >90% of synovial sarcoma subtypes, this marker may be useful for diagnosis. We evaluated the accuracy of a multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of the primary SYT-SSX fusion transcript types in formalin-fixed, paraffin-embedded (FFPE) tissues and frozen tissues. Methods: 17 tumors (7 synovial sarcomas, 4 Ewing’s sarcomas, 5 rhabdomyosarcomas, 1 small round blue-cell tumor), 4 normal tissues, and 4 control samples were tested for SYT-SSX translocations using real-time RT-PCR. Results were compared to those obtained with gel electrophoresis detection of amplified transcripts; discrepant results were confirmed by sequencing. Results: Concordance between real time RT-PCR and gel electrophoresis was 100% (25/25) for internal control genes and SYT-SSX1, and 92% (23/25) for SYT-SSX2. Of the 2 samples with discordant SYT-SSX2 results, 1 was positive by real-time RT-PCR but not gel electrophoresis and 1 was positive by electrophoresis but not real-time RT-PCR; in both cases, DNA sequencing confirmed the real-time RT-PCR results. The minimum percentage of tumor to normal cells required for detection of SYT-SSX fusion transcripts by real-time RT-PCR was 6.25%. Conclusions: This real-time RT-PCR assay appears to provide greater accuracy than gel electrophoresis for identification of SYT-SSX translocation and fusion types. No significant financial relationships to disclose.

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