Development of quantitative RT-PCR assays for detection of three classes of HHV-6B gene transcripts

2012 ◽  
Vol 84 (9) ◽  
pp. 1388-1395 ◽  
Author(s):  
Masaru Ihira ◽  
Yoshihiko Enomoto ◽  
Yoshiki Kawamura ◽  
Hidetaka Nakai ◽  
Ken Sugata ◽  
...  
Keyword(s):  
Rt Pcr ◽  
Gene Transcripts ◽  
Pcr Assays ◽  
6B Gene

Update on the large-scale screening of ALK fusion oncogene transcripts in archival NSCLC tumor specimens using multiplexed RT-PCR assays.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 7594-7594 ◽  
Author(s):  
Tianhong Li ◽  
Eric Huang ◽  
Sonal Desai ◽  
Laurel Beckett ◽  
Craig Stephens ◽  
...  
Keyword(s):  
Large Scale ◽  
Fusion Gene ◽  
Standard Of Care ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fusion Transcripts ◽  
Gene Transcripts ◽  
Pcr Assays ◽  
Alk Positive ◽  
Large Scale Screening

7594 Background: The ALK inhibitor crizotinib offers a new standard of care for advanced NSCLC patients with EML4-ALK fusion oncogenes. We previously reported a 4.0% frequency of EML4-ALK fusion oncogene transcripts detected in 1889 NSCLC specimens in the RGI database (Li et al., ASCO 2011). Methods: Patented single and multiplexed RT-PCR assays suitable for rapid and accurate detection of all variants of ALK fusion oncogene transcripts were used as previously described, including all 9 known EML4-ALK fusion gene transcripts and ALK RNA levels (Danenberg, ASCO 2010). The sensitivity and specificity on archival formalin-fixed, paraffin-embedded tumor specimens are 99% and 100%, respectively. We here update the detection of EML4-ALK fusion transcripts in the RGI database. Results: Between 12/2009 and 09/2011, 4750 NSCLC specimens in the RGI database were tested for the presence of ALK fusion transcripts. We found 152 (3.2%) NSCLC cases with EML4-ALK fusion positivity, including 87 (57.2%) V1, 15 (9.9%) V2, 47 (30.9%) V3, and 3 (2.0%) V5a variants. Median age (range): 61.1 (33-96). Female: 74 (49%). All EML4-ALK-positive tumors were adenocarcinomas. No EGFR or K-Ras mutation was detected in ALK fusion-positive samples. Expression of chemotherapy-related biomarkers was available from 63 (female: 31, 49%) EML4-ALK-positive cases in the database: 43 (68%) had low TS level of <2.33; 40 (63.5%) had low ERCC1 level of <1.7, and 25 (40%) had low RRM1 level of <0.97. Conclusions: This RT-PCR assay provides a tool for rapid, large-scale screening of NSCLC FFPE tissues for EML4-ALK fusion gene transcripts. The relative value of this RT-PCR assay as a companion diagnostic test for drugs targeting ALK merits evaluation in comparison with the FDA approved ALK FISH test.

Development of real-time RT-PCR assays for detection of three classes of HHV-6A gene transcripts

2017 ◽  
Vol 89 (10) ◽  
pp. 1830-1836 ◽  
Author(s):  
Masaru Ihira ◽  
Akiko Urashima ◽  
Hiroki Miura ◽  
Fumihiko Hattori ◽  
Yoshiki Kawamura ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Gene Transcripts ◽  
Pcr Assays

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

scholarly journals Comparison of nine different commercially available molecular assays for detection of SARS-CoV-2 RNA

2021 ◽  
Author(s):  
Ute Eberle ◽  
◽  
Clara Wimmer ◽  
Ingrid Huber ◽  
Antonie Neubauer-Juric ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Pcr Assays ◽  
Laboratory Experiences

AbstractTo face the COVID-19 pandemic, the need for fast and reliable diagnostic assays for the detection of SARS-CoV-2 is immense. We describe our laboratory experiences evaluating nine commercially available real-time RT-PCR assays. We found that assays differed considerably in performance and validation before routine use is mandatory.

scholarly journals Prolonged SARS-CoV-2-RNA Detection from Nasopharyngeal Swabs in an Oncologic Patient: What Impact on Cancer Treatment?

2021 ◽  
Vol 28 (1) ◽  
pp. 847-852
Author(s):  
Anna Ferrari ◽  
Marco Trevenzoli ◽  
Lolita Sasset ◽  
Elisabetta Di Liso ◽  
Toni Tavian ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Treatment ◽  
Cancer Patients ◽  
Clinical Symptoms ◽  
Palliative Radiotherapy ◽  
Rt Pcr ◽  
Cancer Treatments ◽  
Global Challenge ◽  
Viral Culture ◽  
Pcr Assays

The pandemic of SARS-CoV-2 is a serious global challenge affecting millions of people worldwide. Cancer patients are at risk for infection exposure and serious complications. A prompt diagnosis of SARS-CoV-2 infection is crucial for the timely adoption of isolation measures and the appropriate management of cancer treatments. In lung cancer patients the symptoms of infection 19 may resemble those exhibited by the underlying oncologic condition, possibly leading to diagnostic overlap and delays. Moreover, cancer patients might display a prolonged positivity of nasopharyngeal RT-PCR assays for SARS-CoV-2, causing long interruptions or delay of cancer treatments. However, the association between the positivity of RT-PCR assays and the patient’s infectivity remains uncertain. We describe the case of a patient with non-small cell lung cancer, and a severe ab extrinseco compression of the trachea, whose palliative radiotherapy was delayed because of the prolonged positivity of nasopharyngeal swabs for SARS-CoV-2. The patient did not show clinical symptoms suggestive of active infection, but the persistent positivity of RT-PCR assays imposed the continuation of isolation measures and the delay of radiotherapy for over two months. Finally, the negative result of SARS-CoV-2 viral culture allowed us to verify the absence of viral activity and to rule out the infectivity of the patient, who could finally continue her cancer treatment.

scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

scholarly journals Quantitation of replication of the HCV genome in human livers with end-stage cirrhosis by strand-specific real-time RT-PCR assays: Methods and clinical relevance

2009 ◽  
Vol 81 (9) ◽  
pp. 1569-1575 ◽  
Author(s):  
Lan Lin ◽  
Louis Libbrecht ◽  
Jannick Verbeeck ◽  
Chris Verslype ◽  
Tania Roskams ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Relevance ◽  
Rt Pcr ◽  
Pcr Assays ◽  
Human Livers ◽  
End Stage

Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus

2014 ◽  
Vol 201 ◽  
pp. 79-85 ◽  
Author(s):  
Michele Drigo ◽  
Giovanni Franzo ◽  
Ilaria Belfanti ◽  
Marco Martini ◽  
Alessandra Mondin ◽  
...  
Keyword(s):  
Real Time ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
End Point ◽  
Pcr Assays

scholarly journals Influence of estradiol on bovine trophectoderm and uterine gene transcripts around maternal recognition of pregnancy

2021 ◽  
Author(s):  
Emmalee J Northrop-Albrecht ◽  
Jerica J J Rich ◽  
Robert A Cushman ◽  
Runan Yao ◽  
Xijin Ge ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Mrna Level ◽  
Fixed Time ◽  
Beef Cows ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Rt Pcr ◽  
Embryo Survival ◽  
Uterine Fluid ◽  
Gene Transcripts

Abstract Embryo survival and pregnancy success is increased among animals that exhibit estrus prior to fixed time artificial insemination (AI), but there are no differences in conceptus survival to d16. The objective of this study was to determine effects of preovulatory estradiol on uterine transcriptomes, select trophectoderm transcripts, and uterine luminal fluid (ULF) proteins. Beef cows/heifers were synchronized, artificially inseminated (d0), and grouped into either high (highE2) or low (lowE2) preovulatory estradiol. Uteri were flushed (d16); conceptuses and endometrial biopsies (n = 29) were collected. RNA sequencing was performed on endometrium. Real-Time PCR (RT-PCR) was performed on trophectoderm (TE; n = 21) RNA to measure relative abundance of IFNT, PTGS2, TM4SF1, C3, FGFR2, and GAPDH. Uterine fluid was analyzed using 2D LC–MS/MS based iTRAQ method. RT-PCR data were analyzed using the MIXED procedure in SAS. There were no differences in mRNA abundances in TE, but there were 432 differentially expressed genes (DEGs) (253 downregulated, 179 upregulated) in highE2/conceptus versus lowE2/conceptus groups. There were also 48 differentially expressed proteins (DEPs; 19 upregulated, 29 downregulated), 6 of these were differentially expressed (FDR &lt; 0.10) at the mRNA level. Similar pathways for mRNA and proteins included: calcium signaling, protein kinase A signaling, and corticotropin releasing hormone (CRH) signaling. These differences in uterine function, may be preparing the conceptus for improved likelihood of survival after d16 among highE2 animals.

scholarly journals Capacitative Ca2+ entry in agonist-induced pulmonary vasoconstriction

2001 ◽  
Vol 280 (5) ◽  
pp. L870-L880 ◽  
Author(s):  
Sharon S. McDaniel ◽  
Oleksandr Platoshyn ◽  
Jian Wang ◽  
Ying Yu ◽  
Michele Sweeney ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Channel Blocker ◽  
Receptor Potential ◽  
Rt Pcr ◽  
Pulmonary Vasoconstriction ◽  
Store Depletion ◽  
Intracellular Ca ◽  
Gene Transcripts ◽  
Transient Receptor ◽  
Sustained Increase

Agonist-induced increases in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery (PA) smooth muscle cells (SMCs) consist of a transient Ca2+ release from intracellular stores followed by a sustained Ca2+ influx. Depletion of intracellular Ca2+ stores triggers capacitative Ca2+ entry (CCE), which contributes to the sustained increase in [Ca2+]cyt and the refilling of Ca2+ into the stores. In isolated PAs superfused with Ca2+-free solution, phenylephrine induced a transient contraction, apparently by a rise in [Ca2+]cyt due to Ca2+ release from the intracellular stores. The transient contraction lasted for 3–4 min until the Ca2+ store was depleted. Restoration of extracellular Ca2+ in the presence of phentolamine produced a contraction potentially due to a rise in [Ca2+]cyt via CCE. The store-operated Ca2+ channel blocker Ni2+ reduced the store depletion-activated Ca2+ currents, decreased CCE, and inhibited the CCE-mediated contraction. In single PASMCs, we identified, using RT-PCR, five transient receptor potential gene transcripts. These results suggest that CCE, potentially through transient receptor potential-encoded Ca2+ channels, plays an important role in agonist-mediated PA contraction.

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