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Detection of balanced translocations in acute lymphoblastic leukemia by a novel multiplex reverse transcriptase reverse transcription-polymerase chain reaction.

Fusion transcripts detection is essential for subtyping and diagnosis of acute lymphoblastic leukemia (ALL). This enables institution of appropriate therapy and provides a parameter to monitor disease progression and response to therapy. This study endeared to detect and analyze various balanced translocations known in ALL by using a novel polymerase chain reaction (PCR) method. A pilot study was done in which 16 consecutive cases of ALL were analyzed and followed-up for a period of 1 year. Diagnosis of ALL was established after subjecting blood/bone marrow aspirate samples to morphological examination, immunophenotyping, and detection of fusion transcripts by multiplex reverse transcription (RT)-PCR using HemaVision kit. Results were analyzed by correlating with morphology, immunophenotype, and response to therapy. Epi-Info statistical software was used. 43% (seven cases) showed balanced translocations, with all seven cases being B-ALL and t(9;22) being the most common. There was a consistent association of CD25 cases with t(9;22). Analyses of relation to other parameters were as expected by their respective WHO 2008 subtype. No significant correlation in terms of survival benefit was seen between cases with and without balanced translocations (P = 0.7472). The study demonstrated the utility of multiplex RT-PCR in the initial evaluation, subtyping, and monitoring minimal residual disease in ALL cases with balanced translocations, thereby guiding both therapy and prognosis. The consistent association of CD25 in cases of t(9;22) ALL indicated that CD25 could be used as a surrogate marker.

2235 related Products with: Detection of balanced translocations in acute lymphoblastic leukemia by a novel multiplex reverse transcriptase reverse transcription-polymerase chain reaction.

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In situ hybridization detection methods for HPV16 E6/E7 mRNA in identifying transcriptionally active HPV infection of oropharyngeal carcinoma: an updating.

The aim of this study is comparing two in situ hybridization (ISH) detection methods for human papilloma virus (HPV) 16 E6/E7 mRNA, i.e. the RNAscope® 2.0 High Definition (HD) and the upgraded RNAscope® 2.5 HD version. The RNAscope® 2.5 HD has recently replaced the RNAscope® 2.0 HD detection kit. Therefore, this investigation starts from the need to analytically validate the new mRNA ISH assay and, possibly, to refine the current algorithm for HPV detection in oropharyngeal squamous cell carcinoma (OSCC) with the final goal to apply it to daily laboratory practice. The study was based on HPV status and on generated data, interpreted by a scoring algorithm. The results highlighted that the compared RNAscope HPV tests had a good level of interchangeability and enabled to identify OSCC that are truly driven by high risk-HPV infection. This was also supported by the comparison of the RNAscope HPV test with HPV E6/E7 mRNA real time reverse transcriptase-polymerase chain reaction (RT-PCR), in a fraction of cases where material for HPV E6/E7 mRNA real time RT-PCR was available. Furthermore, the algorithm that associates p16 immunohistochemistry (IHC) with the identification of HPV mRNA by RNAscope was more effective than the one that associated p16 IHC with the identification of HPV DNA by ISH.

2509 related Products with: In situ hybridization detection methods for HPV16 E6/E7 mRNA in identifying transcriptionally active HPV infection of oropharyngeal carcinoma: an updating.

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Molecular characterization of a series of solitary fibrous tumors, including immunohistochemical expression of STAT6 and NATB2-STAT6 fusion transcripts, using Reverse Transcriptase(RT)-Polymerase chain reaction(PCR) technique: An Indian experience.

A solitary fibrous tumor (SFT) is characterized by a diverse clinicopathologic spectrum. Recent studies have unraveled STAT6 as a useful diagnostic immunohistochemical (IHC) marker for a SFT and NAB2-STAT6 as its specific gene fusion transcript. Thirty-three SFTs were tested for STAT6 immunostaining by polymer detection technique. STAT6 immunoexpression was further graded, based on intensity (mild, moderate and strong) and percentage of immunopositive tumor cells, ranging from 1 to 25%(1+); 26-50%(2+); 51-75%(3+) and in more than 75%(4+) tumor nuclei. These cases along with 17 other tumors were tested for 8 variants of NAB2-STAT6, using qualitative endpoint reverse-transcriptase (RT)-PCR technique. RNA extraction was performed using Recover All Total nucleic acid extraction kit. The selected cases were screened for all the 8 fusion variants, using 8 primer pairs for NAB2 and STAT6 genes. Thirty-three SFTs occurred in 18 men and 15 women (M: F=1.2:1), with age varying from 13 to 74 years(average=49.6); across various body sites. Immunohistochemically, most SFTs (30/33) (90.9%) displayed moderate to strong immunostaining for STAT6, including 3+ and 4+ immunostaining patterns in 27/33 (81.8%) tumors. By RT-PCR, 30/33(90.9%) cases of SFT were positive for NAB2-STAT6 fusions, including NAB2ex4/STAT6ex2 (7cases), NAB2ex7/STAT6ex2 (7cases), NAB2ex6/STAT6ex3 (6cases), NAB2ex6/SAT6ex16 (4cases), NAB2ex3/STAT6ex19 (4cases), NAB2ex6/STAT6ex17 (single case), NAB2ex4/STAT6ex4 (single case) and NAB2ex6/STAT6ex18 (none). NAB2-STAT6 fusions were not observed in 9 cases of synovial sarcoma, 4 of Ewing sarcoma, 2 of MPNST and 2 cases of dedifferentiated liposarcomas (100% specificity). On comparing with clinical outcomes, more cases (7/11)(63.6%) of classic SFT were associated with favorable outcomes, while more cases(5/8)(62%) of atypical and malignant SFTs were associated with aggressive outcomes. This study reinforces high sensitivity and specificity of NAB2-STAT6 fusion and its correlation with strong and diffuse IHC expression of STAT6 in a SFT, irrespective of its occurrence in various body sites and its histopathologic types. NAB2ex4-STAT6ex2 and NAB2ex7-STAT6ex2 fusions were relatively more frequently observed in our patients. Atypical and malignant SFTs, together, were more frequently associated with relatively aggressive clinical outcomes.

2646 related Products with: Molecular characterization of a series of solitary fibrous tumors, including immunohistochemical expression of STAT6 and NATB2-STAT6 fusion transcripts, using Reverse Transcriptase(RT)-Polymerase chain reaction(PCR) technique: An Indian experience.

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Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer.

Patients with lung cancers harboring an activating anaplastic lymphoma kinase () rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests forrearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation ofexpression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off Δof ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-lengthtranscripts orandfusion variants in discordant cases in whichexpression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-lengthexpression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.

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Diagnostic Validation of the RealStarZika Virus Reverse Transcription Polymerase Chain Reaction Kit for Detection of Zika Virus RNA in Urine and Serum Specimens.

With the Zika virus outbreak in South America starting in 2015 and its potential to cause malformation of the fetus in infected women, the need for diagnostic methods became obvious. Until now, only limited data are available on the diagnostic performance of commercial kits. Here, we present data comparing the RealStarZika Virus RT-PCR Kit 1.0 for detection of Zika virus from 208 serum and urine samples collected in French Guiana with a reference method. Of these, 114 samples tested positive with the RealStarKit and 111 with the reference method.

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High prevalence of norovirus in children with sporadic acute gastroenteritis in Manaus, Amazon Region, northern Brazil.

Norovirus (NoV) is a major cause of acute gastroenteritis (AGE) worldwide, especially in children under five years. Studies involving the detection and molecular characterisation of NoV have been performed in Brazil, demonstrating its importance as an etiological agent of AGE.

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Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.

Virome (viral megagenomics) detection using next generation sequencing has been widely applied in virology, but its methods remain complicated and need optimization. In this study, we detected the viromes of RNA viruses of one mock sample, one pooled duck feces sample and one pooled mink feces sample on the Personal Genome Machine platform using the sequencing libraries prepared by three methods. The sequencing primers were added through random hybridization and ligation to fragmented viral RNA using a RNA-Seq kit in method 1, through random reverse transcription (RT) and polymerase chain reaction (PCR) in method 2 which was developed in our laboratory, and through hybridization and ligation to fragmented amplicons of random RT-PCR using a single primer in method 3. Although the results of these three samples (nine libraries) all showed that more classified viral families and genera were identified using methods 2 and 3 than using method 1, and more classified viral families and genera were identified using method 2 than using method 3, most of the differences were of no statistical significance. Moreover, 11 mammalian viral genera in minks were possibly identified for the first time through this study.

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Clinical evaluation of a new single-tube multiplex reverse transcription PCR assay for simultaneous detection of 11 respiratory viruses, Mycoplasma pneumoniae and Chlamydia in hospitalized children with acute respiratory infections.

Respiratory Pathogen 13 Detection Kit (13× kit) is able to simultaneously detect 11 respiratory viruses, Mycoplasma pneumoniae (MP) and Chlamydia in a single reaction. Using 572 Nasopharyngeal aspirates collected from hospitalized children, the clinical performance of 13× kit for detecting 11 respiratory viruses was evaluated in comparison with a routinely used 2-tube multiplex reverse transcription PCR assay (2-tube assay) at provincial Centers for Disease Control and Prevention in China. The clinical performance of 13× kit for detecting MP and Chlamydia was evaluated by commercial real-time quantitative PCR (qPCR) kits or sequencing. For tested viruses, the assay concordance was 95.98% and the kappa coefficient was 0.89. All the MP and Chlamydia positive samples detected by 13× kit were confirmed as true positives. The utilization of the 13× kit in clinical settings will be helpful for doctors to assess clinical outcome according to virus type or multiple infections, and to limit the use of antibiotics.

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Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II.

Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (10DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.

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Expression Analysis of Previously Verified Fecal and Plasma Dow-regulated MicroRNAs (miR-4478, 1295-3p, 142-3p and 26a-5p), in FFPE Tissue Samples of CRC Patients.

Colorectal cancer (CRC) is one of the most common causes of cancer-related mortality worldwide. Early diagnosis of this neoplasm is critical and may reduce patients' mortality. MicroRNAs are small non-coding RNA molecules whose expression pattern can be altered in various diseases such as CRC.

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