NTRK Gene Fusion Detection in Atypical Spitz Tumors

Atypical Spitz tumors (AST) deviate from stereotypical Spitz nevi for one or more atypical features and are now regarded as an intermediate category of melanocytic tumors with uncertain malignant potential. Activating NTRK1/NTRK3 fusions elicit oncogenic events in Spitz lesions and are targetable with kinase inhibitors. However, their prevalence among ASTs and the optimal approach for their detection is yet to be determined. A series of 180 ASTs were screened with pan-TRK immunohistochemistry and the presence of NTRK fusions was confirmed using FISH, two different RNA-based NGS panels for solid tumors, and a specific real time RT-PCR panel. Overall, 26 ASTs showed pan-TRK immunostaining. NTRK1 fusions were detected in 15 of these cases showing cytoplasmic immunoreaction, whereas NTRK3 was detected in one case showing nuclear immunoreaction. Molecular tests resulted all positive in only two ASTs (included the NTRK3 translocated), RNA-based NGS and real time RT-PCR were both positive in three cases, and FISH and real time RT-PCR in another two cases. In seven ASTs NTRK1 fusions were detected only by FISH and in two cases only by real time RT-PCR. The frequency of NTRK fusions in ASTs is 9%, with a clear prevalence of NTRK1 compared to NTRK3 alterations. Pan-TRK immunohistochemistry is an excellent screening test. Confirmation of NTRK fusions may require the use of different molecular techniques.

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Sensitive SYBR Green—Real Time PCR for the Detection and Quantitation of Avian Rotavirus A

Veterinary Sciences ◽

10.3390/vetsci6010002 ◽

2018 ◽

Vol 6 (1) ◽

pp. 2 ◽

Cited By ~ 2

Author(s):

David De la Torre ◽

Claudete Astolfi-Ferreira ◽

Ruy Chacon ◽

Antonio Piantino Ferreira

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Sybr Green ◽

Molecular Techniques ◽

Economic Losses ◽

Rt Pcr ◽

Chain Reaction ◽

Rotavirus A ◽

Avian Nephritis Virus ◽

Polymerase Chain

Avian rotavirus A (ARtV-A) is a virus that affects young birds, causing acute diarrhea and economic losses in the poultry industry worldwide. The techniques used for the diagnosis of ARtV-A include electron microscopy, isolation in cell culture, and serology, as well as molecular techniques, such as the reverse transcription-polymerase chain reaction (RT-PCR). The objective of this work was to standardize a real-time RT-polymerase chain reaction (RT-qPCR) using SYBR Green chemistry for the rapid detection and quantification of ARtV-A from bird tissues and materials fixed on FTA cards on the basis of the nucleotide sequence of segment 6 (S6), which codes for the structural VP6 protein of ARtV-A. The results show the efficient amplification of the proposed target, with a limit of detection (LoD) of one copy gene (CG) per microliter of cDNA and a limit of quantification (LoQ) of 10 CGs per microliter. The efficiency of the primers was determined to be 95.66% using a standard curve, with an R2 value of 0.999 and a slope of −3.43. The specificity was determined using samples coinfected with ARtV-A, the chicken parvovirus, the chicken astrovirus, and the avian nephritis virus as positive controls and commercially available vaccines of the infectious bronchitis virus, infectious bursa disease virus, avian reovirus and healthy organs as negative controls. This technique, which lacks nonspecific PCR products and dimers, demonstrated greater sensitivity and specificity than conventional RT-PCR, and it reduced the analysis time by more than 50%.

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Lyophilized Matrix Containing Ready-to-Use Primers and Probe Solutions for Standardization of Real-Time PCR and RT-qPCR Diagnostics

Proceedings ◽

10.3390/proceedings2020050007 ◽

2020 ◽

Vol 50 (1) ◽

pp. 7

Author(s):

Remi N Charrel ◽

Laurence Thirion

Keyword(s):

Real Time ◽

Molecular Techniques ◽

Cold Chain ◽

Rt Pcr ◽

Emerging Viruses ◽

Valley Fever ◽

Term Stability ◽

E Gene ◽

Long Term Stability

Real-time molecular techniques have become the reference methods for the direct diagnosis of pathogens. The reduction of steps is a key factor in order to decrease the risk of human errors resulting in invalid series and delayed results. We describe here a process involving the preparation of oligonucleotide primers and a hydrolysis probe in a single tube at predefined optimized concentrations that are stabilized via lyophilization (Lyoph-P&P). Lyoph-P&P was compared to the classic protocol using extemporaneously prepared liquid reagents, assaying (i) sensitivity, (ii) long-term stability at 4 °C, and (iii) long-term stability at 37 °C, mimicking transportation without a cold chain. Two previously published molecular assays were selected for this study. They target two emerging viruses that are listed on the blueprint of the WHO to be considered for preparedness and response actions: chikungunya virus (CHIKV) and Rift Valley fever phlebovirus (RVFV). The results of our study demonstrate that (i) Lyoph-P&P is stable for at least four days at 37 °C, supporting shipping without the need of a cold chain, (ii) Lyoph-P&P rehydrated solution is stable at 4 °C for at least two weeks, (iii) the sensitivity observed with Lyoph-P&P is at least equal to, and often better than, that observed with liquid formulation, and (iv) the validation of results observed with low-copy specimens is rendered easier by higher fluorescence levels. In conclusion, Lyoph-P&P holds several advantages over extemporaneously prepared liquid formulations and merits consideration as a novel real-time molecular assay for implementation into a laboratory with routine diagnostic activity. Since the meeting, this concept has been applied to the COVID-19 situation: two diagnostic assays (E gene and RdRp) have been developed and can be ordered on the European Virus Archive catalog (https://www.european-virus-archive.com/detection-kit/lyophilized-primers-and-probe-rt-pcr-2019-ncov-e-gene; https://www.european-virus-archive.com/detection-kit/lyophilized-primers-and-probe-rt-pcr-sars-cov-2-rdrp-gene).

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Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization

PLoS ONE ◽

10.1371/journal.pone.0248581 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0248581

Author(s):

Clyde S. Manuel ◽

Cassandra Suther ◽

Matthew D. Moore ◽

Lee-Ann Jaykus

Keyword(s):

Detection Limit ◽

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Blot Hybridization ◽

Molecular Techniques ◽

Rt Pcr ◽

Dot Blot ◽

Dot Blot Hybridization ◽

One Step

Human norovirus (NoV) is the leading cause of acute viral gastroenteritis and a major source of foodborne illness. Detection of NoV in food and environmental samples is typically performed using molecular techniques, including real-time reverse transcription polymerase chain reaction (RT-PCR) and less frequently, nested real-time PCR. In this study, we conducted a controlled comparison of two published NoV detection assays: a broadly reactive one-step real-time RT-PCR and a two-step nested real-time PCR assay. A 20% human fecal suspension containing a genogroup II human NoV was serially diluted, genome extracted, and subjected to amplification using the two assays compared via PCR Units. Additional amplicon confirmation was performed by dot blot hybridization using digoxigenin (DIG)-labeled oligonucleotide probes. Both assays displayed similar amplification standard curves/amplification efficiencies; however, the nested assay consistently detected one log10 lower virus. Dot blot hybridization improved the detection limit of the nested real-time PCR by one log10 NoV genome copies but impaired the detection limit of the one-step real-time RT-PCR by one log10 NoV genome copies. These results illustrate the complexities in designing and interpreting molecular techniques having a sufficient detection limit to detect low levels of viruses that might be anticipated in contaminated food and environmental samples.

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Serological and molecular evidence of Brucella species in the rapidly growing pig sector in Kenya

10.21203/rs.2.21914/v1 ◽

2020 ◽

Author(s):

James Miser Akoko ◽

Roger Pelle ◽

Velma Kivali ◽

Esther Schelling ◽

Gabriel Shirima ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Epidemiological Data ◽

Molecular Techniques ◽

Molecular Evidence ◽

Rt Pcr ◽

Growing Pig ◽

Pcr Targeting ◽

The Rose

Abstract BackgroundBrucellosis is an emerging, yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi, Kenya were screened in parallel, using the Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp.. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 gene and real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes.ResultsA prevalence of 0.57% (n=4/700) was estimated using RBT. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a multiple real-time PCR. ConclusionThe detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs indicate the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

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Serological and Molecular Evidence of Brucella Species in the Rapidly Growing Pig Sector in Kenya

10.21203/rs.2.21914/v2 ◽

2020 ◽

Author(s):

James Miser Akoko ◽

Roger Pelle ◽

Velma Kivali ◽

Esther Schelling ◽

Gabriel Shirima ◽

...

Keyword(s):

Real Time ◽

Epidemiological Data ◽

Molecular Techniques ◽

Molecular Evidence ◽

Rt Pcr ◽

Conventional Pcr ◽

Brucella Infection ◽

Growing Pig ◽

Pcr Targeting

Abstract Background: Brucellosis is an emerging yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi region, Kenya were screened in parallel, using both Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 geneand real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes. Results: A prevalence of 0.57% (n = 4/700) was estimated using RBT; none of these samples was positive on cELISA. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a real-time multiplex PCR. Conclusion: The detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs confirm the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

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Serological and molecular evidence of Brucella species in the rapidly growing pig sector in Kenya

10.21203/rs.2.21914/v3 ◽

2020 ◽

Author(s):

James Miser Akoko ◽

Roger Pelle ◽

Velma Kivali ◽

Esther Schelling ◽

Gabriel Shirima ◽

...

Keyword(s):

Real Time ◽

Epidemiological Data ◽

Molecular Techniques ◽

Molecular Evidence ◽

Brucella Species ◽

Rt Pcr ◽

Conventional Pcr ◽

Growing Pig ◽

Pcr Targeting

Abstract Background: Brucellosis is an emerging yet neglected zoonosis that has been reported in Kenya. Epidemiological data on brucellosis in ruminants is readily accessible; however, reports on brucellosis in pigs remain limited. This study sought to detect Brucella infection in pig serum by both serological and molecular techniques. Serum from 700 pigs randomly collected at a centralized abattoir in Nairobi region, Kenya were screened in parallel, using both Rose Bengal Test (RBT) and competitive Enzyme-Linked Immuno-sorbent Assay (cELISA) for antibodies against Brucella spp. All sera positive by RBT and 16 randomly selected negative samples were further tested using conventional PCR targeting bcsp31 gene and real-time PCR (RT-PCR) assays targeting IS711 and bcsp31 genes.Results: A prevalence of 0.57% (n = 4/700) was estimated using RBT; none of these samples was positive on cELISA. All RBT positive sera were also positive by both PCRs, while two sero-negative samples also tested positive on RT-PCR (n = 6/20). Brucella abortus was detected in four out of the six PCR positive samples through a real-time multiplex PCR.Conclusion: The detection of antibodies against Brucella spp. and DNA in serum from slaughterhouse pigs confirm the presence of Brucella in pigs. Therefore, investigation of the epidemiology and role of pigs in the transmission of brucellosis in Kenya is needed. Further targeted studies would be useful to systematically quantify and identify the spp. of Brucella in pigs.

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Prognostic Value of Real-Time PCR BCR-ABL Transcript Monitoring after Allogeneic Stem Cell Transplantation in Chronic Myeloid Leukemia Patients.

Blood ◽

10.1182/blood.v108.11.4824.4824 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4824-4824

Author(s):

Iwona Solarska ◽

Miroslaw Majewski ◽

Barbara Nasilowska-Adamska ◽

Jan Zaucha ◽

Iwona Kania ◽

...

Keyword(s):

Stem Cell ◽

Chronic Myeloid Leukemia ◽

Stem Cell Transplantation ◽

Real Time ◽

Cell Transplantation ◽

Myeloid Leukemia ◽

Molecular Techniques ◽

Rt Pcr ◽

Molecular Remission ◽

Low Level

Abstract Allogeneic hematopoietic stem cell transplantation (alloHSCT) in chronic phase of chronic myeloid leukemia (CML) is associated with long-term disease-free survival and potentially eradication of leukemic cells. The goal of early MRD detection is to allow timely therapeutic intervention before hematologic relapse. The concomitant detection of BCR-ABL mRNA following alloHSCT is strongly associated with relapse, though not absolutely predictive. The relapse risk decreases with increased time after alloHSCT. The detection of BCR-ABL mRNA is the most strongly associated with relapse shortly after HSCT but all patients need to be monitored indefinitely after transplantation by molecular techniques presumably at 3–6 months intervals. Real-time quantitative PCR (RQ-PCR) for BCR-ABL mRNA provides an accurate and reliable measure of response to therapy in CML. In this study we evaluated 412 available samples from 75 patients at 1 month to 10 years after allo HSCT. Quantification of BCR-ABL was performed by RQ-PCR assay according to the Europe Against Cancer (EAC) protocol. Peripheral blood/bone marrow samples were studied every 3–6 months after alloHSCT for the presence of BCR-ABL transcripts using RT-PCR/nested PCR and RQ-PCR. RT-PCR positive patients were analyzed further at monthly intervals. RNA isolation from mononuclear cells was performed by column method. Reverse transcription was performed using Super Script II and random hexamers. BCR-ABL level was normalized with control ABL gene and expressed as the ratio of BCR-ABL/ABL compared to diagnostic sample or median expression values of BCR-ABL/ABL from EAC protocol. In our group BCR-ABL/ABL ratio decreased at least 1000-fold in all patients after alloHSCT. RT-PCR became negative in 64.7% patients after first 90 days. In the group of 65 patients with RQ-PCR tests performed at least 1 year after alloHSCT, 12 (18.5%) patients were always negative (no BCR-ABL/ABL transcripts detected, at least 10−5 test sensitivity), 40 (61.5%) were persistently low-level positive (with the BCR-ABL/ABL ratio less than 0.02%) and 13 (20.0%) patients were stable, high-level positive with transcript levels exceeded 0.02% threshold. The molecular relapse was observed in three patients with 15–80 fold increased of BCR-ABL expression in the first year after SCT. Decreased immunosuppressive therapy allowed achieving molecular remission in two patients. One patient developed hematological relapse despite donor lymphocyte infusion (DLI). One patient developed cytogenetic relapse and was successfully treated with 400 mg imatinib daily dose and achieved molecular remission with a low-level BCR-ABL expression. Standard imatinib therapy was applied in seven patients prior to SCT without negative transplant outcome. We conclude that RQ-PCR is valuable method to quantitate BCR-ABL expression in CML patients after alloHSCT and allows monitoring the kinetics of BCR-ABL mRNA transcipts and is useful in prediction of the hematologic relapse.

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