Study on Bioluminescent Detection of the SARS-CoV-2 ORF1ab Gene by Coupling Isothermal RNA Reverse Transcription Amplification with a Digital PCR: A Novel Approach

Abstract Objective A standardized procedure of fused REarranged during Transfection (RET) gene detection using fluorescence in situ hybridization (FISH) remains to be established in papillary thyroid carcinoma (PTC). Our purpose was to investigate false-negative and false-positive events and their FISH signal characteristics. Methods A total of 111 PTC cases were analyzed using break-apart FISH probe for RET status evaluation. All FISH results were validated using fusion-induced asymmetric transcription assay (FIATA)-based reverse transcription droplet digital PCR (RT-ddPCR). Then, suspected RET-positive cases were tested using quantitative reverse transcription-PCR (RT-qPCR) and following next-generation sequencing (NGS) for recognizing fusion variants. Results Thirty RET+ cases were revealed, including 20 CCDC6-RET (exon1;exon12), 6 NCOA4-RET (exon8;exon12), 1 NCOA4-RET (exon7;exon12), 1 CCDC186-RET (exon7;exon12), 1 ERC1-RET (exon12;exon12) and 1 SPECC1L-RET (exon9;exon12) tumors. All RET fusion cases occurred in the BRAF- population, with a prevalence of 41.7% (30/72). Four cases of 8–13% (cutoff was 7.6%) dominant isolated 3’ green (IG) FISH signals were RET-. One FISH- case with isolated 5’ red (IR) signals with 94% abnormal tumor cells was demonstrated to be positive, harboring the NCOA4-RET (exon7; exon12) variant. Compared to RET fusions characterized by dominant break-apart (BA) signals with 29–100% aberrant cells, RET + with dominant IG-signal patterns all showed more frequent FISH+ cells (84–92%). RET+ PTC with a BA signal pattern was more frequently found in unifocal lesions than in multifocal/bilateral tumors (p=0.049). Conclusions A false-positive or false-negative event may exist for RET status detection in PTCs using the traditional FISH scoring method with BA probes.

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A Novel Approach for Hepatitis Delta Virus Viremia Quantification by Droplet Digital PCR Assay

Journal of Hepatology ◽

10.1016/s0168-8278(16)00518-3 ◽

2016 ◽

Vol 64 (2) ◽

pp. S354

Author(s):

A. Olivero ◽

M.L. Abate ◽

G. Niro ◽

G.P. Caviglia ◽

C. Rosso ◽

...

Keyword(s):

Hepatitis Delta Virus ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Pcr Assay ◽

Hepatitis Delta ◽

Novel Approach ◽

Delta Virus

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Use of RNA-based genotypic approaches for quantification of viable but non-culturable Salmonella sp. in biosolids

Water Science & Technology ◽

10.2166/wst.2008.555 ◽

2008 ◽

Vol 58 (9) ◽

pp. 1823-1828 ◽

Cited By ~ 11

Author(s):

T. Dunaev ◽

S. Alanya ◽

M. Duran

Keyword(s):

Environmental Stress ◽

Reverse Transcription ◽

Rna Isolation ◽

Stress Genes ◽

Viable Cells ◽

Novel Approach ◽

Polymerase Chain ◽

Stress Related Genes ◽

Initial Results ◽

Accurate Quantification

Recent research efforts demonstrated an increase in fecal coliform counts in anaerobically digested biosolids after dewatering. Variety of bacteria enters viable but nonculturable (VNC) state as a survival response when exposed to environmental stress. Increase in coliform concentration after digestion and dewatering processes have been attributed to cells going into a viable but non-culturable state implying that traditional coliform enumeration methods are not sufficient to determine number of viable cells. Therefore, this research has been undertaken to develop a method for rapid and accurate quantification of viable but non-culturable pathogens in biosolids via monitoring and quantifying stress-related genes in Salmonella sp. The proposed method has the potential to allow accurate detection of pathogens in biosolids even when the cells are non-culturable due to environmental stress. The research proposed identification of stress related genes in Salmonella when cells are exposed to heat for different durations by using available Salmonella microarrays. In the context of this study the identified stress genes can be quantified through reverse transcription, complementary DNA (cDNA) synthesis, and amplification of cDNA via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Then quantity of mRNA can be correlated to cell viability and cells ability to grow, i.e., their culturability. Development of a novel approach to understand the pathogen behaviour in biosolids is key to ensure low public health risks from biosolids. Nevertheless, the initial results suggest that intact RNA isolation from biosolids is still challenging task.

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RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia

Clinical Chemistry ◽

10.1373/clinchem.2016.262824 ◽

2017 ◽

Vol 63 (2) ◽

pp. 525-531 ◽

Cited By ~ 38

Author(s):

Mary Alikian ◽

Alexandra S Whale ◽

Susanna Akiki ◽

Kim Piechocki ◽

Celia Torrado ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Reverse Transcription ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Dynamic Range ◽

Residual Disease ◽

Fusion Transcript ◽

Digital Pcr ◽

Analytical Sensitivity

Abstract BACKGROUND Tyrosine kinase inhibitors (TKIs) are the cornerstone of successful clinical management of patients with chronic myeloid leukemia (CML). Quantitative monitoring of the percentage of the fusion transcript BCR-ABL1 (breakpoint cluster region–c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1IS (%BCR-ABL1IS) by reverse transcription–quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to TKIs and classification into prognostic subgroups. However, this approach can be challenging to perform in a reproducible manner. Reverse-transcription digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for truly standardized patient stratification. METHODS BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. 3 commercially available digital PCR platforms (QS3D, QX200 and Raindrop) were compared with the platform routinely used in the clinic for RT-qPCR using the EAC (Europe Against Cancer) assay. RESULTS Measurements on all instruments correlated well when the %BCR-ABL1IS was ≥0.1%. In patients with residual disease below this level, greater variations were measured both within and between instruments limiting comparable performance to a 4 log dynamic range. CONCLUSIONS RT-dPCR was able to quantify low-level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without use of a calibration curve. Adaptions to the protocol to increase the amount of RNA measured are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing on a dPCR platform.

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2626. Rhinovirus in Children Presenting to the Emergency Department: Role of Viral Load in Disease Severity and Co-Infections

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.2304 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S915-S916

Author(s):

Alpana Waghmare ◽

Bonnie Strelitz ◽

Kirsten Lacombe ◽

Garrett Perchetti ◽

Arun Nalla ◽

...

Keyword(s):

Emergency Department ◽

Viral Load ◽

Reverse Transcription ◽

Digital Pcr ◽

Tertiary Hospital ◽

Supplemental Oxygen ◽

Amplification Efficiency ◽

Viral Loads ◽

Syncytial Virus

Abstract Background Rhinovirus (RV) quantitation by reverse transcription-quantitative PCR is limited by variable amplification efficiency across genotypes. We used a precise viral quantitation method, reverse transcription-digital PCR (RT-dPCR), to characterize the role of viral load in clinical outcomes and in viral co-infections in children presenting to a tertiary hospital emergency department (ED). Methods Children < 18 years with respiratory symptoms for ≤ 14 days were enrolled from December 1, 2016 to December 31, 2018. Participants had nasal and throat specimens obtained and multiplex PCR testing with a commercial assay (FilmArray; bioMerieux). RV positive samples were quantified using RT-dPCR. Samples with sufficient viral load were sequenced at a 543 bp fragment of the RV VP4/VP2 region. RV species were assigned by comparison to RV sequences in GenBank using BLAST. Clinical data were collected into REDCap. T-tests were used to compare mean viral loads between groups. Results Of 1703 children enrolled in the ED, 697 were RV/enterovirus positive by FilmArray [median age 18 months (interquartile range 9–39 months)]. Of 590 subjects with viral load available, 276 (47%) were admitted to the hospital. Among RV mono-infections (N = 434), mean viral load did not differ between subjects admitted vs. discharged from the ED (7.03 log copies/mL for both, P = 0.97). Among admitted subjects with RV mono-infection, viral load also did not differ between subjects requiring supplemental oxygen vs. not (7.01 vs. 7.10 log copies/mL, P = 0.6). Subjects with viral co-infections had lower mean RV viral loads (6.31 log copies/mL) compared with those with RV only (7.03 log copies/mL; P < 0.001) (figure). Significantly different RV viral loads were seen with co-infections with respiratory syncytial virus (RSV), metapneumovirus (MPV) and parainfluenza (PIV), but not with influenza, adenovirus or coronavirus. In 525 sequenced samples (46% RV-A, 4% RV-B, 50% RV-C), viral load did not vary between RV viral species (P = 0.09). Conclusion Precise viral quantitation demonstrates children co-infected with RV and RSV, MPV or PIV have lower nasal viral loads than those with RV alone. Among RV mono-infections, RV viral load was not associated with admission or need for supplemental oxygen. Disclosures All authors: No reported disclosures.

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