scholarly journals Development and Validation of Heminested RT-PCR and qRT-PCR Assays for Comprehensive Detection of Rabies Virus in the Suspected Rabid Brain and Saliva Samples

2019 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Fereshteh Amiri ◽  
Zohreh Fadajan ◽  
Azadeh Rasooli ◽  
Iman Salahshourifar ◽  
Rouzbeh Bashar ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Rt Pcr ◽  
Qrt Pcr ◽  
Pcr Assays ◽  
Development And Validation

scholarly journals Evaluation of Two Real-Time, TaqMan Reverse Transcription-PCR Assays for Detection of Rabies Virus in Circulating Variants from Argentina: Influence of Sequence Variation

Viruses ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 23
Author(s):  
Diego A. Caraballo ◽  
María A. Lombardo ◽  
Paula Becker ◽  
María S. Sabio ◽  
Cristina Lema ◽  
...  
Keyword(s):  
Rabies Virus ◽  
False Negative ◽  
Rapid Test ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Nucleoprotein Gene ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Pcr Assays ◽  
Rabies Diagnosis

In rabies diagnosis, it is essential to count on a rapid test to give a quick response. The combined sensitivity and robustness of the TaqMan RT-PCR assays (qRT-PCR) have made these methods a valuable alternative for rabies virus (RABV) detection. We conducted a study to compare the applicability of two widely used qRT-PCR assays targeting the nucleoprotein gene (LysGT1 assay) and leader sequences (LN34 qRT-PCR assay) of RABV genomes, in all variants circulating in Argentina. A total of 44 samples obtained from bats, dogs, cattle, and horses, that were previously tested for rabies by FAT and conventional RT-PCR, were used in the study. All variants were successfully detected by the pan-lyssavirus LN34 qRT-PCR assay. The LysGT1 assay failed to detect three bat-related variants. We further sequenced the region targeted by LysGT1 and demonstrated that the presence of three or more mismatches with respect to the primers and probe sequences precludes viral detection. We conclude that the LysGT1 assay is prone to yield variant-dependent false-negative test results, and in consequence, the LN34 assay would ensure more effective detection of RABV in Argentina.

Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines

2019 ◽  
Vol 270 ◽  
pp. 46-51 ◽  
Author(s):  
Beatriz Lourenç Correia Moreira ◽  
Luciane Aparecida Pereira ◽  
Ana Paula Lappas Gimenez ◽  
Jorge Minor Fernandes Inagaki ◽  
Sonia Mara Raboni
Keyword(s):  
Quality Control ◽  
Real Time ◽  
Rabies Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Development And Validation

scholarly journals Surveillance and correlation of SARS-CoV-2 viral RNA, antigen, virus isolation, and self-reported symptoms in a longitudinal study with daily sampling

2021 ◽  
Author(s):  
Gaston Bonenfant ◽  
Jessica Deyoe ◽  
Terianne Wong ◽  
Carlos G. Grijalva ◽  
H. Keipp Talbot ◽  
...  
Keyword(s):  
Positive Culture ◽  
Viral Rna ◽  
Rt Pcr ◽  
Predictive Values ◽  
Qrt Pcr ◽  
Household Transmission ◽  
Daily Sampling ◽  
Diagnostic Panel ◽  
Pcr Assays ◽  
Novel Coronavirus

The novel coronavirus pandemic incited unprecedented demand for assays that detect viral nucleic acids, viral proteins, and corresponding antibodies. The 320 molecular diagnostics in receipt of FDA emergency use authorization mainly focus on viral detection; however, no currently approved test can be used to infer infectiousness, i.e., the presence of replicable virus. As the number of tests conducted increased, persistent SARS-CoV-2 RNA positivity by RT-PCR in some individuals led to concerns over quarantine guidelines. To this end, we attempted to design an assay that reduces the frequency of positive test results from individuals who do not shed culturable virus. We describe multiplex quantitative RT-PCR (qRT-PCR) assays that detect genomic RNA (gRNA) and subgenomic RNA (sgRNA) species of SARS-CoV-2, including spike (S), nucleocapsid (N), membrane (M), envelope (E), and ORF8. The absolute copy number of each RNA target was determined in longitudinal specimens from a household transmission study. Calculated viral RNA levels over the 14-day follow up period were compared with antigen testing and self-reported symptoms to characterize the clinical and molecular dynamics of infection and infer predictive values of these qRT-PCR assays relative to culture isolation. When detection of sgS RNA was added to the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel, we found a qRT-PCR positive result was 98% predictive of a positive culture (negative predictive value was 94%). Our findings suggest sgRNA presence correlates with active infection, may help identify individuals shedding culturable virus, and that similar multiplex assays can be adapted to current and future variants.

scholarly journals One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1560 ◽  
Author(s):  
Rashi Gautam ◽  
Slavica Mijatovic-Rustempasic ◽  
Mathew D. Esona ◽  
Ka Ian Tam ◽  
Osbourne Quaye ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Wild Type ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Group A ◽  
One Step ◽  
Stool Samples ◽  
Pcr Assays ◽  
Type Strains

Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

Circulating tumor cell detection: A direct comparison between the CellSearch System, the AdnaTest, and CK-19/mammaglobin RT-PCR in patients with metastatic breast cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e22117-e22117
Author(s):  
L. Y. Dirix ◽  
H. Elst ◽  
I. Benoy ◽  
I. Van der Auwera ◽  
A. Prové ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Metastatic Breast Cancer ◽  
Tumor Cells ◽  
Metastatic Breast ◽  
Rt Pcr ◽  
Cellsearch System ◽  
Blood Samples ◽  
Detection Techniques ◽  
Qrt Pcr ◽  
Pcr Assays

e22117 Background: The detection, enumeration and isolation of circulating tumor cells (CTC) has considerable potential to influence the clinical management of patients with breast cancer. There is however a substantial variability in the rates of positive samples using existing detection techniques. Methods: This study was designed to compare three techniques for detecting CTC in blood of 80 patients with metastatic breast cancer (MBC) and 20 healthy controls: the CellSearch System, which is an automated, standardized and regulatory-approved system for the immunocytochemical detection and quantification of CTC in blood; ii) the AdnaTest Breast Cancer Select/Detect, which involves the detection of tumor-associated transcripts by RT-PCR after an immunomagnetically enrichment of tumor cells; iii) an in-house developed multimarker qRT-PCR assay, which involves the quantification of tumor-associated transcripts (CK-19 and MAM) by qRT- PCR. Results: As a result, 23% of patients with MBC were positive by the CellSearch System (≥5 CTC), 22% by the AdnaTest (>0.30 ng/μl for any of the amplicons), 31% by qRT-PCR for CK-19 and 49% by qRT-PCR for MAM. Samples were more likely to be positive by qRT-PCR for at least one mRNA marker than by the CellSearch System (P<0.001) or the AdnaTest (P <0.001). The concordance between samples analyzed by the CellSearch System and the AdnaTest was substantial (κ = 0.667, P <0.001). Agreement between both detection techniques was observed in 88% of blood samples. When the CellSearch System was compared with the qRT-PCR assays for CK-19 and MAM, we observed agreement percentages of 78% and 58%, respectively (κ = 0.462, P <0.001 and κ = 0.159, P = 0.09). Agreement between the AdnaTest and the qRT-PCR assays for CK-19 and MAM was observed in 78% and 53% of blood samples, respectively (κ = 0.443, P <0.001 and κ = 0.05, P = 0.607). Conclusions: We observed a substantial variation in the detection rates of CTC in blood from MBC patients using three different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and MAM, which suggests that this is currently the most sensitive technique for detecting CTC. No significant financial relationships to disclose.

scholarly journals Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus

2017 ◽  
Vol 243 ◽  
pp. 120-130 ◽  
Author(s):  
Martin Faye ◽  
Laurent Dacheux ◽  
Manfred Weidmann ◽  
Sylvie Audrey Diop ◽  
Cheikh Loucoubar ◽  
...  
Keyword(s):  
Real Time ◽  
Rabies Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Development And Validation

scholarly journals Development and validation of a rapid, single-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) system potentially to be used for reliable and high-throughput screening of COVID-19

2020 ◽  
Author(s):  
Minghua Jiang ◽  
Weihua Pan ◽  
Amir Aratehfar ◽  
Wenjie Fang ◽  
Liyan Ling ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
High Throughput ◽  
Predictive Value ◽  
High Throughput Screening ◽  
Lamp Assay ◽  
Single Step ◽  
Clinical Samples ◽  
Qrt Pcr ◽  
Pcr Assays ◽  
Development And Validation

AbstractObjectivesDevelopment and validation of a single-step and accurate reverse transcriptase loop-mediated isothermal amplification technique (RT-LAMP) for rapid identification of SARS COV-2 relative to commercial quantitative reverse transcriptase real-time PCR (qRT-PCR) assays to allow prompt initiation of proper medical care and containment of virus spread.MethodsPrimers showing optimal in-silico features were subjected to analytical sensitivity and specificity to assess the limit of detection (LOD) and cross-reaction with closely- and distantly-related viral species, and clinically prominent bacterial and fungal species. In order to evaluate the clinical utility, our RT-LAMP was subjected to a large number of clinical samples, including 213 negative and 47 positive patients, relative to two commercial quantitative RT-PCR assays.ResultsThe analytical specificity and sensitivity of our assay was 100% and 500 copies/ml when serial dilution performed in both water and sputum. Subjecting our RT-LAMP assay to clinical samples showed a high degree of specificity (99.5%), sensitivity (91.4%), positive predictive value (97.7%), and negative predictive value (98.1%) when used relative to qRT-PCR. Our RT-LAMP assay was two times faster than qRT-PCR and is storable at room temperature. A suspected case that later became positive tested positive using both our RT-LAMP and the two qRT-PCR assays, which shows the capability of our assay for screening purposes.ConclusionsWe present a rapid RT-LAMP assay that could extend the capacity of laboratories to process two times more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes when demand is increasing at critical situations.

Comparison of immunohistochemistry (IHC) and quantitative RT-PCR: ER, PR, and HER2 receptor status.

2012 ◽  
Vol 30 (27_suppl) ◽  
pp. 47-47 ◽  
Author(s):  
Cindy Christopherson ◽  
Monica Chang ◽  
David A. Eberhard ◽  
John J. Sninsky ◽  
Steven M. Anderson ◽  
...  
Keyword(s):  
Oncotype Dx ◽  
Taqman Assay ◽  
Rt Pcr ◽  
Promising Alternative ◽  
Receptor Status ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Alternative Method ◽  
Additional Testing ◽  
Pcr Assays

47 Background: IHC is the present standard for measuring estrogen (ER) and progesterone receptor (PR) expression for breast cancer. However, the lack of concordance between testing laboratories and the critical patient treatment decisions made with results prompted ASCO/CAP to recommend guidelines and proficiency testing requirements to ensure accuracy. Quantitative RT-PCR (qRT-PCR) is an alternative method for ER and PR testing and while concordance with IHC has been reported, additional testing is merited. This study compares ER, PR and HER2 status determined by IHC and qRT-PCR. Methods: FFPE tissues of ER(+) tumors collected and tested at the Blumenthal Cancer Center were studied. Expression levels of ESR1, PGR, and ERBB2 were determined by a multiplex qRT-PCR TaqMan assay and by the Oncotype Dx assay. Pre-established cutpoints were used to determine positivity. Only samples with qRT-PCR and corresponding IHC data were used, resulting in 144 ER, 128 PR, and 107 HER2 comparisons. ESR1 and PGR expression levels determined by the two qRT-PCR assays were also compared. Results: Of the144 IHC ER(+) samples, 142 were positive and 2 were negative by both qRT-PCR assays. All 120 IHC PR(+) samples were positive by both qRT-PCR assays. Of the 8 IHC PR(-) samples, 5 were negative and 3 were positive by both qRT-PCR assays. Of the 107 IHC HER2(-) samples, all but 2 were negative by qRT-PCR. One sample was positive by both qRT-PCR assays; one was positive by Oncotype Dx only. The expression levels determined by the qRT-PCR assays showed good correlation for ESR1 (r=0.85) and PGR (r=0.9). Conclusions: Concordance between qRT-PCR and IHC was 98.6% for ER and 97.7% for PR. Except for a single HER2 determination, there was 100% concordance between the two qRT-PCR assays. The discordant sample was HER2 (+) by multiplex qRT-PCR and “equivocal” by Oncotype Dx. Excellent correlation was also observed in the mRNA expression levels. The absence of HER2(+) and ER(-) samples are limitations of this study. These results suggest that qRT-PCR is a promising alternative method to IHC for determining hormone receptor status. Additional testing of samples with endocrine therapy outcomes including ER(-) samples would be beneficial.

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