scholarly journals COVID-19 infection in the palatine tonsil tissue and detritus: the detection of the virus compartment with RT-PCR

2021 ◽  
Vol 14 (2) ◽  
pp. e239108
Author(s):  
Hamsu Kadriyan ◽  
Bayu Tirta Dirja ◽  
Dewi Suryani ◽  
Didit Yudhanto
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Palatine Tonsil ◽  
Rdrp Gene ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Recurrent Tonsillitis ◽  
Igg Antibody ◽  
Tonsil Tissue

Two patients suffering from chronic recurrent tonsillitis were reported. The first patient was confirmed infected with COVID-19, 3 weeks prior to tonsillectomy. The detritus and tonsil specimen were further analysed through real-time PCR (RT-PCR) and revealed amplification of the fragment N and ORF1ab genes of SARS-CoV-2. The second patient had a negative IgM and positive IgG antibody for COVID-19; however, the nasopharyngeal swab indicated negative for SARS-CoV-2. Tonsillectomy was performed 2 weeks after the swab; the tonsil specimen was analysed through RT-PCR and revealed amplification of the N2 and RdRp gene of SARS-CoV-2. According to both results, the presence of the SARS-CoV-2 gene remains to be detected in tonsil and/or detritus after 2–3 weeks after recovery. Hence, it is suggested that it is necessary to use adequate protection when performing tonsillectomy on early recovered patients with COVID-19. Furthermore, tonsillectomy would be more advisable to be performed after the fourth week after recovery from COVID-19.

scholarly journals Visual Detection of SARS-CoV-2 RNA by Conventional PCR-Induced Generation of DNAzyme Sensor

2020 ◽  
Vol 7 ◽  
Author(s):  
Anbalagan Anantharaj ◽  
Soon Jyoti Das ◽  
Patil Sharanabasava ◽  
Rakesh Lodha ◽  
Sushil K. Kabra ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Visual Detection ◽  
Fluorescence Probe ◽  
Scale Validation ◽  
Unmet Need ◽  
Pilot Scale ◽  
Molecular Diagnostic ◽  
Nasopharyngeal Swab ◽  
Rt Pcr

The gold standard for the diagnosis of SARS-CoV-2, the causative agent of COVID-19, is real-time polymerase chain reaction (PCR), which is labor-intensive, expensive, and not widely available in resource-poor settings. Therefore, it is imperative to develop novel, accurate, affordable, and easily accessible assays/sensors to diagnose and isolate COVID-19 cases. To address this unmet need, we utilized the catalytic potential of peroxidase-like DNAzyme and developed a simple visual detection assay for SARS-CoV-2 RNA using a conventional thermal cycler by the PCR-induced generation of DNAzyme sensor. The performance of RT-PCR DNAzyme-based sensor was comparable to that of real-time PCR. The pilot scale validation of RT-PCR DNAzyme-based sensor has shown ~100% sensitivity and specificity in clinical specimens (nasopharyngeal swab, n = 34), with a good correlation (Spearman r = 0.799) with the Ct-value of fluorescence probe-based real-time PCR. These findings clearly indicate the potential of this inexpensive, sensitive, and specific molecular diagnostic test to extend our testing capabilities for the detection of SARS-CoV-2 to curtail COVID-19 transmission.

scholarly journals Cost-Effective Pooling of DNA from Nasopharyngeal Swab Samples for Large-Scale Detection of Bacteria by Real-Time PCR

2014 ◽  
Vol 53 (3) ◽  
pp. 1002-1004 ◽  
Author(s):  
Sophie Edouard ◽  
Elsa Prudent ◽  
Philippe Gautret ◽  
Ziad A. Memish ◽  
Didier Raoult
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Scale Detection ◽  
The Cost

We investigated the potential of pooling DNA from nasopharyngeal specimens to reduce the cost of real-time PCR (RT-PCR) for bacterial detection. Lyophilization is required to reconcentrate DNA. This strategy yields a high specificity (86%) and a high sensitivity (96%). We estimate that compared to individual testing, 37% fewer RT-PCR tests are needed.

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
Author(s):  
D.M. Valero-Hervás ◽  
P. Morales ◽  
M.J. Castro ◽  
P. Varela ◽  
M. Castillo-Rama ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Low Cost ◽  
Complement C3 ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Dna Amount ◽  
Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

scholarly journals Increased Expression of the Pro-Protein Convertase Furin Predicts Decreased Survival in Ovarian Cancer

2007 ◽  
Vol 29 (4) ◽  
pp. 289-299
Author(s):  
Robert E. Page ◽  
Andrés J. P. Klein-Szanto ◽  
Samuel Litwin ◽  
Emmanuelle Nicolas ◽  
Raid Al-Jumaily ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Tumor Progression ◽  
Real Time ◽  
Cell Lines ◽  
Cancer Cell ◽  
Real Time Pcr ◽  
Ovarian Tumor ◽  
Cancer Cell Lines ◽  
Rt Pcr ◽  
Primary Tumors

Background: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. Methods: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). Results: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). Conclusions: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease.

scholarly journals Μελέτη του μεταβολισμού του σιδήρου στον σκελετικό μυ

2012 ◽  
Author(s):  
Αικατερίνη Πολονύφη
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
T Test ◽  
Rt Pcr ◽  
Densitometric Analysis ◽  
Paired T Test

Ο μεταβολισμός του σιδήρου δεν είναι επαρκώς μελετημένος στον σκελετικό ιστό σε αντίθεση με τον ηπατικό. Σκοπός της μελέτης είναι η σύγκριση της έκφρασης των γονιδίων του μεταβολισμού του σιδήρου στον ανθρώπινο σκελετικό και ηπατικό ιστό. Συλλέχτηκαν βιοψίες μυϊκού και ηπατικού ιστού από έξι φυσιολογικά άτομα. Επιλέχθηκαν να μελετηθούν12 γονίδια που εμπλέκονται στο μεταβολισμό του σιδήρου για την εισαγωγή σιδήρου στο κύτταρο, αποθήκευση και εξαγωγή του καθώς και δύο μόρια ρύθμισης της ομοιοστασίας του σιδήρου: εισαγωγή σιδήρου [οι υποδοχείς της τρανσφερρίνης (TfR1 και TfR2), το μόριο HFE, ο μεταφορέας δισθενών μετάλλων (DMT1,DMT1nonIRE), και το σιδεροφόρο μόριο λιποκαλίνη (NGAL)], αποθήκευση σιδήρου [η βαριά αλυσίδα της φερριτίνης (FTH1)] και εξαγωγή σιδήρου [η φερροπορτίνη (IREG1), η ηφαιστίνη (HEPH) και η σερουλοπλασμίνη (CP)] καθώς και δύο μόρια ρύθμισης της ομοιοστασίας του σιδήρου [η εψιδίνη (HAMP) και η αιμοτζουβελίνη (HJV)]. Ακολούθησαν αλυσιδωτές αντιδράσεις της πολυμεράσης, RT-PCR και ημιποσοτικοποίηση των επιπέδων έκφρασης των γονιδίων με τη μέθοδο της πυκνομετρίας (Densitometric Analysis). Τα αποτελέσματα εκφράζονται με βάση το ποσοστό επί τοις εκατό του γονιδίου της β-ακτίνης. Το γονίδιο της β-ακτίνης χρησιμοποιήθηκε για την κανονικοποίηση των επιπέδων έκφρασης, ως γονίδιο αναφοράς της μελέτης. Αναδεικνυόμενες διαφορές συγκριτικής έκφρασης μεγαλύτερες του 20%, των μελετημένων γονιδίων του μεταβολισμού του σιδήρου με ημιποσοτικοποίηση, αναλύθηκαν περαιτέρω με την μέθοδο της PCR σε αληθινό χρόνο (Real time PCR, qPCR) και ποσοτικοποιήθηκαν (LightCycler, Roche). Η στατιστική ανάλυση των αποτελεσμάτων ποσοτικοποίησης έγινε με το one paired t test και τα αποτελέσματα είναι στατιστικά σημαντικά (p<0,05). H συγκριτική μελέτη μεταξύ ανθρώπινου ηπατικού και σκελετικού ιστού στα επιλεγμένα 12 γονίδια έδειξε ότι: 1. Περισσότερα απο τα γονίδια: HJV, TFR1, HFE, DMT1, DMT1nonIRE, NGAL, HEPH, IREG1 ,DMT1(IRE) , DMT1nonIRE, FTH1 εκφράζονται και στους δύο ιστούς με ποσοστό έκφρασης >70% των επιπέδων έκφρασης της βακτίνης. 2. Εξαίρεση αποτελούν τα HAMP, CP και TfR2 που απουσιάζουν ή παρουσιάζουν ελάχιστη έκφραση (<10% των επιπέδων έκφρασης της βακτίνης) στο σκελετικό μυ αντίστοιχα. 3. Ενώ τα HJV και HEPH παρουσιάζουν μεγαλύτερη έκφραση των επιπέδων mRNA στο σκελετικό μύ συγκριτικά με το ήπαρ (SM/L=2,65±1,1(p<0,05) και SM/L=1,5±0,06(p<0,05 αντίστοιχα στην Q-PCR). (Εικόνα 1). Η εργασία αυτή αφορά φυσιολογικές καταστάσεις ανθρώπινων ιστών, δίνει όμως σημαντικά ερεθίσματα για αντίστοιχες μελέτες του μεταβολισμού του σιδήρου σε παθολογικές καταστάσεις. Υπογραμμίζει δε την σπουδαιότητα του σκελετικού μυϊκού ιστού και την ανάλογη συμμετοχή του στη ομοιοστασία του σιδήρου. Οι ποσοτικές διαφορές που παρατηρούνται στην έκφραση γονιδίων που εμπλέκονται σε διάφορα κυτταρικά μονοπάτια αναδεικνύουν την ανάγκη περαιτέρω έρευνας του κυτταρικού μεταβολισμού στο σκελετικό ιστό.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

O-091 Semen microbiota in patients with astenozoospermia and healthy controls: cluster analysis of real-time PCR data

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Panacheva ◽  
D Pochernikov ◽  
E Voroshilina
Keyword(s):  
Cluster Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Rate ◽  
Bacterial Load ◽  
Rrna Gene ◽  
Rt Pcr ◽  
Silhouette Index ◽  
Enterococcus Spp ◽  
Lactobacillus Spp

Abstract Study question What are the differences in the semen microbiota composition of patients with asthenozoospermia and normospermia according to cluster analysis of PCR data? Summary answer The detection rate of 4 stable semen microbiota clusters and the dominant bacteria groups varied in patients with asthenozoospermia and normospermia. What is known already Most of the research dedicated to analyzing normal and pathological semen microbiota is based on 16S rRNA gene specific Next generation sequencing (NGS). It has shown that microbiota is represented by polymicrobial communities (clusters) that consist of microorganisms from different genera and bacteria phyla. Despite it being highly informative, NGS has several weaknesses: complex sample preparation, difficult sample intake control, long analysis process, complicated results interpretation, high cost of equipment and reagents. These factors make it virtually impossible to use this approach in routine medical practice. Quantitative real-time PCR (RT-PCR) is far more suitable for this. Study design, size, duration Patients included in the study (n = 301) came to the “Garmonia” Medical Center (Yekaterinburg, Russia) either seeking preconception care or for infertility treatment. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 171) — asthenozoospermia, Group 2 (n = 130) — normospermia. Participants/materials, setting, methods Semen microbiota was analyzed using RT-PCR kit Androflor (DNA-Technology, Russia). Cluster analysis was performed for 201 samples with the total bacterial load (TBL) of at least 103 GE/ml (asthenozoospermia = 96, normospermia = 105). Cluster analysis was conducted using the k-means ++ algorithm, scikit-learn. The Silhouette index and the Davies–Bouldin index (DBI) were used to confirm the stability of clusters. Main results and the role of chance Both in the samples with normospermia and asthenozoospermia, four stable microbiota clusters were distinguished. Cluster I was characterized by the prevalence of obligate anaerobes, Lactobacillus spp. were prevalent in Cluster II, Gram-positive facultative anaerobes were prevalent in Cluster III, Enterobacteriaceae/Enterococcus spp. were prevalent in Cluster IV. Cluster I was detected the most often in both groups. However, in normospermia it was represented by various obligate anaerobes without pronounced quantitative predominance of any bacteria group. In samples with asthenozoospermia one of the bacteria groups were prevalent in Cluster I: Bacteroides spp./Porphyromonas spp./Prevotella spp., Peptostreptococcus spp./Parvimonas spp. or Eubacterium spp. In samples with asthenozoospermia Cluster II was characterized by the prevalence of Lactobacillus spp., while in samples with normospermia other bacteria groups were present along with lactobacilli, mainly obligate anaerobes. In samples with normospermia Corynebacterium spp. and Streptococcus spp., typical of normal microbiota of male UGT, were prevalent in Cluster III. In samples with asthenozoospermia Cluster III were characterized by the prevalence of Staphylococcus spp. In samples with asthenozoospermia Lactobacillus spp was present in Cluster IV along with Enterobacteriaceae/Enterococcus spp., which was not typical of the samples with normospermia. Limitations, reasons for caution Cluster analysis was not conducted for the samples with TBL lower than 103 GE/ml, since their results were incompatible with the data received for the negative control samples. Wider implications of the findings Further research could determine the detection rate of the described bacterial clusters in semen with other pathologies. Establishing the relationship between the characteristics of semen microbiota and infertility in men might allow the development of new algorithms for treating patients with reproductive disorders, depending on the composition of semen microbiota. Trial registration number not applicable

Detection of dengue virus serotypes by single-tube multiplex RT-PCR and multiplex real-time PCR assay

2021 ◽  
Author(s):  
Kundan Tandel ◽  
Mahadevan Kumar ◽  
G.S. Bhalla ◽  
S.P.S. Shergill ◽  
Vijaya Swarnim ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Tube ◽  
Dengue Virus Serotypes

scholarly journals The First Quarter of SARS-CoV-2 Testing: the University of Washington Medicine Experience

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Alexander L. Greninger ◽  
Keith R. Jerome
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Real Time Pcr ◽  
Medical Center ◽  
Rt Pcr ◽  
University Of Washington ◽  
Clinical Laboratories ◽  
To Come ◽  
The Many ◽  
The University

ABSTRACT In early March 2020, the University of Washington Medical Center clinical virology laboratory became one of the first clinical laboratories to offer testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). When we first began test development in mid-January, neither of us believed there would be more than 2 million confirmed SARS-CoV-2 infections nationwide or that we would have performed more than 150,000 real-time PCR (RT-PCR) tests, with many more to come. This article will be a chronological summary of how we rapidly validated tests for SARS-CoV-2, increased our testing capacity, and addressed the many problems that came up along the way.

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