Detection of Neuroblastoma Cells During Clinical Follow Up: Advanced Flow Cytometry and RT-PCR for Tyrosine Hydroxylase Using Both Conventional and Real-Time PCR

2011 ◽  
Vol 223 (06) ◽  
pp. 326-331 ◽  
Author(s):  
R. Esser ◽  
W. Glienke ◽  
K. Bochennek ◽  
S. Erben ◽  
A. Quaiser ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tyrosine Hydroxylase ◽  
Real Time ◽  
Real Time Pcr ◽  
Neuroblastoma Cells ◽  
Rt Pcr

The use and correlation with flow cytometry of fluorescent molecular beacons in real time PCR of IgH gene rearrangements for evaluation of minimal residual disease in multiple myeloma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 17552-17552
Author(s):  
O. Kara ◽  
A. Yigin ◽  
B. Sahin ◽  
S. Paydas
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Real Time ◽  
Minimal Residual Disease ◽  
Real Time Pcr ◽  
Residual Disease ◽  
Pcr Detection ◽  
Molecular Beacons ◽  
Rt Pcr ◽  
Minimal Residual

17552 Background: At present, the prognostic value of the amount of residual tumor cells in PB, BM or stem cell harvests and its changes over time is stil not clear. Also the advent of new therapeutic approaches to multiple myeloma made necessary the introduction of novel methods for detection of minimal residual disease. Among others approaches residual disease can be detected by using flow cytometry. The aim of the present study was to evaluate a real time PCR test for the IgH gene using alellespecific molecular beacons as fluorescence probes to quantify residual disease and also correlate flow cytometric detection of plasma cells in MM patients during followup after treatment with high dose of chemotherapy or standart chemotherapy. Methods: After clinical diagnosis of 17 MM patients, the CDR1, CDR2 and CDR3 regions of the IgH gene were analysed and sequenced to identify its clonal nature. Unique sequences of the clonal IgH rearrangement were used to design specific molecular beacon probes for each MM patient. We have also examined the co-expression of CD19, CD38, CD45, CD56, and CD138 molecules in cells of bone marrow aspirates in patients with multiple myeloma by flow cytometry. Results: The active disease had been accepted of whom plasma cell infiltration ratio was over 10% in bone marrow and also of whom labeled by CD38 and CD138 by FCM. The detection of the MRD was positive in 13 patients by RT-PCR, respectively. The infiltration ratio was correlated with CD138 expression (p = 0.009) and RT-PCR detection of plasma cells (p = 0.006) and also significant correlation had been found between RT-PCR detection and CD138 expression respectively. No any correlaton was found between other surface antigens (CD38, CD45, CD56). Conclusion: Our results indicated that real time PCR with specific molecular beacons provides a feasible, accurate and reproducible method for the determination of minimal residual disease in MM. By FCM only CD138 expression may have been used as disease marker in addition of the RT-PCR detection. No significant financial relationships to disclose.

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 Necrotrophic Growth of Legionella pneumophila

2006 ◽  
Vol 72 (6) ◽  
pp. 4323-4328 ◽  
Author(s):  
R. Temmerman ◽  
H. Vervaeren ◽  
B. Noseda ◽  
N. Boon ◽  
W. Verstraete
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Legionella Pneumophila ◽  
Real Time Pcr ◽  
Water Systems ◽  
Saccharomyces Boulardii ◽  
Wall Structure ◽  
Lower Growth ◽  
Average Growth ◽  
Microbial Cells

ABSTRACT This study examined whether Legionella pneumophila is able to thrive on heat-killed microbial cells (necrotrophy) present in biofilms or heat-treated water systems. Quantification by means of plate counting, real-time PCR, and flow cytometry demonstrated necrotrophic growth of L. pneumophila in water after 96 h, when at least 100 dead cells are available to one L. pneumophila cell. Compared to the starting concentration of L. pneumophila, the maximum observed necrotrophic growth was 1.89 log units for real-time PCR and 1.49 log units for plate counting. The average growth was 1.57 � 0.32 log units (n = 5) for real-time PCR and 1.14 � 0.35 log units (n = 5) for plate counting. Viability staining and flow cytometry showed that the fraction of living cells in the L. pneumophila population rose from the initial 54% to 82% after 96 h. Growth was measured on heat-killed Pseudomonas putida, Escherichia coli, Acanthamoeba castellanii, Saccharomyces boulardii, and a biofilm sample. Gram-positive organisms did not result in significant growth of L. pneumophila, probably due to their robust cell wall structure. Although necrotrophy showed lower growth yields compared to replication within protozoan hosts, these findings indicate that it may be of major importance in the environmental persistence of L. pneumophila. Techniques aimed at the elimination of protozoa or biofilm from water systems will not necessarily result in a subsequent removal of L. pneumophila unless the formation of dead microbial cells is minimized.

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 Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested Real-Time RT-PCR Assays for SARS-CoV-2 Detection

2020 ◽  
Vol 21 (16) ◽  
pp. 5674
Author(s):  
Cyril Chik-Yan Yip ◽  
Siddharth Sridhar ◽  
Kit-Hang Leung ◽  
Anthony Chin-Ki Ng ◽  
Kwok-Hung Chan ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Rt Pcr ◽  
Single Tube ◽  
Highly Sensitive ◽  
Testing Algorithms ◽  
Pcr Assays ◽  
Human Coronaviruses ◽  
Pooled Sample

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.

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