scholarly journals Comparison of culture and polymerase chain reaction techniques in the identification of Tannerella forsythia in periodontal health and disease, an in vitro study

2014 ◽  
Vol 18 (2) ◽  
pp. 155 ◽  
Author(s):  
PraveenKumar Bankur ◽  
Kishore Bhat ◽  
Rashmi Bankur ◽  
Reshma Naik ◽  
Aarati Nayak ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Vitro Study ◽  
Vitro Study ◽  
Chain Reaction ◽  
Periodontal Health ◽  
Tannerella Forsythia ◽  
Health And Disease ◽  
Polymerase Chain

scholarly journals Sensitivity of polymerase chain reaction for detection of known aliquots of Trypanosoma cruzi in the blood of mice: an in vitro study

2002 ◽  
Vol 35 (5) ◽  
pp. 487-490 ◽  
Author(s):  
Rozália F. Campos ◽  
Juracy B. Magalhães ◽  
Eliana A.G. Reis ◽  
Mitermayer G. Reis ◽  
Sonia G. Andrade
Keyword(s):  
Polymerase Chain Reaction ◽  
Trypanosoma Cruzi ◽  
In Vitro Study ◽  
High Sensitivity ◽  
Positive Control ◽  
Chain Reaction ◽  
Negative Results ◽  
Polymerase Chain ◽  
Positive Controls

To evaluate the sensitivity of polymerase chain reaction (PCR) to reveal known number of trypomastigote in the blood of mice, three separate experiments were done. First: To eight samples of 500mul of normal mice blood, one aliquot of 1, 2, 3, 4, 5, 10, and 50 trypomastigotes respectively, were added. Second and third: 10 aliquots with 1 and 10 with 2 trypomastigotes were added to samples of 500mul of normal mice blood. Positive control: 500mul of blood containing 100,000 trypomastigotes. For kDNA minicircles amplification by PCR the primers:S35 and S36 were used. PCR revealed products of 330 b.p in the positive controls. When only one sample with the aliquots of 1 or 2 trypomastigotes was examined, results were negative; results were positive with aliquots of 3 to 50 trypomastigotes. In the 2nd and 3rd experiments, 9/10 aliquots with one parasite and 9/10 with 2 trypomastigotes were positive revealing a high sensitivity of this reaction. In conclusion, the presence of one single parasite in 500mul of blood, is enough for a positive PCR. This method could be used as a complement to the various parasitological cure tests in treated mice, when low volumes of blood are individually examined.

Εξέλιξη και βελτίωση της τεχνικής βιοψίας βλαστοκύστεων για μοριακές αναλύσεις και εκτίμηση της επιτυχίας εμφύτευσης και εγκυμοσύνης κατά την εξωσωματική γονιμοποίηση σε σχέση με το μοριακό υπόστρωμα

2013 ◽  
Author(s):  
Γεωργία Κόκκαλη
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Clinical Practice ◽  
Expression Profiles ◽  
Chain Reaction ◽  
Trophectoderm Cells ◽  
Monogenic Diseases ◽  
Blastocyst Biopsy ◽  
Polymerase Chain

IntroductionOne of the most difficult aspects in assisted reproductive technology (ART) is the selection of asuitable embryo for transfer to the patient’s uterus, in order to achieve implantation anddevelopment to term. This study was based on the hypothesis that preimplantation embryosmay have different gene expression profiles that characterize their ability to implant in theuterus and develop to a healthy baby at term.The main aim of this study was to investigate molecular markers associated with developmentalcompetence and successful implantation in ART. The primary aim of the study was to developand optimize a blastocyst biopsy method, suitable for application in clinical practice. Thesecondary aim of the study was to investigate the gene expression of beta Human ChorionicGonadotropin (CGβ) in blastocysts and correlate it with their morphology. Previously to thecurrent study, blastocyst biopsy was not implemented in clinical practice and no prior researchon the existence, quantification and standardization of transcripts of CGβ has been performedin blastocysts.MethodologyThe methodology for trophectoderm cell biopsy from blastocysts was developed and optimizedprimary to be a safe technique for the embryo and secondary to ensure biopsy of a sufficientnumber of cells, in order to allow the application of multiple molecular analyses. The blastocystbiopsy method involved three steps: A., opening of a hole in the zona pellucida using lowfrequency laser, B., blastocyst culture to allow trophectoderm cells to herniate from the holeand C., trophectoderm cell dissection of the blastocyst mass by laser ablation.The methodology for the investigation of CGβ gene expression in blastocysts, included RNAisolation, cDNA synthesis, amplification and quantification of CGβ transcripts. Because CGβ isencoded by a cluster of homologous genes (CGβ1, CGβ2, CGβ3, CGβ5, CGβ7, CGβ8),methodology was designed considering the homology between them into groups (A: CGβ1,CGβ2 and B: CGβ3, CGβ5, CGβ7, CGβ8). For group A, real time polymerase chain reaction (RealTime PCR, RT-PCR) was applied and then transcripts were identified using restriction enzymedigestion. For group B, nested polymerase chain reaction (Nested-PCR) was used incombination with polymerase chain reaction temperature decreasing hybridization (Touch-downPCR). Following amplification, the products were sequenced (DNA sequencing) for theiridentification.ResultsThe biopsy technique did not appear to impact on the blastocyst’s ability to reform a blastocoelecavity and continue to grow and hatch from the zona pellucida, as it was shown followingfurther in vitro culture. No blastocyst showed signs of morphological damage at the lightmicroscopic level. Blastocyst biopsy was applied in clinical practice in two steps: A., 49 couples undergoing IVF had a biopsy in 153 blastocysts. The implantation rate per blastocysttransferred was 34.3% and lead to 23 full-term pregnancies (46.9%) with 37 babies born. B.,24 couples undergoing IVF for PGD of monogenic diseases had biopsy in 144 blastocysts. Thediagnosis success rate was 93%, the implantation rate per blastocyst transferred was 40% andlead to 11 full-term pregnancies (50%) with 15 term newborns. Then, a randomized pilot studywas conducted with the aim to evaluate and compare the diagnosis and implantation successrates between patients undergoing blastomere biopsy and blastocyst transfer and those havingtrophectoderm biopsy and blastocyst transfer for the diagnosis of monogenic diseases. Theresults showed that the diagnosis success rate was superior in the blastocyst biopsy group,while implantation and pregnancy rates were not statistically significant between the twogroups.For the study of CGβ expression profiles 45 blastocysts were donated to research, of which 39generated trophectoderm cells cDNA libraries. RT-PCR revealed the presence of CGB3, CGB5,CGB7, CGB8 transcripts in 5 blastocysts. The transcripts CGB5, CGB7, CGB8 were expressed inone hatched and one hatching blastocysts (fair morphology on day 7 post insemination) and thetranscript CGβ3 was expressed in three hatched blastocysts (excellent morphology on day 5/6post insemination). The transcript CGβ1 was identified in one only blastocyst. Four blastocystswere biopsied in order to investigate whether CGβ expression can be detected at the minimallevel of few trophectoderm cells. No transcript was found in trophectoderm cell samples orbiopsied blastocyst proper.DiscussionIn recent years, many new technologies have been introduced in clinical practice of ART.Blastocyst biopsy since its first announcement in 2005, until today, has been adopted andintegrated into the application of preimplantation genetic diagnosis (Kokkali et al., 2005). Asblastocyst biopsy has the advantage of providing adequate number of cells for multipleanalyses, it has been lately used for the PGD for monogenic diseases in combination withhistocompatibility screening (HLA matching) or PGD for monogenic diseases screening forstructural or numerical chromosomal abnormalities. Besides its clinical application, blastocystbiopsy offers great opportunities for research, such as the study for the expression ofpreimplantation genetic profiles for the identification of the single most viable blastocyst amongthe cohort developing in vitro that will enable single blastocyst transfers without a concomitantreduction in pregnancy rates.In this study, we investigated whether the β HCG may be used as a predictive marker ofdevelopmental competence for human embryos. This study showed that CGβ gene expressionwas diverse and heterogeneous between blastocysts. Further studies need to be accomplishedto investigate this further.ConclusionsBlastocyst biopsy was developed and optimized to serve as powerful tool for diagnostics ofhuman diseases or to identify diagnostic markers of competence to develop to term for humanembryos.

scholarly journals Η χρήση του πεπτιδίου Ec της ισόμορφης IGF-1Ec στην επισκευή χόνδρινων βλαβών

2018 ◽  
Author(s):  
Νικόλαος Αρμακόλας
Keyword(s):  
Polymerase Chain Reaction ◽  
Trypan Blue ◽  
Quantitative Polymerase Chain Reaction ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tgf Β1 ◽  
Invasion Assays

Το πεπτίδιο Ec (PEc) του IGF-1Ec (IGF-1Ec) επάγει την κινητοποίηση των ανθρωπίνων μεσεγχυματικών βλαστικών κυττάρων (hMSC) και ενεργοποιεί την εξωκυτταρική κινάση 1 και 2 (ERK 1/2) διαφόρων κυττάρων. Σκοπός της παρούσας μελέτης ήταν η διερεύνηση της επιδρασης του PEc στην κινητοποίηση και τη διαφοροποίηση των hMSCs, καθώς και η δυνατότητα εφαρμογής του σε συνδυασμό με τον TGF-β1 (TGF-β1) στην επιδιόρθωση του αρθρικού χόνδρου. Τα αποτελέσματα της εξωγενούς χορήγησης του ΡΕc και του ΤGF-β1, ξεχωριστά και σε συνδυασμό, σε hMSCs εκτιμήθηκαν χρησιμοποιώντας trypan blue assay, reverse transcription-quantitative polymerase chain reaction, western blot analysis, Alcian blue staining, wound healing assays και migration/invasion assays. Προσδιορίστηκε ότι το PEc εμπλέκεται στη διαδικασία διαφοροποίησης των hMSCs προς υαλώδη χόνδρο. Η χορήγηση PEc ή / και TGF-β1 σε hMSCs έδειξε συγκρίσιμη εναπόθεση χονδρικής θεμέλειας ουσίας. Ακόμα, η χορήγηση του ΡΕc σε συνδυασμό με τον ΤGF-β1 συσχετίστηκε με μια σημαντική αύξηση στην κινητοποίηση των hMSC σε σύγκριση με την χορήγηση μόνο του TGF-β1 ή του ΡEc (Ρ <0,05). Επομένως, το ΡΕc φαίνεται να διευκολύνει in vitro την κινητοποίηση των hMSC και την διαφοροποίηση τους προς χονδροκύτταρα, ενισχύοντας το ρόλο του ΤGF-β1.

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