O objetivo do estudo foi avaliar, o efeito do laser de Diodo 808nm como coadjuvante ao tratamento periodontal na redução de periodontopatógenos Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia pela técnica da Reação em Cadeia da Polimerase em Tempo Real. Foram selecionados vinte e quatro pacientes portadores de periodontite crônica neste estudo de boca dividida, duplo cego e randomizado. Dois sítios uniradiculares de cada paciente foram utilizados e divididos em dois grupos experimentais: TESTE - raspagem alisamento polimento corono radicular (RAPCR) associado à duas aplicações de laser de Diodo de alta potência (comprimento de onda de 808nm, 1,5 Watts, 597,1 W/cm2, durante 20 segundos no modo contínuo). A primeira aplicação foi realizada 24 horas após RAPCR e a segunda após sete dias; CONTROLE foi realizado o mesmo procedimento porém sem a aplicação do laser. O biofilme subgengival foi coletado antes do tratamento e seis semanas após a segunda aplicação do laser. A avaliação microbiológica foi feita através da Reação em Cadeia da Polimerase em tempo real para a quantificação de Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia. A comparação entre os grupos não demonstram diferenças estatisticamente significantes (p<0,05). Concluiu-se que, dentro dos limites deste estudo, a aplicação do laser de Diodo de 808nm como coadjuvante ao tratamento periodontal não reduziu de forma significativa os periodontopatógenos: Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia quando comparado à RAPCR. / The aim of this study was to evaluate, by real-time polymerase chain reaction, the effect of Diode laser 808nm as an adjuvant in the reduction of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia in to periodontal treatment. Twenty-four patients with chronic periodontitis the study designs was split-mouth, double blind and ramdomized controlled trial. Two sites from uniradicular teeth of each patient were used and divided in two experimental groups: TEST scalling and root planing (SRP) associated with two high power Diode laser application (wavelength of 808nm, 1,5W at the display (597,1 W/cm2) for 20 seconds in the continuous-wave mode) the first laser application was 24 hours after SRP and the second seven days later; CONTROL a similar procedure without laser application. The subgingival biofilm was colleted before treatment and six weeks after the second laser application. The microbiologic evaluation was done by real-time polymerase chain reaction for the quantification of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia. The comparison between the groups did not show significant differences (p<0,05). Within the limits of this study, it can be concluded that Diode laser 808nm application as an adjuvant in the periodontal treatment did not reduce the periodontal pathogens Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia when compared by scaling root planning.

Introduktion: Polymerase chain reaction (PCR) är en biokemisk och molekylärbiologisk laboratorieteknik som används för in vitro amplifiering av specifika gensekvenser. Det finns olika varianter av PCR, en mer utvecklad version är Realtids-PCR, även benämndkvantitativ PCR (qPCR). qPCR mäter fluorescensintensitet i varje qPCR cykel. Metoden delas in i fyra huvudfaser: linjär-, tidig exponentiell-, exponentiell- och platåfas.   Syfte: Syftet med projektet var att utvärdera prestanda hos Quantstudio 7 vid varierande reaktionsvolym och plattposition, samt vid singleplex och duplex, för att öka kvaliteten på resultat och göra metoden mer kostnadseffektiv.   Material &amp; metod: Syntetisk DNA-sekvens (gBlock) späddes och sattes upp i en standardkurva med sju punkter och användes för 20 µl respektive 10 µl reaktionsvolymen, varje punkt bestod av 4 replikat. För att utvärdera Duplex vs Singelplex förbereddes standardkurva i kombination med en konstant koncentration av en annan assay. För att undersöka intra-plate variation sattes upp identiska reaktioner i samtliga brunnar i PCR-plattan.    Resultat: Samtliga experiment gav detekterbara ampliferingsprodukter.  Cq-värdet användes för att beräkna medelvärde och standardavvikelse, samt effektivitet och R2-värde   Slutsats: Resultatet som erhålls från QS instrumentet visade att reaktionsvolymerna 10 µl och 20 µl är jämförbara. Duplex experimentet visade att gener med låg genuttryck kan duplexas med gener som har 10 000x högre genuttryck. Resultatet från intra-plate variation visade att variationen i SD var högre i den högre sidan av PCR-plattan. / Introduction: Polymerase chain reaction (PCR) is a biochemical and molecular laboratory technique that is used for amplification of specific gene sequences. There are different variants of PCR. A more developed version is quantitative PCR (qPCR). In qPCR the fluorescence intensity is measured in realtime during each qPCR cycle.    Aim: The purpose of the project is to evaluate whether the reaction volume can be reduced by half, which leads to using less material and thus make the method more cost-effective.   Matherial &amp; method: Synthetic DNA sequence (gBlock) was diluted and set up in a standard curve with seven standards and used for 20 μl and 10 μl reaction volume, respectively. Each standard consisted of 4 replicates. To evaluate Duplex vs Singelplex, standard curve was prepared in combination with a constant concentration of another assay. To investigate intra-plate variation, identical reactions were set up in all wells of the PCR-plate.   Results: All experiments yielded detectable amplification products. The Cq value was used to calculate the mean and standard deviation, as well as the efficiency and R2 value   Conclusion:  The obtained results showed that the reaction volumes 10 and 20 µl are comparable. In duplex assay, genes with low gene expression can be analyzed with genes that have 10,000x higher gene expression. In intraplate-assay variation, the variation in the standard deviation increased in the right side of PCR-plate.

På grund av planerat tillverkningsstopp av instrumenten som används idag på Octapharma AB, så syftar detta projekt till nyutvecklingen av en kvantitativ PCR analysmetod för detektion av HIV-1 i human blodplasma, genom TaqMankemi. Projektet inkluderade design, testning och utvärdering av olika set av primer och probe sekvenser. För att säkerställa specificiteten hos metoden designades primrarna och proberna för att vara komplementära till olika konservativa regioner av HIV-1:s genom. Primer och probe set:en (P/P) testades både individuellt och kombinerat i spädningsserier och genotypspaneler. Analyserna visade att det inte fanns någon korrelation mellan felmatchning av P/P och referenssekvenser hos subtyper, och detektionsnivå. När P/P testades i kombination hittades falsk-positiva signaler i negativa kontroller (4 falsk-positiva signaler; n= 106). Detta åtgärdades genom att utesluta specifika prober(0 falsk-positiva signaler; n= 152)(p = 0,030, Fisher’s exact test). Kombinationen av primer och prober, med någraprober uteslutna, hade en högre detektionsnivå för HIV-1 subtyper än de individuella set:en (29 positiva prover vs 23.5; n= 64), och lyckadesäven detektera åtminstone ett positivt prov hos varje subtyp A, B, C, D, AE, F, G och H. Avsaknaden av korrelation mellan felmatchning av P/P och detektionsnivå, visar på att orsaken av den suboptimala detektionsnivån av subtyper var inte på grund av antalet felmatchningar mellan primer och probe set:en till målsekvenserna. Den högre specificiteten vid kombination av P/P indikerar även att primrar och prober som riktar in sig på olika regioner avHIV-1 genomet ytterligare ökar specificiteten och detektionsnivån hos metoden. / As a result of future instrument discontinuation by Octapharma AB’s manufacturers, this project sought to develop a new quantitative PCR analysis method for the detection of HIV-1 in human blood plasma using TaqMan chemistry. The project included the design, testing and statistical analysis of different sets of novel primer and probe sequences. To ensure specificity, the primer and probe sequences were designed to target conserved regions of the HIV-1 genome. The primer and probe sets were tested both individually and in combination in dilution series and genotype tests. Linear regression analyses showed no correlation between mismatches between the primer and probe sets and the subtype reference sequences tested, and detection rate. When the primer and probe setswere tested in combination, false positive signals were obtained in negative control samples (4 false positive signals; n= 106). However, this obstacle was overcome by the omission of certain probes, resulting in no false positive signals (0 false positive signals; n= 152)(p = 0.030, Fisher’s exact test).The combination of primer and probe sets, with certain probes omitted, had an increased HIV-1 subtype detection rate compared to the individual P/P (29 positive samples detected versus 23.5; n= 64), and were also able to detect at least one positive sample from each of the HIV-1 subtypes A, B, C, D, AE, F, G, and H. The absence of correlation between primer and probe set mismatch and detection rate, suggests that the cause of the suboptimal detection rate of the subtypes was not the result of primer and probe set mismatch to the target sequences. The increased subtype detection rate upon combining the P/P also indicates that targeting different region of the HIV-1 genome further improves the detection rate and specificity of the method.

MicroRNAs (miRNAs) are a family of small non-coding RNAs that regulate gene expression at both the mRNA and protein levels. MiRNAs have been shown to affect neuronal differentiation, synaptosomal complex localization and synapse plasticity, all functions thought to be disrupted in schizophrenia. We investigated the expression of 667 miRNAs (miRBase v.13) in the prefrontal cortex of individuals with schizophrenia (SZ, N = 35) and bipolar disorder (BP, N =35) using a real-time PCR-based Taqman Low Density Array (TLDA). After extensive QC steps, 441 miRNAs were included in the final analyses. At a FDR of 10%, 22 miRNAs were identified as differentially expressed between cases and controls, 7 dysregulated in SZ and 15 in BP. Using in silico target gene prediction programs, the 22miRNAs were found to target brain-specific genes contained within networks overrepresented for neurodevelopment, behavior, and SZ and BP disease development. Given that miRNAs can bind to their targets with imperfect complementarity, computational prediction of true miRNA:mRNA interactions has been difficult and therefore, functional validation of miRNA:mRNA interactions has been relatively sparse. Thus, it was the goal of this study to demonstrate biological functionality of miRNAs on their targets by evaluating transcriptional and translational levels of gene expression(real-time PCR, western blot) as well as determining miRNA target-site specificity (luciferase reporter gene assays). We investigated two miRNAs, miR-132 and miR-137, both of which have been shown to regulate neuronal function and development, and are believed to be associated with schizophrenia from two distinct avenues of research, miR-132 from expression studies and miR-137 from genetic studies. We demonstrated miR-132 down-regulates NTF3, DISC1, and GRIK5 at the transcript level and down-regulates GRIK5 at the protein level as well. Furthermore, we demonstrated miR-137 down-regulates TCF4, CACNA1C, CDK6, ANK3, and ZNF804A at the transcript level, and down-regulates TCF4, CACNA1C, and CDK6 at the protein level. Going further, we also demonstrated miR-137 binds specifically to target sites in the 3'-UTR of CACNA1C, TCF4, and CDK6, suggesting repression of these genes is directly mediated by miR-137. In total, this study provides strong evidence that miRNA dysregulation may contribute to schizophrenia pathogenesis.

no / Yellow leaf syndrome (YLS) of sugarcane has been associated with Sugarcane yellow leaf virus (ScYLV) and has been reported from most sugarcane growing countries around the world. As sugarcane is vegetatively propagated, it is important to use effective and sensitive detection methods to screen new propagating material. Virus detection in symptomatic tissue is currently achieved using enzyme linked immunosorbent assay (ELISA), tissue blot immunoassay (TBIA) or a conventional RT-PCR based assay. This paper reports the development of an improved assay based on multiplex real-time fluorescent RT-PCR. The new assay is 100-fold more sensitive than conventional RT-PCR, and incorporates a novel `RNA specific¿ internal positive control (based around the intron of the caffeic acid 3-o-methyltransferase gene) to guard against false negative results. The paper also describes the comparison of eight RNA extraction methods for sugarcane tissue giving a number of alternatives for different laboratory situations. The sensitivity of this assay has allowed the detection of ScYLV in many samples that were thought to be healthy following conventional testing (RT-PCR, ELISA or TBIA). The detection of ScYLV using this TaqMan assay can be applied to the production of ScYLV-free plants and prevents its spread through the propagation material.

Die invasive Aspergillose (IA) wird mehr und mehr zu einem lebensbedrohlichen Problem bei immunsupprimierten Patienten. Der Hauptverursacher der IA ist der Aspergillus fumigatus. Es existieren verschiedene DNS-Extraktions-Methoden und PCR Assays, um Aspergillus fumigatus DNS in Bronchiallavagen (BAL) nachzuweisen. Häufig sind diese Methoden langwierig und verdeutlichen den Bedarf an einer klinisch relevanten Methode, mit der der Aspergillus-fumigatus-DNS Nachweis schnell und zuverlässig erbracht werden kann. Wir haben eine schnelle Methode entwickelt, mit der wir quantitative Ergebnisse innerhalb sechs Stunden erhalten können. Es wurde ein Extraktionsverfahren auf mechanischer Basis (FastPrep), in Kombination mit einer real-time-PCR, basierend auf der TaqMan- Technologie, etabliert. Dazu wurde eine Aspergillus-fumigatus-spezifischeSonde entwickelt und ein Aspergillus-fumigatus-spezifisches Primerpaar eingesetzt. Durch den Einsatz einer Standardreihe wurden die Ergebnisse quantifiziert. Es wurden hauptsächlich Bronchiallavagen sowie andere Materialien von 204 Patienten untersucht. Die Patienten wurden hinsichtlich ihres Verdachtes an einer IA erkrankt zu sein, anhand bestimmter Kriterien (EORTC) in bestimmte Gruppen eingeteilt. Die Ergebnisse wurden innerhalb der einzelnen Gruppen analysiert. Bei allen 8 Patienten mit bewiesener IA gelang der Nachweis von Aspergillus-fumigatus-DNS. In dieser Gruppe konnten quantitativ die höchsten Werte gemessen werden. Des Weiteren konnte eine Rückläufigkeit der Werte mit abnehmendem Aspergillose-Verdacht, sowie nach erfolgter antimykotischer Therapie beobachtet werden. Die diagnostische Sensitivität der Methode beträgt 81,3%, die Spezifität liegt bei 93,7%. Der positive prädiktive Wert ist 72,2%, der negative prädiktive Wert beträgt 96,1%. Als zusätzlicher Baustein in einer umfangreichen Gesamtdiagnostik, kann diese Methode einen Beitrag zur Verbesserung molekularer Nachweisverfahren leisten. / Invasive aspergillosis (IA), often caused by Aspergillus fumigatus, is an important cause of death of immunocompromised patients. Several DNA-extraction methods and PCR assays are available for detecting Aspergillus fumigatus DNA in bronchoalveolar lavage (BAL) samples of patients with invasive aspergillosis. These methods are often time consuming and emphasize the need to develop a clinical relevant rapid DNA isolation assay that gives reliable results in a short time. We have developed a new and rapid method which yields results within six hours.This was achieved by combining high-speed cell disruption using a mechanical extraction procedure (FastPrep), with a real-time PCR assay based on TaqMan technology.A newly designed Aspergillus-fumigatus-specific probe and Aspergillus-fumigatus-specific primers were established. This combination also produces quantitative results by comparing the results with a DNA serial dilution used in the real-time PCR. BAL fluids and other material from 204 patients were analyzed. According to the risk for an IA, the patients were devided in different groups, using specific criteria published by the EORTC. The results were related to these groups. For all 8 patients with proven IA, the detection of Aspergillus-fumigatus-DNA succeeded. In this group, the highest amount of Aspergillus-fumigatus-DNA was detected. A decraesing quantitiy of Aspergillus-fumigatus-DNA could be detected after antifungal treatment and connected with the decreasing suspicion of an IA in the other groups. The diagnostic sensitivity of this method is 81,3%, the specifity is 93,7%. The positive predictive value is 72,2%, the negative predicitive value 96,1%. Adjunctive use of these method may be helpful in the diagnosis of IA.

Le thon obèse (Thunnus obesus), le thon alabore (Thunnus albacares) et le listao (Katsuwonus pelamis) comptent parmi les espèces de thons les plus utilisées en conserve. Lors de la fabrication de conserves de thon, la substitution d'espèce et/ou le mélange de différentes espèces de thon sont interdits par la réglementation européenne. L'authentification des espèces de thon reste complexe à cause du degré de similitude élevé entre les espèces de thon, ou encore, lorsque les caractéristiques morphologiques externes sont éliminées au cours du filetage et lors de la mise en conserve. Par conséquent, des substitutions involontaires ou frauduleuses peuvent se produire. Dans cette étude, le marqueur mitochondrial du gène de la sous-unité 2 de la NADH déshydrogénase a été utilisé pour identifier le thon obèse et le gène de la sous-unité II de la cytochrome c oxydase a été utilisé pour identifier le thon albacore et le listao en utilisant la PCR en temps réel basée sur la technologie TaqMan. Deux méthodes différentes basées sur la qPCR ont été développées pour quantifier le pourcentage de chair de chaque espèce présente au sein d'une boîte de thon. La première a été basée sur la quantification absolue avec standard externe réalisée avec les deux marqueurs. La seconde a été basée sur la quantification relative avec standard externe avec le gène endogène de l'ARN 12S. Sur la base de ces résultats, nous pouvons conclure que notre méthode peut s'appliquer pour quantifier les deux espèces de thon albacore et obèse génétiquement très proches lorsqu'elles sont utilisées dans un mélange binaire en conserve. / Bigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus albacares) and skipjack tuna (Katsuwonus pelanis) are among the most widely used tuna species for canning purposes. Not only substitution but also mixing of tuna species is prohibited by the European regulation for canned tuna products. However, it can be difficult to authenticate the tuna species, due to their high degree of similarity or even when the external morphological characteristics are removed due to filleting before canning. Consequently, involuntary or fraudulent substitutions may occur during the canning process. In this study, the mitochondrial marker from NADH dehydrogenase subunit 2 gene was used to identify bigeye tuna and the mitochondrial marker cytochrome c oxidase subunit II gene was used to identify yellowfin tuna and skipjack tuna, utilizing TaqMan qPCR methodology. Two different qPCR-based methods were developed to quantify the percentage of flesh of each species used for can processing. The first one was based on absolute quantification using standard curves realized with these two markers ; the second one was founded on relative quantification with the universal 12S rRNA gene as the endogenous gene. On the basis of our results, we conclude that our methodology could be applied to authenticate the two closely related tuna species (bigeye tuna and yellowfin tuna) when used in a binary mix in tuna cans.

Endometrial adenocarcinoma (EAC) is the most frequently diagnosed gynecological cancer of the female genital tract in the Western world. Research studies in EC is difficult to conduct on human tumor samples due to the complex nature of tumor arousal and genetic heterogeneousness in the human population. Therefore, inbred animal models can be promising tools to use in EC research due to similar histopathology and pathogenesis as humans. Studies performed on MAP2K1 and CCND1 has shown that their altered expression play a crucial role in carcinogenesis. CCND1 has been demonstrated to have oncogenic properties when overexpressed in human neoplasias. The aim of this study is to investigate gene expression levels of MAP2K1 and CCND1 in BDII rat model of endometrial adenocarcinoma cells. Quantitative real-time PCR was used to analyze expression levels of MAP2K1 and CCND1 genes in BDII/Han rat model of endometrial cancer cells using TaqMan approach. The differences in gene expression levels of MAP2K1 and CCND1 between pathologically EAC malignant and nonmalignant cells showed an upregulation of MAP2K1 and CCND1 in EAC malignant cells. The analyzed data presented observable mean differences between MAP2K1 and CCND1 in several endometrial cell lines that were examined. Although no statistical significance was reached, an alteration in gene expression levels in malignant and nonmalignant endometrial cells could be observed. Furthermore, this present study shows observable upregulation of MAP2K1 and CCND1 in endometrial carcinoma cells vs. nonmalignant endometrium cells and encourages further investigation of the role of CCND1 and MAP2K genes in endometrial carcinogenesis.

Acute myeloid leukemia (AML) the most common acute leukemia in adults is characterized by various cytogenetic and molecular abnormalities. However, the genetic etiology of the disease is not yet fully understood. MicroRNAs (miRNAs) are small single- stranded noncoding RNAs that are negative regulators of gene expression. miRNAs influence processes of proliferation, differentiation and apoptosis. Deregulation of miRNAs expression can contribute to human disease. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as breast cancer, colorectal cancer and lung cancer. However, defining a plasma miRNA signature in AML that could serve as a biomarker for diagnosis has been conducted only once. We studied miRNA expression in plasma of 8 AML patients in first detection of the disease and repeatedly after achieving remission using TaqMan miRNA microarray for 750 human miRNA. The plasma expression level of 25 miRNA was down-regulated whilst that of 20 miRNA was up-regulated in the AML group at diagnosis when compared to healthy controls. The plasma expression level of 21 miRNA was down-regulated whilst that of 13 miRNA was up-regulated in the AML group in remission compared to healthy controls. Keywords acute myeloid leukemia (AML), biomarker, microRNA (miRNA), plasma, TaqMan Low...

Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. To date, no real-time, multiplex PCR assay has been developed that can detect and differentiate between B. pseudomallei, B. mallei, and B. thailandensis in a single tube format. Here, we describe the development of such an assay that detects and differentiates the species of the B. pseudomallei complex. A real-time quadruplex qPCR assay, Bcom, was designed to target unique genomic regions of B. pseudomallei, B. mallei, B. thailandensis, and the B. pseudomallei complex that detects and differentiates the three species. A total of 299 isolates within the B. pseudomallei complex was evaluated in this study, as well as 15 near-neighbors and other bacterial species. The results showed that this quadruplex assay was capable of detecting the respective species in a given sample at a sensitivity between 288 fg and 277 pg of genomic DNA. The B. pseudomallei- and B. pseudomallei complex-specific assays tested negative on two presumed B. pseudomallei isolates. In addition, a third presumed B. pseudomallei isolate tested negative by the B. pseudomallei-specific test, but was detected by the B. thailandensis and B. pseudomallei complex-specific assays. After cultural and biochemical characterization, 16s rRNA sequencing, and multiple loci sequencing, it is proposed that B. pseudomallei 34 is B. thailandensis 82172 (Accession No. DQ388536), B. pseudomallei Darwin 175 is Elizabethkingia meningoseptica, and B. pseudomallei 135 is a new strain of B. ubonensis 135.