SYBR Green Method Research Articles

Evaluation of a Real Time PCR Assay and a ELISA Method for the Detection of Walnuts and Almonds Allergen Traces in Food Products

Food allergens are a well acknowledged issue in food industry and are regulated by legislation. The presence of allergens can either origin from the raw material or due to contamination during production. Allergen information on packaging is mandatory although it cannot be accurate in the case of contamination therefore warnings are used. The purpose of the study is the development and validation of a SYBR Green Real Time Polymerase Chain Reaction method using specific primer pairs based on Jug r 1, Jug r 3, and Jug r 4 allergen-coding sequences to improve the sensitivity of Real Time Polymerase Chain Reaction techniques for detection of walnut and almond traces in commercial food products and its comparison with ELISA methodology in terms of detection ability. A total of 100 samples were collected from local markets and were analyzed by Real Time Polymerase Chain Reaction (RT-PCR) and ELISA methods. The results indicated that 16 samples (16%) were found positive in walnut traces and 18 samples (18%) were found positive in almond traces by Real Time Polymerase Chain Reaction of which Elisa identified 14 samples (14%) positive in walnut traces and 15 samples (15%) positive in almond traces. Among them, 4 samples (25%) that contained walnut traces and 6 samples (33.3%) that contained almond traces had no allergen declaration on their label. The improved accuracy of Real Time Polymerase Chain Reaction underlines the importance of this method for allergen detection and quantification in the food industry

Aberrant Expression of the miR-181b/miR-222 after Hematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia.

Recently, dysregulated expression of various micro RNAs has been reported in hematologic malignancies, especially AML disease which affects normal hematopoiesis in these patients and thereby contribute to clinical outcome of AML patients, associated with either poor or favorable prognosis. Herein, we evaluated the expression of miR-181b and miR-222 in acute myeloid leukemia patients and correlation with response to therapy after hematopoietic stem cell transplantation. Eighty newly diagnosed AML patients and 80 healthy controls were recruited. The expression of miR-181b and miR-222 was evaluated by real-time SYBR Green PCR method. miR-181b gene expression was significantly increased (4.7 fold) whereas miR-222 was decreased (18.3 fold) in AML patients compared to controls (P = 0.03 and P < 0.001, respectively). Both miR-181b and miR-222 were not associated with response to treatment (P > 0.05). Also, miR-181b and miR-222 were not differentially expressed in AML patients with M3 compared to non-M3 FAB subtypes (P > 0.05). miR-181b and miR-222 are aberrantly expressed in AML patients and their baseline level is not associated with response to treatment.

Decreased Serum miR-1296 may Serve as an Early Biomarker for the Diagnosis of Non-Alcoholic Fatty Liver Disease.

Circulating microRNA has become a candidate biomarker for many diseases. The purpose of this study was to investigate the significance of miR-1296 as a non-invasive biomarker in nonalcoholic fatty liver disease (NAFLD). Serum samples were collected from normal people and NAFLD patients for biochemical detection. Serum microRNAs were isolated by the NucleoZOL method, and the stem-loop method was used to reverse transcribe the DNA. The relative quantification of miR-1296 was performed by SYBR Green method. Spearman's method was used to analyze the correlation between miR-1296 and serum biochemical parameters. By using 2-∆∆CT method, we found that, compared with the normal control group, the expression of serum miR-1296 increased in patients with normal lipid NAFLD and those with hyperlipidemia NAFLD. At the same time, the expression of microRNA-1296 in the NAFLD hyperlipidemia group increased more significantly than that in the NFALD group with normal lipid. Spearman's correlation assay demonstrated that the correlation between the expression of miR-1296 and blood lipids, including TC, TG, HDL-c, and LDL-c, was TC (r = 0.4951, p = 0.0013), TG (r = 0.054, p = 0.6425), HDL-C (r = 0.3435, p = 0.07522), and LDL-C (r = 0.3307, p = 0.0699. The data showed that miR-1296 was positively correlated with serum TC level. In summary, serum microRNA-1296 is a more sensitive marker of NAFLD than blood lipids, which provides a new method for noninvasive early screening of NAFLD.

APOE hypermethylation is significantly associated with coronary heart disease in males

APOE is involved in exogenous and endogenous lipid metabolism and is a candidate gene for coronary heart disease (CHD). Using the SYBR-Green quantitative methylation-specific PCR method, we measured APOE methylation levels in 640 cases of CHD and 436 controls. Meanwhile, we used the Sequenom MassARRAY platform to genotype APOE rs7412 and rs7259620. In men, we found that CHD patients had significantly lower levels of APOE methylation than non-CHD controls (P = 0.044). In addition, rs7412-T and rs7259620-A were protective factors for CHD in males (rs7412-T: OR = 0.527, allele P = 0.004; rs7259620-A: OR = 0.743, allele P = 0.029). In the CHD group and the non-CHD control group, APOE methylation levels were inversely correlated with age (total patients: r = −0.015, P = 0.009, total controls: r = −0.185, P = 1E−04). In the female control group, the APOE methylation levels were inversely correlated with plasma APOA1 protein levels (female controls: r = −0.188, P = 0.008). In the total controls and female controls, APOE methylation levels were inversely correlated with plasma APOE protein levels (total controls: r = −0.110, P = 0.031, female controls: r = −0.188, P = 0.008). Our GDMR interaction analysis showed that there was an interaction between APOE methylation and rs7412 (P = 0.011). In addition, there was a significant interaction between APOE methylation, rs7259620, gender, smoking, LDL, TC, and APOE protein levels in the risk of CHD (P = 0.011). In summary, our research showed that the methylation of APOE was important for the risk of CHD in men.

Selection of Reference Genes for the Normalization of RT-qPCR Data in Gene Expression Studies in Insects: A Systematic Review.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying expression levels of targeted genes during various biological processes in numerous areas of clinical and biological research. Selection of appropriate reference genes for RT-qPCR normalization is an elementary prerequisite for reliable measurements of gene expression levels. Here, by analyzing datasets published between 2008 and 2017, we summarized the current trends in reference gene selection for insect gene expression studies that employed the most widely used SYBR Green method for RT-qPCR normalization. We curated 90 representative papers, mainly published in 2013–2017, in which a total of 78 insect species were investigated in 100 experiments. Furthermore, top five journals, top 10 frequently used reference genes, and top 10 experimental factors have been determined. The relationships between the numbers of the reference genes, experimental factors, analysis tools on the one hand and publication date (year) on the other hand was investigated by linear regression. We found that the more recently the paper was published, the more experimental factors it tended to explore, and more analysis tools it used. However, linear regression analysis did not reveal a significant correlation between the number of reference genes and the study publication date. Taken together, this meta-analysis will be of great help to researchers that plan gene expression studies in insects, especially the non-model ones, as it provides a summary of appropriate reference genes for expression studies, considers the optimal number of reference genes, and reviews the average number of experimental factors and analysis tools per study.

Differential expression of periostin, sclerostin, receptor activator of nuclear factor-κB, and receptor activator of nuclear factor-κB ligand genes in severe chronic periodontitis.

The aim of the present study was to determine and compare the expression profile of periostin (POSTN), sclerostin (SOST), receptor activator nuclear factor-κB (RANK), and RANK ligand (RANKL) genes in gingival tissue samples collected from healthy gingiva (control) and severe chronic periodontitis sites. Fifty systemically-healthy individuals was enrolled in the present case-control study. Gingival tissue samples were obtained from healthy gingiva (N=25) and sites with severe chronic periodontitis (N=25). Total RNA was isolated from all the tissues. cDNA conversion was then performed using a reverse transcription polymerase chain reaction (PCR) program. Real-time PCR and SYBR green method were used to determine the expression levels of SOST, POSTN, RANK, and RANKL genes. An elevated expression (3.5-4-fold) of SOST, RANK, and RANKL genes, with a concomitant reduced expression of the POSTN gene, was identified in severe chronic periodontitis. The intergroup difference between the mean delta cyclic threshold values showed statistical significance at P<.001. The expression profile of SOST, RANK, RANKL, and POSTN genes observed in gingival tissue samples from sites with severe chronic periodontitis and healthy gingiva suggests that the differential level of the gene expression could serve as an indicator of periodontitis progression/severity.

Anti-Plasmodium falciparum Activity of Extracts from 10 Cameroonian Medicinal Plants.

Background: In the midst of transient victories by way of insecticides against mosquitoes or drugs against malaria, the most serious form of malaria, caused by Plasmodium falciparum, continues to be a major public health problem. The emergence of drug-resistant malaria parasites facilitated by fake medications or the use of single drugs has worsened the situation, thereby emphasizing the need for a continued search for potent, safe, and affordable new antimalarial treatments. In line with this need, we have investigated the antiplasmodial activity of 66 different extracts prepared from 10 different medicinal plants that are native to Cameroon. Methods: Extracts were evaluated for their capacity to inhibit the growth of the chloroquine-sensitive (Pf3D7) and resistant (PfINDO) strains of P. falciparum using the SYBR green fluorescence method. The cytotoxicity of promising extracts against human embryonic kidney cells (HEK293T) mammalian cells was assessed by MTT assay. Results: The antiplasmodial activity (50% inhibitory concentration, IC50) of plant extracts ranged from 1.90 to >100 μg/mL against the two strains. Six extracts exhibited good activity against both Pf3D7 and PfINDO strains, including cold water, water decoction, and ethyl acetate extracts of leaves of Drypetes principum (Müll.Arg.) Hutch. (IC503D7/INDO = 4.91/6.64 μg/mL, 5.49/5.98 μg/mL, and 6.49/7.10 μg/mL respectively), water decoction extract of leaves of Terminalia catappa L. (IC503D7/INDO = 6.41/8.10 μg/mL), and water decoction extracts of leaves and bark of Terminalia mantaly H.Perrier (IC503D7/INDO = 2.49/1.90 μg/mL and 3.70/2.80 μg/mL respectively). These promising extracts showed no cytotoxicity against HEK293T up to 200 μg/mL, giving selectivity indices (SIs) in the range of >31.20–80.32. Conclusions: While providing credence to the use of D. principum, T. catappa, and T. mantaly in the traditional treatment of malaria, the results achieved set the stage for isolation and identification of active principles and ancillary molecules that may provide us with new drugs or drug combinations to fight against drug-resistant malaria.

Decreased regulatory function of CD4+CD25+CD45RA+ T cells and impaired IL-2 signalling pathway in patients with type 2 diabetes mellitus.

In this study, the frequency and function of CD4+CD25+CD45RA+ regulatory T cells (Treg) and intracellular IL-2 signalling molecules in patients with type 2 diabetes mellitus (T2DM) were investigated. Tregs and responder T cells (Tresp, CD4+CD25- T cells) were sorted and suppression assays were performed using flow cytometry. Phosphorylation of signal transducer and activator of transcription-5 (pSTAT5) were assessed using flow cytometry. Gene expression of FOXP3 was performed with the SYBR green Real Time PCR method. Production of IL-2 from cultured cells was assessed using ELISA. We observed a functional defect of CD4+CD25+CD45RA+ Tregs in T2DM patients with higher proliferation of Tresp cells, in response to anti-CD3 and anti CD28 stimulation in the presence of Tregs invitro. The results showed that the proliferation of Tresps in the absence of Treg cells was higher in T2DM patients than in healthy controls. Decreased FOXP3 mRNA expression and pSTAT5 were observed within the Tregs of the patients, whereas the level of secreted IL-2 from PBMCs culture was not statically different between T2DM patients and healthy individuals. Changes in intracellular IL-2 pathways and FOXP3 gene expression may contribute to the defect of Tregs in T2DM patients. These findings indicating that the purified CD4+CD25+CD45RA+ Treg cells have reduced functional capacity together with impaired IL-2 pathway in T2DM, and the Tregs could be used for a potential novel therapeutic target.

HULC and 7SL RNA expression levels in patients with Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by the Crimean-Congo hemorrhagic fever virus. Long non-coding RNAs (lncRNAs) are generally classified as transcripts longer than 200 nucleotides (nt). The various lncRNAs expressed in infected cells are responsible for regulating the expression of viral and host genes. This is the first study to investigate hepatocellular carcinoma upregulated long non-coding RNA (HULC) and 7SL RNA expression levels in patients with CCHF. Blood samples were taken from 100 individuals (60 patients and 40 controls), and total RNA isolation was performed. Quantitative polymerase chain reaction (qPCR) was performed using the SYBR Green method to determine HULC and 7SL RNA expression levels in the study population. Compared the patient and control groups, HULC was upregulated statistically significantly (P = 0.04) and 7SL RNA was downregulated (P = 0.93) in patients. Also, there was a statistically significant difference between fatal cases and surviving patients for HULC and 7SL RNA (P < 0.01 and P = 0.03, respectively). In addition, HULC expression was increased statistically significantly in fatal cases compared with surviving patients in terms of clinical parameters such as aspartate aminotransferase (P < 0.01), alanine aminotransferase (P < 0.01), international normalized ratio (P = 0.05), prothrombin time (P = 0.01), active partial thromboplastin time (P < 0.01), and lactate dehydrogenase (P < 0.01). These findings highlighted that HULC and 7SL RNA could be important mediators for studying the pathogenesis of CCHF and significant therapeutic targets of the disease.

Quantitative distribution of Citrus yellow mosaic badnavirus in sweet orange (Citrus sinensis) and its implication in developing disease diagnostics

Citrus yellow mosaic badnavirus (CMBV) is the etiologic agent of citrus yellow mosaic disease, which has caused serious economic losses to Indian citrus industry. CMBV is a quarantined pathogen that is geographically restricted to India. To prevent unintentional movement of the virus to other major citrus-growing countries in fruits, root stocks or grafted citrus plants and facilitate trade, a sensitive, validated diagnostic tool is needed. In the present study, we developed a SYBR Green real-time PCR-based method to detect and quantify CMBV in different tissues of infected Mosambi sweet orange (Citrus sinensis) and compared its sensitivity to conventional PCR protocols. Primers were designed to recognize a portion of the CMBV capsid protein gene. Conventional and real-time PCR were performed on several different tissues: shoot tips, leaves displaying typical CMBV symptoms, asymptomatic leaves, senescent leaves, thorns, green stems and feeder roots. The detection limit of CMBV by conventional PCR was 2.5 × 104 copies per 5 ng of total genomic DNA, while the detection limit of real-time PCR was found to be 4.6 × 102 virus copies per 5 ng of viral DNA. The viral load varied between different tissues. The highest concentration occurred in feeder roots (3.5 × 108 copies per 5 ng of total genomic DNA) and the lowest in thorns (1 × 106 copies per 5 ng of total genomic DNA). The variation in viral load within different tissues suggests movement of the virus within an infected plant that follows the path of photo-assimilates via the phloem. In symptomatic leaves, the CMBV concentration was highest in the lamella followed by midrib and petiole, suggesting that virus resides inside these sections of a leaf and side by side symptoms develop. On the other hand, in asymptomatic leaves, the petiole contained higher virus load than the lamella and midrib suggesting that the pathogen gets established from the stem through the phloem into petiole then infects the lamella and midrib. In addition to information on virus movement, the distribution of CMBV in different tissues helps with the selection of tissues with relatively higher viral load to sample for early and sensitive diagnosis of the disease, which will be useful for better management of the disease in endemic areas.

ESR1 Gene Amplification in an Iranian Population with Early Onset Invasive Ductal Breast Carcinoma

Aim: The study aimed to evaluate a modified SYBR Green qPCR method to study the inter-relations of ESR1 amplification to other parameters including ER expression status, clinicopathological features and responsiveness to tamoxifen. Materials &amp; methods: 35 breast cancer tissues were assessed for ESR1 amplification by a modified qPCR. Results: Amplification of ESR1 was observed in 31.4% out of 35 samples. ESR1 amplification and overexpression were significantly correlated (Spearman's Rho = 0.658; p &lt; 0.001). ESR1 amplification was also statistically associated with positive response to tamoxifen (p = 0.0005), lower tumor grade (p = 0.027) and lower tumor stage (p = 0.005). Conclusion: Findings showed a positive correlation between ER amplification and ER-α expression and highlighted the potential clinical value of using SYBR Green qPCR to quantify amplification of ESR1. ESR1 amplification could be used as an indicator of positive response to tamoxifen and might have a clinicopathological significance. However, the qPCR data should be confirmed by fluorescence in situ hybridization.

A rapid and sensitive assay to identify HLA-DQ2/8 risk alleles for celiac disease using real-time PCR method.

To perform a simple, rapid and sensitive Real-time PCR based SYBR Green method to determine the human leukocyte antigen (HLA)-DQ 2/8 alleles in celiac disease (CD) patients. Many molecular techniques are available to determine the HLA-DQ2 and DQ8 alleles, but they are too expensive and have many steps that make them difficult to use. To determine the HLA-DQ 2/8 alleles we have developed a new real-time PCR assay, using SYBR Green technique with melting curve analysis on genomic DNA isolated from 75 CD patients and 94 healthy controls. The specific primers to examine HLA-DQA1*05, HLA-DQB1*02 and HLA-DQB1*0302 alleles were used and results were compared with commercially available kits. Using this method, the presence of HLA-DQ2 and HLA-DQ8 alleles were determined with sensitivity and specificity 80% and 100% respectively and compared to low resolution commercially available kits, the results of this method were more efficient. The frequency of DQ2 and DQ8 in patients was 76% and 29%, respectively and overall 96% of patients were carries DQ2 and/or DQ8 alleles. The result of this study showed that Real-time PCR using SYBR Green method with melting curve analysis has good efficiency to identify the HLA-DQ2/8 risk alleles.

Low cost detection of hepatitis C virus RNA in HCV infected patients by SYBR Green I real-time PCR

The prevalence of hepatitis C virus (HCV) is highest in Egypt compared to other countries. Nucleic acid amplification test (NAT) allows detection of HCV early during the course of infection. Unfortunately, NAT is more expensive than ELISA, thus its routine use as a screening tool for blood products or in clinical practice is quite limited. The aim of this study was to compare two common RT-PCR methods, TaqMan probe technique and SYBR Green method in quantitative detection of HCV RNA for diagnosis and follow up of HCV patients. Among the recruited 220 HCV patients, 154 (70%) were HCV-RNA positive by both the techniques, while 24 (10.9%) were negative by both techniques. On the other hand, 40 (18.2%) cases were HCV RNA positive only by SYBR Green technique, and 2 (0.9%) only by TaqMan probe technique. Forty (20.4%) of the 196 chronic HCV cases were HCV-RNA positive by SYBR Green but negative by TaqMan probe technique. ConclusionThis method is useful for rapid qualitative detection of HCV infection and particularly suitable for routine diagnostic applications.

The catalase gene promoter and 5'-untranslated region variants lead to altered gene expression and enzyme activity in vitiligo.

Oxidative stress is considered to be the initial event in the course of vitiligo. The enzyme catalase (CAT) is mainly involved in cellular defence against oxidizing agents through detoxifying H2 O2 . The aims were (i) to assess erythrocyte CAT enzyme activity and lipid peroxidation (LPO) levels as well as CAT mRNA expression in skin and blood; (ii) to investigate CAT gene promoter rs7943316, rs1001179, 5'-untranslated region rs1049982, and exon (rs17886350, rs11032709, rs17880442, rs35677492) polymorphisms; and (iii) to perform genotype/haplotype-phenotype correlation analyses in patients with vitiligo and controls from Gujarat. CAT activity and LPO levels were measured spectrophotometrically. CAT mRNA levels were estimated using real-time polymerase chain reaction (PCR) by the SYBR Green method. Single-nucleotide polymorphism genotyping was performed using PCR-restriction fragment length polymorphism and amplification-refractory mutation system-PCR analyses. Patients with vitiligo showed significantly decreased CAT mRNA expression in lesional and nonlesional skin and in blood, with reduced CAT activity compared with that of controls. CAT -89A/T and -20T/C polymorphisms were significantly associated with patients, especially with active and generalized vitiligo, whereas no association was observed for -262G/A and exon polymorphisms. The A-262 T-89 C-20 haplotype with variant alleles was found to be associated with 6·4-fold risk of vitiligo. Genotype/haplotype-phenotype correlation analyses revealed that individuals with susceptible genotypes/haplotype for CAT -89A/T and -20T/C polymorphisms showed significantly decreased CAT mRNA/activity, and only -89A/T polymorphisms showed significantly increased LPO levels compared with wild-type genotypes/haplotype. The present study proposes the crucial role of CAT and its allelic variants in oxidative stress-mediated pathogenesis of vitiligo.

Cost analysis for somatic and germline SyBr Green PCR method screening for parathyroid gland; a cost analysis from Thailand

Cost analysis for somatic and germline SyBr Green PCR method screening for parathyroid gland; a cost analysis from Thailand

Effect of sub-MIC of vancomycin and clindamycin alone and in combination with ceftazidime on Clostridium difficile surface layer protein A (slpA) gene expression

Clostridium difficile (C.difficile) infection is often established in the presence of antibiotics and probably antibiotics can influence surface layer protein A (slpA) expression as a colonization factor. The aim of this study is to evaluate the effect of vancomycin (VAN), clindamycin (CLI) alone and in combination with ceftazidime (CAZ) on slpA gene expression to determine whether such antibiotics can have any effect on slpA expression. About ∼106 CFU/mL was inoculated to medium containing an appropriate concentration of antibiotics alone and in combination. After 24 and 48 h incubation under anaerobic condition, 3 mL of culture was excluded and centrifuged in 8000 × g per 3 min. The pellet was washed and used for RNA extraction. The RNA extraction, Dnase I treatment and cDNA synthesis was performed by RNA extraction, Dnase I, and cDNA synthesis kits, respectively. The real–time PCR were carried out by sybrGreen methods and data were analyzed based on comparative ΔΔCT. All antibiotics alone and in combination, except VAN/CAZ in clinical isolate, decreased the level of slpA gene expression in the 24-h incubation. While the expression profile of slpA was different in the 48-h incubation period. The VAN and CLI decreased the slpA expression, although the template of expression is closed to control medium. CAZ alone and in combination increased slpA expression. C. difficile may down-regulate slpA expression in the early stages of growth in sub-inhibitory concentration of antibiotics. But, over time C. difficile increases or over expresses the slpA expression level. Consequently C. difficile binds strongly to epithelial cells and continues to survive in the presence of sub-MIC concentration of antibiotics. This effect is observed especially with regard to CAZ and probably other third generation cephalosporins or in combination therapy with VAN or CLI, which are prescribed in the clinic. CAZ can interfere with the anti-down regulatory feature of VAN, CLI, and maybe other antibiotics.

Cyanobacteria as source of marine bioactive compounds: Molecular specific detection based on Δ9 desaturase gene

The blue-green microalga, Arthrospira sp., isolated from the sea of Kssour Essef in Mahdia (Tunisia), was purified and then identified both morphologically and genetically based on 16S rRNA gene sequence. Following physicochemical analysis, the prokaryotic microalga tested represented a competitive source of pigments and showed a considerable rate in protein (64%) which was confirmed by FTIR measurement. The lipid content (4%) was quantified by the gravimetric method and the intracellular lipid bodies were detected with the Nile red staining. Using gas chromatography coupled with flame ionization detector, the fatty acid profile revealed the presence of 27.4% and 32.88% of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs), respectively. Given the richness of the isolated microalga in unsaturated fatty acids, we have developed a SYBR Green real time PCR method for the specific identification of Arthrospira sp. Δ9 desaturase gene. This current method will be of great value for carrying out high-throughput studies like cloning, heterologous expression and structure-function relationship analysis.

Antimalarial Activities of Zea mays Leaf Extract in Ethanol and Selected Solvent Fractions

ABSTRACTZea mays leaf extract and fractions were investigated for antimalarial activity against Plasmodium berghei using a rodent malaria model and in vitro activity against chloroquine-sensitive (Pf 3D7) and -resistant (Pf INDO) strains of P. falciparum using the SYBR green assay method. Median lethal dose (LD50) and cytotoxic activities against HeLa and HEKS cells were also carried out. The most active fraction was analyzed by gas chromatography-mass spectrometry (GCMS). The leaf extract (170–510 mg.kg–1, p.o.), with LD50 of 1,732.05 mg.kg–1, demonstrated antimalarial activity against P. berghei infection in suppressive, prophylactive, and curative tests. The crude extract and the fractions also exerted prominent activity against both chloroquine-sensitive (Pf 3D7) and -resistant (Pf INDO) strains of P. falciparum, with the ethyl acetate fraction exerting the highest activity with IC50 value of 32.31 ± 2.56 μg.mL–1 (Pf 3D7) and 13.60 ± 0.57 μg.mL–1 (Pf INDO). The crude leaf extract and fractions were not cytotoxic to the two cell lines tested, with IC50 of >100 μg.mL–1 against both HeLa and HEKS cell lines.

Novel SYBR Green Real Time PCR Method for Direct Quantification and Detection of Escherichia coli from Clinical Samples

Diagnosis of sepsis in its early stages are key steps for its treatment, however because of the brutal effects of sepsis and the necessity of its immediate treatment, many physicians don’t wait for the conventional microbiological diagnosis and start prescribing antimicrobial drugs. Therefore designing an accurate, fast, reliable and economic method to quantify the total number of Escherichia coli from blood and clinical samples by quantitative SYBR green real time PCR is a key step for early diagnosis and treatment of the problem and is compatible with alternative conventional microbiological methods. The method included artificial inoculation of healthy blood with type strain E. coli used for designing a quantitative standard curve. The protocol was then applied on blood samples collected from suspected patients with sepsis, also used for clinical and environmental samples. SYBR green used as inexpensive indicator to reduce the costs while using probe based methods.

Detection by real-time PCR and pyrosequencing of the cry1Ab and cry1Ac genes introduced in genetically modified (GM) constructs

ABSTRACTThe presence of genetically modified organisms (GMOs) in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in genetically modified (GM) construction, they also constitute excellent screening elements and their use is increasing. In this paper we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time polymerase chain reaction (PCR) and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method can provide a positive signal with a larger number of GM events than do the other existing methods using double dye-probes. The method permits the analysis of results with less ambiguity than the SYBRGreen method recommended by the European Reference Laboratory (EURL) GM Food and Feed (GMFF). A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis can determine the sequence between the primers used for PCR. Pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours with a low percentage GM content and different copy numbers. Improvements in the pyrosequencing protocol provided correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random.