Multiplex touchdown PCR assay to enhance specificity and sensitivity for concurrent detection of four foodborne pathogens in raw milk.

AIMS: The aim of this study was to develop a multiplex touchdown PCR (multiplex TD-PCR) for rapid and simultaneous detection of four major foodborne pathogens to avoid mispriming and unwanted production during gene amplification. Touchdown PCR is the modified form of standard PCR, which enhances specificity, sensitivity. METHODS AND RESULTS: For this reason, a multiplex TD-PCR assay with a pre-enrichment step was developed to detect four foodborne pathogens namely Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica serovar Enteritidis in pure culture and raw milk samples. The results showed that this protocol can eliminate the unwanted band or reduce significantly. The detection sensitivity of the single and multiplex TD-PCR was one cell per ml in pure culture. Furthermore, the detection limit of multiplex TD-PCR was one cell per 25 ml for artificially contaminated raw milk. We obtained similar results for detection of aforementioned pathogens in raw milk, after comparing the multiplex TD-PCR method with the traditional culture, except in one or two samples. CONCLUSIONS: Hence, the proposed multiplex TD-PCR method could be confirmed as an effective way for rapid optimization of PCR reactions to increase specificity, sensitivity during gene amplification. SIGNIFICANCE AND IMPACT OF THE STUDY: Hence, due to its simplicity, cost-effectiveness and being time-saving, it seems that this method is reasonable and economical for rapid optimization of PCR reactions.

Influence of electric fields on the efficiency of multilayer graphene membrane.

Multilayer graphene membranes could be considered as an efficient membrane in water desalination processes based on the reverse osmosis (RO) method. In this study, we designed multilayer graphene channels using the molecular dynamics (MD) simulation approach. The effects of different parameters, such as channel width and length, and the pressure on the operation of the designed channels were examined, in the absence and presence of electric fields with various amplitudes and directions. The results indicated that the ion separation and water flow through the channels were modified under the application of the electric fields. Additionally, it has been shown that salt rejection and water flow could be controlled by the channel's structural parameters mentioned above. The obtained results of this study at the molecular level can improve the knowledge of designing membranes for water purification processes. Graphical abstract Using MD method a multilayer graphene membrane was designed to separate Na+ and Cl- ions from a NaCl solution by the aid of external electric field, which can significantly effect the membrane operation.

Dynamics of the population structure and genetic variability within Iranian isolates of grapevine fanleaf virus: evidence for polyphyletic origin.

To determine the genetic diversity and population structure of grapevine fanleaf virus (GFLV), the complete nucleotide sequence of the coat protein gene of 41 isolates from different regions in Iran was determined. Phylogenetic analyses of these isolates together with those available in the GenBank revealed two evolutionary divergent lineages, designated GFLV-G and GFLV-Ir that reflect origin of the isolates. Analysis of the genetic variability in the coat protein of these isolates revealed 37 genotype groups in GFLV population. Analyses indicate that GFLV-G and GFLV-Ir clades are significantly differentiated populations of GFLV. Also, geographical subpopulations of the virus in Iran were completely distinct from each other. Examination of nonsynonymous/synonymous nucleotide diversity showed that the CP gene has been under purifying selection. The neutrality tests indicate balancing selection operating within isolates of the northwest of Iran and purifying selection within the other populations.

Deformation of water nano-droplets on graphene under the influence of constant and alternative electric fields.

The dynamics of a water nano-droplet on a flexible graphene sheet, in the presence of constant and alternative electric fields with various amplitudes and frequencies, was considered using a molecular dynamics method. It was found that because the water molecules respond to electric field, the nano-droplet elongates in the field direction for a field amplitude larger than 0.08 V Å-1, which is stronger than the predicted value from the Young-Laplace equation. This difference can be described by considering the van der Waals attractions between the droplet molecules and the substrate, which can be calculated by modifying the Young-Laplace equation. Furthermore, under the influence of an alternating field over the GHz frequency range, it was shown that the droplet shape will not change above a threshold frequency, which depends on the relaxation time of the water dipole.

Effect of Adjunctive Celecoxib on BDNF in Manic Patients Undergoing Electroconvulsive Therapy: a Randomized Double Blind Controlled Trial.

INTRODUCTION: The possible effect of inflammatory factors on decreasing BDNF has been proposed in the literature. There is conflicting evidence regarding association between BDNF level alteration and treatment response in depressive patients undergoing electroconvulsive therapy (ECT). This study investigated the effects of celecoxib in manic patients undergoing ECT on treatment response and BDNF levels. METHODS: This randomized, double-blind, clinical trial included 35 manic patients who received either celecoxib (200 mg twice daily) or placebo, from one day before the 1(st) ECT session throughout the 6(th) session. BDNF levels were measured at baseline, 1(st), 3(rd) and 6(th) ECT sessions. Young mania rating scale was used to assess treatment response. RESULTS: Adding celecoxib was not associated with a significant rise in BDNF levels following ECT. No difference was noted between groups in terms of treatment response. No significant association was found between changes in BDNF levels and patients' responses. DISCUSSION: Adjuvant celecoxib did not significantly affect the BDNF level or the treatment response following ECT in manic patients.

Predominance of rotavirus G8 genotype in hospitalized children with acute gastroenteritis in Yasuj, Iran.

BACKGROUND: Rotavirus infection is the most common cause of gastroenteritis among children worldwide. In this study, we were evaluated the occurrence of rotavirus infection and genotypes causing gastroenteritis among children under 5 years in Yasuj, Iran. MATERIALS AND METHODS: This cross sectional-descriptive study was done on 184 fecal samples collected from children aged < 5 years old with acute gastroenteritis. All the stool specimens were tested for group A rotavirus with enzyme immunoassays (EIA). Rotavirus-positive specimens were genotyped by the Nested reverse transcription polymerase chain reaction (RT-PCR) with using different type of specific primers. RESULTS: The prevalence of rotavirus was 28.26% and predominant genotypes were G1 (1.92%), G2 (7.69%), G4 (1.92%), G8 (46.16%), N.T.A (40.39%), mixed infections (1.92%). Predominance of rotavirus G8 genotype in the current survey was reported for the first time in Iran. The highest prevalence of rotavirus infection was observed in autumn (48.08%), and the lowest in spring (5.77%). CONCLUSIONS: The result of study showed that prevalence of rotavirus genotypes in this region of the country is different from that of other regions. The continuous stability of rotavirus in Iran, regarding to the regional differentiates and prevalence of circulating strains, can be useful indication in order to supply and effective vaccine against the rotavirus infections.

Suspension culture of Neospora caninum by Theileria annulata-infected cell line.

There are some limiting aspects of scaling up the Neospora caninum tachyzoites in continuous cell lines, particularly as related to the absence of surface attachment. In this study, suspension cell culture of Theileria annulata-infected lymphoblastoid (TIL) was used as a host cell for the continous production of N. caninum tachyzoites. The numbers of free tachyzoites in the medium supernatant were showed regularly increased up to the day 6 post-cultivation. Transmission electron microscopy demonstrated that N. caninum tachyzoites invaded the TIL cells and multiplied intracellularly. This showed that the tachyzoites were successfully proliferated in TIL cells and were released in complete Dulbecco's modified Eagle's medium. This is a successful report of in vitro cultivation of N. caninum tachyzoites achieved by using suspension host cell culture.

Evaluation of Cross Immunity and Histopathological Findings in Experimentally Infected BALB/c Mice with Neospora caninum and Besnoitia caprae.

BACKGROUND: Caprine besnoitiosis is an economically important disease of goats. Neospora caninum, another coccidian parasite of worldwide distribution, infects several animal species and is a major cause of abortion in cattle. Combined infections of N. caninum and Besnoitia caprae can occur in geographical areas endemic for both species of parasite in goats. This experiment was conducted to investigate the possible cross-immunity between these two infections in experimentally infected BALB/c mice. METHODS: Forty BALB/c mice were divided into four equal groups. The mice of Groups 1 and 4 were inoculated with 1×10(6) live virulent tachyzoites of N. caninum (NC-1), while animals of Groups 2 and 3 were inoculated with sterile tissue culture medium. Each mouse in Groups 1 and 2 was challenged 28 days later with 1×10(6) live virulent bradyzoites of B. Caprae (BC-1). RESULTS: Following the challenge, the mice in Groups 1 and 2 showed 100% morbidity and 100% mortality within 9 days post infection, while all the animals of Groups 3 and 4 remained alive. The dead animals were necropsied. The survivors (mice in Group 3 and 4) were euthanized 9 days after inoculation and the gross and histopathological lesions in different organs were investigated. CONCLUSION: Immunization and challenge experiments with lethal dose of B. caprae in the highly susceptible BALB/c mice showed no cross-protection between N. caninum and B. caprae.

Burden and typing of rotavirus group a in children with acute gastroenteritis in shiraz, southern iran.

BACKGROUND: Human Rotavirus is a significant cause of severe gastroenteritis in infants and young children worldwide. In recent years, Rotavirus genotyping by RT-PCR has provided valuable information about the diversity of Rotaviruses circulating worldwide. The purpose of the present study is to monitor the prevalence of the different G types of Rotaviruses circulating in Shiraz, Southern Iran and detect any uncommon or novel types. METHODS: During the period from December 2007 to November 2008, a total of 138 stool samples were collected from children less than 5 years old who were hospitalized for acute gastroenteritis. Rotavirus-associated diarrhea was investigated in fecal specimens with enzyme immunoassays (EIA). Rotavirus-positive specimens were typed by the Nested RT-PCR and by using different types of specific primers. RESULTS: Out of the 138 collected samples, 34.78% (48 cases) tested positive for Rotavirus. The frequency of G1, G2 and G4 types was 6.25%, 2.08% and 27.08%, respectively. Mixed and non-typeable infections were detected in 33.34% and 31.25% of hospitalized children with acute diarrhea, respectively. This is the first time mixed Rotavirus infections with G1/G3 have been reported in Iran. CONCLUSION: The high frequency of Rotavirus detection indicates the severity and the burden of Rotavirus disease may be able to reduce through the implementation of an effective vaccine and continual surveillance for the detection of Rotavirus genotypes circulating in other regions of Iran. Regarding to the noticeable frequency of non-typeable and mixed infections, it is suggested to use the other specific primers and further studies to detection of other novel and unusual types.

Isolation and characterization of some moderately halophilic bacteria with lipase activity.

Lipases are an important class of enzymes which catalyze the hydrolysis of long chain triglycerides and constitute the most prominent group ofbiocatalysts for biotechnological applications. There are a number of lipases, produced by some halophilic microorganisms. In this study, some lipase producing bacteria from Maharlu salt lake located in south of Iran were isolated. All isolates were screened for true lipase activity on plates containing olive oil. The lipase activity was measured using titrimetric methods. Among thirty three isolates, thirteen strains demonstrating orange zone around colonies under UV light, were selected for identification using the molecular methods and some morphological characteristics. The bacterium Bacillus vallismortis BCCS 007 with 3.41 +/- 0.14 U/mL lipase activity was selected as the highest lipase producing isolate. This is the first report of isolation and molecular identification of lipase producing bacteria from Maharlu lake.

Probiotic inhibits the cytopathic effect induced by Escherichia coli O157:H7 in Vero cell line model.

AIMS: The effectiveness of four strains of Bifidobacteria against enterohemorrhagic Escherichia coli O157:H7 infection was studied using a Vero cell model. METHODS AND RESULTS: E. coli O157 was inoculated on the Vero cell line before and after treatment with probiotic. The cytopathic effect (CPE) was evaluated during 24 h of incubation. The results indicated that Shiga toxin activity was inhibited by the probiotic. To prevent a Stx2 CPE, the probiotic needs one log more than the Stx1. CONCLUSION: The Vero cell assay, in particular, is a good model to evaluate the effect of Bifidobacteria inhibiting bacterial attachment because of soluble substances and the competitive aspect and could be used in a variety of foods like milk and yoghurt to protect pathogen bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Probiotics could control pathogenic bacteria and Vero cell introduce as a model for evaluation of probiotics against pathogen bacteria.

Association between TNF-Alpha (-857) Gene Polymorphism and Susceptibility to Tuberculosis.

BACKGROUND: TNF-α as a pro-inflammatory cytokine plays a key role in host defense against tuberculosis (TB). Presence of mutation in TNF-α gene can influence the effectiveness, performance and capability of immune responses against this infection. The Aim of this study was to investigate the frequency of TNF-α alleles and its relationship with susceptibility to TB and TNF-α gene variations. METHODS: A case-control study was conducted and 103 healthy controls and 93 TB patients were enrolled. Genotype of TNF-238, TNF -244, TNF-308, TNF -857 and TNF-863 were distinguished using PCR-RFLP method. RESULTS: TNF-857 and TNF-863 were in high frequency mutation regions in a population level, and a significant difference at TNF-857 was noticed between the two groups of case and control. CONCLUSION: Presence of mutation in TNF-857 region probably increases the host susceptibility to mycobacterial infection. Genotyping of these regions in combination with other factors can be used for screening of high risk persons. According to high distribution of mutations in TNF-857 and TNF-863 regions, further studies on association of these regions is suggested.

Role of NADPH-insensitive nitroreductase gene to metronidazole resistance of Helicobacter pylori strains.

BACKGROUND AND THE PURPOSE OF THE STUDY: Current anti-H. pylori therapies are based on the use of two antibiotics with a proton pump inhibitor and/or a bismuth component. Metronidazole is a key component of such combination therapies in Iran. The aim of this study was to determine the role of rdxA gene in resistant strains of H. pylori isolated from Shahrekord Hajar hospital to metronidazole. METHODS: This study was a cross-sectional method, which was carried out on 263 patients who referred to endoscopy department of Hajar hospital, in 2007. Biopsy samples were cultured on selective Brucella agar containing 10% blood and incubated under microerophilic condition at 370C for 3-7 days. Suspected colonies were tested by Gram staining, urease, oxidase and catalase activities. Organisms were confirmed to be H. pylori on the basis of the presence of ureC(glmM) gene by PCR.Specific primers were used for detection of rdxA gene mutation. RESULTS: Eighty and four strains of H. pylori determined by PCR method. Of the isolated strains, 49 (58.33%) were resistant, 7 (8.33%) were semi-sensitive to metronidazole and 200bp deletion in rdxA gene was observed in 2 strains. CONCLUSION: Because of the high metronidazole resistance in patients under study it was necessary to replace it by other antibiotics in therapeutic regimens. On the basis of low frequency of resistance mutation in rdxA gene, sequence analysis for identification of other mechanisms is suggested.

The Comparison of Educational Intervention Effect Using BASNEF and Classic Models on Improving Assertion Skill Level.

BACKGROUND: To compare the effectiveness of BASNEF and Classic educational models to improve the assertion skill level of high school boy students. METHODS: The 60 high school male students from Shiraz City, Fars Province Iran, were participated in this study. They were randomly divided in two groups (groups A and B). The group A attended in designed educational planning based on BASNEF model and group B attended in classic educational program. The both groups had participated in six session educational activity (2 hours each session) during the four weeks. The data collected using questionnaire before and after one-month intervention. RESULTS: The mean score of knowledge, attitude, enabling factors, social norms, and Rathus Assertion Test were not significant statistically between two groups before and after intervention. However, the mean scores of all mentioned variables in group A and only knowledge and assertion variables in group B changed significantly after intervention. In addition, the comparison of the mean scores and the means of scores difference of all variables changed significantly between two groups after intervention. DISCUSSION: Performing BASNEF educational model, in accordance with its main parts (knowledge, attitude, social norms, and enabling factors) is more effective than performing classic educational model to improve high school boy students' assertion.

The cyanide-metabolizing enzyme rhodanese in different parts of the respiratory systems of sheep and dog.

Studies on the rhodanese activity of respiratory systems of sheep and dog showed that a significant difference exists in the pattern of distribution of this enzyme in different parts of the respiratory system of both species. In sheep, larynx, trachea, bronchiole, and lung contain higher activity than nasal cavity and pharynx. In dog, significantly greater rhodanese activity was found in nasal cavity than in other parts of the respiratory system. In regions with high rhodanese activity the enzyme was more concentrated in the mucosa than in the underneath tissues. These results are discussed in terms of the possible role of rhodanese in cyanide metabolism in respiratory tract and the efficacy of this organ in inhaled cyanide detoxification in these species of animals.