Detection of mycoplasma contamination of cell culture by a loop-mediated isothermal amplification method

Objective: Mycoplasmas are major contaminants of cell culture and affect in vitro biological and diagnostic tests. Mycoplasma detection is conducted using culture and molecular methods. These methods vary in terms of accuracy, reliably and sensitivity. Loop-mediated isothermal amplification [LAMP] is used to amplify target DNA in a highly specific and rapid manner. This study aimed to develop a LAMP method for rapid detection of Mycoplasma in culture samples

Materials and Methods: In this descriptive laboratory study, for LAMP detection of Mycoplasma contaminations in cell culture, we used primers specifically designed for targeting the 16S rRNA conserved gene of Mycoplasma spp. For a positive control structure, 16S rRNA amplified based on PCR, was cloned in a plasmid vector and sequenced. The assay specificity was evaluated using Mycoplasma genomic DNA and a panel containing genomes of gram-positive and gram-negative organisms

Results: In this study, the method developed for detection of Mycoplasma contamination of cell cultures was a rapid, sensitive and cost-effective LAMP approach. The results demonstrated that this method benefits from high specificity [100%] for amplification of Mycoplasma strains and high speed [multiplication within 60 minutes], while it does not require expensive laboratory equipment compared to those needed for polymerase chain reaction [PCR]-based detection

Conclusion: Our study is the first report about application of LAMP assay based on 16S rRNA gene for detection of Mycoplasma strains; this technique could be considered a useful tool for rapid detection of contamination of cell culture

Biosynthesis of silver nanoparticles using chlorella vulgaris and evaluation of the antibacterial efficacy against staphylococcus aureus

Background: It is well documented that Silver Nanoparticles [SNPs] are potent antimicrobial agents. However, little is known about antimicrobial effects of biologically synthesized SNPs at molecular level. In the present study, efficacy of the green microalgae Chlorella vulgaris in biosynthesis of silver nanoparticles and inhibitory effect of the biosynthesized SNPs on growth and virulence of Staphylococcus aureus [S. aureus] were investigated

Methods: Algal suspension was incubated in the presence of silver nitrate to induce formation of nanoparticles. The experiment was conducted under a pH range to evaluate pH effect on the shape and properties of nanoparticles. Characterization was performed by Transmission Electron Microscopy [TEM], Scanning Electron Microscopy [SEM], Energy Dispersive Spectrometry [EDS] and X-ray diffraction analysis [XRD]. Moreover, concentration of biosynthesized SNPs was measured by high resolution ICP-OES spectrometer. Antibacterial effect of SNPs on growth of S. aureus was evaluated by broth micro-dilution method. Inhibitory effect of SNPs on alpha hemolysin, a well-known virulence factor of S. aureus was investigated through real time PCR assay

Results: Spherical SNPs were produced with characteristic monodispersity at low and neutral pHs; however, in alkaline condition, nanorod structures were formed. SNPs inhibited growth of S. aureus at concentration of 50 microg/ml. Alpha hemolysin expression was also effectively inhibited by SNPs treatment

Conclusion: In general, results revealed formation of spherical silver nanoparticles with inhibitory effects on bacterial growth and antagonist activity on the expression of alpha hemolysin. Moreover, increase in pH to basic condition resulted in aggregation of nanoparticles and formation of rod-like nanostructures

Using recombinant chlamydia trachomatis OMP2 as antigen in diagnostic ELISA test

The obligate intracellular bacterium Chlamydia trachomatis causes sexually transmissible diseases in human. Timely and sensitive detection of this pathogen is very important. There are many cross-reactions in bacteriological and serological methods in detection of this type of pathogens. The aim of this study was to achieve a more specific antigen for serological tests. Blood samples were taken from 192 women with suspected chlamydial infection and sera were isolated. ELISA plate wells were coated with recombinant C. trachomatis OMP2 as antigen. Cut-off system was determined with 40 negative sera. The final results of this research were compared with Euroimmun commercial kit. The ELISA system cut-off was calculated at 0.27 using negative sera samples. ODs of positive samples were higher than 0.27 and negative samples were lower than it. We obtained 30 samples [15.62%] as positive and 162 cases [84.37%] as negative. Sensitivity and specificity of the recombinant antigen were 90% and 86%, respectively. This antigen showed no cross-reactivity with sera of patients infected with Hydatid cyst, HCV, Epstein barr virus, HBV, Helicobacter pylori, Toxoplasma gondii, Cytomegalovirus, Mycoplasma, Measles and Varicella zoster virus. The sensitivity and specificity of rOMP2 in ELISA for detection of C. trachomatis were 90% and 86%, respectively. Though the sensitivity was higher than results of Euroimmun commercial kit, its specificity was calculated lower than reference kit

[Review on the laboratory diagnosis of Q-fever]

Coxiella burnetii is the etiologic agent of a zoonotic disease which named Q-fever in humans and coxiellosis in animals. This bacterium can survive in the environment out of the specific host. Accordingly, it categorized by the CDC in bioterrorism agents 'category B. Consequently, rapid detection of the bacterium administrates the treatment of disease. A review study on the different researches on laboratory diagnosis field in the world and Iran scientific databases was conducted. This paper includes biological safety, cultivation and detection assays. Detection of Coxiella burnetii can be done by classical methods [isolation, cultivation in the appropriate cell line such as P388D1, J774, and L929], serologic tests [immunofluorescence, micro immunofluorescence, complement fixation test and ELISA] and molecular biology methods [PCR, Nested PCR and Real Time PCR]. While have existed kinds of detection methods for this agent, costly, need to specific laboratory and time consuming are the limitation of the mentioned techniques. Molecular methods due to accuracy and high rapidity in detection can be effective

[Design of an improved polymerase chain reaction [PCR] assay for molecular detection of Streptococcus pneumonia]

Streptococcus pneumoniae accounts for bacterial meningitis and is an important cause of morbidity among children and elderly. Control of this disease depends on rapid detection of the causative bacteria. The methods for detection of Streptococcus pneumoniae are gram staining, culture, and serological tests. These tests are time consuming and are limited by antimicrobial agents leading to false negative results. Currently, molecular methods such as PCR are used routinely for detection of infectious organisms. This study was performed with the aim of designing an improved PCR assay for the detection of Streptococcus pneumoniae. The specific diagnostic primers were designed based on ply gene of the bacterium. After amplifying the target gene on the genomic DNA, the PCR product was cloned in pTZ57R/T plasmid and the confirmed pTZ-ply plasmid was used as positive control in next experiments. Sensitivity of the assay was determined by performing the PCR on 10-fold serial dilutions of pTZ-ply. Specificity of the assay was determined using the genomic DNA of other related or unrelated bacterial species. The PCR, as expected, generated a 727bp amplicon. No PCR amplification was observed on the genome of negative controls. These findings indicate high specificity of the PCR. The lowest limit of detection of the assay in the detection of the ply gene was 250 copies in a 25micro l reaction. The high sensitivity, specificity, and rapidity of the designed assay suggested the assay as an appropriate test for use in clinical laboratories. The further evaluation of the assay using clinical samples or artificially contaminated materials will confirm the application of this assay in clinical settings

[Ceramide can induce leukocyte common antigen-related [lar] gene at mrna and protein level in c2c12 muscle cells]

Type 2 diabetes results from two defects, insulin resistance and beta cell dysfunction. At the molecular and cellular levels, there is a connection between fatty acid accumulation and insulin resistance in muscles. Although several mechanisms involved in FFA-induced muscle insulin resistance, the exact mechanism is poorly understood. Recent studies show that the defect in insulin signaling pathway might be underlying mechanism for FFA-induced insulin resistance in the muscle. Protein tyrosine phosphatases like Leukocyte common antigen-related [LAR] are the key elements of insulin signaling and they can be a candidate in FFA induced insulin resistance. Studies have shown that type 2 diabetes involved and obese individuals have had increased levels of LAR in their tissues. However, the responsible factor for LAR overexpression is not well understood. In this study we investigate ceramide effect on LAR expression in the muscle cells. In this laboratory study C2C12 cells [mouse skeletal] after differentiation to myotubes using 2% of horse serum for 4 days, treated with 50 and 100 mMs of C2ceramide for 16h. RNA extracted, cDNA synthesized and Real Time PCR using specific primers for LAR and beta actin used. To detect LAR protein levels western blot was used. 100 mMs Ceramide [45%, P<0.01] significantly induced LAR mRNA expression but there was not significant difference between 50mM ceramide and untreated cells. The results from real time confirmed by protein data. 100 mMs Ceramide [52%, P<0.01] significantly induced LAR protein levels in comparison of control. The data from this study provide evidence that ceramide around pathologic concentration induce LAR expression. Results supported by human studies that were showed that diabetic and obese persons have a high level of LAR expression and revealed ceramide can be one of the responsible factors for inducing LAR expression in diabetic patients. However, further investigations are needed to clarify exact role of LAR in FFA induced insulin resistance.

Design of an improved multiplex PCR method for diagnosis of enterohaemoraghic E.coli and enteropathogic E.coli pathotypes

We aimed to develop a multiplex PCR assay for specific detection of EPEC and EHEC pathotypes based on specific marker genes. About 2.5 million infant's morbidity in developing countries occurs by E.coli pathotypes because of diarrhea and intestinal diseases. The traditional phenotypic methods are time consuming and sometimes detection and differentiation of the pathotypes are not done easily. Multiplex PCR technology is used as a sensitive, specific and rapid molecular method for detection of various pathogens. PCR reactions were performed with primers which targeted the virulence genes selected for each category [stx[1], stx[2] genes for EHEC and bfpA for EPEC].For preparation of a positive control, the PCR products were cloned in pTZ57R/T plasmid. The same PCR reactions were done but in presence of genomes of various negative control bacteria for evaluation of test specificity. As expected, gel agarose electrophoresis of PCR products of the stx1, stx2 and bfpA, showed 329bp, 586bp and459bp bands respectively. Result of amplification using negative control genomes as template was negative. The multiplex PCR assay followed by capillary electrophoresis presented in the present paper provides a simple, reliable, and rapid procedure that in a single reaction identifies the four main pathotypes of E. coli. This assay will replace the previous molecular genetics methods used in our laboratory and work as an important supplement to the more time consuming phenotypic assays

role of cow's milk allergy in pediatric chronic constipation: a randomized clinical trial

Cow's milk allergy has different presentations in children and can cause functional bowel symptoms such as chronic constipation. The aims of this study were to investigate the role of cow's milk allergy as a cause of chronic constipation and effect of cow's milk free diet [CMFD] on its treatment in children. We performed a randomized clinical study comparing CMFD with cow's milk diet [CMD] in two groups each consisting of 70 patients [age range, 1-13 years] with chronic functional constipation [defined as Rome III criteria]. All subjects had been referred to a pediatric gastroenterology clinic and had previously been treated with laxatives for at least 3 months without success; also all 140 patients performed skin prick test. The case group received CMFD for 4 weeks. After that they received CMD for 2 extra weeks. The control group received CMD for whole 6 weeks. A response was defined as decreased in signs and symptoms that not fulfilled Rome III criteria after 4 weeks of CMFD and came back to Rome III criteria after 2 weeks of CMD challenge. After 4 weeks 56 [80%] patients of the case group responded in comparison to 33 [47.1%] patients in the control group [P=0.0001]. In the case group after 2 weeks challenge 24 out of 56 [42.8%] responders developed constipation according to Rome III criteria. With other words, the frequency of cow's milk allergy among constipated patients was 80%. Only one patient had positive skin prick test. In children, chronic constipation can be a manifestation of cow's milk allergy. At present, although several aspects must be further investigated, a therapeutic attempt with elimination diet is advisable in all children with constipation unresponsive to correct laxative treatment

Human tissue plasminogen activator expression in Escherichia coli using cytoplasmic and periplasmic cumulative power

Tissue plasminogen activator [tPA] is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli [E. coli] is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm

Intralenticular foreign body: a case report / 国际眼科杂志(Guoji Yanke Zazhi)

An intralenticular foreign body is a rare condition, but the prevalence is about 5% of all intraocular foreign bodies and it can result in serious complications. We managed them according to the size, location, material type and the risk of infection. This article reported a 51year-old patient with an intralenticular metal foreign body in the left eye, who underwent phacoemulsification lens extraction with removal of the intralenticular foreign body and insertion of a posterior chamber intraocular lens. The visual outcome was good.

Bilateral idiopathic cystoid macular edema:report of four cases / 国际眼科杂志(Guoji Yanke Zazhi)

·We report clinical manifestations,angiographic,and optical coherence tomography (OCT) findings of four cases with bilateral idiopathic cystoid rnacular edema (CME).All were male with age between 30 and 52 years.All of them had compliant of bilateral visual loss during the last week.Fundus examination of their eyes showed macular edema in the posterior pole bilaterally. Fluorescein angiography revealed no specific finding in one of them and "typical petaloid pattern" in others.OCT showed subretinal fluid in all of them.All patients were managed with diagnosis of idiopathic CME,and after 6 weeks they had improved visual acuity.

Cloning of tissue plasminogcn fictivator cdnft in nonpathogenk leishmania

At present, most recombinant proteins are produced in prokaryotes especially E coll. Yeasts and CHO also are used as eukaryotic hosts. Leishmania tarentolae, a parasite of lizards, a member of Trypanosomatidae family is one of the new systems for expression of heterologous proteins. In this system, some of the parasitic protozoa features are used in expression of mammalian proteins. For evaluation of the protozoa for expression of human complex proteins, we cloned cDNA of tPA gene containing native human signal sequence. We used vectors containing 3' and 5' sequences of Leishmania 18s rRNA for integration of the vectors in 18s rRNA gene and severe transcription. RT-PCR test showed production of specific mRNA of tPA gene in the recombinant cells. Southern blot analysis confirmed the cloning of t-PA in the genome of the Leishmania. Conclusion: This study showed native human signal sequence mediate transport and secretion of the protein. Hence, L tarentolae is the first useful biotechnologically protozoan and tPA is the most complex protein expressed in it

Predictive factors for persistent hyperparathyroidism after kidney transplantation

A successful kidney transplantation [KT] corrects the main metabolic abnormalities responsible for secondary hyperparathyroidism [HPT]. Nonetheless, after several months, many patients keep abnormally high parathyroid hormone [PTH] levels and/or become hypercalcemic with persistent HPT. In the present survey, the frequency of high PTH levels and the influence of certain important factors on its evolution among patients with successful KT were investigated within three months posttransplantation. A total of 126 patients, who had successful KT, entered the study between 2000 and 2002. On the day of operation and three months later, demographic data and serum calcium, phosphorus, albumin, creatinine, and immunoreactive PTH [iPTH] [by IRMA] were checked. Hypercalcemic patients, at third month, were followed up for one year after transplantation. With respect to the post-KT iPTH level, patients were divided into two groups; those with iPTH above and below 60 pg/mL. The importance of several factors on the evolution of hyperparathyroidism was determined. Sequential changes in serum calcium were also assessed in hypercalcemic patients up to one year after transplantation. Twenty-one [16.6%] out of 126 patients had a post-KT serum calcium of >10.8 mg/mL. Post-KT iPTH value of > 60 pg/mL was found in 9 [7.1%] out of the 126 cases. There was a statistically significant relationship between the age of patients and duration of dialysis and a post- KT high PTH level [P < 0.001]. Other risk factors did not seem to have a significant correlation with the post-KT high PTH level. In all hypercalcemic patients, PTH levels normalized but hypercalcemia persisted in 14 [88%] out of 16 patients up to 1 year after transplantation. Increased age of the patient as well as the duration of dialysis had significant influences on development of persistent HPT, three months posttransplantation. We believe that it is better to transplant the patients as soon as possible, in order to prevent the devastating complication of persistent HPT and hypercalcemia

effect of acute urinary retention on serum prostate-specific antigen level

Our aim was to evaluate the effect of acute urinary retention on serum prostate-specific antigen [PSA] level. Men aged 50 years and older who presented with acute urinary retention were studied. Patients with urethral stricture, neurogenic bladder, prostate cancer, and those with a history of recent instrumentation or prostate biopsy were excluded. Blood samples for serum PSA measurement were obtained [PSA1], and an indwelling urethral catheter was inserted for 2 weeks. Before catheter removal, a second blood sample for measurement of serum PSA level [PSA2] was obtained. In patients who were able to void, a third sample was obtained 3 weeks later [PSA3]. In the first and second visits, digital rectal examinations [DRE1, DRE2] were performed to assess prostate volume. Mean PSA levels [PSA1, PSA2, and PSA3] and prostate volumes [DRE1, DRE2] were compared. Forty-five patients with a mean age of 70.18 years [range 56 to 85 years] participated in this study. Mean PSA1 and PSA2 levels were 9.8 ng/mL and 5.05 ng/mL, respectively [P<0.001; medians, 6.2 and 4.2 ng/mL]. Mean prostate volumes at the time of retention and 2 weeks later were 43.4 mL and 37.8 mL, respectively [P<0.001; medians, 45 and 40 mL]. PSA3 was measured in 31 patients 2 weeks after catheter removal. In this group of patients, mean PSA2 and PSA3 levels were 5.03 ng/mL and 4.97 ng/mL, respectively [P=0.49; medians, 4.3 and 4.1 ng/mL]. Acute urinary retention can increase serum PSA levels by approximately 2 fold. In this series, we found that this effect may continue up to 2 weeks