Expression of bacillus species isolated phytase gene from soil by PCR method

Recognizing and using of isolated phytase in the soil microorganisms are paramount importance to produce the Phytase enzyme utilized commercially in different industries. This study was conducted to recognize different bacillus j species which are Phytase producers and detection of the gene that can produce this enzyme. Soil samples were gathered through different parts of mountainous areas. The early isolation of bacillus was carried out in Bacillus Medium Agar. After isolating the bacteria and genome extraction, the responsible gene of enzyme producer recognized and amplified by PCR method. The size of this protein and the optimal production situation in supplemental exploitation such as SDS-PAGE and the enzymatic activity of its size were evaluated. Of 40 samples, one bacterium secreting Phytase enzyme was isolated. This bacterium was sequenced and recognized Bacillus Sobtlis species that is classified in STR Genus. The size of protein phytase produced by this gene was about 45 KD and the enzyme activity at 55 degrees was measured about 5.65 in wavelength of 415 I NM. The phytase gene with the size of 1200 bp was propagated. the microorganisms, in natural conditions, produce Phytase enzyme in limited amount and with the quality appropriate to microorganisms. Thus, isolating the bacilli producing Phytase enzyme and purifying this protein are highly significant

[Local anesthesia in vitreoretinal surgery]

To compare general anesthesia [GA] with local anesthesia [LA] in terms of safety and patient satisfaction. In this interventional case series, 928 consecutive patients who underwent vitreoretinal surgery were included. Data for analysis were type of anesthesia and operation, patient compliance, operating conditions and pain scores. General anesthesia was provided with a standard method and local anesthesia was performed through peribulbar or retrobulbar routes. Selection of the type of anesthesia was determined by patient age and co-morbidities based on the surgeon's opinion. Patients were operated under local anesthesia in 343 [36.9%] and general anesthesia in 585 [63.1%] cases. Mean age was 68.8 +/- 7.3 [range 51-78] years in the LA group and 55.9 +/- 6.5 [range 43-70] years in the GA group [P= 0.041]. ASA [American Society of Anesthetics] physical status score in the LA group was higher than GA group. Patients were in appropriate condition in 96.4% and operating conditions were good or excellent in 98.8%. The majority of patients [97%] said they would choose local anesthesia for their next vitreoretinal procedures. Local anesthesia is a useful and flexible method of anesthesia for vitreoretinal surgery, with excellent patient tolerance, especially in old patients and those who suffer from concurrent diseases

Prenatal diagnosis of fetal sex by molecular analysis of maternal plasma after 8 weeks of gestation

Prenatal diagnosis of fetal sex is usually performed by invasive methods such as sampling through amniocentesis or chorionic villus sampling. One potential non-invasive approach involves analysis of cell-free fetal DNA in maternal plasma or serum. The objective of our study was to investigate the feasibility of using fetal DNA in maternal plasma for prenatal diagnosis of fetal sex. In this experimental study, a nested polymerase chain reaction [PCR] techniques was developed for fetal SRY gene identification using cell-free fetal DNA in maternal plasma. Peripheral blood samples were obtained from 32 pregnant women at the gestational period from 8 to 13 weeks and cell-free DNA was extracted by the phenol/chloroform method from plasma. The nested PCR was carried out to amplify the fragment of SRY gene by two sets of PCR primer pairs. Analysis was then performed on the PCR product. Specifically, the presence of Y-chromosome sequences in maternal blood plasma indicated that the fetus is male, whereas lack of signal will indicate that the fetus is female. Among the 32 pregnant women, SRY sequences were detected in 14 plasma samples after nested PCR amplification, while the 18 women bearing female fetuses had the negative results. The sensitivity of this technique was 87.5%. The phenol/chloroform extraction of fetal DNA in maternal plasma is an effective and simple method, and the nested PCR amplification of SRY sequence is a convenient and low-cost approach for the non-invasive early prenatal diagnosis of fetal sex

Quantitative comparison of NASBA-ELISA and RT-PCR-ELISA sensitivities for measurement of the BCR-ABL genes fusion transcript in CML patients

Quantitative comparison of NASBA-ELISA and RT-PCR-ELISA sensitivities for measurement of the BCR-ABL genes fusion transcript in CML patients Nazemi A1, Sadeghizadeh M2, Forouzandeh Moghaddam M3, Javadi Gh4, Hashemi M5 1 Student of PhD of Molecular Genetics, Department of Biology, Islamic Azad University, Science and Research Campus, Tehran, Iran. 2Associate Professor, Department of Genetics, Tarbiat Modarres University, Tehran, Iran. 3 Associate Professor, Department of Medical Biotechnology, Tarbiat Modarres University, Tehran, Iran. 4 Associate Professor, Department of Biology, Islamic Azad University, Science and Research Campus, Tehran, Iran. 5 Assistant Professor,Department of Molecular Genetics, Islamic Azad University, Tehran Medical Branch, Tehran, Iran. Chronic myeloid leukemia [CML] is characterized by neoplastic overproduction of myelocytes and neutrophils. Affected patients have a Philadelphia chromosome which arises following a translocation between long arms of chromosome 9 and 22 [q34; q11]. This results in abelson murine/breakpoint cluster region [BCR/ABL] fusion. Detection of cells carrying BCR/ABL fusion is extremely important in monitoring response to treatment, remission and relapse in CML patients. In this study, we compared RT-PCR and NASBA techniques to determine quantitatively the number of bcr/abl transcripts. Fusion transcripts were synthesized and RNA was extracted from K562 leukemic cell line. A serial dilution of both fusion transcript and RNA was prepared; then sensitivities of both techniques were determined. RT-PCR and NASBA reaction products were labeled using equal ratios of DIG-11-dUTP and DIG-11-UTP respectively. Following denaturation, hybridization reactions were carried out with specific probes. The products were incubated in streptavidin coated microplates. Then, the plates were washed, anti-DIG conjugated with peroxidase added and using ATBS as substrate, enzymatic activity was determined by absorption at 405 nm. The results showed that specificity of two techniques was equal but RT-PCR-ELISA sensitivity was about 100-fold more than NASBA-ELISA as it could detect 100 pg RNA less than NASBA-ELISA [0.006 versus 0.06 pg RNA]. Furthermore, leukemia cell detection precision by RT-PCR-ELISA and NASBA-ELISA was 4 and 400 cells, respectively. While NASBA technique does not need thermal cycler PCR but has less sensitivity than RT-PCR and is not suitable for quantitative assessment

Development of covalent reverse dot-blot technique for rapid diagnosis of two common beta-thalassemia mutations: IVS-I-5 and IVS-I-110 in Iran

Beta-thalassemia is an autosomal recessive disorder that most commonly caused by point mutations in the beta-globin gene. IVS-I-5 and IVS-I-110 are the most frequent mutations found among Iranians comprising 12% of all mutations. In this study, we develop the implementation of the reverse Dot- Blot [RDB] hybridization technique as a rapid and simple method for the detection of the two common mutations in the beta-globin gene. Total genomic DNA was extracted and PCR with specific primer [forward and reverse primer] was performed on region of beta-globin gene consisting the two mutations. Labeled dNTPs with DIG-II-dUTP were used in PCR mixture therefore PCR product contained DIG labeling formed. The oligonucleotide probe was immobilized onto a Biodyne C nylon membrane via activation membrane. The strips were hybridized with 10 microliters of denatured PCR product labeled to DIG at 42°C for 60 minutes. After blocking strip with the blocking buffer, the strip exposed to 5 unit anti-DIG antibody conjugates with alkaline phosphatase for 30 minutes at room temperature. The color detection was performed with nitroblue tetrazolium salt [NBT/BCIP] substrate for 120 minutes. The presence of a particular DNA sequence was detected by the appearance of a dot on the membrane. Normal individuals [N/N] showed dots with each wild-type sequence but not with any mutant probe. Heterozygotic individuals showed the appearance of a single mutation dot in addition to all the normal dots and homozygous individuals revealed dots with each mutant probe but not with any wild-type sequence. We described a rapid and simple strategy to detect beta-thalassemia mutations based on PCR followed by reverse Dot-Blot hybridization

Antimicrobial activity of aqueous and methanol extracts of heracoleum persicum

Medical plants entail numerous therapeutic capabilities due to their antimicrobial activities. During the present study, the antimicrobial activity of aqueous and methanol extracts of Heracoleum Persicum was determined in vitro. We have gathered the aforementioned plant from heights of Ashkour near Tonkabon. The antimicrobial activity of aqueous and methanol extracts was determined against 14 bacterial and 2 fungal specimens according to the disc diffusion, MIC [minimal inhibitory concentration], and MBC [maximum bactericidal concentration]. The aqueous extract of Heracoleum Persicum failed to show antimicrobial activity, however, the methanol extract was effective against bacillus, streptococcous, enterococcous and nocardia. The methanol extract of Heracoleum Persicum has antibacterial activity. Further studies are strongly recommended to find out the possible therapeutic capability of this plant in infectious diseases