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Showing 5 results for Pcr-Rflp

Parisa Tahmasebi, Fatemeh Maryam Sheikolslami , Parisa Farnia , Majid Sadeghizadeh, Rashid Ramazanzadeh, Mahdi Kazempoor, Mohammadreza Masjedi , Ali Akbar Velayati,
Volume 11, Issue 4 (12-2011)

  Background & objectives : Amikacin is one of the key second-line drugs for treatment of tuberculosis. Mutations at the codons 1400, 1401and1483 of the 16srRNA gene are associated with resistance to amikacin. The purpose of this study was to detect these mutations using PCR-RFLP method in multi-drug resistant (MDR) strains of Mycobacterium tuberculosis showing resistance to amikacin.

  Methods : Susceptibility of strains (n=100) against first and second–line anti-tuberculosis drugs was performed by proportional method. Based on antimicrobial resistance pattern 97 strains were analyzed by PCR-RFLP method. rrs1096 and rrs1539 primers were used to amplify a 460bp region of the rrs gene. Then, the PCR products were digested using Tai 1 and Dde1 restriction enzymes. The results were analyzed by the SPSS software using Chi-square test.

  Results : Based on results from proportional method, 63 strains (64.9%) were MDR (Multiple Drug Resistant), 26 (26.8%) and 8 (8.2%) strains were susceptible and non-MDR, respectively. Also, 13.4% and 6.1% of the strains were XDR (Extensively Drug Resistant) and TDR ( Totally drug resistant ) respectively. Using PCR-RFLP method, 7 (7.2%) strains were resistant and 90 (92.7%) strains were susceptible to amikacin respectively. Moreover, we found that the mutation at the codon 1400 was the most frequent mutations responsible for resistance to amikacin.

  Conclusion : The PCR-RFLP method can be used as a supplemental method to detect resistance to amikacin however to increase our knowledge, mutatuions in several number of codons in rrs gene need to be studied.

Gholamreza Irajian , Reza Mirnejad, Mohammad Reza Jalali Nodoshan, Nafiseh Ghorbanpour,
Volume 13, Issue 1 (4-2013)

  Background & Objectives: Prostatitis is relatively one of the common diseases in elderly men. Treatment of this disease is difficult and because of frequent relapses, it provides complicated problems for patients and the physicians. Detection of reservoirs and determination of prevalence of involved microbes in prostatitis are important in the epidemiology and control of the disease. This study was conducted to determine the prevalence of Mycoplasma genitaliumin patients with prostatitis by sequencing and PCR-RFLP techniques.

  Methods : In this cross-sectional study, 200 paraffin-embedded prostate samples from patients with prostatitis during 2008-2011 were checked for M. gentialium. After cutting the tissues and homogenization, the genomic DNA was extracted and used as template in PCR. Primers targeting a 465 bp regions of 16S rRNA of M. genitalium were used in the assay. PCR products were sequenced and Cac8I, Bbs I, EcoRI, AluI, TaqI endonucleases were used in RFLP analysis.

  Results : Of 200 samples, 4 were positive in PCR. The results of DNA sequencing and RFLP confirmed the amplified genes corresponded to M. genitaliumG37.

  Conclusion : The Mycoplasma genome present in tissue samples of prostate showed this bacterium could be one of the risk factors for prostatitis in men. However large studies and control groups are needed to prove this finding.

Ali Pezeshki, Mostafa Rezaeian , Mitra Zarebavani,
Volume 13, Issue 2 (6-2013)

  Background & Objectives: Giardia duodenalis is the most common intestinal parasite with cosmopolitan distribution. This parasite has been found in the intestine of humans and other mammalian hosts including cats, dogs, cattle, sheep, deer, pigs and muskrats. It is postulated that animals maybe reservoir for human infection and viceversa. In present study, the possible genetic similarity between cat and humans Giardia and its probable zoonosis were investigated.

  Methods: Direct examination and formalin-ether concentration techniques were performed on stray and semi stray cat fecal specimens. Gradient sucrose method was applied for collection and purification of cysts and DNA extraction was performed by phenol-chloroform and CTAB (Cetyl trimethyl ammonium bromid ( methods. DNA of cysts could hardly be extracted after repeated freezing and thawing. Polymerase chain reaction (PCR) was performed for DNA amplification. In this study triosephosphate isomerase (tpi) gene was selected as a molecular marker. Two sets of primers (PM 290 and PM 924) were considered. Two restriction enzymes RsaI and AvaI were also used to determine restriction fragment length polymorphism (RFLP) for PCR fragments amplified by both primer sets.

  Results: Ten samples were positive for Giardia cysts which were examined for molecular investigation. Four cat isolates were amplified by PM 290. PCR-RFLP patterns were found to be similar to human isolates AC≠AF 069556 (subgroup of AC≠U 57897) with possibility of cross-transmission.

  Conclusion: Therefore the similarity of genomic characters of isolates of cat and human Giardia implies possibility of zoonosis and transmission of these protozoa from cat to human and vice versa.

B Davoodi, Kh Onsory , M Heydari Nasrabadi,
Volume 15, Issue 2 (6-2015)

  Background & objectives : Ovarian cancer is the most common female reproductive cancer which is caused due to the malignant transformation of ovarian cells. This type of cancer is the fifth most common cancer among women and the primary cause of cancer deaths in the world. Axin2 gene is a tumor suppressor gene of the Axin family in WNT cycle which is essential for embryonic development. WNT proteins in this pathway have important intermediary role in cell messaging and in primary and secondary development of the embryo. Axin2 gene is activated as a negative feedback to prevent excessive proliferation of cells with simultaneous activation of WNT messaging. The aim of this study was to find the frequency of mutation in rs1133683 region of exon 5 in Axin2 gene and its relation with the risk of ovarian cancer.

  Methods : In this case-control study, 100 patients with ovarian cancer together with equal number of same age as controls were collected from Imam Khomeini Hospital. DNAs were extracted from blood and tissue and then were investigated by PCR-RFLP. Data analysis was performed using software SPSS (version 19) using logistic regression.

  Results : The results of study of mutation in rs1133683 region of exon 5 in Axin2 gene between two groups of case and controls indicated that there is no significant association between CT genotype with ovarian cancer (OR=1.26, 95%CI 0.70-2.27,p=0.43). Also no association was observed between TT genotype of Axin2 gene and ovarian cancer risk (OR=1.56, 95%CI 0.49-4.96, p=0.44).

  Conclusion : Study of mutation in rs1133683 region showed that there was no association between TT genotype carriers of Axin2 gene and the risk of ovarian cancer.

Mortaza Nourmohammadi, Hosein Hamidinejat, Mohammadreza Tabandeh, Saad Goraninejad, Somaye Bahrami,
Volume 17, Issue 3 (10-2017)

Background & objectives: Toxoplasma gondii is a zoonotic obligate intracellular protozoan parasite that infects all warm-blooded animals as well as human worldwide. Determining the parasite genotype in intermediate hosts  is crucial in  evaluating the role of these types in human infections as wll as in prevention programs. Therefore, this study aimed to identify and detect the genotypes of Toxoplasma gondii in aborted fetuses of ewes in Lorestan province.
Methods: Identification of the parasite was performed  on the brain and liver tissues of 142 aborted fetuses using  a conventional PCR based on amplification of highly repetitive 529 bp region of the parasite genome. Genotyping of positive samples, which were isolated from the brain and liver, was performed by PCR-RFLP based on SAG2, SAG3 and GRA6 molecular markers.
Results: From a total of 142 samples  obtained from  brain  and fetus, 10 cases (7%) were determined as positive samples based on conventional PCR. The precence of parasite DNA was also confirmed in the liver of  3 positive samples. Evaluation of RFLP pattern of amplified SAG2, SAG3 and GRA6 genes showed the presence of various types of parasites, incuding type I in 3 samples, type II in 2 samples and atypical type in 5 samples.
Conclusion: Isolation of types I, II and atypical type of T. gondii from ewes in  Lorestan province suggests the need for greater attention to parasite transmission from livestock to human, particularly in pregnant women and people with weakened immune system.

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