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Background: One way of embryo preservation is cryopreservation, but this process may damage and lead to the loss of the embryos, and bring about chromosomal abnormality. This has led researchers to seek techniques for short term preservation of embryos in 0-10 ºC temperatures. The aim of this study was to determine the effect of short time exposure to 4°C temperature on the expression profiles of mono-carboxylic transporter genes 1,2 ,3, and 4(MCT1-4) in 4-cell mouse embryos. Materials and Methods: In this fundamental study, forty 4-cell mouse embryos from NMRI strain were randomly divided into two groups. The first group consisted of fresh 4-cell embryos, and the second group included 4-cell mouse embryos that were exposed to 4°C temperature for 24 hours. After RT-PCR, the samples were electro-phoresised for expressing the MTC1-4 genes. Results: The expression of MCT 1-3 was observed in the first group, but the obtained results did not indicate their expression in the second group. Conclusion: Preservation of 4-cell embryos in 4°C for 24 hours inhibits the expression of MCT 1-3 genes. Keeping embryos in 4°C temperature is not a proper way for their short time preservation.

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Background: Helicobacter pylori iceA gene has been reported to be a genetic marker for the development of peptic ulcer in western populations. The aim of this study was to investigate the prevalence of iceA genotypes and their relationship with peptic ulcer in Iran. Materials and Methods: This observational study was carried out on 75 patients. Biopsy specimens were evaluated for the presence of Helicobacter pylori through rapid urease test. GlmM gene and iceA1 and iceA2 genotypes allelic verification and variation culture were determined via PCR. Results: In this study, iceA1 and iceA2 alleles were identified in peptic ulcer disease (PUD) patients. IceA1 genotype (64%) was more prevalent than iceA2 (21.3%). IceA1 strains were more observable in patients with PUD. No significant relationships were seen between iceA genotypes and the clinical outcomes following infection (p= 0.71). Conclusion: This study revealed a significant two-tailed correlation between iceA genotypes and PUD occurrence. The results indicate that iceA1 gene can be used as a reliable marker in predicting the clinical outcomes of Helicobacter pylori infection. Therefore, further in-vitro and in-vivo investigations are needed for reaching general consensus in this regard.

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Background: Familial hypercholesterolemia (FH) is a disorder with autosomal dominant pattern caused mainly by mutations in the low-density lipoprotein receptor (LDLR) and apolipoprotein B-100. The aim of this study was to investigate the frequency and type of LDLR gene mutations in an Iranian population of patients with high blood cholesterol. Materials and Methods: In this descriptive-lab based study, a total of 50 non-related possible FH subjects from Cheharmahal Bakhtiari were studied. All subjects were tested for presence of LDLR gene mutations in 9 exons of the LDLR gene including 2, 4, 6, 7, 8, 9, 10, 12, and 14. The shifted bands were detected on electrophoresis gels and confirmed by subsequent DNA sequencing method. Results: Overall, four different polymorphisms were identified in 18% of the patients. We found 1413G>A, 1725C>T and 1773C>T, 2140+5G>A in 2,23,2 and 2 subjects respectively from which 1413G >A and 1773C>T were detected in both alleles of the gene. Conclusion: The results did not indicate the involvement of LDLR gene mutations of FH in the samples studied.

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Background: Polymerase chain reaction is the most common technique in the field of molecular biology that use for amplification of a specific nucleic acid sequence. Degenerate primers have ability to amplify related but distinct sequences. The aim of current study was to use, two sets of degenerate primers in combination with Hemi Nested PCR for detection V3-Loop sequence of envelope gene from wide spectrum of Human Immunodeficiency Virus (HIV) subtypes. Material and Methods: In this experimental study we designed and optimized, a degenerate primer pair in combination with Hemi Nested PCR, to detect HIV-1 V3 loop from Envelop gene that has wide variations among genotypes. The developed assay was used to check, 40 HIV infected, 10 negative controls as well as 5 samples from each HCV, HBV, HGV, and TTV were analyzed using developed method. Results: after optimization, 35 out of 40 positive controls were positive using our test. None of the negative human and viral control samples showed specific band. Also, in positive samples, non-specific bands were not detected. Conclusion: In this study moreover than standard PCR, we used two degenerate primers that could detect specific region of genome. In fact, first round of PCR product act as a template for second round inner primers and can produce smaller sequence with high sensitivity due to degeneracy. Based on the current investigation, the developed assay had advantages including product confirmation and hence more sensitivity.

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Background: Type III Interferon (IFN) is a novel member of the interferon family, which contains three ligands: IFN-λ1 (IL-29), IFN-λ2 (IL-28A) and IFN-λ3 (IL-28B).These three ligands use the same unique heterodimeric receptor composed of CRF2-12 (IFN-λ-R1/IL-28Ra) and CRF2-4 (IL10-R-b) chains which are completely different from type I & type II IFN receptors. IFNsλ exhibit several features such as antiviral activity, antiproliferative activity, immunomodulatory activity and in vivo antitumour activity. In this work we aimed to clone the ogene of IFN-λ1 obtained from dendritic cells and assess protein production in eukaryotic expression vector. Materials and methods: in thid experimental study, total RNA was extracted from monocyte derived dendritic cells stimulated with 100 ng/ml of LPS. cDNA was synthesized from total RNA .Then cDNA of IFN-λ1 was amplified by PCR with specific primers and cloned into the PTZ57R/Tvector in the E.coli (DH5α). This was subsequently subcloned into plasmid pcDNA3.1+, using KpnI and BamHI restriction endonucleases. After tranfection into HEK293 T, expression of protein was tested by sandwich-ELISA method. Results: The DNA sequence of the insert was identical to the published sequences encoding IFN-λ1 in GeneBank. It was demonstrated that IFN-λ1 gene was markedly transcribed in transfected cells. Expression of IFN-λ1 in HEK293 T cells was confirmed by sandwich ELISA. Conclusion: Successful cloning and expression IFN-λ1 can be the first step for more production and further investigation about other activities of this cytokine and provides grounds for research on obtaining new therapeutic approaches for cancer, viral, autoimmune and allergic disease and designing more effective vaccines.

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Background: Leishmania major and leishmania tropica are the main causes of cutaneous leishmaniasis in Iran, especially in Isfahan and Bam regions. In this study, noticing the effect of diversity of this parasite strains on designing disease control strategies, human isolates were examined through PCR-RFLP to determine the type of strains. Materials and Methods: In this experimental study, 340 samples obtained from CL patients due to Leishmania were cultured and prepared for microscopic study and examined through PCR-RFLP. The products of some of these samples were sequenced and analyzed. ITS1 region of genomic DNA was extracted and amplified with LITSr and L5.8s primers. Data on sequencing the samples were related to ITS1 region that in extracted DNAs with LITSr and L5.8s primers appeared with four kinds of genotype patterns, two for L.major and two for L.tropica in Isfahan and Bam regions. Results: Genotypic groups, LmA and LmB, were detected from L.major isolates while LtA and LtB genotypic groups were indicated for L.tropica in these two regions. The most prevalent genotypes related to isolates of Isfahan were LmA geneotype, whereas LtA geneotype was mostly reported in isolates of Bam. Conclusion: Leishmania major and leishmania tropica, the causative agents of zoonotic cutaneous leishmaniasis (ZCL) in Isfahan and anthroponotic cutaneous leishmaniasis (ACL) in Bam, respectively, are genetically polymorphic species. There exists a relationship between genetic heterogeneousness and clinical manifestation and geographical regions of this disease in human

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Background: HIV-1 and HCV are two of the most important blood-borne infectious agents. Hence, reliable, precise, and sensitive detection of these viruses in infected patients and donated blood units is highly important. Noticing the limitations of serological assays in detection of these infectious agents, this study was to use fast and sensitive molecular assays like real-time PCR. Materials and Methods: In this trial, a home-brewed SYBR green-based multiplex real time PCR, on the basis of melting curve analysis, was developed for the single or simultaneous detection of HCV and HIV-1 infections in plasma samples. Data were analyzed using SPSS software version 16. Results: The results obtained from different reactions on several clinical samples showed that the analytical sensitivities of the developed assay for HIV-1 and HCV were 200 and 100 copies/ml, respectively. It was also shown that the primers designed for each virus had no interaction with each other and other interfering agents. Conclusion: Noticing the good level of sensitivity and specificity, easy handling, relatively low cost, and rapid analysis of samples, this method can be a useful and rapid approach for simple and effective detection of HCV and HIV-1 in plasma samples.

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Background: Brucellosis is one of the most common zoonotic diseases in the world which imposes a great financial burden on the endemic regions. Diagnosis of the human brucellosis is mainly based on blood culture and serological tests. PCR, however, is recommended for diagnosis at greater specificities and sensitivities. This study aims to compare the diagnosis of human brucellosis by PCR method using l7/l12 and 16srRNA genes and serological tests. Materials and Methods: In a cross-sectional study, a total of 700 blood samples were collected from patients suspected to brucellosis who had referred to the hospitals and laboratories of Ilam, Iran. The samples were selected through Rose Bengal test. Then 50 positive samples diagnosed by Rose Bengal test were assayed by Wright, Coombs Wright, and PCR using l7/l12 and 16srRNA genes and 50 negative samples diagnosed by PCR using these two genes were tested. Results: Of the total 700 samples assayed by Rose Bengal test, 125 were positive and the rest 575 were negative. The 50 positive Rose Bengal samples in PCR were shown to be positive by both genes and 50 negative Rose Bengal samples were shown negative by both samples. 47 samples in Wright test and 49 samples in Coombs test had titration levels above 1:60. Conclusion: PCR method has a higher sensitivity and specificity in diagnosis of human brucellosis in comparison with serological tests. Sensitivity of PCR by l7/l12 gene is similar to16srRNA and can be used for diagnosis of human brucellosis.

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Background: Human brucellosis is a significant public health concern in many countries, including Iran. Therefore, the development of new diagnostic techniques, with high sensitivity and minimum risk of laboratory infection are of great importance. PCR is one of the procedures which has these advantages. However, PCR efficiency is largely dependent on DNA extraction methods. In this study, we studied the efficiency of three different extraction methods of brucella DNA in serum samples. Materials and Methods: In this experimental study, microbial suspensions were initially prepared in saline that its turbidity was equivalent to 0.5 McFarland. Human serum samples were spiked with certain concentrations of Brucella melitensis in vitro. DNA was extracted by three methods and tested by a genus-specific PCR method. Results: Our results showed that the cinneagen kit protocol detected brucella DNA in lower serum concentrations compared with the other protocols. Cinnagen kit could detect brucella DNA in ten-fold dilution in comparison with the other two methods. Conclusion: According to the findings of this study, cinnagen kit was the preferred assay method that yields a better sensitivity for isolation of brucella DNA in serum samples.

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Background: Numerous factors, such as Enterococcus antimicrobial resistance and expression of virulence factors, may account for the maintenance and prevalence of Enterococci infection. The aim of the present study was to assess the occurrence of esp and eep genes in the E.faecalis and E.facium strains isolated from the patients with urethral system infections. Materials and Methods: In this experimental study, 214 clinical samples, including 80 catheters and 134 urine samples, were collected from the patients. The identification of the isolated samples was based on the growth on Bilesculin agar culture media, tolerance of 6.5% Nacl, gram staining, and catalase, hydrolysis of hyporate, telorite reduction, arginine hydrolization, and fermentation of the carbohydrates tests. The assessment of genes was done by PCR method. Results: esp gene was present in 83% of the urine samples and in 97% of the catheters while eep gene was present in 100% of the urine samples and 90% of the urine catheters. The results of antibiogram indicated that the multi-antibiotic resistance was about 78.1% against vancomycin and tetracyclin, 75% against cyprofeloxin and tetracyclin, 59.3% against vancomycin and cyprofeloxin, and about 53% against vancomycin and streptomycin. Conclusion: The findings of this study indicate that esp gene plays an important role in formation of biofilm in patients. Due to the presence of eep gene in almost all of the samples, it can used as a rapid identical agent for the assessment of pheromone production and provision of suitable conditions for plasmid transformation between clinical strains and the prevalence of antibiotic resistance.

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Background: Brucellosis is one of the most common zoonotic diseases in Iran. In most cases, the diagnosis of brucellosis is difficult not only because of its clinical similarity to many infectious and noninfectious diseases, but also because diagnostic methods often fail to detect organisms. PCR is a rapid and safe diagnostic method applied to the diagnosis of brucellosis. The purpose of this study was to determine the sensitivity and specificity of PCR for diagnosis of human brucellosis by using serum samples. Materials and Methods: This study which was done to evaluate diagnostic tests included30 serum samples from patients with clinical presentation of brucellosis with positive Wright test and serum samples of30 healthy people with negative Wright test. These samples were examined by PCR. Results: PCR results were positive for 15 samples of the patients group in comparison with 4 samples from the 30 healthy subjects. The sensitivity and specificity of PCR were 50% and 86.6%, respectively. Conclusion: Although in some studies, the preferred sample for diagnosis of brucellosis was serum, in this study, PCR on serum samples did not indicate high sensitivity and specificity in diagnosis of brucellosis. Hence, using a combination of methods for diagnosis of human brucellosisis suggested.

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Background: Cutaneous leishmaniasis is a health problem in many areas of Iran. Cutaneous leishmaniasisis reported mostly in the central county of Qom province, including Ghanavat and Qomrood villages. This study was done to identify parasite species in human and rodents in order to illustrate the epidemiologic picture of the disease and provide an appropriate control program in 2010 Materials and Methods: This cross-sectional study was done on microscopic smears of 45 human samples and rodents samples hunted around Ghanavat and Qomrood villages in the central county of Qom province in 2010.In total,15 human samples and one hunted rodent sample were positive. In this study,the DNA of the parasites were extracted from the slides and analyzed by Leishmania specific premiers using ITS1 PCR-amplification (Internal Transcribed Spacer1). PCR (PolymeraseChain Reaction) products were digested by Haelll enzyme. Results: Overall, 15 human samples and one rodent sample from Merioneslibycus species were evaluated by PCR-RFLP (Restriction Fragment Length Polymorphism). After electrophoresis, it was demonstrated that the parasite was Leishmaniamajor in both human and rodent species. Conclusion: PCR-RFLP technique is an effective method to determine Leishmania parasite species in Geimsa stained slides from human and rodent reservoirs. One of the advantages of this technique is that it is possible to recognize the pathogen species of Leishmania parasite without gene sequencing. Besides, PCR-RFLP technique is a method of quite high sensitivity and specificity which can identify parasite species in addition to the diagnosis of leishmaniasis within 24 hours.

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Background: Reports on microsporidia infections are increasing and some species, such as Enterocytozoon bieneusi and Encephalitozoon intestinalis, have become important causes of chronic malabsorptive diarrhea, especially in HIV infected patients. In this study, Modified Trichrome-Blue (MTS) and Acid-Fast Trichrome (AFT) staining methods as well as PCR technique were used for detecting microsporidia in stool specimens. Materials and Methods: In this experimental study, a total of 71 stool specimens from AIDS patients with chronic diarrhea were collected and sent to laboratory. Two slides were prepared for each stool specimen. The slides were fixed with methanol, stained with MTS and AFT stain, and read by at least three individuals. In addition, PCR with primers directed to a conserved region of the 16s rRNA of intestinal microsporidian spores was used. Results: Totally of 71 patients, 13 patients (18.30%) were positive for microsporidia by MTS and AFT stain methods. In addition, 9 patients (12.67%) were positive for cryptosporidium by AFT stain and 4 (5.63%) of them were positive for microsporidia. Furthermore, 16 patients (22.53%) were positive for intestinal microsporidiosis by PCR technique. Notably, all cases that were positive for microsporidia by staining methods were also positive for PCR technique as well Conclusion: PCR technique was more sensitive than staining methods. Also, MTS and AFT stain methods were equally useful in the diagnosis of microsporidiosis.

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Background: Vibrio cholerae is a gram-negative bacterial pathogen that causes diarrheal disease cholera. One of the most pathogenic factors of Vibrio cholera is pili. Pili plays an important role in colonization and persistence of bacteria in small intestine. Materials and Methods: In this study, pili A (tcpA) gene was amplified by Polymerase chain reaction (PCR) method and sub-cloned into expression vectors such as pGEX4T-1. Escherichia coli competent cells were transformed by recombinant plasmids and the expression of protein with IPTG. The recombinant proteins were purified by affinity chromatography (GST) and immunoblot analysis was used for evaluation of new recombinant proteins antigenicity. The concentration of recombinant proteins was measured according to Bradford assay. Results: The results of this study indicated that recombinant proteins were expressed successfully in competent cell of E. coli, such as E. coli BL21 (DH3). The recombinant protein was purified by affinity chromatography (GST). The immunoreactivity pattern of anti-Tcp antibody with recombinant proteins of TcPA showed that the recombinant proteins had antigenic properties. Conclusion: Because these recombinant proteins are antigenic, these proteins may be considered as tentative candidates for designing cholera vaccine.

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Background: There is abundant evidence indicating that inflammatory mechanisms within the central nervous system contribute to cognitive impairment via cytokine-mediated interactions between neurons and glial cells. BAT1, a member of the DEAD-box family of RNA helicases, appears to regulate the production of inflammatory cytokines associated with AD pathology. In the current study BAT1 -22 promoter polymorphism was analyzed in AD and control subjects.

Materials and Methods: In this case-control study, genomic DNA from peripheral blood samples of 153 Alzheimer’s patients and 153 healthy controls was extracted using salting-out method. DNA analysis was performed by PCR-RFLP method and p<0.05 was considered statistically significant.

Results: After genotyping and statistical analysis the results failed to show any association between BAT1 -22 promoter polymorphism and sporadic Alzheimer’s disease.

Conclusion: BAT1 -22 is not associated with Alzheimer’s disease in Iranian population and so has no effect on predisposition to sporadic Alzheimer’s disease.

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Background: Vulvovaginal candidiasis is a common problem in women. The purpose of this study was to compare two identification methods new PCR analysis (with DNA extracted from samples) and conventional culture technique in detection of Candida species in vulvovaginal samples.

Materials and Methods: In this experimental-analytical study, 150 women of fertility ages participated and two vaginal discharge samples were collected by speculum. One sample used for direct DNA extraction as well as PCR and the other was used for culture and phenotypic evaluations. Phenotypic evaluations waere performed by germ tube and chlamydospore formation and specially culture in chrome agar medium to detect color of the colonies. PCR was performed by DNA extracted from samples as templates and finally size of Candida specific amplicons was determined.

Results: From 150 samples, 87 in culture and 127 in new PCR technique were positive. In culture method, from total 87  Candida species , 73 C. albicans, 12 C. glabrata and 2 C. tropicalis were isolated whereas in new PCR technique, from  total 127 candida species 107  C. albicans, 18 C. glabrata and 2 C. tropicalis were identified. Concordance of the two methods were calculated as 68 percent.

Conclusion: The new molecular method (innovative PCR) has the potential to rapidly and accurately diagnose Candida vulvovaginitis in patients and can be used for diagnosis of  Candida species in clinical specimens.

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Background: Listeria is a gram positive bacterium, facultive intracellular pathogene. Prf A gene has an important role in virulence. The purpose of this study was to determine of the presence of Listeria monocytogenes in various meat products and presence of prf A gene in bacteria isolated from food samples and compare them with clinical samples and standard samples.

Materials and Methods: During 6 months of 60 samples of beef, chicken, ham, cocktail, sausage, from a butchery’s Khoramabad and shops in Khoramabad and Tehran were collected. L. monocytogenes was isolated according to cold enrichment method and prf A gene were analyzed by PCR (polymerase chain reaction) method. Statistical analysis was performed with the software SPSS (statistical package for social science).

Results: From 60 samples of meat and meat products, 8 (13.3%) were positive for Listeria spp. Among in these samples, 4 cases of L. monocytogenes (6.6%), 3 cases (5%), L. innocua and 1 case (1.6%) L. welshimri, were isolated which L. innocua was isolated from meat and poultry samples and L. monocytogens from meat, chicken, ham and L. welshimeri from meat were isolated. Prf A gene was detected in isolated L. monocytogenes and donated bacteria from dairy products, clinical and standard samples, 2 cases of donated samples of vegetable contained prf A genes.

Conclusion: The prf A gene (activator of transcription and regulators the expression of other virulence genes), in the studied samples were observed. This gene plays a role in pathogenesis. Because these bacteria are transmitted through food and is a serious threat to public health. Thus identification of bacteria especially its genes could be possible to find some ways to prevent the bacteria and avoid disease from it.

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Background: Preeclampsia (PE) is a serious problem of pregnancy and its etiology is still unknown. The inheritance of preeclampsia is one of the theories regarding to the etiology of preeclampsia. Methylenetetrahydrofolatereductase (MTHFR) is a key enzyme in folate metabolism and the C677T polymorphism of the MTHFR gene is associated with decrease MTHFR activity, and therefore cause higher blood levels of homocysteine and leads to vascular disease that can be the reason of preeclampsia. The aim of this study was to evaluate the relationship between MTHFR gene C677T polymorphism with PE development in south-west of Iran.

Materials and Methods: This case-control study was performed in 129 preeclamptic pregnant women and 125 control individuals.The C677T polymorphism of the MTHFR gene was determined by PCR-RFLP method.

Results: The CC, CT and TT genotypes frequency of C677T polymorphism of MTHFR gene were 57.4, 38.8 and 3.9 percent in preeclamptic women and 53.6, 40 and 6.4 percent in control group. They were not significantly different (p=0.614). However, the frequency of TT genotype was higher in control group (p=0.36). There was not any significant difference in T allele distribution between preeclamptic women (23.3%) and control group (26.4%).

Conclusion: Our results showed that there was not any correlation between the C677T polymorphism and PE but the TT genotype of C677T polymorphism seems to be a protective factor for preeclampsia.

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Background: Allergy is regarded as a multifactorial condition that its onset and severity are influenced by both genetic and environmental factors. Hence, identification of genetic factors involved in allergic rhinitis development and its related phenotypes is a major task in understanding the genetic background of allergic rhinitis. This study was designed to examine the association between IL-18 -607 A/C promoter polymorphism on chromosome 11q22 and allergic rhinitis.

Materials and Methods: In this analytic study, genomic DNA was obtained from the blood samples of 293 patients with allergic rhinitis and 218 healthy controls by standard phenol chloroform method. The IL-18/-607 A/C polymorphism was analyzed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. To analyze the association between genotypes and alleles and the disease in the case group compared with the control group, X2 test was used.

Results: The frequency of the AC genotype of the IL-18/-607 A/C gene polymorphism was significantly greater in allergic rhinitis patients than in controls (p<0.05). By comparing the frequency of AA genotype with other genotypes, OR was calculated as 2.03.

Conclusion: The results suggest that IL-18/-607 A/C polymorphism gene may be one of the factors participating in the pathogenesis of AR or its intermediary phenotypes.

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Background: The Real-time PCR assay has been established as the standard method for Human Cytomegalovirus (HCMV) quantitation in immunocompromised patients. However, the question of which one of whole blood or plasma specimens is better for viral quantitation is still unresolved for many clinical laboratories. To answer this question, the current study compares HCMV DNA load in whole blood and plasma samples.

Materials and Methods: In this prospective study, the whole blood and plasma samples were obtained from 41 transplantated patients and the viral load was detected using a validated, in-house Real-time PCR assay.

Results: Of the total 193 examined specimens, 174 were negative and 19 samples, from 16 patients, were positive in at least one of whole blood or plasma samples. Based on the results of linear regression analysis, the cytomegalovirus viral load was correlated in whole blood and plasma samples (R2: 0.872). However, the regression equation shows that the HCMV load in whole blood samples is higher than load of this virus in plasma. The validity of the quantitative results was confirmed by repeating the tests and analyzing the results using the repeated measure analysis.

Conclusion: Based on the results of the present study, HCMV quantitation in whole blood samples has a higher analytical sensitivity than in plasma samples.