Enzymes HinfI Research Articles

Genetic Screening of FecXG Polymorphism in Saanen Goat (Capra hircus) Breed in Türkiye

Determination of fecundity gene mutations and utilizing them in farm animals programs offers the opportunity to improve productivity. The BMP15/FecX gene is one of the candidate genes with significant effects on multiple births in sheep. Studies in small ruminant have shown that BMP15 gene mutations increase the rate of multiple births, although the effect of BMP15 gene mutations varies at the breed level. Although there are many studies on sheep fecundity in Türkiye, there are no studies on goat. Therefore, the objective of the current study was to investigate FecXG mutation in the exon 2 region of BMP15 gene in Saanen goats (Capra hircus). A total of 24 samples were used to investigate the FecXG mutation in Saanen goats raised in the Muş Plain of Türkiye. A fragment of 141 bp of BMP15 gene was amplified by PCR and then products subjected to the digestion of restriction enzyme HinfI. This preliminary study’s findings showed that there is no FecXG mutation in Saanen goats.

Molecular approach for identification and validation of Eurycoma longifolia, Labisia pumila, and Orthosiphon stamineus in herbal products

BackgroundEurycoma longifolia (E. longifolia), Labisia pumila (L. pumila), and Orthosiphon stamineus (O. stamineus) are popular species known for their therapeutic properties. An increase in local demand for herbal products makes them susceptible to adulteration, which poses a risk to their safety and efficacy. Current identification methods, such as organoleptic, microscopic, and macroscopic analysis, need to be revised to identify plant species in highly processed herbal products due to their limited ability to detect morphological features and provide comprehensive plant taxonomy information.MethodsThis research objective was to develop a simple, reliable, and accurate DNA molecular identification method based on polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) for E. longifolia, L. pumila, and O. stamineus, used to validate the species identification for herbal products. PCR–RFLP was developed for rapid identification using restriction enzymes TaqI, BamH I, HinfI, EcoRI, EcoRV, Mbol, and Mspl.ResultsThe nuclear DNA internal transcribed spacer 2 (ITS2) sequences were identified and compared between plant specimens of E. longifolia, L. pumila, and O. stamineus and 101 samples of commercial herbal products. Plant specimens of E. longifolia, L. pumila, and O. stamineus were successfully identified with high similarity of 100%, 100%, and 99.33%, respectively, based on National Center for Biotechnology Information (NCBI) GenBank. The recovery of DNA sequences from the herbal products was 60.4%, of which 81.97% were identified, and 18.03% showed no sequence through Basic Local Alignment Search Tool (BLAST) identification.ConclusionA reliable approach for identifying and validating plant species in herbal products has been created using restriction enzymes. This simple and accurate PCR–RFLP approach efficiently identifies E. longifolia, L. pumila, and O. stamineus by analysing ITS2 sequences, assuring consumer health and safety.

Association of the polymorphism of the Pon-1 locus with the concentration of Paraoxonase-1 and post-thaw sperm viability in pig

The objective of the present work was to correlate the expression of the PON-1 locus polymorphism, the concentration of the enzyme paraoxonase 1 in seminal plasma and the effect on post-thawing sperm viability in the pig. Nine ejaculates from 3 boars were used, to obtain seminal plasma; part of the ejaculates was centrifuged at 800 g/10 min; the material obtained was stored at -20°C until use. To freeze the semen, the samples were diluted 1:1 in commercial diluent (Magapor®) at 37°C and the temperature was gradually lowered to 16°C. They were transported to the laboratory protected from light, and kept at the same temperature in the laboratory for 24 h. After this, they were frozen using the glycerolation technique and a portion was cryopreserved by adding 20% of seminal plasma to the conservation medium and another portion was made without seminal plasma. The concentration of PON-1 was determined by an enzyme-linked immunosorbent assay (ELISA). The functional status of the plasma membrane was evaluated by chlortetracycline staining. The amplification of a fragment of the PON-1 gene was carried out by PCR and the identification of allelic variability by the analysis of Restriction Fragment Length Polymorphism (RFLP), with the enzymes HinfI and FokI, from a DNA sample of each individual. The results were analyzed by analysis of variance (ANOVA) and Spearman correlation, with the use of statistical software (Statistica V10 for Windows). In electrophoresis of enzymatic digestion with the enzyme HinfI All the expected fragments were obtained, with the FokI enzyme a mutation was observed in one individual. No correlation was observed (R=-0.1, P>0.05) between the concentration of the paraoxonase-1 enzyme in seminal plasma and the observed mutations. As well as the concentration of the enzyme paraoxonase-1 in the ejaculates was 1.8 to 2.7 ng/mL and this had no statistically significant difference in the seminal plasma of the ejaculates analyzed (P>0.05). As well as with sperm viability in thawed semen (P>0.05).

ПОРОДНІ ОСОБЛИВОСТІ АЛЕЛЬНОГО ПРОФІЛЮ ГЕНІВ PIT1 ТА GH УКРАЇНСЬКОЇ АБОРИГЕННОЇ ЛЕБЕДИНСЬКОЇ ПОРОДИ КОРІВ

The preservation of genetic diversity is a paramount concern in addressing global challenges today. The foundation of biological diversity hinges on its genetic components, and the loss of species and genetic variability poses a genuine threat to the biosphere. This is because the sustainability of natural ecosystems and agroecosystems depends directly on their genetic adaptability to changing environmental conditions.
 In cattle, genes associated with milk yield play a crucial role, including genes related to milk proteins and hormones. Among these, the pituitary-specific transcription factor (PIT1) and growth hormone (GH) genes are of particular significance. In this study, we investigated the polymorphism of these genes in the Lebedyn breed of cattle.
 A total of 2 gene polymorphisms, PIT1 and GH, were examined through molecular and genetic research conducted at the genetics laboratory of the Institute of Animal Breeding and Genetics named after M. V. Zubets, part of the National Academy of Agrarian Science of Ukraine. The study utilized 32 DNA samples extracted from the venous blood of Lebedyn breed cows using the 'DNA Sorb-B' kit from AmpliSens. Genotyping was carried out through polymerase chain reaction (PCR-RFLP) analysis of restriction fragment length polymorphism, employing the “Tertsyk” amplifier for PCR. The resulting DNA restriction analysis products were assessed via electrophoresis in a 2–3% agarose gel containing ethidium bromide, with DNA fragment lengths determined using the GeneRuler TM 50 bp DNA Ladder and Thermo Scientific™ GeneRuler 1 kb Plus DNA Ladder markers.
 For the PIT1 gene analysis, a 451 bp amplified fragment was treated with the enzyme HinfI, yielding the following allele frequencies: A (0.38) and B (0.62). The heterozygous AB genotype was the most prevalent (0.45), while homozygotes for the A allele constituted 0.15 of the population. In the case of the GH gene fragment (223 bp), hydrolysis was performed using the restriction enzyme AluI. Analysis of allele frequency distribution indicated a high frequency of the L allele (0.85), with no animals exhibiting the homozygous GHVV genotype, and the GHLL genotype was present in 0.7 of the population.
 These findings reveal unique features of the allelic spectrum of the PIT1 (V-0.62) and GH (L-0.85) genes specific to the Ukrainian indigenous Lebedyn breed of cows. Selectionally significant genotypes include PIT1AA (15%) and GHLL (70%), while heterozygote carriers, which contribute to genetic diversity within the population, account for 45% and 30%, respectively.
 This study’s results hold particular value due to the alarming decline in the population of local indigenous breeds and the imminent threat to the preservation of agricultural genetic resources.

Identification of SNP g.10428C>T of Stearoyl-CoA Desaturase gene related to meat quality in Bali cattle using PCR-RFLP method

The objectives of this research was to analyze the polymorphisms of SNP g.10428C>T in the SCD gene using PCR-RFLP and its association with meat charactheristics in Bali cattle. The number of 52 Bali cattle from BPTU-HTP Denpasar were used in this study and which were compared to Belgianblue (n=30), Limousine (n=14), Peranakan Ongole (PO) (n=30) and Wagyu (n=7). The polymorphism of the SCD gene was characterized by the use of the PCR-RFLP technique with HinfI enzyme restriction. The characteristics of the meat quality in Bali cattle were estimated using ultrasound imaging method. Genotyphic and allelic frequencies, heterozygosity values, and the Hardy-Weinberg equilibrium have been calculated using PopGen 1.32. The association between genotypes of SNP g.10428C>T SCD gene and meat quality characteristics was calculated using t-test. The results showed that homozygous genotypes (CC and TT) and heterozygous genotypes (CT) of the SCD|HinfI gene with C (0.26) and T (0.74) allele frequencies were and in Bali cattle. The CC genotype was found in Belgian-blue, Limousine, PO, and Wagyu cattle with the allele C frequency of 1.00. The observed heterozygosity (Ho=0.44) higher than expected heterozygosity (He=0.39) showed high diversity in Bali cattle. Genotypes of SNP g.10428C>T SCD gene were associated with backfat thickness, marbling score, and percentage of intramuscular fat. Thus the SNPs g.10428C>T have potential as marker-assisted selection candidates for Bali cattle.

Identification of polymorphism of FecX gene in indigenous sheep of Meghalaya

Fecundity gene such as FecX, govern fertility and litter size in sheep. Selection of animals with the help of molecular markers through identification of polymorphism of fecundity genes is an impactful method of genetic improvement. The present study was carried out to identify the polymorphism of FecX gene in indigenous sheep of Meghalaya. Blood samples were collected from 50 randomly selected ewes from 2 districts of Meghalaya namely, East Khasi Hills and West Khasi Hills. Genomic DNA was extracted from the samples and PCR amplification of FecX gene with specific primer set was done. PCR product size of 141 bp was obtained. Further, digestion of the PCR product with restriction enzyme HinfI through PCR-RFLP technique was conducted to identify the variants in FecX gene. Upon digestion, 2 types of fragment pattern identified, arbitrarily designated as AA and AB genotype, where AA genotype yielded one fragment (141 bp) and AB genotype yielded 2 fragments (100 and 41 bp). Presence of polymorphism can be an important indicator for selection with the help of molecular markers.

De Novo SNP Discovery and Genotyping of Iranian Pimpinella Species Using ddRAD Sequencing

The species of Pimpinella, one of the largest genera of the family Apiaceae, are traditionally cultivated for medicinal purposes. In this study, high-throughput double digest restriction-site associated DNA sequencing technology (ddRAD-seq) was used to identify single nucleotide polymorphisms (SNPs) in eight Pimpinella species from Iran. After double-digestion with the enzymes HpyCH4IV and HinfI, a total of 334,702,966 paired-end reads were de novo assembled into 1,270,791 loci with an average of 28.8 reads per locus. After stringent filtering, 2440 high-quality SNPs were identified for downstream analysis. Analysis of genetic relationships and population structure, based on these retained SNPs, indicated the presence of three major groups. Gene ontology and pathway analysis were determined by using comparison SNP-associated flanking sequences with a public non-redundant database. Due to the lack of genomic resources in this genus, our present study is the first report to provide high-quality SNPs in Pimpinella based on a de novo analysis pipeline using ddRAD-seq. This data will enhance the molecular knowledge of the genus Pimpinella and will provide an important source of information for breeders and the research community to enhance breeding programs and support the management of Pimpinella genomic resources.

Sığır Kappa-Kazeinin A ve B Genetik Varyantlarının PCR-RFLP Analizi için Yeni Bir Primer Dizaynı

İnek sütü yaklaşık %3,4 oranında yüksek kaliteli protein içerir ve insan beslenmesi için oldukça elzemdir. Kappa (κ)kazein, süt proteinlerinden birisidir ve CSN3 geni tarafından kodlanmaktadır. Çalışmalar κ-kazeinin sütün özellikleri veişlenmesi üzerine önemli bir etkiye sahip olduğunu göstermektedir. κ-kazein sütün kesilmesi sürecinde stabilize edici birfaktör olarak rol almaktadır; bu durum, κ-kazeini peynir fabrikalarında istenilir hale getirmektedir. Kappa (κ) kazeinin A ve Bgenetik varyantları iyi bilinmekte ve yoğun çalışılmaktadır. Sığırların genotiplenmesi için PCR-RFLP yöntemi kullanılmaktadır.Bu amaçla, farklı primer çiftleri CSN3 geninin polimorfik bölgesini çoğaltmak için kullanılmaktadır. Önceki bir çalışmada,polimorfik bölgenin HinfI enzimi ile kesimi kısa ve benzer uzunlukta fragmentler oluşturdu. Agaroz jel elektroforezinde çokbenzer DNA fragmentlerinin ayrımı neredeyse imkansızdır ve kısa DNA fragmentlerin yorumlanması bazen zorluklara sebepolmaktadır. Bu sebeple, bu çalışmada yeni bir primerin dizaynı tanımlandı. Yeni primerin kullanılamasıyla daha uzun DNAfragmenti başarılı bir şekilde çoğaltıldı ve bu PCR ürünlerinin HinfI kesimi, agaroz jel elektroforezinde kolaylıkla ayrılabilen veyorumlanabilen çok farklı uzunlukta DNA fragmentlerinin oluşmasını sağladı. Bu çalışmada tanımlanan primer, allel frenkansaraştırmaları ve yetiştirme stratejileri ile ilgili gelecek çalışmalarda kullanılabilir.

Anisakid nematode species identification in harbour porpoises (Phocoena phocoena) from the North Sea, Baltic Sea and North Atlantic using RFLP analysis.

Harbour porpoises (Phocoena phocoena) are the only native cetacean species in the German North and Baltic Seas and the final host of Anisakis (A.) simplex, which infects their first and second gastric compartments and may cause chronic ulcerative gastritis. Anisakis simplex belongs to the family Anisakidae (Ascaridoidea, Rhabditida) as well as the phocine gastric nematode species Pseudoterranova (P.) decipiens and Contracaecum (C.) osculatum. These nematode species are the main causative agents for the zoonosis anisakidosis. The taxonomy of these genus with life cycles including crustaceans and commercially important fish is complex because of the formation of sibling species. Little is known about anisakid species infecting porpoises in the study area. Mature nematodes and larval stages are often identifiable only by molecular methods due to high morphological and genetic similarity. The restriction fragment length polymorphism (RFLP) method is an alternative to sequencing and was applied to identify anisakid nematodes found in harbour porpoises from the North Sea, Baltic Sea and North Atlantic to species level for the first time. In the study areas, five gastric nematodes from different harbour porpoise hosts were selected to be investigated with restriction enzymes HinfI, RsaI and HaeIII, which were able to differentiate several anisakid nematode species by characteristic banding patterns. Anisakis simplex s. s. was the dominant species found in the North Sea and Baltic porpoises, identified by all three restriction enzymes. Additionally, a hybrid of A. simplex s. s. and A. pegreffii was determined by HinfI in the North Sea samples. Within the North Atlantic specimens, A. simplex s. s., P. decipiens s. s. and Hysterothylacium (H.) aduncum were identified by all enzymes. This demonstrates the value of the RFLP method and the chosen restriction enzymes for the species identification of a broad variety of anisakid nematodes affecting the health of marine mammals.

Food Intervention with Folate Reduces TNF-α and Interleukin Levels in Overweight and Obese Women with the MTHFR C677T Polymorphism: A Randomized Trial

Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism associated with body fat accumulation could possibly trigger an inflammatory process by elevating homocysteine levels and increasing cytokine production, causing several diseases. This study aimed to evaluate the effects of food intervention, and not folate supplements, on the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in overweight and obese women with the MTHFR C677T polymorphism. A randomized, double-blind eight-week clinical trial of 48 overweight and obese women was conducted. Participants were randomly assigned into two groups. They received 300 g of vegetables daily for eight weeks containing different doses of folate: 95 µg/day for Group 1 and 191 µg/day for Group 2. MTHFR C677T polymorphism genotyping was assessed by digestion with HinfI enzyme and on 12% polyacrylamide gels. Anthropometric measurements, 24-h dietary recall, and biochemical analysis (blood folic acid, vitamin B12, homocysteine (Hcy), TNF-α, IL-1β, and IL-6) were determined at the beginning and end of the study. Group 2 had a significant increase in folate intake (p < 0.001) and plasma folic acid (p < 0.05) for individuals with the cytosine–cytosine (CC), cytosine–thymine (CT), and thymine–thymine (TT) genotypes. However, only individuals with the TT genotype presented reduced levels of Hcy, TNF-α, IL-6, and IL-1β (p < 0.001). Group 1 showed significant differences in folate consumption (p < 0.001) and folic acid levels (p < 0.05) for individuals with the CT and TT genotypes. Food intervention with folate from vegetables increased folic acid levels and reduced interleukins, TNF-α, and Hcy levels, mainly for individuals with the TT genotype.

A new phytoplasma strain associated with the sugarcane white leafdisease in Vietnam

Sugarcane white leaf (SCWL) disease is one of the most important diseases of sugarcane in Vietnam. The presence of phytoplasmas in phloem tubes of infected plants was confirmed by DAPI staining. A nested PCR assay using R16mF2/R16mR1 and R16F2n/R16R2 primer pairs amplified products with approximate length of 1,250 bp from the symptomatic sugarcane samples. The sequence analysis indicated that the phytoplasma strains from Khanh Hoa province shared 100% sequence identity with previously sequenced SCWL phytoplasma strains that were related to ‘Candidatus Phytoplasma oryzae’ (16SrXI). The virtual RFLP patterns of a 1,127 bp sequence obtained with the restriction enzymes BfaI, BstUI, EcoRI, HaeIII, HinfI, HpaI, RsaI and TaqI indicate that this SCWL-associated phytoplasma from Vietnam is a different strain from those in the 16SrXI described ribosomal subgroups. The two insect species, Matsumuratettix hiroglyphicus and Yamatotettix flavovittatus, identified as the vectors of SCWL in Thailand, were identified and found to be infected also in the SCWL symptomatic fields in Vietnam. The transmission experiments from SCWL-infected leafhoppers to healthy sugarcane and to insect feeding medium failed, suggesting that the infected planting material is the major transmission manner of SCWL disease in Vietnam.

Molecular Typing of ccrB Gene in Methicillin-resistant Staphylococcus aureus by Restriction Fragment Length Polymorphism

Background: Staphylococcus aureus is one of the most important pathogens acquired from the hospital and community. Increasing the resistance of S. aureus to antibiotics is a major health concentration, and thus the study of antibiotic resistance in S. aureus is very important. The aim of this study is to determine the typing of methicillin-resistant S. aureus (MRSA) in the region of the ccrB gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Methods: One hundred and six S. aureus were isolated from urine, blood, sputum, wound, and the trachea of patients hospitalized in Tehran during (March-April) 2016. Antibiotic susceptibility test was done by the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI). In addition, molecular typing for staphylococcal cassette chromosome mec (Sccmec) type I-V was performed in MRSA isolates, followed by conducting PCR-RFLP by restriction enzymes BsmI and HinfI in the ccrB gene area. Results: PCR and typing showed that type II SCCmec was 40% (n=20), followed by types III (28, 56%), IVc (12, 24%), I (11, 22%), V (9, 18%) IVa (7, 14%), and IVb (5, 10%). However, SCCmec type IVd was not observed in the isolates. Finally, after the amplification of ccrB gene and RFLP, all isolates the same as the typing method represented types I, II, III, IVa, IVb, and IVc while no type V was detected by this method. Conclusions: The results of this study demonstrated that SCCmec (type I-IV) can be detected by PCR-RFLP in the ccrB gene, but this method identified no type V SCCmec in MRSA

Diversity of Bradyrhizobium strains isolated from root nodules of the shrubby legume Cytisus villosus growing in the Moroccan Rif

Abstract The genotypic and phenotypic diversity of nineteen bradyrhizobial strains isolated from the root nodules of wild-grown Cytisus villosus in the central-western region of the Moroccan Rif were carried out. PCR-based RFLP (restriction fragment length polymorphism) of the 16S rDNA (ARDRA) of 19 strains using the restriction enzymes AluI, CfoI, DdeI, HinfI, MspI and RsaI showed that they grouped in 3 clusters. The cluster I includes the type strain Bradyrhizobium canariense BTA-1T, the cluster II the type strain Bradyrhizobium japonicum USDA6T and the cluster III the type strains Bradyrhizobium cytisi CTAW11T and Bradyrhizobium rifense CTAW71T. The results from 63 physiological and biochemical tests confirmed the existence of the B. canariense and the B. japonicum groups but also allowed the separation of the B. cytisi and B. rifense strains in two groups. The phenotypic characteristics analysed permitted description of a wide physiological and biochemical diversity among the strains, and showed that the heavy metals resistance test was the most discriminating characteristic.

Investigation of the association between C677T polymorphism of the MTHFR gene and plasma homocysteine level in recurrent fetal miscarriage.

The aim of this study is to determine the association between C677T polymorphism in MTHFR gene and plasma homocysteine concentration in recurrent fetal miscarriages. Overall, 100 women were included in the research, the case group comprised of 50 women who had a history of spontaneously recurrent miscarriage with unspecified cause, and 50 of whom had experienced at least two successful pregnancy, as controls. Methods used in the study included PCR-RFLP with limited effective HinfI enzyme in order to investigate MTHFR polymorphism and enzyme-linked immunosorbent assay analysis to investigate plasma homocysteine concentration. There was a significant increase in the prevalence of mutant TT genotype among women with miscarriage history. Also, the mean homocysteine level in the case group was significantly higher than that in the control group (P = 0.002) and higher level of homocysteine was found in the carriers of T allele. Our data suggest that C677TT genotype may be a risk factor for miscarriage and CC wild type genotype supposed to have protective effect on hyperhomocysteinemia.

Polymorphisms of Growth Differentiation Factor 9 (GDF 9)and Bone Morphogenetic Protein 15 (BMP15) Genes in Barki and Rahmani Sheep Breeds

Blood samples from 100 ewes (Barki and Rhmani breeds, 50 each) were collected from a private farm at Giza Governorate for determination of polymorphisms of bone morphogenetic protein 15 (BMP15) and the growth differentiation factor 9 (GDF9) genes in the two breeds using PCR-RFLP and DNA sequencing.Restriction analysis of 712 bp GDF9 gene (amplicon of exon I) using HhaI enzyme revealed 120, 254 and 338 fragments without any differences between the two breeds being tested. GDF9 gene exon II amplicon(713 bp) showed similar pattern of restriction in the two tested breeds with 4 bands (54, 62, 137 and 460bp) using HinfI enzyme. The restriction enzymes, HinfI, SpeI and XbaI, failed to digest the amplicons of exons I and II(500 bp, each) of BMP15 gene in all tested animals of both breeds suggesting absence of polymorphism. This study concluded that there was no polymorphism in the four exons between Barki and Rahmani sheep and the two breeds have the wild genotype with absence of any mutation.

Identification of Forensically Important Flesh Flies Using the Cytochrome C Oxidase Subunits I and II Genes.

Forensically important flesh flies (Diptera: Sarcophagidae) often are not morphologically distinguishable, especially at the immature stage. In addition, female flies are quite similar in general morphology, making accurate identifications difficult. DNA-based technologies, particularly mitochondrial DNA (mtDNA), have been used for species-level identification. The cytochrome oxidase subunits I and II (COI-COII) sequences of Iranian Sarcophagidae are still unavailable in GenBank. In this study as many as 648 (540 males and 106 females) fly specimens from family Sarcophagidae, representing 10 sarcophagid species, including eight forensically important species were collected from seven locations in five Iranian provinces. Of these, 150 male specimens were identified based on both morphology of male genitalia and DNA sequencing analysis. Sequence data from the COI-COII regions for 10 flesh fly species collected in Iran were generated for the first time. Digestion of COI-COII region by restriction enzymes RsaI, EcoRV, and HinfI provided distinct restriction fragment length polymorphism profiles among the species and can serve as molecular markers for species determination. Phylogenetic analysis represented that the COI-COII sequences are helpful for delimitation of sarcophagid species and implementation in forensic entomology. However, the application of the COI-COII fragment as a species identifier requires great caution and additional species and markers should be studied to ensure accurate species identification in the future.

A Survey of gyrA Target-Site Mutation and qnr Genes among Clinical Isolates of Escherichia coli in the North of Iran

Background: Urinary tract infection (UTIs) caused by Escherichia coli is one of the most common human complications. The discriminate use of antibiotics leads to increasing multiple-drug resistance (MDR) in E. coli. Objectives: This study aimed at investigating the frequency of fluoroquinolones resistance by detecting qnr genes and mutation in gyrA gene in clinical isolates of E. coli in the North of Iran. Methods: This cross-sectional study was conducted on 309 E. coli isolates collected from major hospitals in Rasht, North of Iran. All isolates were identified by standard microbiological methods. Antimicrobial susceptibility testing was conducted by the disk diffusion method for the three quinolone antibiotics. The detection of gyrA, qnrS, qnrB, and qnrA genes was performed by the polymerase chain reaction (PCR) method. PCR products of gyrA gene were digested by HinfI enzyme to identify the mutation in gyrA gene by restriction fragment length polymorphism (RFLP) method. Results: Of 309 tested E. coli isolates, the most common resistance was observed against nalidixic acid (60.2%) and the least common one against norfloxacin (40.5%). The prevalence of qnrS, qnrB, and qnrA genes was 4.5%, 73.1%, and 62.8%, respectively. Mutation in gyrA gene was observed in codon 83 in 56.3% of the strains. Conclusions: In summary, despite the significant association of the investigated genes with fluoroquinolones resistance in these bacteria, the presence of these genes in susceptible strains suggested that some other possible mechanisms may also influence resistance against fluoroquinolones.

Typing of Campylobacter jejuni isolated from poultry on the basis of flaA-RFLP by various restriction enzymes

RFLP analysis of the flagellin (flaA) gene was compared using three different restriction endonucleases i.e DdeI, HinfI and DpnII to determine the genetic diversity among 43 Campylobacter jejuni isolates of poultry origin from the same geographical area. flaA gene was amplified in all the isolates and RFLP analysis showed variations. Dde-based RFLP was found most efficient in discriminating C. jejuni isolates by generating 15 different Dde-RFLP patterns with discriminatory index (D.I) of 0.9258 whereas DpnII produced seven Dpn-RFLP patterns (D.I .= 0.8427). While HinfI enzyme produced only six Hinf-RFLP patterns (D.I .= 0.6977). The discrimination of Dpn-RFLP was comparable to discrimination given by Dde-RFLP analysis, which is generally used to study flaA gene RFLP.

Association of MTHFR polymorphism and periodontitis’ severity in Indonesian males

Periodontitis is an oral disease with a complex etiology and pathogenesis, but with a suspected contribution by genetic factors. This study aimed to assess the association of polymorphism in MTHFR (methylene tetrahydrofolate reductase, C677T) gene and the severity of periodontitis in Indonesian males. Severity of periodontitis was classified as mild, moderate or severe for 100 consenting, 25 to 60 years old male Indonesians. Using PCR amplification for DNA extracted from blood serum samples, the variation at the SNP polymorphism of the MTHFR (C677T) gene was evaluated by using RFLP, cutting by the restriction enzyme HinfI and subjecting the fragments to electrophoresis on agarose gel. Chi-square testing was mainly used for statistical assessment of the results. The CC genotype (wild type) of the tested polymorphism was the most common variant (78%) and TT (mutant) genotype relatively rare (2%), so that C-allele appeared in 88% of the cases and T-allele in 12% of the cases. The results suggest that there is no significant association between MTHFR C677T polymorphism and the severity of periodontitis in the tested Indonesian males.

Epidemiology and Molecular Typing of Candida krusei Based on PCRRFLP of the ITS rDNA Regions

Purpose: Candida krusei strains are intrinsically resistant for the first choice antifungal. Fast identification of C. krusei as an infectious agent will decrease the risk of choice of not correct therapy. The aim of the present work was to study the epidemiology of Candida krusei infections during 10 years. We also attempt to study the phylogeny of these isolates by PCR- RFLP. Methods: Two hundred five cases of C. krusei candidiasis were referred to laboratory of parasitology mycology, UH Habib Bourguiba of Sfax-Tunisia during 10 years (2006 to 2016). Identification of our strains was performed by conventional methods and by PCR-ITS amplification followed by a digestion with three restriction enzymes MspI, HinfI and HincII. Result: The mean frequency of cases of C. krusei candidiasis was 17.08 per year. Invasive infection represented 10.24%. The superficial infections with C. krusei represented 89.76% of cases. Analysis of the phylogeny tree allowed us to deduce that there is a great diversity in C. krusei strains. No particular genotype has been associated with the sampling site, or department or year of infection. We noted that patient P4 was hosted by three strains with the same genotype. Conclusion: The modification in epidemiology of candidiasis emphasizes the necessity to monitor local incidence, species distribution and susceptibility in order to optimize therapy and outcome. Molecular methods are essential for correct identification of the Candida species in order to obtain clues regarding the source of infection and to apply the correct therapy for the infected individual.