Fragment Length Polymorphism Analysis Research Articles

Transmission of multidrug-resistant Mycobacterium tuberculosis is reality in Switzerland

We report the first documented patient-to-patient transmission of Multidrug resistant tuberculosis observed in Swit- zerland between two young, previously healthy patients from Tibet. They both lived in Switzerland for several years and did not originate from the same community. Resistance patterns and restriction fragment length polymorphism-analysis (RFLP-analysis) of both strains were identical.

Molecular Epidemiology and Phylogenetic Analysis of Human Adenovirus Caused an Outbreak in Taiwan during 2011.

An outbreak of adenovirus has been surveyed in Taiwan in 2011. To better understand the evolution and epidemiology of adenovirus in Taiwan, full-length sequence of hexon and fiber coapsid protein was analyzed using series of phylogenetic and dynamic evolution tools. Six different serotypes were identified in this outbreak and the species B was predominant (HAdV-3, 71.50%; HAdV-7, 15.46%). The most frequent diagnosis was acute tonsillitis (54.59%) and bronchitis (47.83%). Phylogenetic analysis revealed that hexon protein gene sequences were highly conserved for HAdV-3 and HAdV-7 circulation in Taiwan. However, comparison of restriction fragment length polymorphism (RFLP) analysis and phylogenetic trees of fiber gene in HAdV-7 clearly indicated that the predominant genotype in Taiwan has shifted from 7b to 7d. Several positive selection sites were observed in hexon protein. The estimated nucleotide substitution rates of hexon protein of HAdV-3 and HAdV-7 were 0.234×10-3 substitutions/site/year (95% HPD: 0.387~0.095×10-3) and 1.107×10-3 (95% HPD: 0. 541~1.604) respectively; those of the fiber protein of HAdV-3 and HAdV-7 were 1.085×10-3 (95% HPD: 1.767~0.486) and 0.132×10-3 (95% HPD: 0.283~0.014) respectively. Phylodynamic analysis by Bayesian skyline plot (BSP) suggested that using individual gene to evaluate the effective population size might possibly cause miscalculation. In summary, the virus evolution is ongoing, and continuous surveillance of this virus evolution will contribute to the control of the epidemic.

Diversity of insecticidal crystal protein genes of Bacillus thuringiensis isolated from soil and cloning of novel haplotypes of cry genes

Bacillus thuringiensis (Bt) strains used in this study were isolated from forestland soil in Chengdu, China. Sodium acetate-antibiotic treatment was used to isolate the strains, and scanning electron microscopy was used to identify crystal morphology. Diversity was observed in the protein and cry gene profiles, and most of the isolated Bt strains produced 130, 90, 80, 70, 66, and 45 kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Polymerase chain reaction (PCR) analysis of these isolates identified 14 cry genotypes and 30 different genotype combinations. Several novel cry fragments were discovered using PCR restriction fragment length polymorphism (RFLP) analysis, and five novel full-length cry genes—cry1Hc1, cry1Ie5, cry30Ea4, cry54Ba2, and cry56Aa4—were cloned. The analysis of insecticidal activity indicated that the majority of the isolates had high activity against Ostrinia furnacalis larvae; the cloned novel genes cry1Hc1 and cry1Ie5 conferred high activity against Ostrinia furnacalis larvae. Forty isolates did not yield PCR products, and eight of these had activity against Ostrinia furnacalis larvae. The results suggest that the genetic resources are rich in these isolates, which have the potential for future use as insecticides.

Evaluation of Genetic Polymorphisms in Clusterin and Tumor Necrosis Factor-Alpha Genes in South Indian Individuals with Pseudoexfoliation Syndrome

Purpose: The aim of this study was to explore the potential association of genetic variants across clusterin (CLU) and tumor necrosis factor-alpha (TNF-α) genes in South Indian individuals with pseudoexfoliation syndrome (PEXS) and pseudoexfoliation glaucoma (PEXG).Materials and Methods: A total of 523 individuals including 299 unrelated cases (150 PEXS and 149 PEXG) and 224 age- and ethnically-matched healthy controls were recruited for genetic analysis. Six single-nucleotide polymorphisms (SNPs) including, five CLU SNPs (rs11136000, rs2279590, rs9331888, rs9331931, rs3087554) and one promoter SNP (rs1800629) of TNF-α were genotyped in all study subjects. Genotyping of CLU SNPs were performed using the TaqMan allelic discrimination assay while TNF-α SNP was genotyped using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis. Association analysis was performed by determining the distributions of genotype and allele frequencies, Hardy–Weinberg equilibrium, and chi-square p values and odds ratios as implemented in the Golden Helix SNP & Variation Suite (SVS).Results: Five CLU SNPs did not show any significant differences in allele frequencies between patients and control subjects (rs3087554, p = 0.919, OR = 1.01, 95% CI: 0.77–1.33; rs2279590, p = 0.432, OR = 1.12, 95% CI: 0.84–1.51; rs9331931, p = 0.310, OR = 1.24, 95% CI: 0.81–1.89; rs11136000, p = 0.072, OR = 1.31, 95% CI: 0.97–1.76; rs9331888, p = 0.911, OR = 1.01, 95% CI: 0.78–1.31). The investigation of TNF-α SNP established a significant association with PEXS and PEXG (p = 0.042, OR = 0.61, 95% CI: 0.38–0.99). However, this association did not remain significant after Bonferroni correction.Conclusions: Our data suggest that genetic variants in CLU and TNF-α genes do not play a major role in the development of PEXS and PEXG in the South Indian population.

Su1138 Concomitant Therapy Appeared Superior to Sequential, Tailored, or PAC, PAM Based Triple Therapy for the Eradicatino of Helicobacter in Prospective, Multi-Center, Radomized Controlled Trial

Objective of the study: Since 2013, H. pylori eradication therapy for atrophic gastritis associated with H. pylori became available for coverage by insurance in Japan. For first-line therapy, triple eradication therapy (proton pump inhibitor (PPI) + amoxicillin (AMPC) + clarithromycin (CAM) is available for H. pylori eradication, in Japan. The resistance of H. pylori strains to CAM accounts for the majority of eradication failures, the incidence of which is currently increasing and it affects H. pylori eradication rate in Japan. Another matter is recurrence of H. pylori infection after eradication. H. pylori infection sometimes recurs after successful eradication. The rate of recurrence after successful eradication is reported to be 0.5% to 2.0% in the western countries. In this study, we investigated about the long term transition of primary drug susceptibility ofH. pylori strains to CAM, and the rate of recurrence after successful eradication in Japan.Methods: Three hundred sixty six patients after successful eradication of H. pylori were followed up by culture, histological examination and urea breath tests. In 22 patients in whom H. pylori recurred during follow-up, the susceptibility of the H. pylori strains to clarithromycin was investigated. And in 17 recurrent patients, H. pylori strains isolated initially and after recurrence were compared using PCR-based restriction fragment length polymorphism (RFLP) analysis. Results: The primary resistance of H. pylori to CAM is increasing, especially in these 6 years, its rate is very high at 35%. Ten of 22 (45.5%) recurrences appeared at 6 months after successful eradication of H. pylori, and after 1 year, the annual recurrence rate of H. pylori infection was 0.29% per patient year. In recurrent cases, 50.0% (5/10) of the strains detected 6 months after successful eradication were found to be susceptible to clarithromycin. And 58.3% (7/12) of the strains detected at 12 months or later were found to be susceptible to clarithromycin. In the 10 patients who showed a recurrence of H. pylori after 12 months, all the patients showed different DNA profiles, and 5 of 7 (71.4%) patients showed different DNA profiles at 6 months after eradication Conclusions: The rate of CAM-resistant H. pylori strains is very high in Japan. Because of that, for the recurrence cases, it occurs with low frequency (0.29% per patient year), drug sensitivity examination may be necessary in Japan.

Mo1811 Clinical Remission Induced by Exclusive Enteral Nutrition (EEN) in Pediatric Crohn's Disease Is Associated With Microbiome Metabolic Changes Toward Increased Xenobiotic Biodegradation and Metabolism

esis of IBD, both Crohn's disease (CD) and ulcerative colitis (UC), is not well known. Most studies of human gut microbiota rely on the non-invasive collection of stool samples. However, the analysis of the fecal microbiota may not reflect the role of the mucosa-associated microbes which live in close proximity to the intestinal epithelium and that are in contact with the cells of the innate immune system directly involved in the inflammatory response. The aim of this study was to investigate the genotypes of Bacteroidetes microbiota from colon biopsies of IBD patients and to determine their relationship with the endoscopic activity of the disease. Methods: A prospective case-control study was designed. Colon biopsies from consecutive IBD patients and healthy controls (HC) (healthy subjects undergoing colonoscopy for colon cancer screening and having a healthy colon mucosa) were included. Inactive UC was defined as a Mayo endoscopic subscore of 0. Inactive CD was defined as a SES-CD score ≤ 2. Microbiota was characterized by using a restriction fragment length polymorphism (RFLP) analysis on PCR products targeting the 16SrRNA genes of Bacteroidetes digested with HinfI, PciI, DpnII and AciI. Results are shown as percentages. Results: 29 consecutive IBD patients (22 UC and 7 CD) and 15 HC were included. 8 patients presented with inactive UC (iUC) and 14 with active UC (aUC). All 7 CD patients presented with endoscopic activity (aCD). A total of 7 genotypes of Bacteroidetes called N1 and C1-C6 (of which N1 genotype is probably a strain of Bacteroides dorei and C1, and maybe C2, strains of B. vulgatus) were detected in all the biopsy samples analyzed. The number of genotypes present in biopsies from IBD patients was higher than that in HC, in whom only the N1 (66%) and C1 (34%) genotypes appear. While the presence of N1 genotypes was relatively constant in patients with active and inactive IBD, the percentage of C1 genotype in patients with aUC and aCD were very low (<5%) compared to controls. The C4 genotype never appeared in control samples and it was present in only 2% of iUC biopsies, whereas it was present in 15% and 16% of patients with aUC and aCD respectively. The C2, C5 y C6 genotypes appeared sporadically in biopsies of IBD with percentages of 1% in iUC (C2), 3% in aUC and 1% in aCD (C5), and 1% in iUC and aCD (C6), but never in HC. Conclusions: Bacteroidetes genotypes in colon biopsies differ between IBD patients and HC. These genotypes, and especially the C4 genotype, are highly specific for active IBD and further studies on their role on IBD pathogenesis are required.

Su1137 The Recurrence and Clarithromycin Resistant Rate of Helicobacter pylori After Successful Eradication Therapy in Japan

Objective of the study: Since 2013, H. pylori eradication therapy for atrophic gastritis associated with H. pylori became available for coverage by insurance in Japan. For first-line therapy, triple eradication therapy (proton pump inhibitor (PPI) + amoxicillin (AMPC) + clarithromycin (CAM) is available for H. pylori eradication, in Japan. The resistance of H. pylori strains to CAM accounts for the majority of eradication failures, the incidence of which is currently increasing and it affects H. pylori eradication rate in Japan. Another matter is recurrence of H. pylori infection after eradication. H. pylori infection sometimes recurs after successful eradication. The rate of recurrence after successful eradication is reported to be 0.5% to 2.0% in the western countries. In this study, we investigated about the long term transition of primary drug susceptibility ofH. pylori strains to CAM, and the rate of recurrence after successful eradication in Japan.Methods: Three hundred sixty six patients after successful eradication of H. pylori were followed up by culture, histological examination and urea breath tests. In 22 patients in whom H. pylori recurred during follow-up, the susceptibility of the H. pylori strains to clarithromycin was investigated. And in 17 recurrent patients, H. pylori strains isolated initially and after recurrence were compared using PCR-based restriction fragment length polymorphism (RFLP) analysis. Results: The primary resistance of H. pylori to CAM is increasing, especially in these 6 years, its rate is very high at 35%. Ten of 22 (45.5%) recurrences appeared at 6 months after successful eradication of H. pylori, and after 1 year, the annual recurrence rate of H. pylori infection was 0.29% per patient year. In recurrent cases, 50.0% (5/10) of the strains detected 6 months after successful eradication were found to be susceptible to clarithromycin. And 58.3% (7/12) of the strains detected at 12 months or later were found to be susceptible to clarithromycin. In the 10 patients who showed a recurrence of H. pylori after 12 months, all the patients showed different DNA profiles, and 5 of 7 (71.4%) patients showed different DNA profiles at 6 months after eradication Conclusions: The rate of CAM-resistant H. pylori strains is very high in Japan. Because of that, for the recurrence cases, it occurs with low frequency (0.29% per patient year), drug sensitivity examination may be necessary in Japan.

Newborn APOE genotype influences maternal lipid profile and the severity of high-risk pregnancy – preeclampsia: Interaction with maternal genotypes as a modulating risk factor in preeclampsia

Aim: To establish the role of the interaction between maternal and newborn apolipoprotein E (APOE) genotypes on the risk, lipid profile and prognosis of preeclampsia (PE). Materials and methods: Forty-seven preeclamptic women and 94 normotensive pregnant women and their newborns were genotyped for APOE using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Results: Maternal APOE-ɛ4 allele was associated with an about eight times higher risk of PE (adjusted OR = 8.4, 95% CI: 2.51–28.17, p = 0.001). The multivariate logistic regression model showed that the newborn APOE-ɛ4 allele was associated with an about six times higher risk of PE (adjusted OR = 5.6, 95% CI: 2.09–15.21, p = 0.001) for the given gestational age levels. Pregnant women with severe PE whose newborns carried the APOE-ɛ4 allele delivered at earlier gestational ages neonates with a lower birth weight compared to pregnant women with newborns negative for this allele. Higher TG and LDL-C levels and lower HDL-C levels were found in pregnant women with severe PE whose newborns were carriers of the APOE-ɛ4 allele compared to preeclamptic women whose newborns were carriers of the ɛ3/ɛ3 genotype. If we checked the combined effect of the mother/newborn genotypes on the risk of PE, we found that the risk to develop PE was 15.4-fold (p < 0.001) increased if mothers or newborns were carriers of the APOE-ɛ4 allele. The risk increased to 20.02 (p < 0.001) if both the mother and newborn were carriers of the APOE-ɛ4 allele. Conclusions: Our study confirms the maternal/newborn APOE genotype interaction influences the risk for PE, as well as prognosis and lipid profile.

Phenotype receptor gene mutations in low-density lipoprotein in patients with familial hypercholesterolemia in Karelia

Familial hypercholesterolemia (FHC) - hereditary dyslipidemia, which is based on mutations in the gene for low-density lipoprotein receptor.However, there is variability in the clinical manifestations of the disease difficult to assess individual risk.Materials and methods. Under our supervision for 10 years were 109 patients with FHC, 17 mutation in the receptor density lipoprotein. FHC diagnosis established by the criteria of the British leadership Simon Broom. To search for mutations in low-density lipoprotein receptor was performed automated fluorescent SSCP-analysis of exons of the gene analysis of restriction fragment length polymorphism and the direct sequencing of DNA on a gel sequencer ALFExpress-2 (Amersham Biosciences) using the program ALFwin Sequence Analyzer.The Results. We analyzed five clinical cases of patients with genetically confirmed diagnosis of FHC. Shows a wide phenotypic variability FHC: the possibility of early debut of coronary heart disease, coronary tropism for the pool some patients and cerebral - others, the possibility of a long asymptomatic disease.Conclusion. The absence of clinical manifestations of atherosclerosis and wide phenotypic variability at FHC require targeted screening for FHC, at least among patients with coronary heart disease in order to timely and adequate preventive measures, especially in cases where the mutation is set low density lipoprotein receptor.

Congruence of community structure between taxonomic identification and T-RFLP analyses in free-living soil nematodes

Abstract Molecular-based methods of community analysis are becoming a popular alternative to the traditional, highly specialized, and time-consuming taxonomic identifications especially for morphologically challenging groups like free-living soil nematodes. In particular, terminal restriction fragment length polymorphism (T-RFLP) analysis has become popular as a quick and efficient tool to provide researchers with broad-scale assessments of community structure. The majority of studies comparing T-RFLP with morphological-based assessments have used cultured or previously characterized communities. Here we compare morphological identification to T-RFLP analyses in a previously unexplored system for nematode diversity, the boreal forest of Ontario, Canada. Samples were collected from five silvicultural treatments over two sampling seasons with nematodes extracted using the Baermann funnel technique. We found significantly greater richness from T-RFLP analyses than morphological identifications at both sampling times, but in both cases silvicultural treatment had no effect. Despite differences in taxonomic richness, community compositional similarities were highly correlated between morphological and molecular methods for both sampling times. We suggest that both methods are reliable in studies of previously undescribed communities where the goal is community assessment under treatment.

Genetic Structure and Morphological Differentiation of Three Western North American Dwarf Mistletoes (&lt;I&gt;Arceuthobium&lt;/I&gt;: Viscaceae)

Abstract— Dwarf mistletoes (Arceuthobium spp., Viscaceae) are ecologically and economically important parasitic flowering plants. Despite detection of morphological and host specialization differences, ribosomal DNA and plastid sequence information have not resolved genetic differentiation among the dwarf mistletoes parasitizing white pines (Pinus subgenus Strobus) in the western U. S. A. We assessed the effectiveness of integrating amplified fragment length polymorphism and morphometric analyses in the delineation of three closely related and morphologically similar species of white pine dwarf mistletoes. Using amplified fragment length polymorphism data for 995 loci, we quantified intra- and inter-specific genetic variation, tested for geographic isolation-by-distance, and visualized genetic patterns using Bayesian-based structure analyses, neighbor-joining tree reconstruction, and principal coordinate analysis ordination. A total of 19 morphological characters were also analyzed using standard statisti...

Association of the genetic polymorphisms in XRCC6 and XRCC5 with the risk of ESCC in a high-incidence region of North China.

The XRCC6 and XRCC5 genes are part of the nonhomologous end-joining (NHEJ) pathway, which is the main mechanism repairing DNA double-strand breaks (DSBs) in human cells. Genetic variations of XRCC6 and XRCC5 might contribute to esophageal squamous cell carcinoma (ESCC) susceptibility. ESCC patients (n = 189) and cancer-free controls (n = 189) were recruited in an ESCC high-risk area of north China. Then the rs2267437 (XRCC6), rs3835 (XRCC5) and rs16855458 (XRCC5) polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. A significant difference in genotype distribution and allele frequency of rs2267437 (XRCC6) was observed between the cases and controls. The CG carriers were at higher risk of ESCC (p = 0.001, odds ratio [OR] = 2.040, 95% confidence interval [95% CI], 1.323-3.147). G allele carriers were also associated with an increased ESCC risk (p = 0.003, OR = 1.868, 95% CI, 1.230-2.836). In the 2 polymorphisms of XRCC5, no significant difference was found between both groups in the distribution of either genotype or allelic frequency. But in the haplotypes established by the single nucleotide polymorphisms (SNPs) of XRCC5, the haplotype AT and CC separately increased by 4.28- and 2.31-fold the risk ratio of ESCC (p = 0.01, OR = 4.28, 95% CI, 1.40-13.05; p = 0.03, OR = 2.31, 95% CI, 1.11-4.80, respectively). In addition, gene-smoking or gene-drinking interactions, and their effect on the risk of ESCC were observed, but no significant gene-environment interaction was demonstrated. In conclusion, both the CG carriers/G allele carriers of rs2267437 (XRCC6) and the haplotype AT/CC established by the SNPs of XRCC5 are associated with ESCC susceptibility.

The Impact of Enterohemorrhagic &lt;i&gt;Escherichia coli&lt;/i&gt; (EHEC) on Ciliate Protozoan Populations in Municipal Sewage

Enterohemorrhagic Escherichia coli strains (EHEC) have caused many foodborne outbreaks. Bacterivorous protozoa could remove bacteria from aquatic systems. We analyzed the ciliate protozoan population changes influenced by EHEC co-culture in activated sludge. EHEC and non-EHEC control E. coli cells were added to activated sludge samples in microcosms. The ciliate population changes were monitored by terminal restriction fragment length polymorphism (T-RFLP) analysis. EHEC and non-EHEC fed ciliate protozoan populations were different from each other and the no bacteria added controls based on the additive main effects and multiplicative interaction model (AMMI) analysis. Ciliate species were identified by 18S rDNA clone libraries. The 18S rDNA clones from the original sludge sample were identified as Epistylis wenrichi (70%) and Prorodon teres (30%), while clones from EHEC treated sludge sample were identified as P. teres (52%), Vorticella fusca (41%), Dexitrichides pangi (5%), and Opisthonecta henneguyi (2%). This study could provide helpful information about ciliate protozoan population changes caused by different E. coli strains in wastewater treatment plants, which could be useful for preventing and tracking E. coli outbreaks.

Association of Protein Tyrosine Phosphatase 1B (PTPN1) Gene Polymorphisms (1023C&gt;A and 467T&gt;C) With Type 2 Diabetes: A Case-Control Study

Background: Type 2 diabetes (T2D) is a heterogeneous disorder that results from a combination of environmental and genetic factors. Insulin resistance (IR) is the core defect in T2D. The molecular mechanisms underlying IR are poorly understood. Protein tyrosine kinases and Protein tyrosine phosphatase 1B (PTPN1) are important regulators of insulin signal transduction. The association of PTPN1single-nucleotide polymorphisms (SNPs) with traits related to T2D has been investigated. The aim of this study was to determine the association of 1023C>A and 467T>C gene polymorphisms with Type 2 diabetes and its related metabolic traits. Method: Polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) analyses were carried out to detect the 1023C>A and 467T>C variants of PTPN1 gene in 100 Egyptian patients with T2D as compared to controls (n=80). Results: The 1023C>A PTPN1genotype was significantly associated with T2DM (X2=7.816, P=0.02). The C allele was more frequent in the T2DM as compared to controls (p=0.001), odds ratio (OR) and 95% CI= 0.282 (0.131-0.606). We did not observe any significant difference in 467T>C PTPN1 genotypes between patients and control groups (X2=2.205, P=0.332). 1023C>A and 467T>C PTPN1 variants showed non-significant association with diabetic metabolic traits in both groups; plasma insulin levels, fasting blood glucose levels (FBG), HOMA-IR, the lipid profile parameters , diastolic blood pressure (DBP), systolic blood pressure (SBP), Waist circumference (WC) and body mass index (BMI). Conclusion: The PTPN1 promoter variant 1023C>A was associated with presence of T2D, but it had no correlation with any of neither metabolic traits nor obesity in this study but we could not detect any association between 467T>C variants of PTPN1 gene with T2D Egyptian patients nor related traits in this study. Further studies must be done on a larger population to detect any potential metabolic association.

Correlation study on susceptible genes of obsessive compulsive disorder of Han population in northeast China

Objective To explore the relationship between Monoamine oxidase A (MAOA) receptor gene, catechol-O- methyltransferase(COMT)receptor gene, dopamine D3 receptor(DRD3)gene and 5-HT2C receptor gene(5-HT2c) of Han population in northern China and obsessive compulsive disorder. Methods Polymerase chain reaction amplification determination of MAOA-T1460C, COMT-Val158Met, DRD3-Ser9Gly, 5-HT2c-C ys23Ser four loci receptor gene polymorphism in 164 patients with OCD patients including 103 core pedigrees of fragment length polymorphism, and association and linkage disequilibrium(TDT)analysis. Results There was no significant difference of MAOA-T1460C, COMT-Val158Met, DRD3-Ser9Gly, 5-HT2c-Cys23Ser four receptor gene in the patient group and the control group of genotype and allele distribution difference(P>0.05), four receptor gene loci were in accordance with the balance of the H-W, the MAOA-T1460C receptor gene in female patients group and control group, the early group and control group, which has forced thinking and difference of compulsive behavior group and the control group, only the obsessional group and the control group of genotype and allele distribution was statistically significant(P 0.05), and with the house group does not exist between the chain (P=0.0622); DRD3-Ser9Gly receptor gene in the control experiments were no significant difference(P>0.05), and with the family groups there is no chain(P=0.1101). Conclusion MAOA-T1460C receptor gene polymorphism and 5-HT2c-Cys23Ser receptor gene polymorphisms may be the susceptible gene of obsessive compulsive disorder. Key words: Obsessive-compulsive disorder; Gene; Monoamine oxidase A receptor; Catechol-O- methyl transferase receptor; Dopamine D3 receptor; 5-serotonin 2C receptor; Association study

FIRST REPORT OF PEPINO MOSAIC VIRUS ON TOMATO IN APULIA, ITALY

In October 2013, unusual chlorotic patches were observed on the middle leaves of a few tomato plants cv. Lotty grown in a greenhouse located in the countryside of Fasano (Apulia, southern Italy). Younger leaves and fruits were symptomless and no aggravation of symptoms was detected as time went by. Electron microscope observations of leaf dips revealed the presence of filamentous virus particles ca. 520 nm in length. Mechanical inoculations with leaf extracts from symptomatic tomatoes elicited a mosaic reaction in Nicotiana benthamiana, but not in tomato plants cv. UC82, which, however, were systemically infected, as shown by RT-PCR. A RT-PCR product of the expected size (ca. 700 bp) was obtained using the degenerate broad-spectrum potexvirus primers Potex5/Potex1RC (van der Vlugt and Berendsen, 2002). The amplicon was custom sequenced (BMR Genomics, Italy) and the sequence deposited in GenBank under the accession No. KM923762. BLAST alignment showed that the 700 bp amplicon shared 97-99% homology with the RNA-dependent RNA polymerase (RdRp) gene of several isolates of Pepino mosaic virus (PepMV) genotype CH2, 82% homology with the LP and EU genotypes, and 79-80% with the US genotype. A RT-PCR-restriction fragment length polymorphism (RFLP) analysis of the RdRp amplicon with EcoRI and BglII restriction endonucleases confirmed that our isolate, designated PUG1, belongs to the CH2 genotype (Hanssen, 2010). Our observations and assays are consistent with the presence of PepMV in the tomato plants tested, and the fact that PUG1 is a mild isolate of the virus. PepMV has previously been recorded from tomato in three Italian regions, i.e. Sardinia, Sicily and Campania, but this the first report from Apulia.

IDENTIFICATION OF GYMNEMA SPECIES BY RANDOM AMPLIFIED POLYMORPHIC DNA TECHNIQUE AND CHLOROPLAST trnK GENE

Gymnema is one of the important anti-diabetic medicinal plants used from ancient times and is commonly known as ‘sugar killer’. Most of its species have been used in many applications in Indian traditional medicine. Nevertheless, their efficiency is critically dependent on the use of the correct material. The sharing of similar vernacular name and morphological features make confusion in the usage of Gymnema species. In the present study, Gymnema sp. were identified through random amplified polymorphic DNA (RAPD) technique and species specific markers were generated for easy identification of G. elegans, G. montanum and G. sylvestre. Using the RAPD techniques of 3 species specific markers for G. sylvestre, 7 markers for G. elegans and 4 markers for G. montanum had been generated. Highest genetic identity was found between G. sylvestre and G. montanum and highest genetic distance was found between G. sylvestre and G. elegans. Further, DNA barcode was developed by sequencing chloroplast partial trnK DNA of these three species. No significant variation was found in partial trnK gene sequences between Gymnema species. But these sequences can efficiently differentiate the Gymnema and Mandevilla species. In-silico sequence–restriction fragment length polymorphism (RFLP) analysis revealed three fragments measuring G. sylvestre - 204, G. elegans - 174, and G. montanum - 168 bp Gymnema species. The present study concluded that RAPD markers were highly efficient for species detection than the partial trnK gene sequences. This could be used to confirm the Gymnema sp. identities and to ensure their safe application in pharmaceuticals.

Cluster of differentiation 14 gene polymorphism and its association with incidence of clinical mastitis in Karan fries cattle

Aim: The present study was undertaken with the objectives to characterize, identify DNA polymorphism in cluster of differentiation 14 (CD14) gene in Karan Fries (KF) cattle and to analyze association between genetic variants with incidence of clinical mastitis in National Dairy Research Institute (NDRI) herd, Karnal. Materials and Methods: Genomic DNA was extracted using blood of randomly selected hundred KF lactating cattle by phenol-chloroform method. After checking its quality and quantity, polymerase chain reaction (PCR) was carried out using reported primers to amplify 832 base pair region covering nucleotide base position number 1012 to 1843 (part of promoter, 5’UTR, exon 1, intron 1 and part of exon 2) of bovine CD14 gene. The PCR amplified target product was purified, sequenced and further ClustalW analysis was done to align edited sequence with reported Bos taurus sequence (EU148610.1). The restriction fragment length polymorphism (RFLP) analysis was performed for each KF cow using HinfI restriction enzyme (RE). Cows were assigned genotypes obtained by PCR-RFLP analysis and association study was done using Chi-square (χ 2 ) test. Results: After PCR amplification, DNA sequencing of amplicon confirmed the 832 bases covering 1012 to 1843 nucleotide base position of bovine CD14 gene. ClustalW multiple sequence alignment program for DNA revealed six nucleotide changes in KF cows at positions T1117D, T1239G, T1291C, G1359C, G1361A, and G1811A. Cows were also screened using PCR-RFLP with HinfI RE, which revealed three genotypes CC, CD and DD that differed significantly regarding mastitis incidence. Within CC genotype, 72.73% of cows were in a mastitis non-affected group whereas, those in CD and DD genotypes 69.44% and 60.38% respectively were mastitis affected.

Correlation between ApoE gene polymorphisms and the occurrence of urolithiasis.

The aim of the present study was to investigate the correlation between apolipoprotein E (ApoE) gene polymorphisms and the occurrence of urolithiasis and dyslipidemia. A total of 180 Uyghur individuals, including 90 urolithiasis patients and 90 healthy controls, were enrolled in this study. The blood lipid profiles of the patients and controls were investigated and compared, and the composition of the urinary calculi was determined. The polymorphisms of the ApoE alleles were analyzed using polymerase chain reaction-restriction fragment length polymorphism analysis. Three common genotypes of the ApoE gene, E3/3, E3/4 and E4/4, were detected in the urolithiasis patients and control group. In the patient group, 28 patients with the E3/3 genotype (30.1%), 58 patients with the E3/4 genotype (64.4%) and four patients with the E4/4 genotype (4.5%) were identified. By contrast, in the control group, 52 patients with the E3/3 genotype (57.8%), 35 patients with the E3/4 genotype (38.9%) and three patients with the E4/4 genotype (3.3%) were identified. The frequency of the E3/4 genotype was found to be significantly higher in the patient group when compared with the control group (χ2=12.96; P<0.001). In addition, the frequency of the E4 allele was significantly higher in the patient group when compared with the control group (χ2= 6.61; P=0.025). In conclusion, the occurrence of urolithiasis was found to be associated with ApoE gene polymorphisms, and the E4 allele may be a potential susceptibility factor for urolithiasis.

Intra-habitat Differences in the Composition of the Methanogenic Archaeal Community between the Microcystis-Dominated and the Macrophyte-Dominated Bays in Taihu Lake

A regime shift between a macrophyte-dominated clear state and a phytoplankton-dominated turbid state can have considerable impact on ecosystem structure and function of shallow lakes. However, very little is known about the response of the methanogenic archaeal community in the sediment during this regime shift. We investigated the methanogenic archaeal community at two sites in the large, shallow, eutrophic Taihu Lake over the course of one year. One site is located in Meiliang Bay and is dominated by Microcystis blooms, and the other site is located in East Taihu Bay and is dominated by aquatic macrophytes. Terminal restriction fragment length polymorphism (T-RFLP) and phylogenetic analyses of archaeal 16S rRNA genes were used to analyze the methanogenic community. Higher ratio of methanogens in Archaea was observed in East Taihu Bay than in Meiliang Bay. The methanogenic archaeal community was dominated by the Methanobacteriales and the LDS cluster in macrophytes-dominated East Taihu Bay, while it was dominated by the Methanosarcinaceae, Methanobacteriales, and the LDS cluster in Microcystis-dominated Meiliang Bay. Clustering analysis of all of the samples revealed differences in the composition of the methanogenic archaeal communities between the two sites that were independent of seasonal variations. Further statistical analysis indicated that the chlorophyll a (Chla) concentration had a profound impact on the composition of the methanogenic archaeal community in Meiliang Bay, whereas it was primarily influenced by total organic carbon (TOC) levels in East Taihu Bay. Overall, this investigation demonstrates that intra-habitat differences in the composition of methanogenic archaeal communities are likely driven by changes in the available organic materials.