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Abstract The paper presents data on phytosanitary monitoring of garden cenoses for fire blight in the Turkestan, Zhambyl, and Almaty regions of Kazakhstan. The purpose of this study is to assess the phytosanitary situation in various regions of Kazakhstan, determine the extent of fire blight spread, and isolate and identify the fire blight pathogen. During the study, methods such as hypersensitivity, pathogenicity, and fluorescent simplification-based specific hybridization polymerase chain reaction (FLASH-PCR) were used. It was found that in all the surveyed areas, disease foci were identified. For the first time, the fire blight pathogen was detected on fruit crops such as plum, peach, cherry plum, and quince, as well as on wild apricots. 274 plant samples were collected from which microorganisms were isolated. Isolates related to the fire blight pathogen Erwinia amylovora were identified by methods of hypersensitivity, pathogenicity, and FLASH-PCR diagnostics. Of the 156 isolates of microorganisms isolated from apple tree plant samples, 21 inhibited the in vitro growth of E. amylovora to varying degrees. Isolates 16.2 and 19.2 with maximum antagonistic activity were selected, where the pathogen growth inhibition zones were 52.2 ± 2.58 mm and 45.6 ± 0.55 mm, respectively. Based on the obtained sequence of nucleotides of the 16SpRNA gene site, it was found that the selected isolates with high antagonistic activity belonged to the Pseudomonas genus. In the future, based on these isolates, a new biological product for fire blight control can be created and adapted to the natural and climatic conditions of Kazakhstan.

Sex identification plays an important role in avian production. Hitherto, it is difficult to distinguish the sexes of monomorphic birds based on their external features. The chromo-helicase-DNA-binding genes contain CHD-W gene and CHD-Z gene, which are located on the W chromosome and Z chromosome, respectively. Since CHD-W gene is unique to females, the polymerase chain reaction can be used for sex identification. However, extracting DNA procedures for verifying the sex is tedious and expensive. To address these disadvantages, the objective of this study was to develop a simple DNA extraction assay to efficiently process blood, liver, and feather samples. The results showed that 2% dimethylsulfoxide was suitable for processing blood, and phosphate-buffered saline was suitable for processing liver and feather samples. The specific primers were designed, and the length of the targets is 474 bp on Z chromosome and 319 bp on W chromosome. The pigeons were identified as females based on the presence of two bands on the gel, and as males based on the presence of one band. Taken together, our results suggested that feather samples were more appropriate than blood or liver for sex identification of pigeons. Compared to the traditional DNA extraction, this method shortened the assay time and reduced the cost. [ABSTRACT FROM AUTHOR]

the Aim Of the Study: Human papillomavirus (HPV) is an oncogenic virus and the high-risk genotype HPV 16 and 18 are the most commonly associated with carcinoma. The aim of the study is to determine the prevalence of HPV 16 and 18 in normal oral mucosa, potentially malignant oral disorders (PMOD), and in oral squamous cell carcinoma (OSCC) in South Indian population and whether it can be used as a biological marker to identify the severity of the disease in patients.Materials and Methods: Cytological samples from buccal mucosa were obtained from ten OSCC patients, ten patients with PMOD, and ten from control group. The samples were subjected to polymerase chain reaction.Results: The prevalence of HPV 16 in control, PMOD, and OSCC was 80%, 50%, and 70%, respectively. The prevalence of HPV 18 in control, PMOD, and OSCC was 70%, 60%, and 50%, respectively.Conclusion: HPV 16 and 18 was noticed in normal oral mucosa, potentially malignant oral lesions, and SCC. The absence of sequential increase or decrease of HPV 16 and 18 in the three groups in this study prevents its use from being used as a marker to identify the progression of the disease. [ABSTRACT FROM AUTHOR]

RT-qPCR is the accepted technique for the quantification of microRNA (miR) expression: however, stem-loop RT-PCR, the most frequently used method for quantification of miRs, is time- and reagent-consuming as well as inconvenient for scanning. We established a new method called ‘universal stem-loop primer’ (USLP) with 8 random nucleotides instead of a specific sequence at the 3′ end of the traditional stem-loop primer (TSLP), for screening miR profile and to semi-quantify expression of miRs. Peripheral blood samples were cultured with phytohaemagglutinin (PHA), and then 87 candidate miRs were scanned in cultured T cells. By USLP, our study revealed that the expression of miR-150-5p (miR-150) decreased nearly 10-fold, and miR-155-5p (miR-155) increased more than 7-fold after treated with PHA. The results of the dissociation curve and gel electrophoresis showed that the PCR production of the USLP and TSLP were specificity. The USLP method has high precision because of its low ICV (ICV<2.5%). The sensitivity of the USLP is up to 103 copies/µl miR. As compared with the TSLP, USLP saved 75% the cost of primers and 60% of the test time. The USLP method is a simple, rapid, precise, sensitive, and cost-effective approach that is suitable for screening miR profiles. [ABSTRACT FROM AUTHOR]

We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5′ end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are endogenous small non coding RNAs acting as negative regulators. miRNA are involved in lung development and pulmonary diseases. Measurement of their levels by qPCR is directly influenced by the stability of normalization gene(s), which can be affected by the experimental conditions. The developing lung is a changing tissue and one normalization gene showing stability on one developmental day may be modulated over time. Moreover, some developmental events are affected by sex, which also has to be considered. In this study, we compared stability of five putative control genes in the lung between sexes from the pseudoglandular to the alveolar stages and in adult lungs. Expression of sno135, sno142, sno202, sno234, and sno251 was studied by qPCR in male and female lung samples collected at seven time points from GD 15.5 to PN 30. Cq values of sno251 showed the highest variation across the different developmental stages, while sno234 was the most stable gene. Gene expression stability was studied by geNorm, NormFinder and BestKeeper. Our data showed that ranking of genes based on expression stability changed according to developmental time and sex. sno135/sno234 and sno142/sno234 were proposed as best combinations of normalization genes when both sexes and all the studied developmental stages are considered. Normalization of let7-a RNA levels with different pairs of control genes proposed by geNorm and NormFinder gave similar data, while the use of less stable genes introduced a statistically significant difference on PN 0. In conclusion, variations in stability of normalization gene expression are observed over time and according to sex during lung development. Best pairs of normalization genes are presented for specific developmental stages, and for the period extending from the pseudoglandular to the alveolar stages. The use of normalization genes selected for their expression stability is essential in lung development studies. [ABSTRACT FROM AUTHOR]

Background: Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation. Methods: TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC). Results: The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA−TcdB+, and TcdA−TcdB− types), TcdB-producing strains (TcdA+TcdB+ and TcdA−TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA−TcdB− strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota. Conclusion: Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile. [ABSTRACT FROM AUTHOR]

The µ opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing in rodents and humans, with dozens of alternatively spliced variants of the OPRM1 gene. The present studies establish a SYBR green quantitative PCR (qPCR) assay to more accurately quantify mouse OPRM1 splice variant mRNAs. Using these qPCR assays, we examined the expression of OPRM1 splice variant mRNAs in selected brain regions of four inbred mouse strains displaying differences in µ opioid-induced tolerance and physical dependence: C56BL/6J, 129P3/J, SJL/J and SWR/J. The complete mRNA expression profiles of the OPRM1 splice variants reveal marked differences of the variant mRNA expression among the brain regions in each mouse strain, suggesting region-specific alternative splicing of the OPRM1 gene. The expression of many variants was also strain-specific, implying a genetic influence on OPRM1 alternative splicing. The expression levels of a number of the variant mRNAs in certain brain regions appear to correlate with strain sensitivities to morphine analgesia, tolerance and physical dependence in four mouse strains. [ABSTRACT FROM AUTHOR]

The presence of selected tetracycline resistance (TcR) genes was studied in different Greek seawater habitats, originated from wastewater treatment facilities, fishfarm, and coastal environments. The methods employed included assessment of the presence of twelve gene clusters by PCR, followed by hybridization with specific probes, in habitat extracted DNA, TcR bacteria, and exogenous isolated plasmids conferring TcR. The direct DNA-based analysis showed that tet(A) and tet(K) genes were detected in all habitats, whilst tet(C) and tet(E) were present in fishfarm and wastewater effluent samples and tet(M) was detected in fish-farm and coastal samples. Resistance genes tet(h), tet(C), tet(K), and tet(M) were detected in 60 of the 89 isolates screened. These isolates were identified by fatty acid methyl ester analysis (FAME) as Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Staphylococcus strains. The presence of the TcR genes in 15% of the bacterial isolates coincided with the presence of IncP plasmids. A habitat-specific dissemination of IncP alpha plasmids in wastewater effluent isolates and of IncP beta plasmids in fishfarm isolates was observed. Exogenous isolation demonstrated the presence of plasmids harbouring TcR genes in all the habitats tested. Plasmids were shown to carry tet(h), tet(C), tet(E), and tet(K) genes. It is concluded that TcR genes are widespread in the seawater habitats studied and often occur on broad host range plasmids that seem to be well disseminated in the bacterial communities. [ABSTRACT FROM AUTHOR]

A significant increase in the growth of genetically modified organisms has been observed in recent years. As a result of the market introduction of these genetically modified (GM) crops in foods, legislation worldwide was prepared to deal with the question of the use and labelling requirements on GM crops and their derivatives. The aim of this work was to detect and to quantify GM soybean in soy-derived food products sold commercially in Florianópolis, Brazil, by polymerase chain reaction-based analysis. In total sixty-two samples of texturized soy protein and powdered soymilk were analysed and forty samples have been shown to contain Roundup Ready (RR) soy. Quantitative analysis revealed that only two out of these forty samples presented RR soybean content above 1%. These findings confirm the presence of RR soybean in Brazilian soy-derived food products and that the amount of samples with % RR content above 1% decreases when compared with previously published data. [ABSTRACT FROM AUTHOR]

The article presents a study on the evaluation of polymerase chain reaction (PCR) and DNA isolation protocols for detection of genetically modified soybean. Samples of soybeans were obtained from Brazil and U.S. Using three protocols, DNA were extracted and its concentration were estimated with a spectrophotometer. The nested PCR using primers genetically modified organism (GMO) was considered accurate in distinguishing Roundup Ready (RR) soybean from non-genetically modified soybean.

The utilities at a major government office facility include both condenser and chilled water systems. Though they are similar, the water in the condenser system is regularly aerated, while the chilled water is a closed system. Piping in both is largely carbon steel, with a minor amount of cast iron. Both systems use Victaulic mechanical couplings (Victaulic Company) rather than welded joints. The joints in the condenser section of the system are deeply corroded, while the chilled water system is in good condition. The cause appears to be initial system pressure testing without biocide, followed by several months of no use at all. Microbially-influenced corrosion (MIC) started below the gaskets in the joints and could not be stopped. Polymerase chain reaction analysis allowed identification of the bacteria involved after the system was dismantled. The entire condenser water system had to be replaced. [ABSTRACT FROM AUTHOR]

A comprehensive multiphasic survey of the prevalence and transfer of gentamicin resistance (Gmr) genes in different non-clinical environments has been performed. We were interested to find out whether Gmr genes described from clinical isolates can be detected in different environmental habitats and whether hot spots can be identified. Furthermore, this study aimed to evaluate the impact of selective pressure on the abundance and mobility of resistance genes. The study included samples from soils, rhizospheres, piggery manure, faeces from cattle, laying and broiler chickens, municipal and hospital sewage water, and coastal water. Six clusters of genes coding for Gm-modifying enzymes (aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6′)-II/Ib, ant(2″)-I, aph(2″)-I) were identified based on a database comparison and primer systems for each gene cluster were developed. Gm-resistant bacteria isolated from the different environments had a different taxonomic composition. In only 34 of 207 isolates, mainly originating from sewage, faeces and coastal water polluted with wastewater, were known Gmr genes corresponding to five of the six clusters detected. The strains belonged to genera in which the genes had previously been detected (Enterobacteriaceae, Pseudomonas, Acinetobacter) but also to phylogenetically distant bacteria, such as members of the CFB group, α- and β-Proteobacteria. Gmr genes located on mobile genetic elements (MGE) could be captured in exogenous isolations into recipients belonging to α-, β- and γ-Proteobacteria from all environments except for soil. A high proportion of the MGE, conferring Gm resistance isolated from sewage, were identified as IncPβ plasmids. Molecular detection of Gmr genes, and broad host range plasmid-specific sequences (IncP-1, IncN, IncW and IncQ) in environmental DNA indicated a habitat-specific dissemination. A high abundance and diversity of Gmr genes could be shown for samples from faeces (broilers, layers, cattle), from sewage, from seawater, collected close to a wastewater outflow, and from piggery manure. In the latter samples all six clusters of Gmr genes could be detected. The different kinds of selective pressure studied here seemed to enhance the abundance of MGE, while an effect on Gmr genes was not obvious. [Copyright &y& Elsevier]