966 resultados para |Nested-PCR
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In the present study, EA-CATH1 and EA-CATH2 were identified from a constructed lung cDNA library of donkey (Equus asinus) as members of cathelicidin-derived antimicrobial peptides, using a nested PCR-based cloning strategy. Composed of 25 and 26 residues, respectively, EA-CATH1 and EA-CATH2 are smaller than most other cathelicidins and have no sequence homology to other cathelicidins identified to date. Chemically synthesized EA-CATH1 exerted potent antimicrobial activity against most of the 32 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, and minimal inhibitory concentration values against Gram-positive bacteria were mostly in the range of 0.3-2.4 mu g center dot mL-1. EA-CATH1 showed an extraordinary serum stability and no haemolytic activity against human erythrocytes in a dose up to 20 mu g center dot mL-1. CD spectra showed that EA-CATH1 mainly adopts an alpha-helical conformation in a 50% trifluoroethanol/water solution, but a random coil in aqueous solution. Scanning electron microscope observations of Staphylococcus aureus (ATCC2592) treated with EA-CATH1 demonstrated that EA-CATH could cause rapid disruption of the bacterial membrane, and in turn lead to cell lysis. This might explain the much faster killing kinetics of EA-CATH1 than conventional antibiotics revealed by killing kinetics data. In the presence of CaCl2, EA-CATH1 exerted haemagglutination activity, which might potentiate an inhibition against the bacterial polyprotein interaction with the host erythrocyte surface, thereby possibly restricting bacterial colonization and spread.
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Latex beads were sensitized with monoclonal antibodies (MAb) rose against VP28 of WSSV. The optimum concentration of MAb required to sensitize the latex beads was 125 g/ml. The sensitized latex beads were used to detect WSSV from PCR-positive stomach tissue homogenates obtained from infected shrimp. Stomach tissue homogenates from WSSV-infected shrimp agglutinated the sensitized latex beads within 10 minutes, while uninfected samples did not produce any agglutination, although non-specific agglutinations were observed in some samples. The analytical sensitivity, analytical specificity, diagnostic sensitivity and diagnostic specificity of the (LAT) agglutination test were assessed. The analytical sensitivity of the test was 40 ng of purified WSSV (2 g/ml). The sensitized latex beads did not agglutinate with normal shrimp tissue or MBV-infected tissue homogenate. The test has a diagnostic sensitivity of 70 and 45%, respectively, compared to single-step and nested PCR. The diagnostic specificity of the test was 82%. This test is a simple and rapid on-farm test which can be used to corroborate clinical signs for the detection of WSSV in grow-out ponds.
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Acid-sensing ion channels (ASICs) are ligand-gated cation channels activated by extracellular protons. In periphery, they contribute to sensory transmission, including that of nociception and pain. Here we characterized ASIC-like currents in dorsal horn neurons of the rat spinal cord and their functional modulation in pathological conditions. Reverse transcriptase-nested PCR and Western blotting showed that three ASIC isoforms, ASIC1a, ASIC2a, and ASIC2b, are expressed at a high level in dorsal horn neurons. Electrophysiological and pharmacological properties of the proton-gated currents suggest that homomeric ASIC1a and/or heteromeric ASIC1a + 2b channels are responsible for the proton-induced currents in the majority of dorsal horn neurons. Acidification-induced action potentials in these neurons were compatible in a pH-dependent manner with the pH dependence of ASIC-like current. Furthermore, peripheral complete Freund's adjuvant-induced inflammation resulted in increased expression of both ASIC1a and ASIC2a in dorsal horn. These results support the idea that the ASICs of dorsal horn neurons participate in central sensory transmission/modulation under physiological conditions and may play important roles in inflammation-related persistent pain.
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(HAART)AIDS HIVHIVHAART 2003 HIV-1 (WHO)HIV HIV-1 HIV (PBMC) HIV-1 BED-CEIA (VCT) , (nested-PCR)pol ( 199 1242 )PCR HIV (Los Alamos HIV Database) (Standford HIV Drug Resistance Database) 20052006 52 HIV-1 49 PBMC HIV CRF08_BC (51.0%), CRF07_BC (24.5%), CRF01_AE (20.4%)B (4.1%)PI6 (11.7%)7 PI 9 (18.4%)10 (NRTI) 1 (2.0%) (NNRTI) PI/NRTI/NNRTI 1 PBMC 27 VCT 20062007 10310 WB BED-CEIA 63 50 VCT 19 69 VCT HIV B (95.7%)CRF01_AE(2.9%) C(1.4%)PI 26 (37.7%) 27 PI 3 (4.3%)6 NRTI 7 (10.1%)8 NNRTI PI 2 (2.8%)NRTI 1 M184V (3TC)(FTC) 1 T215YM41LL210W (ABC) (ddI)(TDF)(AZT)(d4T) NNRTI 3 (4.3%)1 K103N (NVP)(DLV)(EFV)1 Y188L NVP EFV 1 K101E G190A NVP DLVEFV (ETR) HIV-1 CRF_BC VCT B
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Heterodera avenae,,,(Triticum aestivum L.) E-10Aegilops tauschiiCre3Rccn4,,SON-PCR(single oligonucleotide nested PCR),E-10DNA1264 bp(Rccn-L),,Rccn431209 bp,Cre3861026 bp,,3425.1938112.6Da113332NBS-LRRLRR,XXLXXLXXLLRR17Cre3LRR8978SON-PCR ,Cre3NBS-LRRNBSLRRDNA532bp1175bp32bpNBS-LRR1675bpRCCN1673bp557pI5.3963537.5DaCre387.877RCCNNBSkinase2ILDDkinase3ESKILVTTRSKKGSPLAARTVGGRRCFAYCSEGFRCCN NBSCre3 NBS96.494274548LRRaXXLXXLXXLaIVLFM15.6Cre3LRR80.874 Cereal cyst nematode (CCN) is a damaging pathogen of broad acre cereal crops in Australia, Europe, North America, India and China. It affects wheat, barley, oat and triticale and causes yield loss of up to 80%. At present, Transferring resistance genes against CCN into wheat cultivars and breeding varieties are considered one of the most effective methods for controlling the CCN. However, there are very limited reports concerning the cloning studies of resistance genes against the cereal cyst nematode. According to the sequence of Rccn4 which had high similarity to the nucleotide binding site (NBS) coding region of cereal cyst nematode resistance gene, Cre3 We designed three 3 nested primers. Using single oligonucleotide nested PCR (SON-PCR) we successfully amplified one band, Rccn-L, of 1264bp from E-10 which is the wheat-Ae.variabilis translocation line containing the cereal cyst nematode resistance gene of Ae.variabilis. We found that this band of interesting is the 3 flanking sequence of 1209bp in size of Rccn4. The coding region was 1026bp, which contained an incomplete open reading frame and a terminator codon, without initiation codon and intron, encoding a peptide of 342 amino acid residues, and shared 86nucleotide sequence identity with Cre3. This peptide had a conserved LRR domain, containing the imperfect repeats,XXLXXLXXL, which contains 17 leucine residues and shares, respectively, 89 nucleotide sequence and 78 amino acid sequence identity with the LRR sequence of Cre3 locus. This research firstly used SON-PCR in the research of plant genome successfully, which indicated that SON-PCR is another method of cloning plant gene. At the same time, According to the conversed motif of NBS and LRR region of cereal cyst nematode resistance gene Cre3 from wild wheat (Triticum tauschlii L.) and the known NBS-LRR group resistance genes, we designed two pairs of specific primers for NBS and LRR region respectively. One band of approximately 530bp was amplified using the specific primers for conversed NBS region and one band of approximately 1200bp was amplified with the specific primers for conversed LRR region. After sequencing, we found that these two sequences included 32bp common nucleotide sequence and have 1675 bp in total, which was registered as RCCN in the Genbank. RCCN contained a NBS-LRR domain and an incomplete open reading frame without initiation codon, terminator codon and inxon. Its exon encodes a peptide of 557 amino acid residues. The molecular weight of the protein from the amino acid was 63.537 KDa. The amino acid sequence of RCCN contained conserved motif: ILDD, ESKILVTTRSK, KGSPLAARTVGG, RRCFAYCS, EGFLRR. RCCN shares 87.8 nucleotide sequence and 77 amino acid sequence identity with cereal cyst nematode gene Cre3. It might be a novel cereal cyst nematode resistance gene. These research results of cloning the resistance genes against cereal cyst nematode bring a great promise for transferring resistance genes into wheat cultivars and breeding new wheat varieties against cereal cyst nematode by gene engineering. And these results also lay the hard foundation for the expressing researches of these genes.
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We studied the possible role that marine microalgae may play during the outbreaks of WSS (white spot syndrome). In order to elucidate the possibility of marine microalgae carrying WSSV (white spot syndrome virus), six marine microallgae (Isochr.vsis galbana, Skeletonema costatum, Chlorella sp., Heterosigma akashiwo, Scrippsiella trochoidea, Dunaliella salina) were co-cultured with adult Marsupenaeus japollicus infected with WSSV and were assayed daily by nested-PCR to study whether they could carry WSSV. Further experiments were conducted to investigate whether the virus carried by microalgae could re-infect juvenile M. japonicus. Results showed that all of the experimental microalgae, except H. akashiwo could carry WSSV, and among them, Chlorella sp. and S. trochoidea had the strongest WSSV-carrying ability. Unlike other invertebrate carriers of WSSV, the WSSV detections in microalgae, which were positive after I and 3 days, were negative after 10 days of incubation. WSSV detection results in juvenile M. japonicus showed that the juvenile shrimp were re-infected by co-cultured Chlorella sp., although the juvenile M. japonicus carried so small an amount of WSSV that it could only be detected by nested-PCR. The results of this experiment suggest that microalgae might be one possible horizontal transmission pathway for WSSV. Further research, however, is required to better understand the factors behind the different carrying abilities and virus-carrying mechanisms of different microalgae. (c) 2007 Elsevier Inc. All rights reserved.
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Using the LAMP method, a highly specific and sensitive detection system for genetically modified soybean (Roundup Ready) was designed. In this detection system, a set of four primers was designed by targeting the exogenous 35S epsps gene. Target DNA was amplified and visualized on agarose gel within 45 min under isothermal conditions at 65 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by the addition of SYBR Green I for naked-eye inspection. The detection sensitivity of LAMP was 10-fold higher than the nested PCR established in our laboratory. Moreover, the LAMP method was much quicker, taking only 70 min, as compared with 300 min for nested PCR to complete the analysis of the GM soybean. Compared with traditional PCR approaches, the LAMP procedure is faster and more sensitive, and there is no need for a special PCR machine or electrophoresis equipment. Hence, this method can be a very useful tool for GMO detection and is particularly convenient for fast screening.
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We used nested-polymerase chain reaction (PCR) to detect Roundup Ready soybean in aquatic feeds and feeding tilapias. A template concentration of 10(-10) g mu L-1 DNA solution could be detected with a dilute degree of 0.01%. Most (90.6%) of the aquatic feeds containing soybean byproduct included exogenous DNA segments. We also compared genetically modified (GM) soybean with non-GM soybean diets in feeding tilapias (Oreochromis niloticus, GIFT strain) and examined the residual fragments (254 bp) of GM soybeans. Tilapias receiving GM soybean diets had DNA fragments in different tissues and organs, indicating that exogenous GM genes were absorbed systemically and not completely degraded by the tilapia's alimentary canal.
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<p>The prothrombin G20210A polymorphism is associated with a threefold-increased risk of venous thrombosis. There is considerable variation in the reported prevalence of this polymorphism within normal populations, ranging from 0 to 6.5%. The prevalence within the Irish population has not been determined. A restriction fragment length polymorphism (RFLP)-based assay is commonly used for the detection of the prothrombin 20210A allele. This assay does not include a control restriction digest fragment and, consequently, failure of the enzyme activity or lack of addition of enzyme to the sample cannot be distinguished from wild-type prothrombin. We developed a RFLP-based assay, which incorporates an invariant digest site, resulting in the generation of a control digest fragment. Furthermore, we developed a nested polymerase chain reaction (PCR) method for the amplification and digestion of poor-quality or low-concentration DNA. In the Irish population studied, five of 385 (1.29%) were heterozygous and one patient was homozygous for the prothrombin 20210A polymorphism. This is the first reported data on an Irish or Celtic population and suggests that the allele frequency is similar to Anglo-Saxon populations. The nested PCR method successfully amplified and digested 100/100 (100%) of the archived samples; none of these samples could be analyzed by the standard single-round PCR method. In conclusion, nested PCR should be considered in the analysis of archived samples. Single-round PCR is appropriate for recently collected samples; however, an invariant control digest site should be incorporated in RFLP-based assays to validate the integrity of the digestion enzyme and limit the risk of false-negative results.</p>
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Dissertao de Mestrado, Biologia Marinha, Faculdade de Cincias e Tecnologia, Universidade do Algarve, 2015
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A infeco urogenital causada por Chlamydia trachomatis a doena bacteriana sexualmente transmissvel mais comum na Europa, tanto em homens como em mulheres. Constitui um grave problema de sade pblica devido elevada percentagem de portadores assintomticos, s complicaes clnicas que da podem resultar e possibilidade de transmisso vertical. O presente trabalho teve como principais objectivos: i) avaliar a prevalncia da infeco por C. trachomatis e por Neisseria gonorrhoeae num grupo de grvidas de 36 semanas atendidas na consulta externa de Obstetrcia do Hospital Amadora Sintra e nos recm-nascidos de mes infectadas, ii) identificar os serovares responsveis pelas infeces por C. trachomatis, iii) verificar a distribuio da prevalncia da infeco por C. trachomatis em funo da idade e iv) avaliar a utilidade de uma tcnica de PCR multiplex e de PCR multiplex em tempo real no diagnstico desta infeco. Foram testadas 1201 amostras de urina do primeiro jacto de grvidas e 18 exsudados oculares provenientes de recm-nascidos cujas mes estavam infectadas com C. trachomatis. Cada amostra foi testada pelas tcnicas de PCR multiplex e de PCR multiplex em tempo real, tendo como alvos de amplificao um fragmento do plasmdio crptico e outro do gene omp1. Todos os resultados positivos foram confirmados com uma tcnica de nested PCR e posteriormente enviados para sequenciao para identificao dos serovares envolvidos. Em todas as amostras foi ainda pesquisada a presena de ADN de N. gonorrhoeae atravs de tcnicas de PCR e de PCR em tempo real sendo que, na primeira, o alvo a amplificar foi um fragmento do gene ccpB do plasmdio pJDI e na segunda o pseudogene porA. Os resultados positivos foram confirmados por RFLP. A prevalncia da infeco por C. trachomatis e por N. gonorrhoeae foi de 3,7% (45/1201) e de 0,08% (1/1201), respectivamente. Nos recm-nascidos, a prevalncia foi de 0% para ambas as infeces, embora o nmero de recm-nascidos estudados (18/45) dificilmente seja representativo. O serovar mais prevalente foi o E (31,1%), seguido do G (15,6%), do D/Da (13,3%), do F, I/Ia e do J (11,1%). O serovar K foi identificado em 4,4% das amostras infectadas e o H em apenas 2,2%. A tcnica de PCR multiplex em tempo real parece ser mais adequada para o diagnstico da infeco por C. trachomatis do que a tcnica de PCR multiplex, tendo a primeira detectado 100% dos casos de infeco por este microrganismo (45/45), enquanto que a segunda detectou apenas 71% (32/45) dos mesmos.
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To determine viral subtypes and resistance mutations to antiretroviral treatment (ART) in untreated HIV-1 acutely infected subjects from Southwest Switzerland. Clinical samples were obtained from the HIV primary infection cohort from Lausanne. Briefly, pol gene was amplified by nested PCR and sequenced to generate a 1?kb sequence spanning protease and reverse transcriptase key protein regions. Nucleotide sequences were used to assess viral genotype and ART resistance mutations. Blood specimens and medical information were obtained from 30 patients. Main viral subtypes corresponded to clade B, CRF02_AG, and F1. Resistant mutations to PIs consisted of L10V and accessory mutations 16E and 60E present in all F1 clades. The NNRTI major resistant mutation 103N was detected in all F1 viruses and in other 2 clades. Additionally, we identified F1 sequences from other 6 HIV infected and untreated individuals from Southwest Switzerland, harboring nucleotide motifs and resistance mutations to ART as observed in the F1 strains from the cohort. These data reveal a high transmission rate (16.6%) for NNRTI resistant mutation 103N in a cohort of HIV acute infection. Three of the 5 resistant strains were F1 clades closely related to other F1 isolates from HIV-1 infection untreated patients also coming from Southwest Switzerland. Overall, we provide strong evidence towards an HIV-1 resistant transmission network in Southwest Switzerland. These findings have relevant implications for the local molecular mapping of HIV-1 and future ART surveillance studies in the region.
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Cryptosporidium spp. est un protozoaire parasite du systme gastro-intestinal largement rpandu chez les vertbrs et causant la cryptosporidiose, une zoonose occasionnant des troubles digestifs svres pouvant entrainer la mort chez les individus immunodficients. Au Canada, la dclaration de cette maladie est obligatoire depuis lan 2000. Ainsi, il est pertinent de mieux comprendre linfection chez les animaux de compagnie, puisquils sont potentiellement un rservoir du parasite. Durant lanne 2008, des chantillons fcaux provenant de 1 202 chats (n = 371) et chiens (n = 831) de la province du Qubec ont t analyss par comptage des ookystes de Cryptosporidium spp. au moyen de la technique de centrifugation en solution de sulfate de zinc. Dans cette tude,la prvalence de Cryptosporidium spp. chez les chats (28/371 : 7,55 %) et chez les chiens(88/831 : 10,59 %) de compagnie confirme leur potentiel en tant que rservoir du parasite. Au Qubec, de par leur nombre, les chats sont potentiellement un rservoir zoonotique du parasite plus important que celui des chiens, bien quil nexiste pas de diffrence significative entre la prvalence du parasite chez le chat et le chien pour lanne 2008. Lge (p = 0,0001) et linfection concomitante par Giardia spp. (p = 0,0001) se sont avrs tre des facteurs associs avec la prsence de Cryptosporidium spp. chez le chien. Parmi lensemble des variables testes chez le chat (lge, le sexe, la saison et linfection concomitante par Giardia spp.), aucune na t associe de manire significative la prsence du parasite chez le chat. Ceci peut tre d au nombre limit dindividus tests pour cette espce. Un suivi de lexcrtion des ookystes de Cryptosporidium spp. chez deux chats suggre que lexcrtion des ookystes peut se faire sur une priode de sept mois et que le taux dexcrtion varie dans le temps. Le diagnostic molculaire des espces et gnotypes de Cryptosporidium spp. isols partir des chantillons de matires fcales devait tre ralis par la technique de PCR embote des fragments des gnes ARNr 18S et HSP70 et du squenage des produits de PCR. Aucun rsultat positif na toutefois t obtenu. Afin daugmenter la puissance statistique des analyses pidmiologiques sur la prvalence de Cryptosporidium spp., il serait ncessaire lavenir de travailler sur un nombre danimaux beaucoup plus important.
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The carcass of an adult male beluga (Delphinapterus leucas) was found beach cast in 2008 on the shore of the St. Lawrence Estuary at Rivire-Ouelle, Quebec, Canada. The carcass was transported to the Facult de mdecine vtrinaire of the Universit de Montral for postmortem examination. Aspiration pneumonia was the probable cause of death. Necropsy revealed a focal papilloma-like penile lesion, characterized by focal mucosal thickening with disorganization of the epithelial layers and lymphoplasmacytic infiltration. A pan-herpesvirus nested PCR assay on frozen tissue from the penile lesion was positive. The PCR product sequencing revealed a partial herpesvirus DNA polymerase (DPOL) gene sequence of 600 nucleotides. Its nearest nucleotide identity was with the partial DPOL gene of an alphaherpesvirus, bovine herpesvirus 5 (79.5% identity). It also shared high identity with several other marine mammal herpesviruses (50.2 to 77.3% identity). This new herpesvirus was tentatively named beluga whale herpesvirus (BWHV). Virus isolation was unsuccessful. The pathogenic potential of BWHV is unknown, but the evaluation of archived tissues suggests that the virus is endemic in the St. Lawrence Estuary beluga population.
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This thesis covers various aspects of viral diseases affecting shrimp aquaculture. The research component of this thesis can be divided into four areas. The areas covered are: I) A study to determine the prevalence of WSSV among the crustaceans in the Vembanad estuary, the shrimp aquaculture farms surrounding the estuary, and the sea off Cochin coast, India using two , sets of nested PCR primers. 2) An investigation to compare the sequence of six major structural proteins of WSSV; vp28, vp26, vp 19, vp68, vp281, vp466 from different geographical locations with that of an isolate from India. 3) Simultaneous occurrence of HPV, IHHNV, MBV and WSSV in postlarvae of P. monodon from hatcheries in India was monitored by Polymerase Chain Reaction. 4) A real time PCR procedure was developed for the quantitative analysis of WSSV infection. The viral load of postlarvae from hatcheries in Kerala meant for aquaculture was also determined using the quantitative PCR.
