scholarly journals Molecular identification of the ompL1 gene within Leptospira interrogans standard serovars

2014 ◽  
Vol 8 (06) ◽  
pp. 688-693 ◽  
Author(s):  
Mehrangiz Dezhbord ◽  
Majid Esmaelizad ◽  
Pejvak Khaki ◽  
Fariba Fotohi ◽  
Athena Zarehparvar Moghaddam
Keyword(s):  
Nucleotide Sequence ◽  
Vaccine Candidate ◽  
Pcr Amplification ◽  
Specific Primers ◽  
Pathogenic Leptospira ◽  
Porin Protein ◽  
Amino Acid Alignment ◽  
Leptospira Serovars ◽  
Cloned Gene ◽  
Leptospira Species

Introduction: Leptospirosis, caused by infection with pathogenic Leptospira species, is one of the most prevalent zoonotic diseases in the world. Current leptospiral vaccines are mainly multivalent dead whole-cell mixtures made of several local dominant serovars. Therefore, design and construction of an efficient recombinant vaccine for leptospirosis control is very important. OmpL1 is an immunogenic porin protein that could be of special significance in vaccination and serodiagnosis for leptospirosis. Methodology: Three strains belonging to pathogenic L. interrogans were analyzed. The specific primers for proliferation of the ompL1 gene were designed. The amplified gene was cloned. In order to investigate the ompL1 nucleotide sequence and homological analysis of this gene, ompL1 genes cloned from standard vaccinal Leptospira serovars prevalent in Iran were sequenced and cloned. Results: PCR amplification of the ompL1 gene using the designed primers resulted in a 963 bp ompL1 gene product. The PCR based on the ompL1 gene detected all pathogenic reference serovars of Leptospira spp. tested. Based on alignment and phylogenetic analysis, although the ompL1 nucleotide sequence was slightly different within three vaccinal serovars (100%-85% identity), amino acid alignment of the OmpL1 proteins revealed that there would be inconsiderable difference among them. Conclusion: The ompL1 gene of the three isolates was well conserved, differing only by a total of 6 bp and the proteins by 2 amino acids. The cloned gene could be further used for expression and recombinant OmpL1 as an efficient and conserved antigen, and may be a useful vaccine candidate against leptospirosis in our region.

A comparative hybridization analysis of yeast DNA with Paramecium parafusin- and different phosphoglucomutase-specific probes

2000 ◽  
Vol 78 (6) ◽  
pp. 683-690 ◽  
Author(s):  
Elzbieta Wyroba ◽  
Birgit H Satir
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Genomic Dna ◽  
Pcr Amplification ◽  
Molecular Size ◽  
Molecular Probes ◽  
Hybridization Analysis ◽  
Yeast Genome ◽  
Specific Primers ◽  
Genome Database

Molecular probes designed for the parafusin (PFUS), the Paramecium exocytic-sensitive phospho glyco protein, gave distinct hybridization patterns in Saccharomyces cerevisiae genomic DNA when compared with different phosphoglucomutase specific probes. These include two probes identical to segments of yeast phosphoglucomutase (PGM) genes 1 and 2. Neither of the PGM probes revealed the 7.4 and 5.9 kb fragments in Bgl II-cut yeast DNA digest detected with the 1.6 kb cloned PFUS cDNA and oligonucleotide constructed to the PFUS region (insertion 3 – I-3) not found in other species. PCR amplification with PFUS-specific primers generated yeast DNA-species of the predicted molecular size which hybridized to the I-3 probe. A search of the yeast genome database produced an unassigned nucleotide sequence that showed 55% identity to parafusin gene and 37% identity to PGM2 (the major isoform of yeast phosphoglucomutase) within the amplified region.Key words: parafusin, phosphoglucomutase, yeast, hybridization, PCR.

scholarly journals Serologic and urinary survey of exposure to Leptospira species in a feral cat population of Prince Edward Island, Canada

2021 ◽  
pp. 1098612X2110010
Author(s):  
Emilia Bourassi ◽  
Christine Savidge ◽  
Peter Foley ◽  
Sunny Hartwig
Keyword(s):  
Prince Edward Island ◽  
Urine Samples ◽  
Pathogenic Leptospira ◽  
Feral Cat ◽  
Feral Cats ◽  
Antibody Titers ◽  
Leptospira Serovars ◽  
Blood And Urine Samples ◽  
Leptospira Species

Objectives Recent studies show that cats could play an important role in the transmission of Leptospira species. There are few reports of leptospirosis on Prince Edward Island (PEI) and none in cats. The objective of this study was to determine the prevalence of serum antibodies against Leptospira serovars and of Leptospira DNA in the urine of a population of free-roaming cats. Methods Paired blood and urine samples were collected from 200 cats brought to a trap–neuter–return program. Antibody titers against six Leptospira serovars (Bratislava, Canicola, Gryppotyphosa, Hardjo, Pomona, Icterohaemorrhagiae) were determined by microscopic agglutination test. PCR was performed on urine samples to identify urine shedding of Leptospira DNA. Results Antibodies were detected in 20/200 cats (10%) for at least one serovar, with titers ranging from 1:50 to 1:6400 (all serovars tested, except Hardjo). Urine samples of 7/200 cats (3.5%) were PCR-positive. Conclusions and relevance Feral cats in Prince Edward Island (PEI) had a higher than expected exposure to leptospirosis and can shed DNA from pathogenic Leptospira species in urine. Further studies are needed to determine the prevalence of exposure to leptospirosis in other species on PEI and the potential role of feral cats in transmission of the disease.

scholarly journals Isolation and Molecular Cloning of Cellulase Gene from Bovine Rumen Bacteria

2017 ◽  
Vol 1 (1) ◽  
pp. 29-36
Author(s):  
Rahadian Pratama ◽  
I Made Artika ◽  
Tetty Chaidamsari ◽  
Herti Sugiarti ◽  
Soekarno Mismana Putra
Keyword(s):  
Nucleotide Sequence ◽  
Cellulosic Biomass ◽  
Nucleotide Sequence Analysis ◽  
Rumen Bacteria ◽  
Cellulase Gene ◽  
Specific Primers ◽  
Total Rna ◽  
Bovine Rumen ◽  
Degrading Bacteria ◽  
Cloned Gene

Cellulases are the enzymes that hydrolyze cellulosic biomass and are produced by the microorganisms that grow over cellulosic matters. The objective of this research was to isolate and clone cellulase gene from cellulose-degrading bacteria of bovine rumen. Cellulose-degrading bacteria was isolated from rumen fluid using a selective medium. Total RNA was isolated from selected colony having cellulose degrading activity and was used as a template for cDNA construction using reverse transcriptase polymerase chain reaction (RT-PCR) technique. The resulted cDNA was employed as a template for PCR amplification of cellulase gene using specific primers. The cellulase gene candidate obtained was cloned into the pGEM-T-Easy vector followed by determination of its nucleotide sequence. The sequence was then aligned with sequences of cellulase genes from GenBank. Results showed that a number of isolates of rumen bacteria exhibit cellulase activity and the CR-8 isolate was selected for further analysis. The successful isolation of total RNA from CR-8 was indicated by the presence of two intense bands of ribosomal RNA (23S and 16S). The reverse transcription process was successful and the amplification of cellulase gene using the specific primers F1 and R1 resulted in a DNA fragment of 1900 bp as a candidate of cellulase gene. The fragment was successfully cloned into the pGEM-T-Easy vector, and the resulted recombinant plasmid was successfully introduced into the E. coli cells. Nucleotide sequence analysis suggested that the cloned gene is cellulase gene and shares 99% homology with the endo-1,6-beta-glucanase of T. harzianum.

Organization, Expression and Evolution of a Disease Resistance Gene Cluster in Soybean

Genetics ◽  
2002 ◽  
Vol 162 (4) ◽  
pp. 1961-1977
Author(s):  
Michelle A Graham ◽  
Laura Fredrick Marek ◽  
Randy C Shoemaker
Keyword(s):  
Disease Resistance ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Developmental Stages ◽  
Gene Evolution ◽  
Pcr Amplification ◽  
Disease Resistance Genes ◽  
Disease Resistance Gene ◽  
R Genes ◽  
Specific Primers

Abstract PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar “Williams 82” [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca2+-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.

scholarly journals Distribution of the leptospiral immunoglobulin-like (lig) genes in pathogenic Leptospira species and application of ligB to typing leptospiral isolates

2009 ◽  
Vol 58 (9) ◽  
pp. 1173-1181 ◽  
Author(s):  
Gustavo M. Cerqueira ◽  
Alan J. A. McBride ◽  
Mathieu Picardeau ◽  
Samuel G. Ribeiro ◽  
Ângela N. Moreira ◽  
...  
Keyword(s):  
Clinical Isolates ◽  
Pathogenic Leptospira ◽  
Reliable Identification ◽  
Specific Pcr ◽  
The Family ◽  
Leptospira Species

The family of leptospiral immunoglobulin-like (lig) genes comprises ligA, ligB and ligC. This study used PCR to demonstrate the presence of lig genes among serovars from a collection of leptospiral strains and clinical isolates. Whilst ligA and ligC appeared to be present in a limited number of pathogenic serovars, the ligB gene was distributed ubiquitously among all pathogenic strains. None of the lig genes were detected among intermediate or saprophytic Leptospira species. It was also shown that, similar to the previously characterized secY gene, a short specific PCR fragment of ligB could be used to correctly identify pathogenic Leptospira species. These findings demonstrate that ligB is widely present among pathogenic strains and may be useful for their reliable identification and classification.

scholarly journals First Report of Mosaic Disease Caused by Colombian datura virus on Solanum lycopersicum Plants Commercially Cultivated in Japan

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 698-698 ◽  
Author(s):  
Y. Tomitaka ◽  
T. Usugi ◽  
R. Kozuka ◽  
S. Tsuda
Keyword(s):  
Nucleotide Sequence ◽  
Solanum Lycopersicum ◽  
Mosaic Disease ◽  
Causal Agent ◽  
Cultivated Plants ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Specific Primers ◽  
Tomato Plants ◽  
First Report

In 2009, some commercially grown tomato (Solanum lycopersicum) plants in Chiba Prefecture, Japan, exhibited mosaic symptoms. Ten plants from a total of about 72,000 cultivated plants in the greenhouses showed such symptoms. To identify the causal agent, sap from leaves of the diseased plants was inoculated into Chenopodium quinoa and Nicotiana benthamiana plants. Local necrotic lesions appeared on inoculated leaves of C. quinoa, but no systemic infection was observed. Systemic mosaic symptoms were observed on the N. benthamiana plants inoculated. Single local lesion isolation was performed three times using C. quinoa to obtain a reference isolate for further characterization. N. benthamiana was used for propagation of the isolate. Sap from infected leaves of N. benthamiana was mechanically inoculated into three individual S. lycopersicum cv. Momotaro. Symptoms appearing on inoculated tomatoes were indistinguishable from those of diseased tomato plants found initially in the greenhouse. Flexuous, filamentous particles, ~750 nm long, were observed by electron microscopy in the sap of the tomato plants inoculated with the isolate, indicating that the infecting virus may belong to the family Potyviridae. To determine genomic sequence of the virus, RT-PCR was performed. Total RNA was extracted from the tomato leaves experimentally infected with the isolate using an RNeasy Plant Mini kit (QIAGEN, Hilden, Germany). RT-PCR was performed by using a set of universal, degenerate primers for Potyviruses as previously reported (2). Amplicons (~1,500 bp) generated by RT-PCR were extracted from the gels using the QIAquick Gel Extraction kit (QIAGEN) and cloned into pCR-BluntII TOPO (Invitrogen, San Diego, CA). DNA sequences of three individual clones were determined using a combination of plasmid and virus-specific primers, showing that identity among three clones was 99.8%. A consensus nucleotide sequence of the isolate was deposited in GenBank (AB823816). BLASTn analysis of the nucleotide sequence determined showed 99% identity with a partial sequence in the NIb/coat protein (CP) region of Colombian datura virus (CDV) tobacco isolate (JQ801448). Comparison of the amino acid sequence predicted for the CP with previously reported sequences for CDV (AY621656, AJ237923, EU571230, AM113759, AM113754, and AM113761) showed 97 to 100% identity range. Subsequently, CDV infection in both the original and experimentally inoculated plants was confirmed by RT-PCR using CDV-specific primers (CDVv and CDVvc; [1]), and, hence, the causal agent of the tomato disease observed in greenhouse tomatoes was proved to be CDV. The first case of CDV on tomato was reported in Netherlands (3), indicating that CDV was transmitted by aphids from CDV-infected Brugmansia plants cultivated in the same greenhouse. We carefully investigated whether Brugmansia plants naturally grew around the greenhouses, but we could not find them inside or in proximity to the greenhouses. Therefore, sources of CDV inoculum in Japan are still unclear. This is the first report of a mosaic disease caused by CDV on commercially cultivated S. lycopersicum in Japan. References: (1) D. O. Chellemi et al. Plant Dis. 95:755, 2011. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) J. Th. J. Verhoeven et al. Eur. J. Plant. Pathol. 102:895, 1996.

scholarly journals Specific DNA identification of Pheretima in the Naoxintong capsule

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Xiaoxiao Zhu ◽  
Hoi-Yan Wu ◽  
Pang-Chui Shaw ◽  
Wei Peng ◽  
Weiwei Su
Keyword(s):  
Pcr Amplification ◽  
Cerebrovascular Diseases ◽  
Coi Gene ◽  
Chemical Marker ◽  
Dna Identification ◽  
Specific Primers ◽  
Blast Search ◽  
Pcr Products ◽  
Crude Drugs ◽  
Nucleotide Database

Abstract Background Pheretima is a minister drug in Naoxintong capsule (NXTC), a well-known traditional Chinese medicine (TCM) formula for the treatment of cardiovascular and cerebrovascular diseases. Owing to the loss of morphological and microscopic characteristics and the lack of recognized chemical marker, it is difficult to identify Pheretima in NXTC. This study aims to evaluate the feasibility of using DNA techniques to authenticate Pheretima, especially when it is processed into NXTC. Methods DNA was extracted from crude drugs of the genuine and adulterant species, as well as nine batches of NXTCs. Based on mitochondrial cytochrome c oxidase subunit I (COI) gene, specific primers were designed for two genera of genuine species, Metaphire and Amynthas, respectively. PCR amplification was performed with the designed primers on crude drugs of Pheretima and NXTCs. The purified PCR products were sequenced and the obtained sequences were identified to species level with top hit of similarity with BLAST against GenBank nucleotide database. Results Primers MF2R2 and AF3R1 could amplify specific DNA fragments with sizes around 230–250 bp, both in crude drugs and NXTC. With sequencing and the BLAST search, identities of the tested samples were found. Conclusion This study indicated that the molecular approach is effective for identifying Pheretima in NXTC. Therefore, DNA identification may contribute to the quality control and assurance of NXTC.

scholarly journals Molecular characterisation of Leptospira strains in Pakistan

2016 ◽  
Vol 60 (4) ◽  
pp. 417-421
Author(s):  
Muhammad Luqman Sohail ◽  
Muhammad Sarwar Khan ◽  
Muhammad Avais ◽  
Muhammad Yasir Zahoor ◽  
Irfan Khattak ◽  
...  
Keyword(s):  
16S Rrna ◽  
Animal Species ◽  
Molecular Detection ◽  
Molecular Evidence ◽  
Rrna Gene ◽  
Intermediate Species ◽  
Specific Primers ◽  
Pathogenic Leptospira ◽  
Wide Range ◽  
Serological Studies

Abstract Introduction: Leptospirosis affects a wide range of mammals, humans, and even a few poikilothermic animal species. In Pakistan, serological studies of equine leptospirosis have reported a prevalence of over 40%, but no study has ever been conducted towards molecular detection of Leptospira in horses. Material and Methods: Blood samples from 128 horses were screened using ELISA and 41 positive samples were examined for the presence of leptospiral DNA using specific primers for 16S rRNA gene. Results: Out of 41 tested samples, 20 samples were found to be PCR-positive, revealing a fragment of 306 bp after gel electrophoresis. Sequencing and phylogenetic analysis of positive samples revealed circulation of pathogenic Leptospira spp. in Pakistani horses. No evidence of circulation of intermediate species was found in this study. Conclusion: This study reports the first molecular evidence of equine leptospirosis in Pakistan and lays ground for further research in this area. It also confirms the efficiency of 16S rRNA for the diagnosis of equine leptospirosis.

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