scholarly journals Detection and characterization of clustered regularly interspaced short palindromic repeat-associated endoribonuclease gene variants in Vibrio parahaemolyticus isolated from seafoods and environment

2019 ◽  
Vol 12 (5) ◽  
pp. 689-695
Author(s):  
Pallavi Baliga ◽  
Malathi Shekar ◽  
Moleyur Nagarajappa Venugopal
Keyword(s):  
Polymerase Chain Reaction ◽  
Vibrio Parahaemolyticus ◽  
Genotypic Variation ◽  
Gene Variants ◽  
Chain Reaction ◽  
Pcr Products ◽  
Short Palindromic Repeat ◽  
Polymerase Chain

Aim: In Vibrio parahaemolyticus, the clustered regularly interspaced short palindromic repeat (CRISPR)-associated cas6 endoribonuclease gene has been shown to exhibit sequence diversity and has been subtyped into four major types based on its length and composition. In this study, we aimed to detect and characterize the cas6 gene variants prevalent among V. parahaemolyticus strains isolated from seafoods and environment. Materials and Methods: Novel primers were designed for each of the cas6 subtypes to validate their identification in V. parahaemolyticus by polymerase chain reaction (PCR). In total, 38 V. parahaemolyticus strains isolated from seafoods and environment were screened for the presence of cas6 gene. Few representative PCR products were sequenced, and their phylogenetic relationship was established to available cas6 gene sequences in GenBank database. Results: Of the 38 V. parahaemolyticus isolates screened, only about 40% of strains harbored the cas6 endoribonuclease gene, among which 31.6% and 7.9% of the isolates were positive for the presence of the cas6-a and cas6-d subtypes of the gene, respectively. The subtypes cas6-b and cas6-c were absent in strains studied. Sequence and phylogenetic analysis also established the cas6 sequences in this study to match GenBank sequences for cas6-a and cas6-d subtypes. Conclusion: In V. parahaemolyticus, the Cas6 endoribonuclease is an associated protein of the CRISPR-cas system. CRISPR-positive strains exhibited genotypic variation for this gene. Primers designed in this study would aid in identifying the cas6 genotype and understanding the role of these genotypes in the CRISPR-cas immune system of the pathogen.

scholarly journals Detection and Molecular Characterization of Vibrio Parahaemolyticus in Shrimp Samples

2018 ◽  
Vol 12 (1) ◽  
pp. 46-50 ◽  
Author(s):  
Daryoush Asgarpoor ◽  
Fakhri Haghi ◽  
Habib Zeighami
Keyword(s):  
Public Health ◽  
Polymerase Chain Reaction ◽  
Vibrio Parahaemolyticus ◽  
Foodborne Diseases ◽  
Chain Reaction ◽  
Biochemical Tests ◽  
Global Issue ◽  
Retail Outlets ◽  
Polymerase Chain

Background:Food safety has emerged as an important global issue with international trade and public health implications. Bacterial pathogens asVibrio parahaemolyticusrecognized as an important cause of foodborne diseases related to the consumption of raw, undercooked or mishandled seafood worldwide.Methods:A total of 70 individual wild shrimp samples were collected from shrimp retail outlets in Zanjan, Iran and investigated for the presence of potentially pathogenic strains ofV. parahaemolyticus.The shrimp samples were immediately homogenized and cultured on TCBS agarand subjected to confirmatory biochemical tests. Polymerase Chain Reaction (PCR) was performed for detection of total and pathogenicV. parahaemolyticusby amplification ofvp–toxR,tdhandtrhgenes.Results:The conventional method indicated that 16 (22.8%) of samples were positive forV. parahaemolyticus. However, PCR verified that only 12 (17.1%) shrimp samples were positive forV. parahaemolyticus.Of the 70 shrimp samples in our study, only 2 (2.8%)tdhand 1 (1.4%)trhpositive strains were identified.Conclusion:Detection oftdhand/ ortrhpositiveV. parahaemolyticusin shrimp marketed in Zanjan, Iran shows a probable risk for public health. Therefore, the reliable molecular methods for monitoring of potentially pathogenicV. parahaemolyticusare strongly recommended for the routine seafood examination.

scholarly journals Characterization of clone-specific rearrangement T-cell receptor gamma- chain genes in lymphomas and leukemias by the polymerase chain reaction and DNA sequencing

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 574-581 ◽  
Author(s):  
M Kneba ◽  
I Bolz ◽  
B Linke ◽  
J Bertram ◽  
D Rothaupt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
T Cell ◽  
Dna Sequencing ◽  
Cell Lines ◽  
Chain Reaction ◽  
Gamma Chain ◽  
Pcr Products ◽  
Polymerase Chain

The structures of rearranged gamma-chain T-cell antigen receptor (TCR) genes were analyzed in 5 cases of T-cell acute lymphoblastic leukemia (T-ALL), in 15 cases of peripheral T-cell non-Hodgkin's lymphoma (T- NHL), in 1 case with large granular CD8 lymphocytosis, 1 case with CD8 lymphocytosis after autologous bone marrow transplantation for Hodgkin's disease, and in 2 cases with nonneoplastic diseases. Rearranged V-J TCR gamma-gene segments were amplified by the polymerase chain reaction (PCR). Because most of the biopsy tissue or bone marrow samples contained significant amounts of admixed nonmalignant T-cells, direct DNA sequencing of the PCR products yielded mixed sequence data because of coamplification of clonal together with polyclonal TCR gamma V-N-J junctions. Reliable data could only be obtained by cloning the V gamma-J gamma PCR products and sequencing several (4 to 10) randomly chosen clones. In the polyclonal samples, all PCR-derived clones differed in their specific V-N-J junctions, as expected. In the two T- cell lines and in most of the T-cell malignancies, monoclonal PCR products could be identified by the demonstration of clonally restricted V-N-J junctions. In most cases, this information yielded the desired clone-specific sequence and showed a background population of polyclonal TCR gamma cells in each specimen, except for those that were obtained from the T-ALL samples, the cell lines, or the NHL samples with high tumor cell fraction. The results obtained by PCR-directed sequencing were confirmed by temperature-gradient gel electrophoresis (TGGE) that showed distinct DNA bands only with the PCR products containing predominant (ie, monoclonal) TCR gamma V-N-J junctions. By combined sequence and TGGE analysis, it was found that PCR/TGGE is able to distinguish between monoclonal and polyclonal TCR gamma-PCR products. This finding prompted us to complete the analysis of the TCR gamma locus in the samples by PCR/TGGE using primer mixes which covered all possible V gamma and J gamma recombinations. Monoclonality was shown with all mixes by PCR/TGGE in 21 of 24 (87%) of the lymphoproliferations. In summary, the present study shows that the combination of amplifying TCR gamma V-N-J junctions by PCR with the identification of clonal PCR products by TGGE and DNA sequencing is a reliable method for the characterization of clonal TCR gamma sequences.

scholarly journals Characterization of clone-specific rearrangement T-cell receptor gamma- chain genes in lymphomas and leukemias by the polymerase chain reaction and DNA sequencing

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 574-581 ◽  
Author(s):  
M Kneba ◽  
I Bolz ◽  
B Linke ◽  
J Bertram ◽  
D Rothaupt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
T Cell ◽  
Dna Sequencing ◽  
Cell Lines ◽  
Chain Reaction ◽  
Gamma Chain ◽  
Pcr Products ◽  
Polymerase Chain

Abstract The structures of rearranged gamma-chain T-cell antigen receptor (TCR) genes were analyzed in 5 cases of T-cell acute lymphoblastic leukemia (T-ALL), in 15 cases of peripheral T-cell non-Hodgkin's lymphoma (T- NHL), in 1 case with large granular CD8 lymphocytosis, 1 case with CD8 lymphocytosis after autologous bone marrow transplantation for Hodgkin's disease, and in 2 cases with nonneoplastic diseases. Rearranged V-J TCR gamma-gene segments were amplified by the polymerase chain reaction (PCR). Because most of the biopsy tissue or bone marrow samples contained significant amounts of admixed nonmalignant T-cells, direct DNA sequencing of the PCR products yielded mixed sequence data because of coamplification of clonal together with polyclonal TCR gamma V-N-J junctions. Reliable data could only be obtained by cloning the V gamma-J gamma PCR products and sequencing several (4 to 10) randomly chosen clones. In the polyclonal samples, all PCR-derived clones differed in their specific V-N-J junctions, as expected. In the two T- cell lines and in most of the T-cell malignancies, monoclonal PCR products could be identified by the demonstration of clonally restricted V-N-J junctions. In most cases, this information yielded the desired clone-specific sequence and showed a background population of polyclonal TCR gamma cells in each specimen, except for those that were obtained from the T-ALL samples, the cell lines, or the NHL samples with high tumor cell fraction. The results obtained by PCR-directed sequencing were confirmed by temperature-gradient gel electrophoresis (TGGE) that showed distinct DNA bands only with the PCR products containing predominant (ie, monoclonal) TCR gamma V-N-J junctions. By combined sequence and TGGE analysis, it was found that PCR/TGGE is able to distinguish between monoclonal and polyclonal TCR gamma-PCR products. This finding prompted us to complete the analysis of the TCR gamma locus in the samples by PCR/TGGE using primer mixes which covered all possible V gamma and J gamma recombinations. Monoclonality was shown with all mixes by PCR/TGGE in 21 of 24 (87%) of the lymphoproliferations. In summary, the present study shows that the combination of amplifying TCR gamma V-N-J junctions by PCR with the identification of clonal PCR products by TGGE and DNA sequencing is a reliable method for the characterization of clonal TCR gamma sequences.

scholarly journals A Comparative Study of 5S rDNA Non-Transcribed Spacers in Elaeagnaceae Species

Plants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 4
Author(s):  
Oleg S. Alexandrov ◽  
Olga V. Razumova ◽  
Gennady I. Karlov
Keyword(s):  
Polymerase Chain Reaction ◽  
Molecular Markers ◽  
Dna Markers ◽  
5S Rdna ◽  
Chain Reaction ◽  
Closely Related Species ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Species Specific ◽  
Shepherdia Canadensis

5S rDNA is organized as a cluster of tandemly repeated monomers that consist of the conservative 120 bp coding part and non-transcribed spacers (NTSs) with different lengths and sequences among different species. The polymorphism in the 5S rDNA NTSs of closely related species is interesting for phylogenetic and evolutional investigations, as well as for the development of molecular markers. In this study, the 5S rDNA NTSs were amplified with universal 5S1/5S2 primers in some species of the Elaeagnaceae Adans. family. The polymerase chain reaction (PCR) products of five Elaeagnus species had similar lengths near 310 bp and were different from Shepherdia canadensis (L.) Nutt. and Sh. argentea (Pusch.) Nutt. samples (260 bp and 215 bp, respectively). The PCR products were cloned and sequenced. An analysis of the sequences revealed that intraspecific levels of NTS identity are high (approximately 95–96%) and similar in the Elaeagnus L. species. In Sh. argentea, this level was slightly lower due to the differences in the poly-T region. Moreover, the intergeneric and intervarietal NTS identity levels were studied and compared. Significant differences between species (except E. multiflora Thunb. and E. umbellata Thunb.) and genera were found. Herein, a range of the NTS features is discussed. This study is another step in the investigation of the molecular evolution of Elaeagnaceae and may be useful for the development of species-specific DNA markers in this family.

Characterization of Giardia duodenalis by polymerase-chain-reaction fingerprinting

1998 ◽  
Vol 84 (9) ◽  
pp. 707-714 ◽  
Author(s):  
Wieger L. Homan ◽  
Margriet Gilsing ◽  
Hafida Bentala ◽  
Louis Limper ◽  
Frans van Knapen
Keyword(s):  
Polymerase Chain Reaction ◽  
Giardia Duodenalis ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Rhodococcus equi Infections in Goats: Characterization of Virulence Plasmids

2017 ◽  
Vol 55 (2) ◽  
pp. 273-276 ◽  
Author(s):  
Lauren W. Stranahan ◽  
Quinci D. Plumlee ◽  
Sara D. Lawhon ◽  
Noah D. Cohen ◽  
Laura K. Bryan
Keyword(s):  
Polymerase Chain Reaction ◽  
Lymph Nodes ◽  
Rhodococcus Equi ◽  
Caseous Lymphadenitis ◽  
Chain Reaction ◽  
Disseminated Infection ◽  
Virulence Plasmids ◽  
Visceral Organs ◽  
Polymerase Chain

Rhodococcus equi is an uncommon cause of systemic pyogranulomatous infections in goats with macroscopic similarities to caseous lymphadenitis caused by Corynebacterium pseudotuberculosis. Caprine cases have previously been reported to be caused by avirulent R. equi strains. Six cases of R. equi infection in goats yielding 8 R. equi isolates were identified from 2000 to 2017. Lesions varied from bronchopneumonia, vertebral and humeral osteomyelitis, and subcutaneous abscesses, to disseminated infection involving the lungs, lymph nodes, and multiple visceral organs. Isolates of R. equi from infected goats were analyzed by polymerase chain reaction for R. equi virulence-associated plasmid ( vap) genes. Seven of 8 isolates carried the VapN plasmid, originally characterized in bovine isolates, while 1 isolate lacked virulence plasmids and was classified as avirulent. The VapN plasmid has not been described in isolates cultured from goats.

scholarly journals Macrophage migration inhibitory factor may play a protective role in osteoarthritis

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Ming Liu ◽  
Zikun Xie ◽  
Guang Sun ◽  
Liujun Chen ◽  
Dake Qi ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Migration Inhibitory Factor ◽  
Macrophage Migration Inhibitory Factor ◽  
Protective Role ◽  
Macrophage Migration ◽  
Chain Reaction ◽  
Protein Levels ◽  
Tnf Α ◽  
Polymerase Chain

Abstract Background Osteoarthritis (OA) is the most prevalent form of arthritis and the major cause of disability and overall diminution of quality of life in the elderly population. Currently there is no cure for OA, partly due to the large gaps in our understanding of its underlying molecular and cellular mechanisms. Macrophage migration inhibitory factor (MIF) is a procytokine that mediates pleiotropic inflammatory effects in inflammatory diseases such as rheumatoid arthritis (RA) and ankylosing spondylitis (AS). However, data on the role of MIF in OA is limited with conflicting results. We undertook this study to investigate the role of MIF in OA by examining MIF genotype, mRNA expression, and protein levels in the Newfoundland Osteoarthritis Study. Methods One hundred nineteen end-stage knee/hip OA patients, 16 RA patients, and 113 healthy controls were included in the study. Two polymorphisms in the MIF gene, rs755622, and -794 CATT5-8, were genotyped using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and PCR followed by automated capillary electrophoresis, respectively. MIF mRNA levels in articular cartilage and subchondral bone were measured by quantitative polymerase chain reaction. Plasma concentrations of MIF, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) were measured by enzyme-linked immunosorbent assay. Results rs755622 and -794 CATT5-8 genotypes were not associated with MIF mRNA or protein levels or OA (all p ≥ 0.19). MIF mRNA level in cartilage was lower in OA patients than in controls (p = 0.028) and RA patients (p = 0.004), while the levels in bone were comparable between OA patients and controls (p = 0.165). MIF protein level in plasma was lower in OA patients than in controls (p = 3.01 × 10−10), while the levels of TNF-α, IL-6 and IL-1β in plasma were all significantly higher in OA patients than in controls (all p ≤ 0.0007). Multivariable logistic regression showed lower MIF and higher IL-1β protein levels in plasma were independently associated with OA (OR per SD increase = 0.10 and 8.08; 95% CI = 0.04–0.19 and 4.42–16.82, respectively), but TNF-α and IL-6 became non-significant. Conclusions Reduced MIF mRNA and protein expression in OA patients suggested MIF might have a protective role in OA and could serve as a biomarker to differentiate OA from other joint disorders.

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