Detection and identification of Clavibacter michiganensis subsp. sepedonicus and Clavibacter michiganensis subsp. michiganensis by nonradioactive hybridization, polymerase chain reaction, and restriction enzyme analysis

1994 ◽  
Vol 40 (12) ◽  
pp. 1007-1018 ◽  
Author(s):  
J. L. W. Rademaker ◽  
J. D. Janse
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Product ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Bacterial Strains ◽  
Clavibacter Michiganensis ◽  
Chain Reaction ◽  
Clavibacter Michiganensis Subsp ◽  
Detection And Identification ◽  
Polymerase Chain

To develop a rapid and reliable detection and identification method for Clavibacter michiganensis subsp. sepedonicus and C. michiganensis subsp. michiganensis, two biotinylated probes and derived primer sets were evaluated for specificity using a large number of bacterial strains. Detection in dot blot analysis using the Diagen probe against C. michiganensis subsp. sepedonicus was possible with all 32 C. michiganensis subsp. sepedonicus strains tested. Cross-hybridization occurred with all nine C. michiganensis subsp. insidiosus strains tested. No hybridization occurred with any of 54 other related and unrelated bacterial strains including C. michiganensis subsp. michiganensis, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, C. iranicus, C. rathayi, and C. tritici and potato saprophytes. Hybridization of the MIC 1 probe against C. michiganensis subsp. michiganensis was obtained with 22 out of 24 C. michiganensis subsp. michiganensis strains. A weak hybridization signal occurred only with two strains of C. michiganensis subsp. insidiosns. No hybridization occurred with any of the 71 other related and unrelated bacterial strains tested including tomato saprophytes. Restriction fragment length polymorphisms detected with the Diagen probe allowed differentiation between C. michiganensis subsp. sepedonicus and the related C. michiganensis subsp. insidiosus. Restriction fragment length polymorphism analysis using the MIC 1 probe and BamH1 showed at least two groups of patterns within C. michiganensis subsp. michiganensis. By using a primer set derived from the Diagen probe, a DNA sequence could be amplified with all C. michiganensis subsp. sepedonicus strains tested. Only the nontarget organism C. michiganensis subsp. insidiosus yielded a similar polymerase chain reaction product. Restriction enzyme analysis of the polymerase chain reaction product enabled rapid distinction between the subspecies. With a CMM primer set derived from the MIC 1 probe a DNA sequence was amplified from the same 22 out of 24 C. michiganensis subsp. michiganensis strains that showed hybridization with the MIC 1 probe. The polymerase chain reaction product could be verified by restriction enzyme analysis. The Diagen and MIC 1 probes and derived primer sets were shown to be useful for the detection and identification of C. michiganensis subsp. sepedonicus and C. michiganensis subsp. michiganensis. The MIC 1 probe, however, failed to detect two strains of the latter subspecies.Key words: biotin, PCR, REA, potato bacterial ring rot, bacterial canker of tomato, RFLP, Clavibacter michiganensis subsp. insidiosus.

scholarly journals Genome Analysis of North American Small Ruminant Lentiviruses by Polymerase Chain Reaction and Restriction Enzyme Analysis

1995 ◽  
Vol 7 (4) ◽  
pp. 437-443 ◽  
Author(s):  
Sergio Rosati ◽  
Jimmy Kwang ◽  
James E. Keen
Keyword(s):  
Polymerase Chain Reaction ◽  
North American ◽  
Small Ruminant ◽  
Control Variable ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Small Ruminant Lentiviruses

The polymerase chain reaction (PCR) was used to amplify portions of the gag and env structural genes of 8 ovine and 1 caprine lentivirus isolates of North American origin. Three sets of primers were used to amplify p16, p25, and Nî-gp40 gene fragments, and 1 set, annealing highly conserved portions of long terminal repeat (LTR) among small ruminant lentiviruses, was used as a positive control. Variable PCR amplification efficiency was observed. Different stringency conditions of hybridization with specific DNA probes were used to maximize detection of the PCR product. The p25 primers detected all strains, the gp40 primers detected 1 ovine and the caprine strain, and the p16 primers detected only 1 ovine isolate. All strains were detected by LTR primers. Restriction endonuclease analysis of 5 amplified p25 and 2 Nî-gp40 gene fragments revealed extensive heterogeneity among these North American small ruminant lentiviruses.

scholarly journals Demonstration of osteonectin mRNA in megakaryocytes: the use of the polymerase chain reaction

Blood ◽  
1991 ◽  
Vol 78 (5) ◽  
pp. 1216-1222 ◽  
Author(s):  
XC Villarreal ◽  
BW Grant ◽  
GL Long
Keyword(s):  
Polymerase Chain Reaction ◽  
Cell Population ◽  
Bone Cells ◽  
Single Cells ◽  
Northern Analysis ◽  
Enzyme Analysis ◽  
Cell Populations ◽  
Restriction Enzyme Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Platelets have been shown to release osteonectin on thrombin stimulation. The origin of platelet osteonectin was unclear as it may have been synthesized by megakaryocytes or it may have been endocytosed from plasma as other platelet alpha-granule constituents are. Platelet osteonectin has a larger apparent molecular size than the bone species, although the molecular basis for this difference has not been elucidated. These two issues have been addressed here by (1) examining the potential for osteonectin biosynthesis in human megakaryocytes by demonstrating the presence of osteonectin mRNA in purified megakaryocytes, and (2) comparing the coding portion of osteonectin transcript in megakaryocytes to the size of its bone counterpart. Because of the limitations of cell population purity and in obtaining sufficient numbers of megakaryocyte cells for Northern analysis, we have used the polymerase chain reaction (PCR) to detect the presence of human osteonectin mRNA in megakaryocyte and megakaryocyte-depleted bone marrow cells. Isolation of RNA, cDNA synthesis, and PCR were performed on human osteosarcoma SaOS-2 cells, enriched megakaryocytes, and megakaryocyte-depleted cells. Restriction enzyme analysis of PCR DNA products confirmed the identity of the products as those encoding osteonectin for all three cell populations studied. In addition, the sizes of DNA indicate that osteonectin genomic DNA, nuclear RNA, or altered transcript were not amplified, and that the transcript from megakaryocytes is the same size as that from bone cells. These data suggest that the difference in protein size between platelet and bone osteonectin is due to posttranslational modification. To overcome the possibility that megakaryocyte signal originated from contaminating cells (less than 5% by cell count), all three cell populations were diluted to less than one cell per tube and PCR amplification was performed. Limiting dilution analyses demonstrated the presence of osteonectin mRNA in single megakaryocytes as well as in single cells from the cell population depleted of megakaryocytes, suggesting the capacity for osteonectin biosynthesis in all cells studied. The procedure we describe in this report can be used to examine specific characteristics of mRNA molecules in heterogeneous cell populations and in situations where only small quantities of cells can be obtained.

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