Identification and application of AFLP-derived genetic markers for quantitative PCR-based tracking of Bacillus and Paenibacillus spp. released in soil

2009 ◽  
Vol 55 (10) ◽  
pp. 1166-1175 ◽  
Author(s):  
Miguel A. Providenti ◽  
Melissa Begin ◽  
Samielle Hynes ◽  
Christine Lamarche ◽  
David Chitty ◽  
...  
Keyword(s):  
Detection Limit ◽  
Quantitative Pcr ◽  
Genetic Markers ◽  
Paenibacillus Polymyxa ◽  
Soil Core ◽  
Environmental Persistence ◽  
Natural Systems ◽  
Length Polymorphisms ◽  
Amplified Fragment Length Polymorphisms ◽  
Different Strains

In this study, we show that noncoding sequences from amplified fragment length polymorphisms (AFLPs) can provide robust and sensitive genetic markers suitable for PCR-based discrimination of closely related strains of Bacillus and Paenibacillus , and quantitative PCR (qPCR)-based tracking of the strains in complex natural systems like soil. Quantitative PCR was accurate in the ~1 × 109 to ~1 × 104 colony forming units (CFU)/g soil range. The detection limit was improved to ~1 × 102 CFU/g when amplicons were analyzed by gel electrophoresis. Studies with laboratory-contained intact soil-core microcosms indicated that environmental persistence trends vary among different strains. For example, Bacillus circulans ATCC 9500, Bacillus amyloliquefaciens DSL 13563-0, Bacillus licheniformis ATCC 12713, Paenibacillus polymyxa NRRL B-4317, and 3 Bacillus subtilis strains (ATCC 6051A, ATCC 55405, and NRRL B-941) died down to below the 1 × 102 CFU/g detection limit by days 28–105. In contrast, over a 105-day period, B. licheniformis ATCC 55406, Bacillus megaterium NRRL B-14308, and P. polymyxa strains ATCC 55407 and DSL 13540-4 died down but persisted at levels just above the detection limit, whereas Bacillus thuringiensis ATCC 13367 experienced a less than 10-fold decrease in cell numbers.

scholarly journals Genetic markers for Clostridium difficile lineages linked to hypervirulence

Microbiology ◽  
2011 ◽  
Vol 157 (11) ◽  
pp. 3113-3123 ◽  
Author(s):  
Cornelis W. Knetsch ◽  
Marjolein P. M. Hensgens ◽  
Céline Harmanus ◽  
Madelon W. van der Bijl ◽  
Paul H. M. Savelkoul ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Genetic Markers ◽  
Large Strain ◽  
Rapid Identification ◽  
Sequence Comparisons ◽  
Banding Patterns ◽  
Length Polymorphisms ◽  
Strain Collection ◽  
Amplified Fragment Length Polymorphisms ◽  
Geographical Locations

Rapid identification of hypervirulent Clostridium difficile strains is essential for preventing their spread. Recent completion of several full-length C. difficile genomes provided an excellent opportunity to identify potentially unique genes that characterize hypervirulent strains. Based on sequence comparisons between C. difficile strains we describe two gene insertions into the genome of hypervirulent PCR ribotypes 078 and 027. Analysis of these regions, of 1.7 and 4.2 kb, respectively, revealed that they contain several interesting ORFs. The 078 region is inserted intergenically and introduces an enzyme that is involved in the biosynthesis of several antibiotics. The 027 insert disrupts the thymidylate synthetase (thyX) gene and replaces it with an equivalent, catalytically more efficient, thyA gene. Both gene insertions were used to develop ribotype-specific PCRs, which were validated by screening a large strain collection consisting of 68 different PCR ribotypes supplemented with diverse 078 and 027 strains derived from different geographical locations and individual outbreaks. The genetic markers were stably present in the hypervirulent PCR ribotypes 078 and 027, but were also found in several other PCR ribotypes. Comparative analysis of amplified fragment length polymorphisms, PCR ribotype banding patterns and toxin profiles showed that all PCR ribotypes sharing the same insert from phylogenetically coherent clusters. The identified loci are unique to these clusters, to which the hypervirulent ribotypes 078 and 027 belong. This provides valuable information on strains belonging to two distinct lineages within C. difficile that are highly related to hypervirulent strains.

A Demonstration of a 1:1 Correspondence Between Chiasma Frequency and Recombination Using a Lolium perenne/Festuca pratensis Substitution

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 307-314 ◽  
Author(s):  
J King ◽  
L A Roberts ◽  
M J Kearsey ◽  
H M Thomas ◽  
R N Jones ◽  
...  
Keyword(s):  
Lolium Perenne ◽  
Chiasma Frequency ◽  
Chiasma Formation ◽  
Grass Species ◽  
Festuca Pratensis ◽  
Single Chromosome ◽  
Length Polymorphisms ◽  
Amplified Fragment Length Polymorphisms ◽  
Frequency Counts

Abstract A single chromosome of the grass species Festuca pratensis has been introgressed into Lolium perenne to produce a diploid monosomic substitution line (2n = 2x = 14). The chromatin of F. pratensis and L. perenne can be distinguished by genomic in situ hybridization (GISH), and it is therefore possible to visualize the substituted F. pratensis chromosome in the L. perenne background and to study chiasma formation in a single marked bivalent. Recombination occurs freely in the F. pratensis/L. perenne bivalent, and chiasma frequency counts give a predicted map length for this bivalent of 76 cM. The substituted F. pratensis chromosome was also mapped with 104 EcoRI/Tru91 and HindIII/Tru91 amplified fragment length polymorphisms (AFLPs), generating a marker map of 81 cM. This map length is almost identical to the map length of 76 cM predicted from the chiasma frequency data. The work demonstrates a 1:1 correspondence between chiasma frequency and recombination and, in addition, the absence of chromatid interference across the Festuca and Lolium centromeres.

Corrigendum: Identification and application of AFLP-derived genetic markers for quantitative PCR-based tracking of Bacillus and Paenibacillus spp. released in soil

2010 ◽  
Vol 56 (1) ◽  
pp. 87-87
Author(s):  
Miguel A. Providenti ◽  
Melissa Begin ◽  
Samielle Hynes ◽  
Christine Lamarche ◽  
David Chitty ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Genetic Markers

scholarly journals Amplified fragment length polymorphisms (AFLP's)

2017 ◽  
pp. 119 ◽  
Author(s):  
June Simpson
Keyword(s):  
Molecular Marker ◽  
Restriction Fragment ◽  
Restriction Enzyme ◽  
Fragment Length ◽  
Polymorphic Loci ◽  
Length Polymorphisms ◽  
Amplified Fragment Length Polymorphisms ◽  
Pcr Conditions

AFLP is a combination restriction fragment/PCR molecular marker technique which detects polymorphisms due to changes at or in the vicinity of restriction enzyme sites. The technique detects multiple polymorphic loci throughout the genome and may be used for fingerprinting and mapping purposes. The main advantages of the method are the consistency and reliability of the technique due to stringent PCR conditions and the ability to rapidly detect many polymorphic loci.

Sign in / Sign up

Export Citation Format

Share Document