Advantage of MALDI-TOF-MS over biochemical-based phenotyping for microbial identification illustrated on industrial applications.

UNLABELLED: Fast microbial identification is becoming increasingly necessary in industry to improve microbial control and reduce biocide consumption. We compared the performances of two systems based on MALDI-TOF MS (VITEK MS and BIOTYPER) and two based on biochemical testing (BIOLOG, VITEK 2 Compact) with genetic methods for the identification of environmental bacteria. At genus level both MALDI-TOF MS-based systems showed the lowest number of false (4%) and approx. 60% correct identifications. In contrast, the biochemical-based systems assigned 25% of the genera incorrectly. The differences were even more apparent at the species level. The BIOTYPER was most conservative, where assigning a species led to the lowest percentage of species identifications (54%) but also to the least wrong assignments (4%). The other three systems showed higher levels of false assignments: 8·7, 40 and 46% respectively. The genus identification performance on four industrial products of the BIOTYPER could be increased up to 94·3% (average 88% of 167 isolates) by evolving the database in a product specific manner. Comparison of the bacterial population in the example of paints, and raw materials used therein, at different production steps demonstrated unequivocally that the contamination of the final paint product originated not from the main raw material. SIGNIFICANCE AND IMPACT OF THE STUDY: MALDI-TOF-MS has revolutionized speed and precision of microbial identification for clinical isolates outperforming conventional methods. In contrast, few performance studies have been published so far focusing on suitability for particularly industrial applications, geomicrobiology and environmental analytics. This study evaluates the performance of this proteomic phenotyping on such industrial isolates in comparison with biochemical-based phenotyping and genotyping. Further the study exemplifies the power of MALDI-TOF-MS to trace cost-efficiently the dominating cultivable bacterial species throughout an industrial paint production process. Vital information can be retrieved to identify the most crucial contaminating source for the final product.

Evaluation of an upgraded version of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test for HIV-1 load quantification.

We evaluated the performance of the prototype Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0, using prospective and archived clinical samples initially underquantitated by the Cobas AmpliPrep/Cobas TaqMan HIV-1 test. The performance of the new test was significantly improved, and the majority of the underquantitation observed with the first-version test was eliminated.

Genetic structure inside a declining red oak community in old-growth forest.

Problems with oak regeneration have been documented in the last 50 years at numerous sites in the Midwestern United States. We applied nuclear microsatellites to examine the demographic and fine-scale spatial genetic structure of red oaks in two old-growth stands in Indiana. Oaks in one stand have declined in numbers over the past several decades whereas oaks in the other, smaller stand have increased. Large amounts of genetic variation were maintained within stands, and there was slight but significant differentiation among stands. There was significant but weak isolation by distance genetic structure within the large stand, likely reflecting family structure. No significant differences exist in allele frequencies or in levels of genetic diversity between cohorts that remain well represented within each stand, even between medium-sized adults and those antedating European settlement of the area. However, a virtual absence of smaller size classes in the forest interior of the large stand represents the early stages of a genetic bottleneck in what had been the core habitat of this stand. Whether future generations of this old-growth stand will retain the present genetic character depends on the oaks regenerating at the forest margins, absent any major changes in disturbance regimes. Similar demographic and genetic dynamics are likely occurring in a large number of remnant oak forests across the Midwest.

Dinucleotide microsatellites from Eucalyptus sieberi: inheritance, diversity, and improved scoring of single-base differences.

Eight dinucleotide microsatellites were developed in Eucalyptus sieberi L. Johnson (silvertop ash), a member of the subgenus Eucalyptus. Transfer of six of these to the subgenus Symphyomyrtus and their Mendelian inheritance are demonstrated using a full-sib cross in Eucalyptus nitens. Genetic diversity parameters are presented for the eight loci based on a sample of 100 old-growth E. sieberi trees from a single natural stand. One locus, Es266, had an atypically high fixation index, and significantly deviated from Hardy-Weinberg equilibrium genotypic proportions, indicating the likely presence of null alleles. Two of the loci, Es076 and Es140, had many alleles that differed in size by only a single base pair, possibly because of short poly(A) or poly(T) stretches in their flanking regions. These two loci were by far the most polymorphic, but were difficult to score reliably on a capillary DNA sequencer. Reliability of scoring of these two one-base microsatellite loci was markedly improved by the incorporation of internal reference alleles into each sample analysed.

RNA editing in the mitochondria of a conifer.

A 712-base portion of the mitochondrial gene coxI and the corresponding portion of the coxI transcript were amplified by PCR and by RT-PCR, respectively, from the gymnosperm western red cedar. Sequence comparison of amplified coxI DNA and mRNA revealed 26 C-to-U differences that are best explained by RNA editing of the type known to occur in angiosperms. This finding suggests that mitochondrial RNA-editing of the C-to-U type arose before the divergence of gymnosperms and angiosperms and can be considered a feature common to all higher plants.

Single tree genetic linkage mapping in conifers using haploid DNA from megagametophytes.

We report a unique application of the Random Amplified Polymorphic DNA (RAPD) technique for genetic linkage mapping of a single spruce tree using haploid DNA from megagametophyte tissue of individual seeds. Sixty-one segregating loci were analysed for reproducibility, inheritance and linkage. Forty-seven of the 61 markers were distributed into 12 linkage groups and covered 873.8 cM. The 14 markers not associated with any linkage group should be assigned to linkage groups as more markers are added to the map. This new approach quickly provides molecular genetic markers that are simple to evaluate for constructing genetic linkage maps and for other related genetic studies in forest tree species.

Segregation of random amplified DNA markers in F1 progeny of conifers.

The recently developed approach to deriving genetic markers via amplification of random DNA segments with single primers of arbitrary nucleotide sequence was tested for its utility in genetic linkage mapping studies with conifers. Reaction conditions were optimized to reproducibly yield clean and specific amplification products. Template DNA from several genotypes of Douglas-fir (Pseudotsuga menziesii) and white spruce (Picea glauca) were tested against eight ten-base oligonucleotide primers. Most of the tested primer/parent tree combinations yielded polymorphic PCR products ("RAPD" markers). Selected primers were then used in PCR reactions with template DNA isolated from offspring in Douglas-fir and black spruce diallel crosses among the same parental lines. The diallel study confirmed the appropriate inheritance of RAPD markers in the F1 generation. The value of these dominant RAPD markers for genetic linkage mapping in trees was established from both theoretical and applied perspectives.