Enumeration of Mycobacterium avium subsp. paratuberculosis by quantitative real-time PCR, culture on solid media and optical densitometry

<p>Abstract</p> <p>Background</p> <p>Different approaches are used for determining the number of <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) cells in a suspension. The majority of them are based upon culture (determination of CFU) or visual/instrumental direct counting of MAP cells. In this study, we have compared the culture method with a previously published F57 based quantitative real-time PCR (F57qPCR) method, to determine their relative abilities to count the number of three different MAP isolates in suspensions with the same optical densities (OD). McFarland turbidity standards were also compared with F57qPCR and culture, due to its frequent inclusion and use in MAP studies.</p> <p>Findings</p> <p>The numbers of MAP in two-fold serial dilutions of isolates with respective OD measurements were determined by F57qPCR and culture. It was found that culture provided lower MAP CFU counts by approximately two log₁₀, compared to F57qPCR. The McFarland standards (as defined for <it>E. coli</it>) showed an almost perfect fit with the enumeration of MAP performed by F57qPCR.</p> <p>Conclusions</p> <p>It is recommended to use culture and/or qPCR estimations of MAP numbers in experiments where all subsequent counts are performed using the same method. It is certainly not recommended the use of culture as the standard for qPCR experiments and <it>vice versa</it>.</p

Replication of fifteen loci involved in human plasma protein N-glycosylation in 4,802 samples from four cohorts

Human protein glycosylation is a complex process, and its in vivo regulation is poorly understood. Changes in glycosylation patterns are associated with many human diseases and conditions. Understanding the biological determinants of protein glycome provides a basis for future diagnostic and therapeutic applications. Genome-wide association studies (GWAS) allow to study biology via a hypothesis-free search of loci and genetic variants associated with a trait of interest. Sixteen loci were identified by three previous GWAS of human plasma proteome N-glycosylation. However, the possibility that some of these loci are false positives needs to be eliminated by replication studies, which have been limited so far. Here, we use the largest set of samples so far (4,802 individuals) to replicate the previously identified loci. For all but one locus, the expected replication power exceeded 95%. Of the sixteen loci reported previously, fifteen were replicated in our study. For the remaining locus (near the KREMEN1 gene) the replication power was low, and hence replication results were inconclusive. The very high replication rate highlights the general robustness of the GWAS findings as well as the high standards adopted by the community that studies genetic regulation of protein glycosylation. The fifteen replicated loci present a good target for further functional studies. Among these, eight genes encode glycosyltransferases: MGAT5, B3GAT1, FUT8, FUT6, ST6GAL1, B4GALT1, ST3GAL4, and MGAT3. The remaining seven loci offer starting points for further functional follow-up investigation into molecules and mechanisms that regulate human protein N-glycosylation in vivo

Comparison of filtering methods, filter processing and DNA extraction kits for detection of mycobacteria in water

Introduction and objective Mycobacteria have been isolated from almost all types of natural waters, as well as from man-made water distribution systems. Detection of mycobacteria using PCR has been described in different types of water; however, currently, there is no standardised protocol for the processing of large volumes of water. Material and Methods In the present study, different filtering methods are tested and optimised for tap or river water filtration up to 10 L, as well as filter processing and DNA isolation using four commercially available kits. Results The PowerWater DNA isolation kit (MoBio, USA), together with a kit used for soil and other environmental samples (PowerSoil DNA isolation kit, MoBio), had the highest efficiency. Filtration of 10 L of water and elution of the filter in PBS with the addition of 0.05% of Tween 80 is suggested. Conclusions The described protocol for filter elution is recommended, and the use of the PowerWater DNA isolation kit for the highest mycobacterial DNA yield from water samples. The described protocol is suitable for parallel detection of mycobacteria using cultivation

Culture- and Quantitative IS <i>900</i> Real-Time PCR-Based Analysis of the Persistence of Mycobacterium avium subsp. paratuberculosis in a Controlled Dairy Cow Farm Environment

ABSTRACT The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 10 3 were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 10 2 after 24 months. M. avium subsp. paratuberculosis -positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable. </jats:p