Development of a Fluorogenic Probe-Based PCR Assay for Detection of Bacillus cereus in Nonfat Dry Milk

A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5′-to-3′ exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereus strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.     

Numerical method for detection of linkage between genes for two metrical traits

A homozygous population derived from hybrids between two homozygous parents may be used for genetic analysis of metrical traits. The paper describes the use of doubled haploids (DH) and single seed descent (SSD) lines for detection of linkage between genes conditioning two quantitative traits. A computational algorithm is presented, which facilitates matching various variants of relations between variances, covariances and means of DH and SSD populations so as to make it possible to conclude on the presence/absence of linkage. The suggested methodology is illustrated with an example concerning three quantitative traits of barley: length of the third internode, stem wall thickness, and 1000-grain weight.     

Gynecomasty as a first sign of adrenal carcinoma--case report

We present a case of 50 year-old man with feminizing adrenal carcinoma. He was admitted to the hospital because of weakness and one year history of gynecomastia and high blood pressure. Examinations revealed a large left adrenal mass and increased levels of estradiol. Patient underwent adrenalectomy and followed by mitotan therapy as the result of histopathological examination was adrenocortical carcinoma. One year after operation patient stays free from the recurrence of the disease and his estradiol, androstendion and DHEA levels are below the detection limits. We report this case because feminizing adrenal carcinoma is a very rare but serious disease and gynecomastia that could be its manifestation is quite frequent symptom in men's population and thus it could easily be missed. In every case of gynecomastia related to estradiol excess feminizing tumors of testis and adrenal gland should be ruled out.     

Maternal serum proinflammatory cytokines in preterm labor with intact membranes: neonatal outcome and histological associations

Our aim was to compare maternal serum concentrations of interleukin(IL)-1alpha IL-1beta, IL-6 and IL-8 in pregnancies complicated by preterm labor (PTL), with the levels in healthy controls at comparable gestational age, and to determine if these assays have any value in the prediction of early-onset neonatal infection or histological chorioamnionitis. The study population consisted of 65 women with new-onset PTL, and 31 healthy controls. Maternal serum concentrations of IL-6 (8.40 versus 3.30 pg/mL; p = 0.002) and IL-1beta (2.20 versus 0.50 pg/mL; p = 0.003) were significantly higher in patients with PTL as compared to healthy pregnant women. The IL-1beta concentration (13.60 versus 1.20 pg/mL; p = 0.02) was significantly higher in the serum of mothers whose babies developed early-onset infections, than in mothers of newborns that were healthy. However, its predictive value, and the value of the other cytokines studied, was poor. In addition, IL-1beta levels (28.79 versus 5.19 pg/mL; p = 0.001) were significantly higher in patients with histological chorionamnionitis, than in those without the condition,. The cut-off value of >or= 14 pg/mL predicted inflammatory changes with a sensitivity of 80%, specificity of 86%, PPV of 80% and NPV of 86%. IL-1beta seems to be of moderate value in the prediction of histological chorioamnionitis.     

A solid phase fluorescent capillary immunoassay for the detection of Escherichia coli O157:H7 in ground beef and apple cider

A solid phase fluorescence-based immunoassay was developed for the detection of Escherichia coli O157:H7 using an antigen down competition format. A soft glass capillary tube served as the solid support, to which heat-killed E. coli O157:H7 were adsorbed. Polyclonal anti- E. coli O157:H7 antibody, conjugated with biotin, was used and the bound antigen-antibody complex was detected using avidin molecules labelled with Cy5, a fluorescent cyanine dye. Any E. coli O157:H7 in the sample would compete with the formation of this complex, reducing fluorescence. This assay was tested for sensitivity with spiked ground beef and apple cider samples. The minimum detectable number of cells present in the initial inoculum was calculated to be approximately 1 colony-forming unit (cfu) per 10g of ground beef when samples were enriched in modified EC broth for 7 h at 37°C. The minimum detectable number of cells for the apple cider samples was calculated to be ∼0.5 cfu ml^-1 The E. coli cells in the cider samples were captured with immunomagnetic beads, incubated for 7 h in the enrichment broth, and detected with the solid phase fluorescence immunoassay.     

Analyzing multi-environment variety trials using randomization-derived mixed models

Of interest is the analysis of results of a series of experiments repeated at several environments with the same set of plant varieties. Suppose that the experiments, multi-environment variety trials, are all conducted in resolvable incomplete block (IB) designs. Following the randomization approach adopted in Caliński and Kageyama (2000, Lecture Notes in Statistics, 150), two models for analyzing such trial data can be considered. One is derived under a complete additivity assumption, the other takes into account possible different responses of the varieties to variable environmental conditions. The analysis under the first, the standard model, does not provide answers to questions related to the performance of the individual varieties at different environments. These can be considered when using the more general second model. The purpose of this article is to devise interesting parameter estimation and hypothesis testing procedures under that more realistic model. Its application is illustrated by a thorough analysis of a set of data from a winter wheat series of trials.     

Development of a fluorogenic probe-based PCR assay for detection of Bacillus cereus in nonfat dry milk

A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5'-to-3' exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereus strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.     

Is mitochondrial DNA content a potential biomarker of mitochondrial dysfunction?

Mitochondrial dysfunction is central to numerous diseases of oxidative stress. Changes in mitochondrial DNA (MtDNA) content, often measured as mitochondrial genome to nuclear genome ratio (Mt/N) using real time quantitative PCR, have been reported in a broad range of human diseases, such as diabetes and its complications, obesity, cancer, HIV complications, and ageing. We propose the hypothesis that MtDNA content in body fluids and tissues could be a biomarker of mitochondrial dysfunction and review the evidence supporting this theory. Increased reactive oxygen species resulting from an external trigger such as hyperglycaemia or increased fat in conditions of oxidative stress could lead to enhanced mitochondrial biogenesis, and increased Mt/N. Altered MtDNA levels may contribute to enhanced oxidative stress and inflammation and could play a pathogenic role in mitochondrial dysfunction and disease. Changes in Mt/N are detectable in circulating cells such as peripheral blood mononuclear cells and these could be used as surrogate to predict global changes in tissues and organs. We review a large number of studies reporting changes in MtDNA levels in body fluids such as circulating blood cells, cell free serum, saliva, sperm, and cerebrospinal fluid as well as in tumour and normal tissue samples. However, the data are often conflicting as the current methodology used to measure Mt/N can give false results because of one or more of the following reasons (1) use of mitochondrial primers which co-amplify nuclear pseudogenes (2) use of nuclear genes which are variable and/or duplicated in numerous locations (3) a dilution bias caused by the differing genome sizes of the mitochondrial and nuclear genome and (4) template preparation protocols which affect the yields of nuclear and mitochondrial genomes. Development of robust and reproducible methodology is needed to test the hypothesis that MtDNA content in body fluids is biomarker of mitochondrial dysfunction.