Duchenne Muscular Dystrophy Newborn Screening: Evaluation of a New GSP® Neonatal Creatine Kinase-MM Kit in a US and Danish Population.

Duchenne muscular dystrophy (DMD/Duchenne) is a progressive X-linked disease and is the most common pediatric-onset form of muscular dystrophy, affecting approximately 1:5000 live male births. DNA testing for mutations in the dystrophin gene confirms the diagnosis of this disorder. This study involves assessment of screening newborns for DMD using an immunoassay for muscle-type (MM) creatine kinase (CK) isoform-the GSP Neonatal CK-MM kit. Comparisons were made with CK activity determination by fluorescence measurement. In addition, the study evaluated the effect of gestational age, age of infant at time of sampling and how stable the CK-MM was over time. This assay discriminates well between normal, unaffected and Duchenne affected populations and is suitable for Duchenne newborn screening.

Self-reported oral health and associated factors in the North Finland 1966 birth cohort at the age of 31.

BACKGROUND: The Northern Finland 1966 birth cohort (NFBC 1966) is an epidemiological study where the participants have been controlled since pregnancy both in field tests and using questionnaires. This study aimed to evaluate cross-sectionally the association of self-reported oral symptoms (dental caries and bleeding of gums) with sociodemographic and health behavior factors among the subjects. METHODS: Of the 11,541 original members of the cohort, 8,690 (75%) responded to the questionnaire on oral health (dental decay, gingival bleeding and self-estimated dental treatment need) and sociodemographic factors, general health and health behavior. Cross-tabulation and chi-squared tests as well as multiple logistic regression analysis were used to analyze the association between the outcome and explanatory variables. RESULTS: The study group was equally distributed between the genders. One third of the subjects reported having dental decay, one fourth gingival bleeding and a half a dental treatment need. As compared to women, men reported significantly more frequently symptoms (p < 0.05). Logistic regression analysis revealed low tooth brushing frequency increasing the odds most for all oral symptoms ((OR 1.57 (1.39-1.78) for dental decay, 1.94 (1.68-2.24) for gingival bleeding and 1.42 (1.26-1.61) for dental treatment need). Frequent smoking was associated with dental decay (OR 1.63 (1.44-1.84)) and treatment need OR (1.39 (1.23-1.56)), whereas poor general health (OR 1.71 (1.48-1.96)) and high BMI (OR 1.19 (1.03-1.36)) both were associated with gingival bleeding. CONCLUSIONS: Males with single marital status, BMI over 25, poor general health and poor oral health behaviors are at risk for self-reported poor oral health and dental treatment need.

Genotyping of celiac disease-related-risk haplotypes using a closed-tube polymerase chain reaction analysis of dried blood and saliva disk samples.

Expansion of molecular diagnostics more widely into clinical routines requires simplified methods allowing automation. We developed a homogeneous, multilabel polymerase chain reaction (PCR) method based on time-resolved fluorometry, and studied the use of dried disk samples in PCR. Celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 genotyping was used to verify the method with blood and saliva samples dried on S&S 903 and IsoCode sample collection papers. Three sample preparation procedures, including manufacturer's manual elution, an automated elution, and direct use of disk samples, were compared using dried disk samples. The three procedures gave successful amplification and correct genotyping results. Owing to the simplicity of the direct use of disk samples in PCR, this method was chosen for the subsequent homogeneous analysis of blood (n=194) and saliva (n=30) disk samples on S&S 903 paper. The results revealed that, in addition to DNA samples (n=29), both blood and saliva disk samples were successfully amplified and genotyped using the homogeneous PCR assays for HLA-DQA1 and HLA-DQB1. The homogeneous PCR assays developed provide a useful tool to genotype celiac disease-related HLA-DQA1*05, HLA-DQB1*02, and HLA-DQB1*0302 alleles. Furthermore, the method provides a direct way to perform a closed-tube PCR analysis of dried blood and saliva disk samples enabling simple automation.

Time-resolved detection probe for homogeneous nucleic acid analyses in one-step format.

We report here an extension of homogeneous assays based on fluorescence intensity and lifetime measuring on DNA hybridization. A novel decay probe that allows simple one-step nucleic acid detection with subnanomolar sensitivity, and is suitable for closed-tube applications, is introduced. The decay probe uses fluorescence resonance energy transfer (FRET) between a europium chelate donor and an organic fluorophore acceptor. The substantial change in the acceptor emission decay time on hybridization with the target sequence allows the direct separation of the hybridized and unhybridized probe populations in a time-resolved measurement. No additional sample manipulation or self-hybridization of the probes is required. The wavelength and decay time of a decay probe can be adjusted according to the selection of probe length and acceptor fluorophore, thereby making the probes applicable to multiplexed assays. Here we demonstrate the decay probe principle and decay probe-based, one-step, dual DNA assay using celiac disease-related target oligonucleotides (single-nucleotide polymorphisms [SNPs]) as model analytes. Decay probes showed specific response for their complementary DNA target and allowed good signal deconvolution based on simultaneous optical and temporal filtering. This technique potentially could be used to further increase the number of simultaneously detected DNA targets in a simple one-step homogeneous assay.

Homogeneous assay based on anti-Stokes' shift time-resolved fluorescence resonance energy-transfer measurement.

We report here a novel, time-resolved, lanthanide-based energy-transfer assay utilizing nonoverlapping acceptor fluorophores, which have their absorption energetically at a higher level than the emittive transitions of the donor. The technique was studied by comparing a series of nonoverlapping acceptors in a homogeneous DNA model assay utilizing Eu3+ chelate as a donor. The assay provides strong energy-transfer enhanced acceptor emission and enables the anti-Stokes' shift FRET measurement, in which the induced acceptor emission is at shorter wavelength than the donor emission. This results in high sensitivity, and 0.8 pM detection limit was measured for the DNA target. The acceptor signal of the assay is characterized by exceptional lifetime properties and is not strictly following the Forster's theory. The mechanism of nonoverlapping energy transfer is considered, and we propose that when nonoverlapping acceptors are utilized, the energy transfer arises from the upper 5D2 and 5D1 excited states of europium. The assumption was studied using a simplified energy level scheme of the Eu3+ donor and the acceptors, and a correlation between the acceptor emission behavior and the energy level scheme was found.

Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase.

We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energy-transfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z' factors.