Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae.

AIMS: We describe the development and interlaboratory study of modified Saccharomyces cerevisiae as a candidate material to evaluate a full detection workflow including DNA extraction and quantitative polymerase chain reaction (qPCR). METHODS AND RESULTS: S. cerevisiae NE095 was prepared by stable insertion of DNA sequence External RNA Control Consortium-00095 into S. cerevisiae BY4739 to convey selectivity. For the interlaboratory study, a binomial regression model was used to select three cell concentrations, high (4 × 10(7) cells ml(-1)), intermediate (4 × 10(5) cells ml(-1)) and low (4 × 10(3) cells ml(-1)), and the number of samples per concentration. Seven participants, including potential end users, had combined rates of positive qPCR detection (quantification cycle <37) of 100%, 40%, and 0% for high, intermediate, and low concentrations, respectively. CONCLUSIONS: The NE095 strain was successfully detected by all participants, with the high concentration indicating a potential target concentration for a reference material. SIGNIFICANCE AND IMPACT OF THE STUDY: The engineered yeast has potential to support measurement assurance for the analytical process of qPCR, encompassing the method, equipment, and operator, to increase confidence in results and better inform decision-making in areas of applied microbiology. This material can also support process assessment for other DNA-based detection technologies.

Ultrastructural relationship of quadriceps muscle degeneration with a distant peroneal nerve conduction in human myotonia dystrophica.

The association of motor nerve conduction velocity (MNCV) to (1) duration of symptoms, (2) deep tendon reflex responses, (3) clinical muscle atrophy, and (4) ultrastructure of quadriceps muscle was studied in 18 patients with myotonia dystrophica of Steinert and nine normal controls. These patients had neither diabetes mellitus nor any other type of muscle dystrophy. Ultrastructural features of muscle fibers and intercellular spaces between atrophic fibers provided a basis for identifying degenerative changes and evaluating them semi-quantitatively. Our study indicates presence of an association between the pattern of muscle degeneration and both MNCV (correlation coefficients, gamma = +0.60) and duration of symptoms (gamma = -0.62), but not between MNCV and duration of symptoms (gamma = +0.28). Further analysis of the association between the degeneration of quadriceps and the MNCV of a distant peroneal nerve (which does not innervate quadriceps) suggested that the systemic nerve degeneration occurred in some groups of myotonia patients. Our study indicates that while in some patients the muscle degeneration may have been associated with the impairment of neurogenic elements, in others it occurred in the absence of any MNCV abnormality. Our findings favor the role of both neuropathic and myopathic factors in the muscle degeneration seen in myotonia dystrophica.

Thin muscle capillary basement membranes in myotonic dystrophy.

Muscle capillary basement membrane width (MCBMW) was measured in 18 myotonic dystrophy patients and compared with that in age- and sex-matched normal and diabetic subjects. The MCBMW in myotonic dystrophy patients (773 +/- 258 A) was significantly thinner than in normal subjects (925 +/- 181 A, P less than 0.05) or in diabetics (1224 +/- 614 A, P less than 0.01). An increase in MCBMW with advancing age was present in all groups but was greatest in the myotonic dystrophy groups (r = +0.59, P less than 0.01). There was no relation between MCBMW and either the degree of glucose intolerance or insulin hypersecretion in the myotonic dystrophy group, though none had fasting hyperglycemia. This is the first report of a condition associated with thinner-than-normal capillary basement membrane.

Peripheral neuropathy in myotonic dystrophy. Relation to glucose intolerance.

Motor nerve conduction velocity (MNCV) was measured in three peripheral nerves in each of 24 patients with myotonic dystrophy having various degress of associated glucose intolerance and was compared with results in a control group. Both groups had similar glucose tolerance and were matched for age and adiposity. The mean MNCV in the ulnar, median, and peroneal nerves in the myotonic dystrophy group were all significantly slowed (P less than .01). This peripheral nerve dysfunction, usually subclinical, is yet another manifestation of this multisystem disease and is unrelated to the associated glucose intolerance.