An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.

Nanosecond-level time synchronization of autonomous radio detector stations for extensive air showers

To exploit the full potential of radio measurements of cosmic-ray air showers at MHz frequencies, a detector timing synchronization within 1 ns is needed. Large distributed radio detector arrays such as the Auger Engineering Radio Array (AERA) rely on timing via the Global Positioning System (GPS) for the synchronization of individual detector station clocks. Unfortunately, GPS timing is expected to have an accuracy no better than about 5 ns. In practice, in particular in AERA, the GPS clocks exhibit drifts on the order of tens of ns. We developed a technique to correct for the GPS drifts, and an independent method is used to cross-check that indeed we reach a nanosecond-scale timing accuracy by this correction. First, we operate a "beacon transmitter" which emits defined sine waves detected by AERA antennas recorded within the physics data. The relative phasing of these sine waves can be used to correct for GPS clock drifts. In addition to this, we observe radio pulses emitted by commercial airplanes, the position of which we determine in real time from Automatic Dependent Surveillance Broadcasts intercepted with a software-defined radio. From the known source location and the measured arrival times of the pulses we determine relative timing offsets between radio detector stations. We demonstrate with a combined analysis that the two methods give a consistent timing calibration with an accuracy of 2 ns or better. Consequently, the beacon method alone can be used in the future to continuously determine and correct for GPS clock drifts in each individual event measured by AERA.