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Mismatches between template sequences and reverse transcription (RT) or polymerase chain reaction (PCR) primers can lead to underestimation or false negative results during detection and quantification of sequence-diverse viruses. We performed an in silico inclusivity analysis of a widely used RT-PCR assay for detection of hepatitis A virus (HAV) in food, described in ISO 15216-1. One of the most common mismatches found was a single G (primer) to U (template) mismatch located at the terminal 3′-end of the reverse primer region. This mismatch was present in all genotype III sequences available in GenBank. Partial HAV genomes with common or potentially severe mismatches were produced by in vitro transcription and analysed using RT-ddPCR and RT-qPCR. When using standard conditions for RT-qPCR, the mismatch identified resulted in underestimation of the template concentration by a factor of 1.7–1.8 and an increase in 95% limit of detection from 8.6 to 19 copies/reaction. The effect of this mismatch was verified using full-length viral genomes. Here, the same mismatch resulted in underestimation of the template concentration by a factor of 2.8. For the partial genomes, the presence of additional mismatches resulted in underestimation of the template concentration by up to a factor of 232. Quantification by RT-ddPCR and RT-qPCR was equally affected during analysis of RNA templates with mismatches within the reverse primer region. However, on analysing DNA templates with the same mismatches, we found that ddPCR quantification was less affected by mismatches than qPCR due to the end-point detection technique.

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Migratory birds wintering in Africa face the challenge of passing the Sahara desert with few opportunities to forage. During spring migration birds thus arrive in the Mediterranean area with very low energy reserves after crossing the desert. Since early arrival to the breeding grounds often is of importance to maximize reproductive success, finding stopover sites with good refuelling possibilities after the Saharan passage is of utmost importance. Here we report on extensive fuelling in the great reed warbler Acrocephalus arundinaceus on the south coast of Crete in spring, the first land that they encounter after crossing the Sahara desert and the Mediterranean Sea in this area. Birds were studied at a river mouth and due to an exceptional high recapture rate (45 and 51% in two successive years), we were able to get information about stopover behaviour in 56 individual great reed warblers during two spring seasons. The large proportion of trapped great reed warbler compared to other species and the large number of recaptures suggest that great reed warblers actively choose this area for stopover. They stayed on average 3–4 d, increased on average about 3.5 g in body mass and the average rate of body mass increase was 4.8% of lean body mass d–1. Wing length affected the rate of increase and indicated that females have a slower increase than males. The results found show that great reed warblers at this site regularly deposit larger fuel loads than needed for one continued flight stage. The low body mass found in great reed warblers (also in birds with high fat scores) is a strong indication that birds staging at Anapodaris still had not been able to rebuild their structural tissue after the strenuous Sahara crossing, suggesting that rebuilding structural tissue may take longer time than previously thought.

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