Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods

• Knowing the detection sensitivity of techniques used to detect pathogens is important.
• We tested the trypanosome detection in replicates of blood samples of known content.
• Automated and manual extraction methods and different primer sets were compared.
• Manual extraction outperformed automated extraction methods.
• We emphasise the importance of testing laboratory procedures to avoid false negatives.

Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results.

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