Introduction
Our laboratory performs nucleic acid extraction for diagnostic PCR tests. The efficiency of the extraction process is currently not assessed. Thus, we evaluated an RNA Extraction Control 670 (REC) (Bioline Inc, USA) which can fulfil this purpose for RT-PCR tests across varying sample types.
Method
A total of 80 patient samples which were submitted for reverse transcriptase (RT)-PCR tests were from four main sample types: cerebrospinal fluid (CSF), swabs, rectal swabs or stool, and blood samples. These samples were extracted and the RT-PCR master mix was prepared according to the respective laboratory protocols. Each sample was processed in duplicate. One replicate was spiked with 2 µl REC RNA prior to the extraction process. This replicate’s RT-PCR master mix tube was prepared with additional 1 µl REC control mix. All the samples were run on real-time thermocyclers with an additional detection channel at 705 nm for REC detection.
Results
REC was detected in all RT-PCR runs with varying median cycling threshold (Ct) values across different sample types. The median Ct values were 11.30 (10.45-12.61) for CSF, 21.97 (12.45-26.01) for swabs, 13.33 (12.93-20.99) for rectal/stool samples and 15.38 (14.94-17.36) for blood. Out of 80 samples tested, 10 were positive for their respective main targets and the corresponding REC-spiked reaction tubes displayed similar Ct values to the non-spiked reaction tubes, with a mean difference of 0.56 Ct values.
Discussion
The results showed that the REC control is suitable for monitoring the extraction process of all four different sample types. The difference in median Ct values in different sample types may be attributed to the different RT-PCR cycling conditions of the tests involved. Inhibition of REC amplification was not detected in any of the REC-spiked samples, suggesting that REC did not interfere with the RT-PCR detection.