The length of the polyprobes and the position of the individual probes in them determine the sensitivity in the detection of viruses affecting pepper crops

Polyprobes have great potential for simultaneous multipathogen detection and have been successfully used for detecting a large number of plant viruses and/or viroids in a single assay. However, how the length of the polyprobe or the position of the corresponding single probes within them influence their sensitivity has not been previously addressed. In this study, we have developed three polyprobes with the capacity to detect 9 (poly9), 12 (poly12) or 21 (poly21) viruses representing the least prevalent common viruses, the most prevalent viruses or a combination of both types of viruses, respectively, affecting pepper crops. By using known amounts of complementary transcripts and serially diluted extracts from different individually infected pepper plants, we observed that, overall, the detection limit of poly12 and poly21 polyprobes was 5 times and 25/125 times lower than that of the single probes, respectively. An exception was the detection of cucumber mosaic virus and tomato mosaic virus, which were better detected by using poly21 than poly12, possibly due to the more central position of these two probes within the corresponding polyprobes. The analysis of 85 field samples using both poly12 and poly21 also revealed more positives samples with the former, confirming, in general, a higher detection limit for poly12 than poly21. The optimal polyprobe size and temperature for efficient polyvalent virus and/or viroid detection using this technology are discussed.