Current RT-LAMP reactions for Potato Virus Y (PVY)

We already have working homemade RT-LAMP reactions for the detection of PVY RNA, which specifically detect a conserved region of the virus.

This can be monitored using indirect colorimetric methods based on the use of the pH indicator neutral red, as well as using more specific detection methods like QUASR-LAMP, which relies on a quenching probe that binds to a fluorescent-tagged primer for sequence-specific and endpoint monitoring of reaction results. QUASR also has the advantage of being less prone to false positives than other indirect or real-time monitoring methods, which makes it ideal for its use on-field.

 

NTC: non-template control; M1 & M2: two PVY RNA samples extracted from infected plants.

Another LAMP monitoring method we would like to explore is LAMP-OSD, which relies on a toehold-mediated strand exchange that separates a pre-hybridized hemiduplex OSD probe. This allows for sequence-specific detection with the advantages of real-time monitoring for settings that require it. We aim to compare the sensitivity and specificity ranges of OSD and QUASR techniques while also exploring if OSD could become a better alternative for freeze-dried LAMP reactions in the future.

PS: RT-LAMP stands for "reverse-transcription LAMP" and is used for RNA amplification, whereas common non-RT LAMP only amplifies DNA sequences.

Below you can find schematic diagrams of both sequence-specific LAMP detection methods:

 

Principle of QUASR detection in LAMP or RT-LAMP. One of the loop primers (LF or LB) or inner primers (FIP or BIP) is labeled with a dye. The reaction mixture also contains a short probe, labeled with a dark quencher at the 3′ end, and complementary to 7–13 bases at the 5′ end of the dye labeled primer. The quench probe is present at slight excess relative to the labeled primer and has Tm > 10 °C below the temperature of the LAMP reaction, such that it remains dissociated during the amplification. After incubation, the reaction is cooled to ambient temperature, resulting in dark quenching of fluorescent primers (negative reactions) or highly fluorescent amplicons (positive reactions). Image from https://doi.org/10.1021/acs.analchem.5b04054. 

 

LAMP-OSD schematic. (A) Schematic depicting LAMP mechanism. (B) Schematic depicting OSD design and toehold-mediated strand exchange process, where the strand labeled A represents the LAMP loop sequence and the B.C complex represents the hemiduplex OSD probe. F and Q on the OSD denote fluorophore and quencher, respectively. OSD and subsequent strand exchange intermediates are denoted by numbered domains, which represent short (usually < 12-nucleotide) sequences in an otherwise continuous oligonucleotide. Complementary domains are indicated by asterisk. Image from 10.1128/mSphere.00911-20