This method provides a fast and reliable diagnostic tool for the identification of Bemisia tabaci, an invasive insect pest affecting the production of vegetables and ornamental crops in many countries around the world. Due to its ease, the method can be performed on-site directly at points of entry for plant import products, such as harbors and airports, and has thereby the potential to reduce the dispersal of Bemisia tabaci along trading networks. To begin, use scissors to cut an eight-tube LAMP strip into two, four-tube LAMP strips.
Then, label the tubes according to the scheme outlined in figure one of the text protocol. After this, prepare enough B.tabaci LAMP reaction master mix for 80 reactions. Then, dispense 22.5 microliters of the master mix into each of the tubes of the four-tube LAMP strips.
Pulse centrifuge, and put them on ice. Quickly vortex and pulse centrifuge B.tabaci LAMP PAC. Then, add 2.5 microliters into the tube labeled PAC in each of the four-tube LAMP strips.
Next, close the lids, and store the ready-to-use B.tabaci LAMP kit units at minus 20 degrees Celsius. To begin DNA extraction, use sterile toothpicks to transfer the insect specimens into 0.5-milliliter microcentrifuge tubes with 30 microliters of DNA extraction solution. Then, incubate the samples for five minutes at 95 degrees Celsius in a ThermoMixer set to 300 rpm.
Briefly vortex and pulse centrifuge the samples. Thaw one of the ready-to-use B.tabaci LAMP kits. Then, vortex the kit quickly, and pulse centrifuge it.
Add 2.5 microliters of sample DNA extract to the S-one and S-two tubes. Then, add 2.5 microliters of pure alkaline DNA extraction solution to the NAC tube. After vortexing and pulse centrifuging the kit, insert the kit into a LAMP analysis device, and perform isothermal DNA amplification analysis at 65 degrees Celsius for 60 minutes.
In the same run, perform a melting temperature analysis of the DNA amplification products by first heating the kit to 98 degrees Celsius and cooling it to 75 degrees Celsius. Validate the LAMP readout using the automated LAMP validation application. First, define the target species and the number of tested samples before clicking the Generate Report button.
Finally, transfer the readout to the appropriate input fields of the validation application. Immediately after entering the data, the result of the validation will be displayed. In this protocol, B.tabaci insect specimens intercepted in the course of the regular Swiss import control process were analyzed via LAMP assay.
From a total of 80 specimens analyzed, 75 specimens were correctly identified as true positives, and two specimens were correctly identified as true negatives. Three specimens, however, were false negatives and were incorrectly identified as not being B.tabaci. A subset of the assays was performed on-site by plant health inspectors at Zurich Airport.
In the representative results, samples one and two were correctly identified as B.tabaci via DNA amplification after approximately 30 minutes in the expected annealing temperature of approximately 82 degrees Celsius. After its development, this method paved the way for a fast and easy-to-perform on-site identification of Bemisia tabaci. The protocol is designed in such as way that it can be performed by plant health inspectors with limited laboratory training.
After successful on-site validation of the method at a Swiss point of entry for plant import products, the method could be adapted to other plant pathogens but also, if need be, to other human or veterinary pathogens.
