Development of Molecular Diagnostic Protocols for Simultaneous Identification of Common Bed Bugs (Cimex lectularius) and Tropical Bed Bugs (Cimex hemipterus) 

Abstract Background The resurgence of two bed bug species, the common bed bug (Cimex lectularius Linnaeus, 1758) and tropical bed bug (Cimex hemipterus Fabricius, 1803), in the same geographical regions has been frequently reported recently. Consequently, the rapid identification of these species at infestation sites is crucial for implementing targeted control measures, owing to differences in genetic and physiological traits. Methods To develop molecular diagnostic methods, distinct protocols were established for multiplex PCR and loop-mediated isothermal amplification (LAMP) using species-specific primers based on species-specific segments of internal transcribed spacer 2 sequences. These methods were optimized for rapid and accurate identification of the two bed bug species. Results Both multiplex PCR and LAMP protocols were effective in simultaneously identifying the two bed bug species, even when utilizing DNA released from dead specimens. Notably, the straightforward procedure and minimal time commitment of LAMP make it highly applicable for rapid and accurate diagnosis of bed bugs at infestation sites. The diagnostic accuracy of these methods was validated through a blind test. Conclusions The multiplex PCR and LAMP protocols enable rapid and accurate on-site identification of bed bug species, facilitating the timely implementation of species-specific control strategies. This approach ensures effective management tailored to the unique characteristics of each bed bug species.