Rapid and Accurate Detection of Chrysomya megacephala (Diptera: Calliphoridae) Using Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick

Abstract

Estimating the postmortem interval (PMI) is critical in the field of forensic science, and necrophagous insects play a significant role in this process. Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) is a common necrophagous insect species, making its rapid and accurate identification essential. However, commonly used molecular biology methods, such as DNA barcode, still have some limitations in identifying necrophagous insects as they are often complex, time-consuming, and reliant on laboratory instruments. Therefore, in this study, we have developed an innovative detection system for the rapid and accurate identification of C. megacephala based on the Cytochrome b gene using recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) in combination. The developed RPA-LFD detection system achieved complete amplification in just 15 min at 37 °C with good sensitivity and specificity. Only 7.8 × 10^-4 ng or more of target DNA fragments were required, and a positive detection rate of 100% was achieved in 18 C. megacephala samples from actual cases. In addition, the ability of the developed RPA-LFD detection system in combination with rapid DNA extraction methods to enable on-site detection was preliminarily explored. The results suggested that when the RPA-LFD detection system was combined with the grinding ddH₂O extraction method (a rapid DNA extraction method), the process from species acquisition to visualization of detection results could be completed in less than 20 min. In conclusion, this innovative RPA-LFD detection system outperforms commonly used molecular biology methods for C. megacephala identification in terms of speed, sensitivity and convenience, making it suitable for direct application at crime scenes, promising to provide important assistance in estimating PMI and expanding the impact of forensic entomological evidence.

Keywords: Chrysomya megacephala; forensic entomology; insect species identification; lateral flow dipstick; recombinase polymerase amplification.

Figure 1

Results of RPA-LFD detection system at different reaction temperatures and reaction times. (A) Results of RPA-LFD detection system at different reaction temperatures. Temperatures are shown at the top of each test strip. (B) Results of RPA-LFD detection system at different reaction times. The reaction times are shown at the top of each strip. The positions of the quality control and test lines are shown on the right side of the test strip. The color depth of the bands reflects the RPA reaction product content. The NC strips are template-free controls.

Figure 2

Specificity results of RPA-LFD detection system. The corresponding species name abbreviations were shown on the upper side, including Chrysomya megacephala (C.m), Lucilia sericata (L.s), Sarcophaga crassipalpis (S.c), Chrysomya rufifacies (C.r), Calliphora vicina (C.v), Megaselia scalaris (M.s) and Sarcophaga peregrina (S.p). The positions of the quality control and test lines are shown on the right side of the test strip. The color depth of the bands reflects the RPA reaction product content. The NC strips are template-free controls.

Figure 3

Sensitivity results of the RPA-LFD detection system. Synthetic target DNA fragment inputs were shown on the upper side, and dilutions were 2-fold. The positions of the quality control and test lines are shown on the right side of the test strip. The color depth of the bands reflects the RPA reaction product content. The NC strips are template-free controls.

Figure 4

Effectiveness results of RPA-LFD detection system. The positions of the quality control and test lines are shown on the right side of the test strip. The color depth of the bands reflects the RPA reaction product content. The NC strips are template-free controls.

Figure 5

RPA-LFD detection system combined with rapid DNA extraction. (A) Grinding ddH₂O extraction method. (B) Water bath ddH₂O extraction method. (C) One-step extraction method. The corresponding species name abbreviations were shown on the upper side, including Chrysomya megacephala (C.m), Lucilia sericata (L.s), Sarcophaga crassipalpis (S.c), Chrysomya rufifacies (C.r), Calliphora vicina (C.v), Megaselia scalaris (M.s) and Sarcophaga peregrina (S.p). The positions of the quality control and test lines are shown on the right side of the test strip. The color depth of the bands reflects the RPA reaction product content. The NC strips are template-free controls.