A triple rpa-lfa rapid detection and visualization method for necrophagous flies
By employing the triple RPA-LFA method, combined with specific primers and probes, the problem of rapid and accurate identification of necrophilic fly species has been solved, enabling simple and visualized multi-species detection at the scene of death and enhancing the on-site application value of forensic evidence.
Patent Information
- Application Number
- CN202610476266.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-13
- Publication Date
- 2026-07-03
AI Technical Summary
Existing methods for identifying necrophilic fly species are difficult to use for rapid and accurate multi-species identification at the scene of death, and they rely on sequencers and complex experimental equipment, which cannot meet the needs of field applications.
A rapid detection and visualization method using triple RPA-LFA was developed. By designing specific primers and probes, and combining recombinase polymerase amplification (RPA) technology with lateral flow chromatography (LFA) strips, rapid and visual identification of *Gymnocladus macrocarpa*, *Gymnocladus filamentosa*, and *Vallisneria browni* was achieved.
It enables rapid, simple, and accurate identification of necrophilic fly species at the scene of death, is applicable to multi-species detection, does not require a sequencer, and provides visualized results, thereby improving the on-site application value of forensic evidence.
Smart Images
Figure CN122326762A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of triple RPA-LFA rapid detection technology, and specifically relates to a triple RPA-LFA rapid detection and visualization method for necrophilic flies. Background Technology
[0002] At death sites where necrophilic insects are present, the giant golden fly (Golden Fly) Chrysomya megacephala ), Green leaf flies ( Lucilia sericata ) and brown-tailed flesh fly ( Sarcophaga peregrina Species identification is common and dominant, and the ability to quickly and accurately identify them directly affects the accuracy and speed of time-of-death estimation. Various species identification methods, including morphological identification (10.1186 / s13071-024-06441-z), molecular amplification and sequencing identification (10.1016 / j.actatropica.2022.106798), and the combination of physicochemical information and machine learning methods frequently used in recent studies (10.1016 / j.actatropica.2018.04.025, 10.3390 / insects16020144), are difficult to apply rapidly in the field due to their respective drawbacks (high requirements, dependence on sequencers, and lack of practical application).
[0003] Therefore, developing a molecular instant detection method that can be quickly completed on-site, provides visualized results, and is adaptable to the needs of multiple species is of great significance for enhancing the on-site application value of forensic insect evidence. Summary of the Invention
[0004] To overcome the above technical problems, the present invention aims to provide a rapid detection and visualization method for triple RPA-LFA in necrophilic flies. This method is characterized by being independent of sequencers, simple to operate, rapid in detection, and visualized.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A rapid detection and visualization method for triple RPA-LFA targeting necrophilic flies, comprising the following steps; Step 1: Insect DNA extraction; 1.1 DNA was extracted from *Gynostemma pentaphyllum* (Cm), *Gynostemma spp.* (Ls), *Variegata spp.* (Sp), *Bulbus rubiginii* (Cv), *Gynostemma pentaphyllum* (Cr), *Flyflies spp.* (Sc), and *Heterodon spp.* (Ms) using the SteadyPure Universal Genomic DNA Extraction Kit (AG21010, Hunan Aikerui Biotechnology Co., Ltd.); 1.2 The concentration and purity of the genomic DNA of the giant golden fly were assessed using a Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific, USA); Step 2 RPA-LFD detection;
[0006] Perform the procedure according to the instructions of the DNA Isothermal Rapid Amplification Kit (#WLN8203KIT, Anpu Future Biotechnology Co., Ltd.); 2.1.1 Reaction system: A 10 µL reaction system was used, consisting of 5.8 µL of Buffer A (containing dry powder) and 1.6 µL of a primer mixture consisting of 3 pairs of primers. The primer mixture was prepared as follows: the concentration of each primer pair was 10 µM, and the primer mixture ratio was Cm:Ls:Sp = 3:3:2. The mixture was incubated in a metal bath at 37°C for 15 minutes. Prepare an additional 0.3 µL of probe mixture, with each probe (10 µM) combined at a Cm:Ls:Sp ratio of 1:1:1. The reaction also includes 1 µL of DNA template (1 ng / µL), 0.5 µL of buffer B, and 0.8 µL of pure water. 2.1.2 Reaction conditions: Incubate the mixture in a metal bath at 37°C for 15 min.
[0007] Mixed amplification products: After amplification, 5 µL of the product was mixed with 95 µL of dd water.
[0008] Visualization of test results: 80 µL of the mixture was dropped onto the sample pad of the LFD test strip (#WLFS8208, Anpu Future Biotechnology Co., Ltd.). The results were displayed after 15 min.
[0009] Result interpretation: The interpretation criteria are as follows: If both the test line (T line) and the control line (C line) are visible, the result is considered positive; if only the C line is visible, the result is negative; if there is no C line, the result is invalid.
[0010] All the steps together constitute this detection method. The core of this detection method lies in the design of primers and probes, which is also the focus of this invention. It mainly includes two reaction steps: one is the RPA reaction, which is a DNA amplification reaction; the second is visualization detection, where the amplification products are added to the test strip and the results are awaited. To demonstrate the usability of the detection system, specificity and sensitivity analyses are performed. Furthermore, to highlight the detection system's effectiveness in detecting mixed samples, mixed sample analysis is conducted.
[0011] The beneficial effects of the present invention.
[0012] The combination of recombinase polymerase amplification (RPA) technology and lateral flow chromatography strip visualization (LFA) is considered an ideal point-of-care testing (POCT) tool, and has been widely used in clinical pathogen detection, food safety, and environmental monitoring. Therefore, this method, based on RPA-LFA, successfully established a triple RPA-LFA detection system for *Gynostemma pentaphyllum*, *Gynostemma spp.*, and *Vallisneria natans*, by designing specific primers based on different gene loci, enabling rapid and visual identification of forensic insect species. Attached Figure Description
[0013] Figure 1 Interpretation method of flow chromatography test strips and species information at different positions of the test line.
[0014] Figure 2 Specific test results.
[0015] Figure 3 Sensitivity test results.
[0016] Figure 4 Detection results of mixed samples. Detailed Implementation
[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0018] Step 1: Insect DNA extraction; 1.1 DNA was extracted from *Gynostemma pentaphyllum* (Cm), *Gynostemma spp.* (Ls), *Variegata spp.* (Sp), *Bulbus rubra* (Cv), *Gynostemma pentaphyllum* (Cr), *Flyflies spp.* (Sc), and *Heterodontia spp.* (Ms) using the SteadyPure Universal Genomic DNA Extraction Kit (AG21010, Hunan Aikerui Biotechnology Co., Ltd.).
[0019] 1.2 The concentration and purity of the genomic DNA of the giant golden fly were assessed using a Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific, USA).
[0020] Step 2 RPA-LFD detection; 2.1 RPA reaction Perform the procedure according to the instructions of the DNA Isothermal Rapid Amplification Kit (#WLN8203KIT, Anpu Future Biotechnology Co., Ltd.).
[0021] 2.1.1 Reaction System: A 10 µL reaction system was used, consisting of 5.8 µL of Buffer A (containing dry powder) and 1.6 µL of a primer mixture comprising three pairs of primers. The primer mixture was prepared as follows: each primer pair was 10 µM, and the primer mixture ratio was Cm:Ls:Sp = 3:3:2. This mixture was incubated in a metal bath at 37°C for 15 minutes; these reaction conditions were based on our previous studies on single-species identification. An additional 0.3 µL of probe mixture was prepared, with each probe (10 µM) combined at a Cm:Ls:Sp ratio of 1:1:1. The reaction also included 1 µL of DNA template (1 ng / µL), 0.5 µL of Buffer B, and 0.8 µL of purified water. Primer and probe information can be found in [link to primer and probe information]. Table 1:
[0022] 2.1.2 Reaction conditions: Incubate the mixture in a metal bath at 37°C for 15 min.
[0023] 2.2 LFD Visualization 2.2.1 Mixing amplification products: After amplification, 5 µL of the product was mixed with 95 µL of dd water.
[0024] 2.2.2 Visualization of Detection Results: 80 µL of the mixture was dropped onto the sample pad of the LFD test paper (#WLFS8208, Anpu Future Biotechnology Co., Ltd.). The results were displayed after 15 min.
[0025] 2.2.3 Result Interpretation: For example... Figure 1 As shown on the left, the interpretation criteria are as follows: if both the test line (T line) and the control line (C line) are visible, the result is considered positive; if only the C line is visible, the result is negative; if there is no C line, the result is invalid. Figure 1 The right side shows the species information represented by different positions of the detection line (T-line).
[0026] Step 3 Specificity Analysis 3.1 Preparation of Necrophilic Insect DNA: Genomic DNA from seven necrophilic fly species was obtained according to Step 1 to evaluate the specificity of the multiplex RPA-LFA detection system for target species, including *Gynostemma pentaphyllum* (Cm), *Glechoma heliotropium* (Ls), and *Vallisneria natans* (Sp). The DNA input for each reaction was normalized to 1 ng, and all tests were repeated three times. The method was considered specific if only the target detection line for the target species appeared, and there was no cross-reactivity with non-target species.
[0027] 3.2. RPA-LFD detection: The detection method is the same as in Step 2.
[0028] 3.3. Result Interpretation: For example... Figure 2 As shown, only the target species (Cm, Ls, Sp) showed the corresponding detection lines, while the other necrophilic flies were negative. This indicates that the method has good specificity for the detection of *Gnaphalium affine*.
[0029] Step 4 Sensitivity Analysis 4.1 The limit of detection (LOD) of this method was evaluated by detecting genomic DNA of the target species (Golden Fly, Green Botrytis cinerea, and Sarcoptes brownis) at different dilutions. Each dilution was tested three times to determine the minimum amount of DNA required to produce a visible positive signal on the LFA.
[0030] RPA-LFD detection: The detection method is the same as in Step 2.
[0031] 4.3 Result Interpretation: Figure 3 The Levels of Detection (LODs) for the three target species are shown. At a DNA input of 62 pg, *C. m.* (Golden Fly) showed a negative result with an LOD of 125 pg; *L. spp.* (Golden Green Fly) and *V. spp.* (Brown-tailed Flesh Fly) had LODs of 250 pg and 62 pg, respectively. These results demonstrate that the multiplex RPA-LFA detection system has high detection sensitivity.
[0032] Step 5: Mixed Insect DNA Detection 5.1 Sample Preparation: To simulate mixed samples commonly encountered in forensic scenarios, five multi-species DNA mixtures (M1-M5) were prepared to evaluate the detection capability of the multiplex RPA-LFA detection system for mixed samples. The DNA composition and mixing ratio of mixtures M1-M5 are as follows: M1:Cm:Ls:Sp = 1:1:1.
[0033] M2:Cm:Cv:Cr:Sc:ms = 1:1:1:1:1.
[0034] M3: Ls: Cv: Cr: Sc: ms = 1:1:1:1:1.
[0035] M4: Sp: Cv: Cr: Sc: ms = 1:1:1:1:1.
[0036] M5: Contains DNA from 7 species, mixed in a ratio of 1:1:1:1:1:1:1:1.
[0037] These mixed samples were then subjected to multiplex RPA-LFA testing to validate their ability to detect and distinguish species in mixtures containing DNA from multiple species. The steps in Section 2.4 were followed when testing the mixtures.
[0038] The concentration for each species was 1 ng / µL.
[0039] 5.2 RPA-LFD detection: The detection method is the same as in Step 2.
[0040] 5.3 Result Interpretation: The target species present in each mixture were successfully identified, with no cross-reactivity. This indicates that the multiplex RPA-LFA system can accurately and effectively detect *Gymnocladus macrocarpa*, *Gymnocladus filamentosa*, and *Vallisneria brownis* from complex and multivariate mixed samples.
[0041] Except for the blank control (NC), the target species in each mixture were successfully detected.
Claims
1. A rapid detection and visualization method using triple RPA-LFA for necrophilic flies, characterized in that, Includes the following steps; Step 1: DNA amplification reaction; Step 1 Insect DNA Extraction: 1.1 DNA was extracted from *Gynostemma pentaphyllum* (Cm), *Gynostemma spp.* (Ls), *Variegata spp.* (Sp), *Bulbus rubra* (Cv), *Gynostemma pentaphyllum* (Cr), *Flyflies spp.* (Sc), and *Heterodontia spp.* (Ms) using the SteadyPure Universal Genomic DNA Extraction Kit; 1.2 The concentration and purity of genomic DNA from the above species were assessed using a Nano Drop 2000 spectrophotometer; Step 2 RPA-LFD detection; 2.1 RPA reaction: Perform according to the instructions for the DNA isothermal rapid amplification kit; 2.1.1 Reaction system: A 10 µL reaction system was used, consisting of 5.8 µL of Buffer A (containing dry powder) and 1.6 µL of a primer mixture consisting of 3 pairs of primers; 2.1.2 Reaction conditions: The mixture was incubated in a metal bath at 37°C for 15 min to carry out the RPA reaction and obtain the amplified product; Step 2: Visual inspection; Mixed amplification product: 5 µL of the amplified product was mixed with 95 µL of dd water to obtain the mixed amplification product; Visualization of detection results: 80 µL of the mixed amplification product was dropped onto the LFD test paper sample pad, and the results were displayed after 15 min.
2. The rapid detection and visualization method for necrophilic flies using triple RPA-LFA according to claim 1, characterized in that, The primer mixture in the reaction system described in 2.1.1 is prepared as follows: The concentration of each primer pair was 10 µM, and the primer mixture was prepared in the ratio of Cm:Ls:Sp = 3:3:2; the mixture was incubated in a metal bath at 37°C for 15 minutes. Prepare an additional 0.3 µL of probe mixture, with each probe (10 µM) combined in a Cm:Ls:Sp ratio of 1:1:1; the reaction also includes 1 µL of DNA template (1 ng / µL), 0.5 µL of buffer B and 0.8 µL of pure water.
3. The rapid detection and visualization method for necrophilic flies using triple RPA-LFA according to claim 1, characterized in that, In step two, the results are interpreted according to the following criteria: if both the detection line (t-line) and the control line (c-line) are visible, the result is considered positive; if only the c-line is visible, the result is negative; if there is no c-line, the result is invalid.