Primer, probe, kit and method for visually and rapidly detecting RPA-LFD of chrysomyia megacephala

Abstract

The invention belongs to the field of molecular biological detection, and particularly discloses an RPA-LFD primer, a probe, a kit and a method for visually and rapidly detecting chrysomyia megacephala, the primer sequence is as shown in SEQ ID NO: 1-2, and the probe sequence is as shown in SEQ ID NO: 3. According to the primer and probe composition provided by the invention, through an RPA-LFD technology, the whole process from sample DNA obtaining to visual detection result obtaining can be realized within 20 minutes. The RPA-LFD primer, probe, kit and method for visually and rapidly detecting chrysomyia megacephala, provided by the invention, have the advantages of high specificity, good sensitivity, high detection rate and the like.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to an RPA-LFD primer, a probe, a kit and a method for visually and rapidly detecting Chrysomyia megaloblasta. Background Art

[0002] Chrysomya megacephala is the most frequent necrophilous fly at the scene of death and is also one of the first necrophilic flies to arrive. The accuracy and time required for its species identification directly affect the accuracy and speed of the time of death inference. Limited by the difficulty of morphological identification and the ineffectiveness of most larvae and pupae, forensic workers rely more on DNA-barcoding molecular identification. However, molecular identification relies on laboratory PCR equipment, and the currently commonly used DNA-barcoding method requires sequencing and comparison, which takes a long time to identify. It is very necessary to find a fast, accurate, visual, and field-application-worthy Chrysomya species identification technology.

[0003] Recombinase polymerase amplification (RPA) is a constant temperature nucleic acid amplification technology that can complete exponential amplification at 37℃~42℃ without complex equipment, providing a new idea for species identification based on molecular means. It can be combined with lateral flow dipstick (LFD) to achieve naked eye visualization detection, which is very suitable for on-site detection and analysis. Therefore, this method is based on recombinase polymerase amplification technology combined with lateral flow dipstick visualization method (RPA-LFD), and a simple, rapid, highly specific and sensitive species identification method for Chrysocytis megacephala was established, which can promote the grassroots promotion and application of forensic entomology evidence. Summary of the invention

[0004] In view of the above-mentioned shortcomings, the present invention provides a RPA-LFD primer, probe, kit and method for visually and rapidly detecting Chrysopa longipes, which has the advantages of being fast, simple and accurate.

[0005] In order to achieve the above object, the present invention provides the following technical solutions.

[0006] In the first aspect, the present invention provides a pair of RPA-LFD primers for visual rapid detection of Chrysopa longituba, wherein the nucleotide sequence of the primers is:

[0007] The forward primer as shown in SEQ ID NO: 1; wherein the forward primer is: 5'-TACCATTTATTGTTCTAGCTGCACGTCGT-3';

[0008] The reverse primer as described in SEQ ID NO: 2; wherein the reverse primer is: 5'-[Biotin]-TTGAATATGAACTGGAGTAACTAAAGGATT-3';

[0009] In a second aspect, the present invention provides a pair of RPA-LFD probes for visually and rapidly detecting Chrysomyia megacephala, wherein the nucleotide sequence of the probes is:

[0010] The nucleotide sequence of the probe is:

[0011] The probe as described in SEQ ID NO: 3; wherein the probe is: 5'-[FAM]-ATTTTATTAGTATTAATTAATCCTTACTTAC[TNF]TTGGTGACCCTG ATAAT-[C3spacer]-3'.

[0012] In a third aspect, the present invention also provides a combination of the aforementioned primers and probes.

[0013] In a fourth aspect, the present invention also provides an RP A-LFD kit for visually and rapidly detecting Chrysopa longipes, wherein the kit protects the aforementioned primers and probes.

[0014] Furthermore, the kit also includes RPA amplification reaction system reagents.

[0015] Furthermore, the kit also includes a lateral flow chromatography test strip.

[0016] In a fifth aspect, the present invention also provides a RPA-LFD visualization rapid detection method for Chrysopa longituba for non-diagnostic purposes, characterized in that it comprises the following steps:

[0017] (1) Extracting DNA from the sample to be tested;

[0018] (2) using the DNA extracted in step (1) as a template, performing an RPA-LFD reaction using the kit described in any one of claims 4 to 6 to obtain an RPA double-labeled amplicon;

[0019] (3) Using lateral flow chromatography test strips, visually determine the results of the RPA double-labeled amplicon in step (2).

[0020] Furthermore, the RPA double-labeled amplicon is an amplicon with FAM and Biotin double labels.

[0021] Furthermore, the detection method of the lateral flow chromatography reagent strip is: dilute the RPA double-labeled amplicon 40 times with deionized water, mix well, drop into the sample well of the lateral flow chromatography test paper, let stand at room temperature, and observe the result within 5 minutes.

[0022] Beneficial effects of the present invention:

[0023] The present invention uses RPA and LFD technology in combination with cytb gene to construct a rapid and accurate visual detection system for the identification of Chrysopidae species. The system includes primer pairs and probes for detecting Chrysopidae, and can complete amplification in just 15 minutes at 37°C. It has good specificity and a sensitivity of 7.8×10 -4 ng, the positive detection rate of the large-headed golden fly samples in the case reached 100%. Using LFD for visualization, the test results can be stably read in 3 minutes, which is simple and intuitive. In general, the present invention can achieve the whole process from obtaining sample DNA to visualizing the detection results within 20 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A diagram showing a method for reading the lateral flow chromatography paper of the present invention;

[0025] Figure 2 The results of the RPA-LFD rapid visualization detection method of the present invention are shown in the figure; the left side shows the detection results of Chrysocytidae (CM), and the right side shows the detection results of the blank control (NC);

[0026] Figure 3 It is the specific detection result of the RPA-LFD visual rapid detection method described in the present invention;

[0027] Figure 4 It is the sensitivity result of the RPA-LFD visual rapid detection method described in the present invention;

[0028] Figure 5 This is an evaluation of the detection rate of the RPA-LFD visual rapid detection method described in the present invention. DETAILED DESCRIPTION

[0029] To make the present invention easier to understand, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in the field; unless otherwise specified, the reagents and the like involved in this article can be purchased from the market or prepared by known methods.

[0030] Example 1: Primers and probes used in the present invention

[0031] The primer and probe sequence information for RPA amplification used in the present invention are shown in Table 1 below:

[0032] Table 1:

[0033]

[0034] The 5' end of the reverse primer is biotinylated; the 5' end of the probe is labeled with a fluorescent group, a dspacer is marked in the middle of the probe as a recognition site for the exonuclease, and the 3' end of the probe is labeled with a C3spacer.

[0035] Example 2: RPA-LFD visualization rapid detection method for Chrysopa longipes

[0036] (1) Insect DNA extraction

[0037] The DNA of Chrysobacteria melanogaster was extracted using the SteadyPure universal genomic DNA extraction kit (Accurate Biotechnology (Hunan) Co., Ltd, China).

[0038] The concentration and purity of the genomic DNA of Chrysocytidae were assessed using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA).

[0039] (2)RPA detection

[0040] RPA reaction

[0041] According to the instructions of the RPAnfo kit (Genenode, China), 32.9 μl ABuffer was first added to the dry powder tube to dissolve the dry powder to prepare a "dry powder-ABuffer" mixture.

[0042] in,

[0043] Reaction system: Use 10 μL reaction system, 6.5 μL dry powder-ABuffer "mixture, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 0.2 μL probe (10 μM), 2 μL DNA template (1 ng / μL), 0.5 μL BBuffer. Wherein, the forward primer, reverse primer and probe are as shown in Example 1.

[0044] Reaction conditions: 37°C, incubate for 15 min to obtain the amplified product.

[0045] (3) LFD visualization

[0046] Mixing amplification products: 5 μL of amplification products were mixed with 195 μL of diluent (Tris-HCL 0.05 M, pH 8.0) (Genenode, China) to obtain a mixture.

[0047] Visualization of test results: 100 μL of the mixture was dropped onto the sample pad of LFD test paper (Genenode, China).

[0048] 3.3 Interpretation of results: Figure 1 As the interpretation method, if both C line and T line are visible, it is positive; if C line is visible and T line is invisible, it is negative; if C line is invisible, it is invalid.

[0049] Example 3, detection results of the RPA-LFD detection method of the present invention

[0050] The DNA of the big-headed golden fly was extracted by the method of insect DNA extraction in Example 2 and the RPA-LFD detection in Example 2 was performed; the RPA-LFD detection in Example 2 was performed using double distilled water as a negative control. The detection results are as follows: Figure 2 As shown. Figure 2 As shown, the test result is positive, that is, this method successfully detects the big-headed golden fly.

[0051] Example 4: Specificity of the RPA-LFD detection method of the present invention

[0052] (1) Extraction of DNA from sarcophagous insects: The method for extracting insect DNA in Example 2 was used to extract DNA from Chrysopa longituba and six other common sarcophagous flies, including Lucilia sericata (LS), Spheniscus fattoris (SC), Chrysopa longituba (CR), Calliphora rubripes (CV), Heterozoa maggots (MS) and Sarcophagus spheniscus (SP), and the DNA concentration of all fly samples was diluted to 1 ng / μL.

[0053] (2) RPA-LFD detection: The detection method is the same as in Example 2.

[0054] (3) Interpretation of results: Figure 3 As shown, only the detection result of Chrysopa longituba was positive, and the other six sarcophagous flies were negative. That is, the method of the present invention has good specificity for the detection of Chrysopa longituba.

[0055] Example 5: Sensitivity of the RPA-LFD detection method of the present invention

[0056] (1) The target mtDNA fragment of Chrysopa longituba was synthesized using a recombinant plasmid and diluted to a series of different concentrations / contents.

[0057] (2) RPA-LFD detection: The detection method is the same as in Example 2.

[0058] (3) Interpretation of results: Figure 4 As shown, 7.8×10 -4 The test strips with concentrations below ng only have C line (negative). That is, the sensitivity of this system for detecting the DNA of the big-headed golden fly is 7.8×10 -4 ng.

[0059] Example 6: Evaluation of the detection rate of the RPA-LFD detection method of the present invention

[0060] (1) Sample preparation: 18 DNA samples of Chrysopa longituba from 8 real cases were selected and DNA extraction was performed according to Step 1.

[0061] (2)RPA-LFD detection: The detection method is the same as Step 2.

[0062] (3) Interpretation of results: Figure 5 As shown in the figure, all 18 samples of Chrysopa longituba were positive, which means that the detection rate of this system for Chrysopa longituba was 100%.

[0063] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A combination of primers and probes for visually rapid detection of Chrysopa longituba by RPA-LFD, characterized in that: The nucleotide sequence of the primer is: The forward primer as shown in SEQ ID NO: 1; wherein the forward primer is: 5'-TACCATTTATTGTTCTAGCTGCACGTCGT-3'; The reverse primer as described in SEQ ID NO: 2; wherein the reverse primer is: 5'-[Biotin]-TTGAATATGAACTGGAGTAACTAAAGGATT-3'; The nucleotide sequence of the probe is: The probe as described in SEQ ID NO: 3; wherein the probe is: 5'-[FAM]-ATTTTATTAGTATTAATTAATCCTTACTTAC[TNF]TTGGTGACCCTG ATAAT-[C3spacer]-3'.

2. A RPA-LFD kit for visual rapid detection of Chrysopa longituba, characterized in that: The kit comprises the primers and probes as claimed in claim 1.

3. The RPA-LFD kit for visual rapid detection of Chrysopa longituba according to claim 2, characterized in that: The kit also includes RPA amplification reaction system reagents.

4. The RPA-LFD kit for visual rapid detection of Chrysopa longituba according to claim 2, characterized in that: The kit also includes a lateral flow chromatography test strip.

5. A RPA-LFD visualization rapid detection method for Chrysopa longituba for non-diagnostic purposes, characterized in that: The following steps are involved: (1) Extracting DNA from the sample to be tested; (2) using the DNA extracted in step (1) as a template, performing an RPA-LFD reaction using the kit described in any one of claims 2 to 4 to obtain an RPA double-labeled amplicon; (3) Using lateral flow chromatography test strips, visually determine the results of the RPA double-labeled amplicon in step (2).

6. The RPA-LFD visualization rapid detection method of Chrysopa longituba according to claim 5, characterized in that: The RPA double-labeled amplicon is an amplicon with FAM and Biotin double labels.

7. The RPA-LFD visualization rapid detection method of Chrysopa longituba according to claim 5, characterized in that: The detection method of the lateral flow chromatography reagent strip is as follows: the RPA double-labeled amplicon is diluted 40 times with deionized water, mixed evenly, dripped into the sample well of the lateral flow chromatography test paper, left to stand at room temperature, and the result is observed within 5 minutes.

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