A kit for identifying or aiding in the identification of anobiid beetles and a set of primers specific thereto

Abstract

The application discloses a kit for identifying or assisting in identifying Anobiidae and a special complete primer set thereof. The complete primer set is composed of primer-F3, primer-B3, primer-FIP and primer-BIP, and nucleotide sequences are shown in sequence 1-sequence 4 in the sequence table in sequence. The complete primer set of the application can be applied to identifying whether the to-be-tested Anobiidae is or is a candidate of Anobiidae, can be applied to detecting whether the to-be-tested sample contains or contains a candidate of Anobiidae, and can be applied to distinguishing Anobiidae from other Anobiidae. Experiments prove that the complete primer set is used for identifying Anobiidae, has high specificity and high sensitivity, and can realize simple, rapid and accurate detection. The application has great application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a kit for identifying or assisting in the identification of the spotted dermestid beetle and its dedicated primer set. Background Technology

[0002] The variabile (Trogoderma variabile) is widely distributed across North America, Europe, Asia, and Oceania, with localized distributions in Central and South America and Africa. It is one of the most widespread and common destructive storage pests in the world. Among the variabile genus, the variabile is second only to the grain beetle in terms of danger. Its main hosts include over 160 types of stored grains and their products, silk, medicinal herbs, and other animal-shaped collectibles. The larvae are the primary stage of damage, exhibiting a varied diet, often feeding in groups, pupating, and contaminating the damaged goods with their molted skin, carcasses, and excrement.

[0003] In recent years, the number of spotted dermestid beetles intercepted at ports around the world has steadily increased, and their harm has attracted increasing attention from various countries. Since most of the dermestid beetles intercepted at ports are non-adult stages that are extremely similar in morphology, traditional morphological identification is difficult, demanding, and inefficient, making the need for quarantine identification, especially rapid identification, increasingly urgent.

[0004] Molecular identification is considered an effective supplement to morphological identification because it is not affected by the individual developmental state and morphological integrity of the dermestid beetle sample. While existing PCR molecular identification techniques can achieve rapid and accurate identification, they require expensive equipment, resulting in high identification costs and making them unsuitable for widespread application in grassroots departments.

[0005] Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification method developed by Dr. Notomi in 2000. This method utilizes four primers that specifically recognize six regions of the target sequence and a Bst DNA polymerase with strand displacement activity to perform exponential amplification of nucleic acids at 60-65°C, achieving an amplification efficiency of up to 10-1. 9 -10 10 The number of copies is on the order of one, and the entire reaction takes only 1 hour. This method has the advantages of isothermal amplification, fast detection speed, high sensitivity, low cost, and visualized results. Summary of the Invention

[0006] The purpose of this invention is to provide a method for identifying or assisting in the identification of the mottled dermatophyte and its dedicated set of primers.

[0007] In a first aspect, the present invention protects a set of primers for identifying or assisting in the identification of the mottled dermestid beetle.

[0008] The primer set protected by this invention for identifying or assisting in the identification of the spotted dermestid beetle consists of primer-F3, primer-B3, primer-FIP, and primer-BIP;

[0009] The primer-F3 is either a1) or a2) as follows:

[0010] a1) The single-stranded DNA molecule shown in sequence 1 of the sequence listing;

[0011] a2) A single-stranded DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 1 and has the same function as sequence 1;

[0012] The primer-B3 is either a3) or a4) as follows:

[0013] a3) The single-stranded DNA molecule shown in sequence 2 of the sequence listing;

[0014] a4) A single-stranded DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 2 and has the same function as sequence 2;

[0015] The primer-FIP is either a5) or a6) as follows:

[0016] a5) The single-stranded DNA molecule shown in sequence 3 of the sequence listing;

[0017] a6) A single-stranded DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 3 and has the same function as sequence 3;

[0018] The primer-BIP is either a7) or a8) as follows:

[0019] a7) The single-stranded DNA molecule shown in sequence 4 of the sequence listing;

[0020] a8) A single-stranded DNA molecule that has the same function as sequence 4 by substitution and / or deletion and / or addition of one or more nucleotides of sequence 4.

[0021] In the above primer set, the molar ratio of primer-F3, primer-B3, primer-FIP and primer-BIP is 1:1:8:8.

[0022] Secondly, this invention protects new uses for the aforementioned primer set.

[0023] This invention protects the use of the above-described primer set in any of the following b1)-b4):

[0024] b1) Identification or auxiliary identification of the mottled dermestid beetle;

[0025] b2) To identify or assist in the identification of whether the dermestid beetle to be tested is a mottled dermestid beetle;

[0026] b3) Detect or assist in the detection of whether the sample to be tested contains the mottled dermestid beetle;

[0027] b4) To distinguish or help distinguish the spotted dermatophyte from other dermatophytes.

[0028] Thirdly, the present invention protects a reagent kit containing the above-mentioned primer set;

[0029] The kit has the function of any one of the following c1)-c4):

[0030] c1) Identification or auxiliary identification of the mottled dermestid beetle;

[0031] c2) Identify or assist in identifying whether the dermestid beetle to be tested is a spotted dermestid beetle;

[0032] c3) Detect or assist in the detection of whether the sample to be tested contains the mottled dermestid beetle;

[0033] c4) To differentiate or assist in differentiating the mottled dermatophyte from other dermatophytes.

[0034] Furthermore, the kit may also include other reagents for detecting the mottled dermestid beetle. In this invention, the other reagents for detecting the mottled dermestid beetle include at least one of the following: WarmStart LAMP 2X Master Mix, WarmStart Colorimetric LAMP 2X Master Mix, and LAMP Fluorescent Dye.

[0035] Furthermore, the kit also includes a negative control (such as sterile ultrapure water) and a positive control (such as genomic DNA from the mottled dermestid beetle).

[0036] The preparation method of the above-mentioned reagent kit is also within the scope of protection of this invention.

[0037] The preparation method of the kit is as follows (d1) or (d2):

[0038] d1) Package each primer in the above primer set separately;

[0039] d2) Mix the primers in the above primer set together in proportion.

[0040] In step d2), primer-F3, primer-B3, primer-FIP, and primer-BIP in the primer set are mixed together in a molar ratio of 1:1:8:8.

[0041] Fourthly, this invention protects a method for identifying or assisting in the identification of whether a test beetle is a spotted beetle.

[0042] The method for identifying or assisting in the identification of whether a carnivorous beetle is a spotted carnivorous beetle, protected by this invention, is as follows: S1), S2), or S3):

[0043] S1) includes the following steps: extracting nucleic acid from the beetle to be tested; using the nucleic acid as a template, performing loop-mediated isothermal amplification using the aforementioned primer set; after the loop-mediated isothermal amplification reaction, determining whether the beetle to be tested is *Desmodium styracifolium* by detecting whether the reaction product shows ladder-like bands after electrophoresis: if the reaction product shows ladder-like bands after electrophoresis, the beetle to be tested is or is a candidate for *Desmodium styracifolium*; if the reaction product does not show ladder-like bands after electrophoresis, the beetle to be tested is not or is not a candidate for *Desmodium styracifolium*.

[0044] S2) includes the following steps: extracting nucleic acid from the dermestid beetle to be tested; using the nucleic acid as a template, performing loop-mediated isothermal amplification using the aforementioned primer set; after completing the loop-mediated isothermal amplification, determining whether the dermestid beetle to be tested is a mottled dermestid beetle by observing the color of the reaction system: if the reaction system is yellow, the dermestid beetle to be tested is or is a candidate for mottled dermestid beetle; if the reaction system is pink, the dermestid beetle to be tested is not or is not a candidate for mottled dermestid beetle.

[0045] S3) includes the following steps: extracting nucleic acid from the beetle to be tested, using the nucleic acid as a template, and performing loop-mediated isothermal amplification using the above-mentioned primer set; after completing the loop-mediated isothermal amplification, determining whether the beetle to be tested is *Dermestole spp.* by observing whether a significant amplification peak appears in the real-time fluorescence PCR results: if a significant amplification peak appears, the beetle to be tested is or is a candidate for *Dermestole spp.*; if no significant amplification peak appears, the beetle to be tested is not or is not a candidate for *Dermestole spp.*

[0046] Fifthly, the present invention protects a method for detecting or assisting in the detection of whether a sample to be tested contains the mottled dermatophyte.

[0047] The method for detecting or assisting in detecting whether a sample contains the mottled carpenter beetle, protected by this invention, is as follows: T1), T2), or T3):

[0048] The T1 step includes the following steps: extracting nucleic acid from the sample to be tested; using the nucleic acid of the sample to be tested as a template, performing loop-mediated isothermal amplification using the above-mentioned primer set; after the loop-mediated isothermal amplification reaction is completed, determining whether the sample to be tested contains *Dermestrum cristatum* by detecting whether ladder-like bands appear after electrophoresis of the reaction products: if ladder-like bands appear after electrophoresis of the reaction products, the sample to be tested contains or is a candidate for containing *Dermestrum cristatum*; if no ladder-like bands appear after electrophoresis of the reaction products, the sample to be tested does not contain or is a candidate for not containing *Dermestrum cristatum*.

[0049] The T2 step includes the following steps: extracting nucleic acid from the sample to be tested; using the nucleic acid of the sample to be tested as a template, performing loop-mediated isothermal amplification using the above-mentioned primer set; after completing the loop-mediated isothermal amplification, determining whether the sample to be tested contains *Dermestrum cristatum* by observing the color of the reaction system: if the reaction system is yellow, the sample to be tested contains or is a candidate for containing *Dermestrum cristatum*; if the reaction system is pink, the sample to be tested does not contain or is a candidate for not containing *Dermestrum cristatum*.

[0050] The T3 step includes the following steps: extracting nucleic acid from the sample to be tested; using the nucleic acid of the sample to be tested as a template, performing loop-mediated isothermal amplification using the above-mentioned primer set; after completing the loop-mediated isothermal amplification, determining whether the sample to be tested contains *Dermestrum cristatum* by observing whether a significant amplification peak appears in the real-time fluorescence PCR results: if a significant amplification peak appears, the sample to be tested contains or is a candidate to contain *Dermestrum cristatum*; if no significant amplification peak appears, the sample to be tested does not contain or is a candidate to not contain *Dermestrum cristatum*.

[0051] Sixthly, the present invention protects a method for distinguishing or assisting in distinguishing the spotted dermatitis from other dermatitis.

[0052] The method protected by this invention for distinguishing or assisting in distinguishing the spotted carpenter beetle from other carpenter beetles is as follows: U1), U2), or U3):

[0053] The U1 step includes the following steps: extracting nucleic acid from the beetle to be tested; using the nucleic acid as a template, performing loop-mediated isothermal amplification using the aforementioned primer set; after the loop-mediated isothermal amplification reaction, determining whether the beetle to be tested is *Dermestrum spp.* by detecting whether the reaction product shows ladder-like bands after electrophoresis: if the reaction product shows ladder-like bands after electrophoresis, the beetle to be tested is or is a candidate for *Dermestrum spp.*; if the reaction product does not show ladder-like bands after electrophoresis, the beetle to be tested is or is a candidate for other beetles.

[0054] The U2 step includes the following steps: extracting nucleic acid from the beetle to be tested; using the nucleic acid as a template, performing loop-mediated isothermal amplification using the aforementioned primer set; after completing the loop-mediated isothermal amplification, determining whether the beetle to be tested is a spotted beetle by observing the color of the reaction system: if the reaction system is yellow, the beetle to be tested is or is a candidate spotted beetle; if the reaction system is pink, the beetle to be tested is or is a candidate other beetle.

[0055] The U3 method includes the following steps: extracting nucleic acid from the beetle to be tested, using the nucleic acid as a template, and performing loop-mediated isothermal amplification using the above-mentioned primer set; after completing the loop-mediated isothermal amplification, determining whether the beetle to be tested is *Dermestole spp.* by observing whether a significant amplification peak appears in the real-time fluorescence PCR results: if a significant amplification peak appears, the beetle to be tested is or is a candidate for *Dermestole spp.*; if no significant amplification peak appears, the beetle to be tested is or is a candidate for other beetles.

[0056] In any of the methods described above, the loop-mediated isothermal amplification reaction system can specifically be System 1, with a total volume of 25 μL, consisting of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL template, primer mixture and sterile ultrapure water.

[0057] When using System 1, the loop-mediated isothermal amplification reaction conditions can be: 61-67℃ (e.g., 61-63℃, 63-65℃, 65-67℃, 61℃, 63℃, 65℃, or 67℃) for 50 min, followed by 85℃ for 3 min. The reaction products are subjected to 2% agarose gel electrophoresis.

[0058] In any of the methods described above, the loop-mediated isothermal amplification reaction system can specifically be system 2, with a total volume of 24 μL, consisting of 12.5 μL WarmStart Colorimetric LAMP 2X Master Mix, 2.5 μL template, and primer mixture.

[0059] When using System 2, the loop-mediated isothermal amplification reaction conditions can be: 61-67℃ (e.g., 61-63℃, 63-65℃, 65-67℃, 61℃, 63℃, 65℃ or 67℃) for 30 min, and 4℃ for 3 min.

[0060] The primer mixture is a mixture of the primers in the aforementioned primer set. The final concentration of primer-F3 and primer-B3 in system 1, system 2, or system 3 can be 0.2 μM, and the final concentration of primer-FIP and primer-BIP in system 1, system 2, or system 3 can be 1.6 μM.

[0061] In any of the methods described above, the loop-mediated isothermal amplification reaction system can specifically be system 3, with a total volume of 25 μL, consisting of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL template, 0.5 μL LAMP Fluorescent Dye, primer mixture, and sterile ultrapure water. The reaction system should be prepared under light-protected conditions.

[0062] When using System 3, the PCR amplification instrument used can be the ABI QuantStudio 6Flex real-time fluorescence PCR amplification instrument from Thermo Fisher Scientific. The loop-mediated isothermal amplification reaction conditions can be: 65℃ for 10 min; 65℃ for 15 s, 65℃ for 45 s, 40 cycles; 85℃ for 3 min.

[0063] In any of the methods described above, the nucleic acid is genomic DNA.

[0064] In any of the above-described applications, kits, or methods, the test beetle may specifically be the egg, larva, pupa, and / or adult of the test beetle.

[0065] In any of the above-described applications, kits, or methods, the test sample includes various substances that can be harmed by the mottled dermestid beetle, such as various stored grains and their products, silk, Chinese medicinal materials, and other animal-shaped collectibles.

[0066] In any of the above-described applications, kits, or methods, the other dermestids may be at least one of the following dermestids: Trogoderma granarium, Trogoderma glabrum, Dermestesater, Thylodrias contractus, and Dermestes frischii.

[0067] In any of the above-described applications, kits, or methods, the variabile (Trogoderma variabile) can be a variabile from different geographical populations, including variabile from Henan, China; variabile from Shanxi (Taiyuan and Linfen), China; variabile from Shaanxi, China; and variabile from Australia.

[0068] This invention establishes a rapid identification method for the spotted ground beetle based on loop-mediated isothermal amplification (LAMP) technology. Four specific primers were designed targeting six specific regions within the full-length COI gene sequence of the spotted ground beetle. Through optimization of the reaction system and conditions, and taking into account the characteristics of the system's pH changing from alkaline to acidic during the reaction and the different color development of the staining agent at different pH levels, a LAMP molecular identification technique and visualization detection method were established. This effectively avoids potential sample contamination during the detection process caused by opening the container. This LAMP technique for identifying the spotted ground beetle enables rapid identification of eggs, larvae, pupae, and adults. It boasts advantages such as speed, efficiency, high specificity, high sensitivity, low cost, ease of operation, and no need for special instruments. It is suitable for field application and easy to promote at the grassroots level, providing a reliable technical basis for the control of the spotted ground beetle and holding significant importance for the grain storage industry and commodity grain trade. Attached Figure Description

[0069] Figure 1 The results are the detection results of the TV-LAMP primer set in step two of Example 1.

[0070] Figure 2This is the visualized detection result of step two in Example 2.

[0071] Figure 3 This is the result of real-time fluorescence PCR detection in step three of Example 2.

[0072] Figure 4 This is the specific detection result in Example 3.

[0073] Figure 5 The results are from the sensitivity experiment based on agarose gel electrophoresis in Example 4.

[0074] Figure 6 The results are based on the visualization-based sensitivity experiment in Example 4.

[0075] Figure 7 The results are from the sensitivity experiment based on real-time fluorescence PCR in Example 4. Detailed Implementation

[0076] The following embodiments are provided to better understand the present invention, but are not limited to the present invention.

[0077] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0078] The micro-sample genomic DNA extraction kits used in the following examples are products of Tiangen Biotech, catalog number DP316. WarmStart LAMP 2X Master Mix, LAMP Fluorescent Dye, and WarmStart Colorimetric LAMP 2X Master Mix are all products of NEW ENGLAND BioLabs. Specifically, WarmStart LAMP 2X Master Mix and LAMP Fluorescent Dye are products of the WarmStart LAMP Kit (catalog number E1700), and WarmStart Colorimetric LAMP 2X Master Mix has a catalog number of M1800.

[0079] The sample number, species name and collection location information of the tested dermestid beetle samples in the following examples are shown in Table 1. All samples were placed in anhydrous ethanol and stored at -20°C for later use.

[0080] Table 1

[0081]

[0082] Example 1: Obtaining the primer set and kit for identifying the spotted dermestid beetle.

[0083] I. Primer design for identifying the spotted dermestid beetle

[0084] 1. Using the full-length COI gene sequence from the mitochondrial genome of the spotted ground beetle as the target gene, a primer set TV-LAMP for identifying the spotted ground beetle was designed and synthesized. The primer sequences of the primer set are detailed in Table 2.

[0085] Table 2

[0086]

[0087]

[0088] II. Validation of the primer set used to identify the spotted dermestid beetle

[0089] 1. Genomic DNA was extracted from the dermestidus samples using a micro-sample genomic DNA extraction kit. The dermestidus samples tested were the following dermestidus samples listed in Table 1: *Dermestidus spectabilis* (numbered 1), *Dermestidus spectabilis* (numbered 2), *Dermestidus spectabilis* (numbered 3), *Dermestidus spectabilis* (numbered 4), *Dermestidus spectabilis* (numbered 5), *Dermestidus spectabilis* (numbered 6), *Dermestidus chinensis* (numbered 7), *Dermestidus variegata* (numbered 13), and *Dermestidus niger* (numbered 14).

[0090] 2. Using the genomic DNA of the dermatophyte sample as a template, loop-mediated isothermal amplification was performed using the primer set TV-LAMP to obtain the amplification product.

[0091] The reaction system consisted of 25 μL of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL template, primer mixture, and sterile ultrapure water. The primer mixture was a mixture of all the primers in the primer set. The final concentrations of outer primers F3 and B3 were both 0.2 μM, and the final concentrations of inner primers FIP and BIP were both 1.6 μM.

[0092] Reaction conditions: 65℃ for 50 min, 85℃ for 3 min.

[0093] 4. Perform 2% agarose gel electrophoresis on the amplification products.

[0094] Following the above method, replace the template with sterile ultrapure water, keeping all other steps unchanged, as a negative control.

[0095] The detection results of the primer set TV-LAMP are shown below. Figure 1(M is DNA Marker, 1-6 are different geographical populations of the spotted ground beetle, 7 is the grain-spotted ground beetle, 8 is the black-spotted ground beetle, 9 is the strange ground beetle, N is the negative control).

[0096] The results showed that the TV-LAMP primer set exhibited ladder-like bands in the identification of the spotted ground beetle, while the negative control did not show any bands. Therefore, the TV-LAMP primer set can be used to identify the spotted ground beetle.

[0097] III. Preparation of a reagent kit for identifying the spotted dermestid beetle

[0098] The kit for identifying the spotted dermestid beetle includes the primer set TV-LAMP.

[0099] Application of the kits prepared in Example 2 and Example 1 in identifying whether the test dermestid beetle is or is a candidate for the mottled dermestid beetle.

[0100] I. Identification of whether the tested dermestid beetle is or is a candidate for *Dermestidus variegata* using agarose gel electrophoresis results.

[0101] 1. Genomic DNA was extracted from the test dermestid beetles using a micro-sample genomic DNA extraction kit.

[0102] 2. Using the genomic DNA of the dendritic beetle to be tested as a template, loop-mediated isothermal amplification was performed using primer set TV-LAMP to obtain the amplification product.

[0103] The reaction system consisted of 25 μL of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL template, primer mixture, and sterile ultrapure water. The primer mixture was a mixture of all primers in the TV-LAMP primer set. The final concentrations of outer primers F3 and B3 were both 0.2 μM, and the final concentrations of inner primers FIP and BIP were both 1.6 μM.

[0104] Reaction conditions: 65℃ for 50 min, 85℃ for 3 min.

[0105] 3. Perform 2% agarose gel electrophoresis on the amplified products.

[0106] Following the above method, replace the template with sterile ultrapure water, keeping all other steps unchanged, as a negative control.

[0107] Following the method described above, the template was replaced with genomic DNA from the spotted dermestid beetle, while all other steps remained unchanged, serving as a positive control.

[0108] 4. Based on the electrophoresis results, make the following judgments:

[0109] When the positive control shows ladder-like bands and the negative control shows no bands, if the test beetle shows ladder-like bands, then the test beetle is or is a candidate for *Dermestia spp.*; if the test beetle does not show ladder-like bands (e.g., no bands), then the test beetle is not or is not a candidate for *Dermestia spp.*.

[0110] If the positive control does not show ladder-like bands (e.g., no bands) or the negative control shows bands, the identification result of the tested dermestid beetle is invalid.

[0111] II. Visual identification of whether the tested dermestid beetle is or is a candidate for the mottled dermestid beetle.

[0112] 1. Genomic DNA was extracted from the test dermestid beetles using a micro-sample genomic DNA extraction kit.

[0113] 2. Using the genomic DNA of the dendritic beetle to be tested as a template, loop-mediated isothermal amplification was performed using primer set TV-LAMP.

[0114] The reaction system consisted of 24 μL of 12.5 μL WarmStart Colorimetric LAMP 2X Master Mix, 2.5 μL template, and primer mixture. The primer mixture was a mixture of all primers in the TV-LAMP primer set. The final concentrations of outer primers F3 and B3 were both 0.2 μM, and the final concentrations of inner primers FIP and BIP were both 1.6 μM.

[0115] Reaction conditions: 65℃ for 30 min, 4℃ for 3 min.

[0116] Following the above method, replace the template with sterile ultrapure water, keeping all other steps unchanged, as a negative control.

[0117] Following the method described above, the template was replaced with genomic DNA from the spotted dermestid beetle, while all other steps remained unchanged, serving as a positive control.

[0118] 3. After completing step 2, observe the color of the reaction system with the naked eye, and then make the following judgments:

[0119] When the positive control is yellow and the negative control is pink, if the test dermestid beetle is yellow, then the test dermestid beetle is or is a candidate for the mottled dermestid beetle; if the test dermestid beetle is pink, then the test dermestid beetle is not or is not a candidate for the mottled dermestid beetle.

[0120] If the positive control does not turn yellow or the negative control does not turn pink, the identification result of the test dermestid beetle is invalid.

[0121] The inventors of this invention used the method in step two to identify whether the tested dermestid beetle was a spotted dermestid beetle. The tested dermestid beetles were the spotted dermestid beetle numbered 1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13, and 14 of the black-spotted dermestid beetle, as listed in Table 1.

[0122] See results Figure 2 (1-12 are, in order: *Desmodium spp.* 1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 14, 15, and 13; N is the negative control). The results showed that the reaction system for *Desmodium spp.* from different geographical populations was yellow, while the reaction system for non-*Desmodium spp.* was pink.

[0123] III. Identifying whether the tested dermestid beetle is or is a candidate for *Dermestidus variegata* using real-time fluorescence PCR results.

[0124] 1. Genomic DNA was extracted from the test dermestid beetles using a micro-sample genomic DNA extraction kit.

[0125] 2. Using the genomic DNA of the dendritic beetle to be tested as a template, loop-mediated isothermal amplification was performed using primer set TV-LAMP to obtain the amplification product.

[0126] The reaction system consisted of 25 μL of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL template, 0.5 μL LAMP Fluorescent Dye, primer mixture, and sterile ultrapure water. The primer mixture was a mixture of all primers in the TV-LAMP primer set. The final concentrations of outer primers F3 and B3 were both 0.2 μM, and the final concentrations of inner primers FIP and BIP were both 1.6 μM.

[0127] Reaction conditions: 65℃ for 10 min; 65℃ for 15 s, 65℃ for 45 s, 40 cycles; 85℃ for 3 min.

[0128] Following the above method, replace the template with sterile ultrapure water, keeping all other steps unchanged, as a negative control.

[0129] Following the method described above, the template was replaced with genomic DNA from the spotted dermestid beetle, while all other steps remained unchanged, serving as a positive control.

[0130] 3. After completing loop-mediated isothermal amplification, make the following judgments based on the real-time fluorescence PCR results:

[0131] If the positive control shows a significant amplification peak and the negative control does not show a significant amplification peak, then if the test dermestid beetle shows a significant amplification peak, the test dermestid beetle is or is a candidate for *Dermestidus variegata*; if the test dermestid beetle does not show a significant amplification peak, then the test dermestid beetle is not or is not a candidate for *Dermestidus variegata*.

[0132] If the positive control does not show a significant amplification peak or the negative control shows a significant amplification peak, the identification result of the tested dermestid beetle is invalid.

[0133] The inventors of this invention used step three to identify whether the tested dermestid beetle was or was a candidate for *Dermestidus fasciatus*. The tested dermestid beetles were, as listed in Table 1, *Dermestidus fasciatus* numbered 1, 2, 3, 5, 6, 7, 8, 11, *Dermestidus fasciatus*, 12, and 14.

[0134] See results Figure 3 (1-10 are, in order, the spotted dermatitis beetle (1), spotted dermatitis beetle (2), spotted dermatitis beetle (3), spotted dermatitis beetle (5), spotted dermatitis beetle (6), spotted dermatitis beetle (7), spotted dermatitis beetle (8), spotted dermatitis beetle (11), spotted dermatitis beetle (12), and spotted dermatitis beetle (14); N is the negative control). The results showed that the reaction systems of spotted dermatitis beetles from different geographical populations all exhibited significant amplification peaks, while the reaction systems of non-spotted dermatitis beetles did not show significant amplification peaks.

[0135] Example 3, Specificity Experiment

[0136] The dermestid beetle to be tested, numbered 1, is the spotted dermestid beetle.

[0137] The test beetle 2 is the spotted beetle, numbered 2.

[0138] The test beetle 3 is the spotted beetle, numbered 3.

[0139] The test beetle 4 is the spotted beetle, numbered 4.

[0140] The tested dermestid beetle 5 is the spotted dermestid beetle, numbered 5.

[0141] The test specimen, 6, is the spotted dermestid beetle, numbered 6.

[0142] The bark beetle to be tested, number 7, is the bark beetle with the bark beetle mollusks.

[0143] The test beetle 8 is the grain-spotted beetle, numbered 8.

[0144] The bark beetle to be tested, numbered 9, is the bark beetle with the bark beetle mollusks.

[0145] The test beetle 10 is the spotted beetle, numbered 10.

[0146] The test beetle 11 is the hook-shaped beetle numbered 11.

[0147] The test beetle 12 is the white-bellied beetle, numbered 12.

[0148] The tested dermestid beetle 13 is the 13th of the Hundred Strange Dermestid beetles.

[0149] The test beetle 14 is the black-spotted beetle numbered 14.

[0150] The following steps were performed on each of the dermestid beetles to be tested:

[0151] 1. Genomic DNA was extracted from the test dermestid beetles using a micro-sample genomic DNA extraction kit.

[0152] 2. Using the genomic DNA of the dendritic beetle extracted in step 1 as a template, loop-mediated isothermal amplification was performed using primer set TV-LAMP to obtain the amplification product.

[0153] The reaction system consisted of 25 μL of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL template, primer mixture, and sterile ultrapure water. The primer mixture was a mixture of all primers in the TV-LAMP primer set. The final concentrations of outer primers F3 and B3 were both 0.2 μM, and the final concentrations of inner primers FIP and BIP were both 1.6 μM.

[0154] Reaction conditions: 65℃ for 50 min, 85℃ for 3 min.

[0155] 3. Perform 2% agarose gel electrophoresis on the amplified products.

[0156] Following the above method, replace the template with sterile ultrapure water, keeping all other steps unchanged, as a negative control.

[0157] Following the method described above, the template was replaced with genomic DNA from the spotted dermestid beetle, while all other steps remained unchanged, serving as a positive control.

[0158] Some test results can be found Figure 4 (1-14 are the test dermestid beetles 1-14 respectively, N is the negative control). The results showed that when the test dermestid beetles were spotted dermestid beetles, they all showed ladder-like bands; when the test dermestid beetles were non-spotted dermestid beetles, they all showed no bands (i.e., no ladder-like bands); the negative control also showed no bands.

[0159] Therefore, it can be seen that the use of primer set TV-LAMP for the identification of spotted dermestid beetles has good specificity.

[0160] Example 4, Sensitivity Experiment

[0161] I. Sensitivity Experiment Based on Agarose Gel Electrophoresis

[0162] 1. Genomic DNA was extracted from the spotted dermestid beetle using a micro-sample genomic DNA extraction kit.

[0163] 2. Genomic DNA was collected from the spotted ground beetle and serially diluted with sterile water to obtain genomic DNA concentrations of 10.0 ng / μL, 1.0 ng / μL, and 1.0 × 10⁻⁶ ng / μL. -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4 A dilution of ng / μL.

[0164] 3. Using the dilution solution obtained in step 2 as a template, loop-mediated isothermal amplification was performed using primer set TV-LAMP to obtain the amplification product.

[0165] The reaction system consisted of 25 μL of 12.5 μL WarmStart LAMP 2X Master Mix and 1 μL of dilution buffer (each μL of dilution buffer contained 10.0 ng, 1.0 ng, and 1.0 × 10⁻⁶ ng of genomic DNA from the mottled dermestid beetle). -1 ng, 1.0×10 -2 ng, 1.0×10 -3 ng or 1.0×10 -4 The reaction system consists of ng), primer mixture, and sterile ultrapure water. The primer mixture is a mixture of the primers in the TV-LAMP primer set. In the reaction system, the final concentrations of outer primer F3 and outer primer B3 are both 0.2 μM, and the final concentrations of inner primer FIP and inner primer BIP are both 1.6 μM.

[0166] Reaction conditions: 65℃ for 50 min, 85℃ for 3 min.

[0167] 4. Perform 2% agarose gel electrophoresis on the amplification products.

[0168] Following the above method, replace the template with sterile ultrapure water, keeping all other steps unchanged, as a negative control.

[0169] Following the method described above, the template was replaced with genomic DNA from the spotted dermestid beetle, while all other steps remained unchanged, serving as a positive control.

[0170] If the reaction produces ladder-like bands, it indicates that the corresponding genome content in the reaction system can be detected. If the reaction does not produce ladder-like bands (e.g., no bands), it indicates that the corresponding genome content in the reaction system cannot be detected.

[0171] Some test results can be found Figure 5 (The DNA template concentrations for 1-6 were 10.0 ng / μL, 1.0 ng / μL, and 1.0 × 10⁻⁶, respectively.) - 1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4 (N is the negative control). The results showed that the sensitivity of the primer set TV-LAMP for identifying the spotted dermestid beetle was 1.0 × 10⁻⁶. -1 ng / μL.

[0172] Therefore, it can be seen that the primer set TV-LAMP mediated by agarose gel electrophoresis has high sensitivity for identifying the spotted dermestid beetle.

[0173] II. Sensitivity Experiment Based on Visualization

[0174] 1. Genomic DNA was extracted from the spotted dermestid beetle using a micro-sample genomic DNA extraction kit.

[0175] 2. Genomic DNA was collected from the spotted ground beetle and serially diluted with sterile water to obtain genomic DNA concentrations of 10.0 ng / μL, 1.0 ng / μL, and 1.0 × 10⁻⁶ ng / μL. -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4 A dilution of ng / μL.

[0176] 3. Using the dilution solution obtained in step 2 as a template, loop-mediated isothermal amplification was performed using primer set TV-LAMP to obtain the amplification product.

[0177] The reaction system consisted of 24 μL of 12.5 μL WarmStart Colorimetric LAMP 2X Master Mix, 2.5 μL of dilution buffer (each μL of dilution contained 10.0 ng, 1.0 ng, and 1.0 × 10⁻⁶ genomic DNA of the spotted dermestid beetle). -1 ng, 1.0×10 -2 ng, 1.0×10 -3 ng or 1.0×10 -4The reaction system consists of primers (ng) and a primer mixture. The primer mixture is a mixture of the primers in the TV-LAMP primer set. In the reaction system, the final concentrations of outer primer F3 and outer primer B3 are both 0.2 μM, and the final concentrations of inner primer FIP and inner primer BIP are both 1.6 μM.

[0178] Reaction conditions: 65℃ for 30 min, 4℃ for 3 min.

[0179] Following the above method, replace the template with sterile ultrapure water, keeping all other steps unchanged, as a negative control.

[0180] Following the method described above, the template was replaced with genomic DNA from the spotted dermestid beetle, while all other steps remained unchanged, serving as a positive control.

[0181] If the reaction system is yellow, it indicates that the corresponding genome content in the reaction system can be detected. If the reaction system is pink, it indicates that the corresponding genome content in the reaction system cannot be detected.

[0182] Some test results can be found Figure 6 (The DNA template concentrations for 1-6 were 10.0 ng / μL, 1.0 ng / μL, and 1.0 × 10⁻⁶, respectively.) - 1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4 ng, N is the negative control; the top and bottom figures show two parallel replicate experiments. The results indicate that the sensitivity of the primer set TV-LAMP for identifying the spotted dermestid beetle is between 1.0 ng / μL and 1.0 × 10⁻⁶. - 1 Between ng / μL.

[0183] Therefore, the identification of the spotted dermestid beetle using the visualization-mediated primer set TV-LAMP has low sensitivity.

[0184] III. Sensitivity Experiment Based on Real-Time Fluorescence PCR

[0185] 1. Genomic DNA was extracted from the spotted dermestid beetle using a micro-sample genomic DNA extraction kit.

[0186] 2. Genomic DNA was collected from the spotted ground beetle and serially diluted with sterile water to obtain genomic DNA concentrations of 10.0 ng / μL, 1.0 ng / μL, and 1.0 × 10⁻⁶ ng / μL. -1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4ng / μL, 1.0×10 -5 A dilution of ng / μL.

[0187] 3. Using the dilution solution obtained in step 2 as a template, loop-mediated isothermal amplification was performed using primer set TV-LAMP to obtain the amplification product.

[0188] The reaction system consisted of 25 μL of 12.5 μL WarmStart LAMP 2X Master Mix and 1 μL of dilution buffer (each μL of dilution buffer contained 10.0 ng, 1.0 ng, and 1.0 × 10⁻⁶ ng of genomic DNA from the mottled dermestid beetle). -1 ng, 1.0×10 -2 ng, 1.0×10 - 3 ng, 1.0×10 -4 ng or 1.0×10 -5 The reaction mixture consisted of 0.5 μL of LAMP Fluorescent Dye, primer mixture, and sterile ultrapure water. The primer mixture was a mixture of the primers in the TV-LAMP primer set. In the reaction system, the final concentrations of outer primer F3 and outer primer B3 were both 0.2 μM, and the final concentrations of inner primer FIP and inner primer BIP were both 1.6 μM.

[0189] Reaction conditions: 65℃ for 10 min; 65℃ for 15 s, 65℃ for 45 s, 40 cycles; 85℃ for 3 min.

[0190] Following the above method, replace the template with sterile ultrapure water, keeping all other steps unchanged, as a negative control.

[0191] Following the method described above, the template was replaced with genomic DNA from the spotted dermestid beetle, while all other steps remained unchanged, serving as a positive control.

[0192] The presence of a distinct amplification peak indicates that the corresponding genomic content in the reaction system can be detected. The absence of a distinct amplification peak indicates that the corresponding genomic content in the reaction system cannot be detected.

[0193] Some test results can be found Figure 7 (The DNA template concentrations for 1-7 were 10.0 ng / μL, 1.0 ng / μL, and 1.0 × 10⁻⁶, respectively.) - 1 ng / μL, 1.0×10 -2 ng / μL, 1.0×10 -3 ng / μL, 1.0×10 -4 ng, 1.0×10 -5(ng, N is the negative control). The results showed that the sensitivity of the primer set TV-LAMP for identifying the spotted dermestid beetle was 1.0 × 10⁻⁶. -1 ng / μL.

[0194] Therefore, it can be seen that the identification of the spotted dermestid beetle using real-time fluorescent PCR-mediated primer set TV-LAMP has high sensitivity.

[0195] The above description is only a preferred embodiment of the present invention. For those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> China Agricultural University <120> A kit for identifying or assisting in the identification of the spotted dermestid beetle and its dedicated primer set. <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 ggtacatccc ttagagtc 18 <210> 2 <211> twenty two <212> DNA <213> Artificial Sequence <400> 2 gaagccaaaa tcttatgttg tt 22 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <400> 3 gtgcagtgac aattacattg aagactaatc cgaacagaac ttggg 45 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <400> 4 ggtggatttg gaaattgact tgctatccgt gggaatgcc 39

Claims

1. A kit of primers for identifying or assisting in identifying Trogoderma variabile, comprising primer-F3, primer-B3, primer-FIP and primer-BIP; the primer-F3 is a single-stranded DNA molecule as shown in SEQ ID NO: 1 in the sequence listing; the primer-B3 is a single-stranded DNA molecule as shown in SEQ ID NO: 2 in the sequence listing; the primer-FIP is a single-stranded DNA molecule as shown in SEQ ID NO: 3 in the sequence listing; the primer-BIP is a single-stranded DNA molecule as shown in SEQ ID NO: 4 in the sequence listing.

2. The set of primer according to claim 1, characterized in that: the molar ratio of the primer-F3, the primer-B3, the primer-FIP and the primer-BIP is 1: 1: 8:

8. 3.The kit of primers of claim 1 or 2 for use in any one of b1) to b4): b1) identifying or assisting in identifying Trogoderma variabile; b2) identifying or assisting in identifying whether a test insect is Trogoderma variabile; b3) detecting or assisting in detecting whether a test sample contains Trogoderma variabile; b4) distinguishing or assisting in distinguishing Trogoderma variabile from other insects; the other insects can be at least one of Trogoderma granarium, Trogoderma glabrum, Dermestes ater, Thylodrias contractus and Dermestes frischii. 4.A kit comprising the kit of primers of claim 1 or 2; the kit is for any one of c1) to c4): c1) identifying or assisting in identifying Trogoderma variabile; c2) identifying or assisting in identifying whether a test insect is Trogoderma variabile; c3) detecting or assisting in detecting whether a test sample contains Trogoderma variabile; c4) distinguishing or assisting in distinguishing Trogoderma variabile from other insects; the other insects can be at least one of Trogoderma granarium, Trogoderma glabrum, Dermestes ater, Thylodrias contractus and Dermestes frischii. 5.A method for preparing the kit of claim 4, being d1) or d2): d1) individually packaging each primer in the kit of primers of claim 1 or 2; d2) mixing each primer in the kit of primers of claim 1 or 2 together in proportion.

6. The method of claim 5, wherein: in d2), the primer-F3, the primer-B3, the primer-FIP and the primer-BIP in the kit of primers are mixed together in proportion of 1: 1: 8:

8. 7.A method for identifying or assisting in identifying whether a test insect is Trogoderma variabile, being S1) or S2) or S3): The S1) comprises the following steps: extracting nucleic acid of the to-be-tested hide beetle, taking the nucleic acid of the to-be-tested hide beetle as a template, and performing loop-mediated isothermal amplification by using the complete primer set in claim 1; after the loop-mediated isothermal amplification reaction is completed, whether the to-be-tested hide beetle is the Dermestes maculates is judged by detecting whether the reaction product presents ladder bands after electrophoresis; if the reaction product presents ladder bands after electrophoresis, the to-be-tested hide beetle is or is a candidate for the Dermestes maculates; if the reaction product does not present ladder bands after electrophoresis, the to-be-tested hide beetle is not or is not a candidate for the Dermestes maculates. The S2) comprises the following steps: extracting nucleic acid of the to-be-tested hide beetle, taking the nucleic acid of the to-be-tested hide beetle as a template, and performing loop-mediated isothermal amplification by using the complete primer set in claim 1; after the loop-mediated isothermal amplification is completed, whether the to-be-tested hide beetle is the Dermestes maculates is judged by observing the color of the reaction system; if the reaction system is yellow, the to-be-tested hide beetle is or is a candidate for the Dermestes maculates; if the reaction system is pink, the to-be-tested hide beetle is not or is not a candidate for the Dermestes maculates. The S3) comprises the following steps: extracting nucleic acid of the to-be-tested hide beetle, taking the nucleic acid of the to-be-tested hide beetle as a template, and performing loop-mediated isothermal amplification by using the complete primer set in claim 1; after the loop-mediated isothermal amplification is completed, whether the to-be-tested hide beetle is the Dermestes maculates is judged by observing whether obvious amplification peaks appear in the real-time fluorescent PCR result; if obvious amplification peaks appear, the to-be-tested hide beetle is or is a candidate for the Dermestes maculates; if no obvious amplification peaks appear, the to-be-tested hide beetle is not or is not a candidate for the Dermestes maculates.

8. A method for detecting or assisting in detecting whether a to-be-tested sample contains the Dermestes maculates, which is T1) or T2) or T3) as follows: The T1) comprises the following steps: extracting nucleic acid of the to-be-tested sample, taking the nucleic acid of the to-be-tested sample as a template, and performing loop-mediated isothermal amplification by using the complete primer set in claim 1; after the loop-mediated isothermal amplification reaction is completed, whether the to-be-tested sample contains the Dermestes maculates is judged by detecting whether the reaction product presents ladder bands after electrophoresis; if the reaction product presents ladder bands after electrophoresis, the to-be-tested sample contains or is a candidate for containing the Dermestes maculates; if the reaction product does not present ladder bands after electrophoresis, the to-be-tested sample does not contain or is not a candidate for containing the Dermestes maculates. The T2) comprises the following steps: extracting nucleic acid of the to-be-tested sample, taking the nucleic acid of the to-be-tested sample as a template, and performing loop-mediated isothermal amplification by using the complete primer set in claim 1; after the loop-mediated isothermal amplification is completed, whether the to-be-tested sample contains the Dermestes maculates is judged by observing the color of the reaction system; if the reaction system is yellow, the to-be-tested sample contains or is a candidate for containing the Dermestes maculates; if the reaction system is pink, the to-be-tested sample does not contain or is not a candidate for containing the Dermestes maculates. The T3) comprises the following steps: extracting nucleic acid of the to-be-tested sample, taking the nucleic acid of the to-be-tested sample as a template, and performing loop-mediated isothermal amplification by using the complete primer set in claim 1; after the loop-mediated isothermal amplification is completed, whether the to-be-tested sample contains the Dermestes maculates is judged by observing whether obvious amplification peaks appear in the real-time fluorescent PCR result; if obvious amplification peaks appear, the to-be-tested sample contains or is a candidate for containing the Dermestes maculates; if no obvious amplification peaks appear, the to-be-tested sample does not contain or is not a candidate for containing the Dermestes maculates.

9. A method for distinguishing or assisting in distinguishing Trogoderma variabile from other dermestid beetles, being as follows U1) or U2) or U3): said U1) comprising the following steps: extracting nucleic acid of the dermestid beetle to be tested, using the nucleic acid of the dermestid beetle to be tested as a template, and performing loop-mediated isothermal amplification using the kit of primers according to claim 1; after the loop-mediated isothermal amplification reaction is completed, judging whether the dermestid beetle to be tested is Trogoderma variabile by detecting whether the reaction product presents ladder-like bands after electrophoresis: if the reaction product presents ladder-like bands after electrophoresis, the dermestid beetle to be tested is or is a candidate for Trogoderma variabile; if the reaction product does not present ladder-like bands after electrophoresis, the dermestid beetle to be tested is or is a candidate for other dermestid beetles; said U2) comprising the following steps: extracting nucleic acid of the dermestid beetle to be tested, using the nucleic acid of the dermestid beetle to be tested as a template, and performing loop-mediated isothermal amplification using the kit of primers according to claim 1; after the loop-mediated isothermal amplification reaction is completed, judging whether the dermestid beetle to be tested is Trogoderma variabile by observing the color of the reaction system: if the reaction system is yellow, the dermestid beetle to be tested is or is a candidate for Trogoderma variabile; if the reaction system is pink, the dermestid beetle to be tested is or is a candidate for other dermestid beetles; said U3) comprising the following steps: extracting nucleic acid of the dermestid beetle to be tested, using the nucleic acid of the dermestid beetle to be tested as a template, and performing loop-mediated isothermal amplification using the kit of primers according to claim 1; after the loop-mediated isothermal amplification reaction is completed, judging whether the dermestid beetle to be tested is Trogoderma variabile by observing whether obvious amplification peaks appear in the real-time fluorescence PCR results: if obvious amplification peaks appear, the dermestid beetle to be tested is or is a candidate for Trogoderma variabile; if no obvious amplification peaks appear, the dermestid beetle to be tested is or is a candidate for other dermestid beetles. The other dermestid beetles can be at least one of the following dermestid beetles: Trogoderma granarium, Trogoderma glabrum, Dermestes ater, Thylodrias contractus, and Dermestes frischii. The final concentration of the primer-F3 and the primer-B3 in the reaction system is 0.2 μM, and the final concentration of the primer-FIP and the primer-BIP in the reaction system is 1.6 μM. ​ ​ 10. The method according to any of claims 7-9, characterized by: ​

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