LAMP primer set for identifying tinea larva and application thereof

Abstract

The application discloses a LAMP primer set for identifying Trogoderma granarium and application thereof. The LAMP primer set is composed of primer-F3, primer-B3, primer-FIP and primer-BIP, and the nucleotide sequences are shown as sequence 1-sequence 4 in the sequence table. The LAMP primer set can be applied to identify whether the to-be-tested skin beetle is or is a candidate of Trogoderma granarium, can be applied to detect whether the to-be-tested sample contains or contains a candidate of Trogoderma granarium, and can be applied to distinguish Trogoderma granarium from other skin beetles. Experiments prove that the LAMP primer set has high specificity and high sensitivity, and can realize simple, rapid and accurate detection. The application has great application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a LAMP primer set for identifying Trogoderma granarium and application thereof. BACKGROUND

[0002] Trogoderma belongs to Coleoptera, Dermestidae, and is widely distributed in the world. Among them, there are about 130 species of Trogoderma, of which more than 20 species have economic significance. Trogoderma has a wide range of hosts and can seriously damage various plant and animal products. In addition to damaging the appearance and quality of the host, it also seriously affects foreign trade and causes huge economic losses. Trogoderma (non-Chinese species) is an imported plant quarantine pest in China, and its invasion and control are highly concerned by quarantine departments.

[0003] Trogoderma granarium is an imported quarantine pest in China and one of the most important quarantine pests in the world, and is mainly distributed in Africa, South Asia and parts of Europe. Its main hosts are various grains, dried fruits, vegetable seeds, gum and other plant products, or milk powder, leather, dried animal carcasses and other dry animal products. The larvae are the main insect stages that damage the goods, with the characteristics of voracious and crushing food. After hatching, the larvae first feed on the germ of the grain seeds, and then feed on the endosperm, causing irregular damage to the grain seed coat. For large quantities of goods, Trogoderma granarium often damages the surface layer, causing the surface of the goods to be covered with a large number of larval exuviae, broken setae and feces, which poses a great threat to the storage of raw grains and causes serious pollution and economic losses. The loss of grains is generally 5%-30%, and sometimes as high as 73%-100%.

[0004] Trogoderma granarium larvae prefer to hide in crevices, and the adults do not have the ability to fly, so the main mode of transmission is human transportation in trade. In recent years, the number of Trogoderma granarium intercepted at ports around the world has increased steadily, and its damage has attracted increasing attention from countries. Because the dermestid beetles intercepted at ports are mostly non-adult insect stages that are very similar in morphology, traditional morphological identification is difficult, requires high standards, and is low in efficiency.

[0005] Molecular identification is considered an effective supplement to morphological identification because it is not affected by the developmental state and morphological characteristics of the dermestid sample. Although existing PCR molecular identification techniques can achieve rapid and accurate identification, they require expensive equipment and have high identification costs, which are not suitable for popularization and application in grassroots departments.

[0006] Loop-mediated isothermal amplification (LAMP) is a nucleic acid denaturation and automatic cycle chain displacement nucleic acid amplification reaction under isothermal conditions using a Bst DNA polymerase with chain displacement activity and cascade nucleic acid amplification function. LAMP designs four specific primers for six regions of the target sequence, and uses a DNA polymerase with chain displacement function to continuously replicate and amplify DNA at a constant temperature. In order to improve the reaction efficiency, two loop primers can be added to the reaction system to bind to the stem-loop structure, start chain displacement synthesis, and cycle replication. LAMP is a simple and efficient nucleic acid isothermal amplification technology, which can amplify reaction products in a short time, has higher sensitivity, and is simple to operate without special instruments. The detection result can be observed by naked eye The LAMP is suitable for popularization and application in basic-level departments. The primers are the key to determine sensitivity and specificity of detection results in the LAMP . SUMMARY

[0007] The technical problem to be solved by the present application is how to effectively and quickly and accurately identify the grain borer.

[0008] In order to solve the above technical problems, the present application first provides a complete primer set for identifying or assisting in identifying the grain borer.

[0009] The complete primer set for identifying or assisting in identifying the grain borer provided by the present application consists of primer-F3, primer-B3, primer-FIP and primer-BIP;

[0010] The primer-F3 is as follows a1) or a2):

[0011] a1) a single-stranded DNA molecule represented by SEQ ID NO: 1 in the sequence table;

[0012] a2) a single-stranded DNA molecule which is obtained by substituting and / or deleting and / or adding one or more nucleotides to SEQ ID NO: 1 and has the same function as SEQ ID NO: 1;

[0013] The primer-B3 is as follows a3) or a4):

[0014] a3) a single-stranded DNA molecule represented by SEQ ID NO: 2 in the sequence table;

[0015] a4) a single-stranded DNA molecule which is obtained by substituting and / or deleting and / or adding one or more nucleotides to SEQ ID NO: 2 and has the same function as SEQ ID NO: 2;

[0016] The primer-FIP is as follows a5) or a6):

[0017] a5) a single-stranded DNA molecule represented by SEQ ID NO: 3 in the sequence table;

[0018] a6) a single-stranded DNA molecule having the same function as sequence 3, which is obtained by substitution and / or deletion and / or addition of one or several nucleotides of sequence 3;

[0019] The primer-BIP is as follows a7) or a8):

[0020] a7) a single-stranded DNA molecule as shown in sequence 4 in the sequence listing;

[0021] a8) a single-stranded DNA molecule having the same function as sequence 4, which is obtained by substitution and / or deletion and / or addition of one or several nucleotides of sequence 4.

[0022] In the above complete primer set, the molar ratio of the primer-F3, the primer-B3, the primer-FIP and the primer-BIP is 1:1:8:8.

[0023] To solve the above technical problems, the application further provides a new use of the above complete primer set.

[0024] The application provides an application of the above complete primer set in any one of the following b1) to b4):

[0025] b1) identifying or assisting in identifying Rhyzopertha dominica;

[0026] b2) identifying or assisting in identifying whether a to-be-tested Rhyzopertha dominica is Rhyzopertha dominica;

[0027] b3) detecting or assisting in detecting whether a to-be-tested sample contains Rhyzopertha dominica;

[0028] b4) distinguishing or assisting in distinguishing Rhyzopertha dominica from other Rhyzopertha dominica.

[0029] To solve the above technical problems, the application further provides a kit containing the above complete primer set;

[0030] The kit has the following functions as any one of c1) to c4):

[0031] c1) identifying or assisting in identifying Rhyzopertha dominica;

[0032] c2) identifying or assisting in identifying whether a to-be-tested Rhyzopertha dominica is Rhyzopertha dominica;

[0033] c3) detecting or assisting in detecting whether a to-be-tested sample contains Rhyzopertha dominica;

[0034] c4) distinguishing or assisting in distinguishing Rhyzopertha dominica from other Rhyzopertha dominica.

[0035] Further, the kit can further comprise other reagents for detecting the Rhyzopertha dominica. In the present application, the other reagents for detecting the Rhyzopertha dominica comprise at least one of the following: WarmStart LAMP 2X Master Mix, WarmStart Colorimetric LAMP 2X Master Mix, LAMP Fluorescent Dye.

[0036] Still further, the kit further comprises a negative control (such as sterile ultrapure water) and a positive control (such as genomic DNA of the Rhyzopertha dominica).

[0037] The preparation method of the kit is also within the protection scope of the present application.

[0038] The preparation method of the kit can be d1) or d2) as follows:

[0039] d1) each primer in the above complete primer set is packaged separately;

[0040] d2) each primer in the above complete primer set is mixed together in proportion.

[0041] In the d2), the primer-F3, the primer-B3, the primer-FIP and the primer-BIP in the complete primer set are mixed together in a molar ratio of 1:1:8:8.

[0042] To solve the above technical problems, the present application further provides a method for identifying or assisting in identifying whether a to-be-tested bark beetle is the Rhyzopertha dominica.

[0043] The method for identifying or assisting in identifying whether a to-be-tested bark beetle is the Rhyzopertha dominica provided by the present application is S1), S2) or S3) as follows:

[0044] The S1) comprises the following steps: extracting nucleic acid of the to-be-tested bark beetle, taking the to-be-tested bark beetle nucleic acid as a template, and performing loop-mediated isothermal amplification by using the above complete primer set; after the loop-mediated isothermal amplification reaction is completed, whether the to-be-tested bark beetle is the Rhyzopertha dominica is determined by detecting whether the reaction product electrophoresis presents ladder-like bands: if the reaction product electrophoresis presents ladder-like bands, the to-be-tested bark beetle is or is a candidate for the Rhyzopertha dominica; if the reaction product electrophoresis does not present ladder-like bands, the to-be-tested bark beetle is not or is not a candidate for the Rhyzopertha dominica.

[0045] 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; after the loop-mediated isothermal amplification is completed, whether the to-be-tested hide beetle is the Dermestes maculatus 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 maculatus; if the reaction system is pink, the to-be-tested hide beetle is not or is not a candidate for the Dermestes maculatus.

[0046] 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; after the loop-mediated isothermal amplification is completed, whether the to-be-tested hide beetle is the Dermestes maculatus is judged by observing whether an obvious amplification peak appears in the real-time fluorescent PCR result: if the obvious amplification peak appears, the to-be-tested hide beetle is or is a candidate for the Dermestes maculatus; if there is no obvious amplification peak, the to-be-tested hide beetle is not or is not a candidate for the Dermestes maculatus.

[0047] In order to solve the above technical problems, the application further provides a method for detecting or assisting in detecting whether a to-be-detected sample contains The method of the Dermestes maculatus.

[0048] The method for detecting or assisting in detecting whether the to-be-tested sample contains the Dermestes maculatus provided by the application is as follows T1) or T2) or T3):

[0049] 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; after the loop-mediated isothermal amplification reaction is completed, whether the to-be-tested sample contains the Dermestes maculatus is judged by detecting whether ladder-shaped bands appear after electrophoresis of the reaction product: if the reaction product appears ladder-shaped bands after electrophoresis, the to-be-tested sample contains or is a candidate for containing the Dermestes maculatus; if there is no ladder-shaped band after electrophoresis of the reaction product, the to-be-tested sample does not contain or is not a candidate for containing the Dermestes maculatus.

[0050] 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; after the loop-mediated isothermal amplification is completed, whether the to-be-tested sample contains the Dermestes maculatus 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 maculatus; if the reaction system is pink, the to-be-tested sample does not contain or is not a candidate for containing the Dermestes maculatus.

[0051] 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; after the loop-mediated isothermal amplification is completed, whether the to-be-tested sample contains the Dermestes maculatus is judged by observing whether an obvious amplification peak appears in the real-time fluorescent PCR result: if the obvious amplification peak appears, the to-be-tested sample contains or is a candidate for containing the Dermestes maculatus; if there is no obvious amplification peak, the to-be-tested sample does not contain or is not a candidate for containing the Dermestes maculatus.

[0052] To solve the above technical problems, the application finally provides a method for distinguishing or assisting in distinguishing Tinea setosa from other tineas.

[0053] The method for distinguishing or assisting in distinguishing Tinea setosa from other tineas provided by the application is as follows U1) or U2) or U3):

[0054] The U1) comprises the following steps: extracting nucleic acid of a tinea to be tested, using the nucleic acid of the tinea to be tested as a template, and performing loop-mediated isothermal amplification by using the complete primer set; after the loop-mediated isothermal amplification reaction is completed, whether the tinea to be tested is Tinea setosa is determined by detecting whether the reaction product presents ladder bands after electrophoresis; if the reaction product presents ladder bands after electrophoresis, the tinea to be tested is or is a candidate for Tinea setosa; if the reaction product does not present ladder bands after electrophoresis, the tinea to be tested is or is a candidate for other tineas.

[0055] The U2) comprises the following steps: extracting nucleic acid of a tinea to be tested, using the nucleic acid of the tinea to be tested as a template, and performing loop-mediated isothermal amplification by using the complete primer set; after the loop-mediated isothermal amplification is completed, whether the tinea to be tested is Tinea setosa is determined by observing the color of the reaction system; if the reaction system is yellow, the tinea to be tested is or is a candidate for Tinea setosa; if the reaction system is pink, the tinea to be tested is or is a candidate for other tineas.

[0056] The U3) comprises the following steps: extracting nucleic acid of a tinea to be tested, using the nucleic acid of the tinea to be tested as a template, and performing loop-mediated isothermal amplification by using the complete primer set; after the loop-mediated isothermal amplification is completed, whether the tinea to be tested is Tinea setosa is determined by observing whether an obvious amplification peak appears in the real-time fluorescence PCR result; if an obvious amplification peak appears, the tinea to be tested is or is a candidate for Tinea setosa; if no obvious amplification peak appears, the tinea to be tested is or is a candidate for other tineas. The to-be-detected skin beetle is or is a candidate for The to-be-detected skin beetle is or is a candidate for

[0057] In any of the above methods, the loop-mediated isothermal amplification reaction system can be system 1, and the total volume is 25 μL, which is composed of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL template, primer mixture and sterile ultrapure water.

[0058] When system 1 is used, the loop-mediated isothermal amplification reaction condition can be 63-67 ℃ (such as 63-65 ℃, 65-67 ℃, 63 ℃, 65 ℃ or 67 ℃) constant temperature for 50 min, and 85 ℃ constant temperature for 3 min. The reaction product is subjected to 2% agarose gel electrophoresis.

[0059] In any of the above methods, the loop-mediated isothermal amplification reaction system can be system 2, and the total volume is 24 μL, which is composed of 12.5 μL WarmStart Colorimetric LAMP 2X Master Mix, 2.5 μL template and primer mixture.

[0060] When system 2 is used, the loop-mediated isothermal amplification reaction conditions can be: 63-67℃ (such as 63-65℃, 65-67℃, 63℃, 65℃ or 67℃) for 35 min, 4℃ for 3 min.

[0061] In any of the above methods, the loop-mediated isothermal amplification reaction system can specifically be system 3, and the total volume is 25μL, which is composed of 12.5μL WarmStart LAMP 2X Master Mix, 1μL template, 0.5μL LAMP Fluorescent Dye, primer mixture and sterile ultrapure water. The preparation of the reaction system should be carried out in the dark.

[0062] When system 3 is used, the PCR amplification instrument used can specifically be ABIQuantStudio 6Flex real-time fluorescent PCR amplification instrument of Thermo Fisher company. 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] The primer mixture is a mixture composed of each primer in the above complete primer set. The final concentration of the primer-F3 and the primer-B3 in system 1 or system 2 or system 3 can specifically be 0.2μM, and the final concentration of the primer-FIP and the primer-BIP in system 1 or system 2 or system 3 can specifically be 1.6μM.

[0064] In any of the above methods, the nucleic acid is genomic DNA.

[0065] In any of the above applications or kits or methods, the to-be-tested hide beetle can specifically be the egg, larva, pupa and / or adult of the to-be-tested hide beetle.

[0066] In any of the above applications or kits or methods, the to-be-tested sample can include various substances that can be damaged by Trogoderma granarium, such as various grains, dried fruits, vegetable seeds, gum and other plant products, milk powder, leather, dried animal carcasses and other dried animal products.

[0067] In any of the above applications or kits or methods, the other hide beetle can be at least one of the following hide beetles: Trogoderma variabile, Trogoderma glabrum, Dermestes ater, Thylodrias contractus and Dermestes frischii.

[0068] In any of the above described applications or kits or methods, the Trogoderma granarium can be Trogoderma granarium from different geographical populations, including Trogoderma granarium from Czech, The to-be-detected skin beetle is or is a candidate for Trogoderma granarium, Figure 1 Trogoderma granarium from Czech, Trogoderma granarium from Suzhou (intercepted in warehouse) and Trogoderma granarium from USA.

[0069] The present application is based on loop-mediated isothermal amplification technology, and establishes a rapid identification method for Trogoderma granarium. Four specific primers are designed for six regions in the specific segment of the full-length sequence of the COI gene of Trogoderma granarium. Through optimization of the reaction system and reaction conditions, a LAMP molecular identification technology and visual detection method are established according to the characteristics that the PH of the system changes from alkaline to acid during the reaction, and the color of the dyeing agent is different at different PH. The method effectively avoids the sample positive contamination that may be caused in the detection link due to the opening of the cover operation. The loop-mediated isothermal amplification technology for identifying Trogoderma granarium can realize the rapid identification of Trogoderma granarium eggs, larvae, pupae / adult individuals. The technology has the advantages of rapidity, high efficiency, strong specificity, high sensitivity, low cost, simple operation, no need for special instruments, and is suitable for on-site use, easy to popularize at the grassroots level, and can provide reliable technical basis for the prevention and control of Trogoderma granarium, which has important significance for the storage grain industry and commodity grain trade. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 2 The detection results of the primer set TG-LAMP in step two in Example 1.

[0071] Figure 3 The visual detection results in step two in Example 2.

[0072] Figure 4 The real-time fluorescent PCR detection results in step three in Example 2.

[0073] Figure 5 The specific detection results in Example 3.

[0074] Figure 6 The sensitivity experiment results based on agarose gel electrophoresis in Example 4.

[0075] Figure 7 The sensitivity experiment results based on visualization in Example 4.

[0076] Figure 1 The sensitivity experiment results based on real-time fluorescent PCR in Example 4. DETAILED DESCRIPTION

[0077] The following examples facilitate a better understanding of the present application, but are not intended to limit the present application.

[0078] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged.

[0079] The micro-sample genomic DNA extraction kit in the following examples is a product of Tiangen, with product 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. Among them, WarmStart LAMP 2X Master Mix and LAMP Fluorescent Dye belong to the products in WarmStart LAMP Kit (product catalog number E1700), and the product catalog number of WarmStart Colorimetric LAMP 2X Master Mix is M1800.

[0080] The sample numbers, species names, and collection site information of the test Dermestes spp. samples in the following examples are shown in Table 1. The samples are placed in anhydrous ethanol and stored at -20°C for standby use.

[0081] Table 1

[0082]

[0083]

[0084] Example 1, Obtaining of primer set and kit for identifying Dermestes maculatus

[0085] I. Design of primer set for identifying Dermestes maculatus

[0086] 1. Taking the full-length sequence of COI gene in the mitochondrial genome of Dermestes maculatus as the target gene, a primer set TG-LAMP for identifying Dermestes maculatus was designed and synthesized. The primer sequences of the primer set are shown in Table 2.

[0087] Table 2

[0088]

[0089] II. Verification of primer set for identifying Dermestes maculatus

[0090] 1. Extracting genomic DNA of the sample of the hide beetle by using the kit for extracting genomic DNA of trace sample, obtaining the genomic DNA of the sample of the hide beetle. The sample of the hide beetle for the test is the hide beetle of the valley spot hide beetle numbered 1, the valley spot hide beetle numbered 2, the valley spot hide beetle numbered 3, the valley spot hide beetle numbered 4, the flower spot hide beetle numbered 10, the hook pattern hide beetle numbered 12 and the hundred strange hide beetle numbered 13 in Table 1.

[0091] 2. Using the primer group TG-LAMP to perform loop-mediated isothermal amplification with the genomic DNA of the sample of the hide beetle as the template, obtaining the amplification product.

[0092] The reaction system is 25 μL, which is composed of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL template, primer mixture and sterile ultrapure water. The primer mixture is a mixture of each primer in the primer group TG-LAMP. In the reaction system, the final concentration of the outer primer F3 and the outer primer B3 is 0.2 μM, and the final concentration of the inner primer FIP and the inner primer BIP is 1.6 μM.

[0093] The reaction condition is 65℃ for 50 min and 85℃ for 3 min.

[0094] 3. The amplification product is subjected to 2% agarose gel electrophoresis.

[0095] According to the above method, the template is replaced with sterile ultrapure water, and the other steps are unchanged, which is used as a negative control.

[0096] The detection results of the primer group TG-LAMP are shown in The genomic DNA of the to-be-detected skin beetle 12 is the genomic DNA of the hook skin beetle numbered 12. (M is DNA Marker, 1-4 is the valley spot hide beetle of different geographical populations, 5 is the flower spot hide beetle, 6 is the hundred strange hide beetle, 7 is the hook pattern hide beetle, and N is the negative control).

[0097] The results show that the primer group TG-LAMP shows ladder-like bands in identifying the valley spot hide beetle, and the negative control does not appear bands. Therefore, the primer group TG-LAMP can be used to identify the valley spot hide beetle.

[0098] III. Preparation of the kit for identifying the valley spot hide beetle

[0099] The kit for identifying the valley spot hide beetle comprises the primer group TG-LAMP.

[0100] Example 2, application of the kit prepared in Example 1 in identifying whether the test hide beetle is or is a candidate for the valley spot hide beetle

[0101] I. Identifying whether the test hide beetle is or is a candidate for the valley spot hide beetle by agarose gel electrophoresis

[0102] 1. Extract the genomic DNA of the to-be-tested hide beetle by using the micro-sample genomic DNA extraction kit, to obtain the genomic DNA of the to-be-tested hide beetle.

[0103] 2. Perform loop-mediated isothermal amplification by using the primer set TG-LAMP with the genomic DNA of the to-be-tested hide beetle as the template, to obtain the amplification product.

[0104] The reaction system is 25 μL, which is composed of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL template, primer mixture, and sterile ultrapure water. The primer mixture is a mixture of primers in the primer set TG-LAMP. In the reaction system, the final concentration of the outer primer F3 and the outer primer B3 is 0.2 μM, and the final concentration of the inner primer FIP and the inner primer BIP is 1.6 μM.

[0105] The reaction condition is 65℃ for 50 min and 85℃ for 3 min.

[0106] 3. Perform 2% agarose gel electrophoresis on the amplification product.

[0107] According to the above method, replace the template with sterile ultrapure water, and the other steps remain unchanged, as a negative control.

[0108] According to the above method, replace the template with the genomic DNA of the Trogoderma variabile, and the other steps remain unchanged, as a positive control.

[0109] 4. According to the electrophoresis results, the following judgments are made:

[0110] When the positive control shows ladder-like bands and the negative control has no bands, if the to-be-tested hide beetle shows ladder-like bands, the to-be-tested hide beetle is or is a candidate for Trogoderma variabile; if the to-be-tested hide beetle does not show ladder-like bands (such as no bands), the to-be-tested hide beetle is not or is not a candidate for Trogoderma variabile.

[0111] When the positive control does not show ladder-like bands (such as no bands) or the negative control has bands, the identification result of the to-be-tested hide beetle is invalid.

[0112] II. Visual identification of whether the to-be-tested hide beetle is or is a candidate for Trogoderma variabile

[0113] 1. Extract the genomic DNA of the to-be-tested hide beetle by using the micro-sample genomic DNA extraction kit, to obtain the genomic DNA of the to-be-tested hide beetle Figure 2

[0114] 2. Perform loop-mediated isothermal amplification by using the primer set TG-LAMP with the genomic DNA of the to-be-tested hide beetle as the template.

[0115] The reaction system was 24 μL, which was composed of 12.5 μL WarmStart Colorimetric LAMP 2X Master Mix, 2.5 μL template and primer mixture. The primer mixture was a mixture of each primer in the primer group TG-LAMP. In the reaction system, the final concentration of the outer primer F3 and the outer primer B3 was 0.2 μM, and the final concentration of the inner primer FIP and the inner primer BIP was 1.6 μM.

[0116] The reaction condition was 65℃ for 35 min and 4℃ for 3 min.

[0117] According to the above method, the template was replaced with sterile ultrapure water, and the other steps were unchanged, as a negative control.

[0118] According to the above method, the template was replaced with the genomic DNA of the Rhyzopertha dominica, and the other steps were unchanged, as a positive control.

[0119] 3. After completing step 2, the color of the reaction system was observed with the naked eye, and then the following judgment was made:

[0120] When the positive control was yellow and the negative control was pink, if the test Dermestes was yellow, the test Dermestes was or was a candidate for Rhyzopertha dominica; if the test Dermestes was pink, the test Dermestes was not or was not a candidate for Rhyzopertha dominica;

[0121] When the positive control was not yellow or the negative control was not pink, the identification result of the test Dermestes was invalid.

[0122] The inventor of the present application used the method of step two to identify whether the test Dermestes was Rhyzopertha dominica. The test Dermestes was Rhyzopertha dominica numbered 1, Rhyzopertha dominica numbered 2, Rhyzopertha dominica numbered 3, Rhyzopertha dominica numbered 4, Rhyzopertha dominica numbered 5, Dermestes maculatus numbered 6, Dermestes maculatus numbered 7, Dermestes maculatus numbered 8, Dermestes maculatus numbered 10, Dermestes aegyptius numbered 11, Dermestes marginatus numbered 12, Dermestes adustus numbered 13, and Dermestes sp numbered 14.

[0123] The results are shown in The final concentration of the inner primer FIP is 0.2 μM, and the final concentration of the inner primer BIP is 1.6 μM. (1-13 are Rhyzopertha dominica numbered 1, Rhyzopertha dominica numbered 2, Rhyzopertha dominica numbered 3, Rhyzopertha dominica numbered 4, Rhyzopertha dominica numbered 5, Dermestes maculatus numbered 6, Dermestes maculatus numbered 7, Dermestes maculatus numbered 8, Dermestes maculatus numbered 10, Dermestes aegyptius numbered 11, Dermestes marginatus numbered 12, Dermestes adustus numbered 13, and Dermestes sp numbered 14, and N is a negative control). The results show that the reaction system of Rhyzopertha dominica of different geographical populations is yellow, and the reaction system of non-Rhyzopertha dominica is pink.

[0124] III. Identifying whether the to-be-tested hide beetle is or is a candidate for Rhyzophaga humeralis by real-time fluorescent PCR result

[0125] 1. Extracting the genomic DNA of the to-be-tested hide beetle by using the micro-sample genomic DNA extraction kit to obtain the genomic DNA of the to-be-tested hide beetle.

[0126] 2. Using the genomic DNA of the to-be-tested hide beetle as a template and adopting the primer group TG-LAMP to perform loop-mediated isothermal amplification to obtain an amplification product.

[0127] The reaction system is 25 μL, which is composed 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 is a mixture of primers in the primer group TG-LAMP. In the reaction system, the final concentration of the outer primer F3 and the outer primer B3 is 0.2 μM. Figure 3 The to-be-detected skin beetle 12 is the hook skin beetle numbered 12.

[0128] The reaction condition is: 65℃ for 10 min; 65℃ for 15 s, 65℃ for 45 s, 40 cycles; 85℃ for 3 min.

[0129] According to the above method, the template is replaced with sterile ultrapure water, and the other steps are unchanged, serving as a negative control.

[0130] According to the above method, the template is replaced with the genomic DNA of Rhyzophaga humeralis, and the other steps are unchanged, serving as a positive control.

[0131] 3. After completing the loop-mediated isothermal amplification, the following judgment is made according to the real-time fluorescent PCR result:

[0132] When the positive control appears obvious amplification peak and the negative control does not appear obvious amplification peak, if the to-be-tested hide beetle appears obvious amplification peak, the to-be-tested hide beetle is or is a candidate for Rhyzophaga humeralis; if the to-be-tested hide beetle does not appear obvious amplification peak, the to-be-tested hide beetle is not or is not a candidate for Rhyzophaga humeralis.

[0133] When the positive control does not appear obvious amplification peak or the negative control appears obvious amplification peak, the identification result of the to-be-tested hide beetle is invalid.

[0134] The inventor of the present application identifies whether the to-be-tested hide beetle is Rhyzophaga humeralis by the method of step three. The to-be-tested hide beetle is Rhyzophaga humeralis numbered 1, Rhyzophaga humeralis numbered 2, Rhyzophaga humeralis numbered 3, Rhyzophaga humeralis numbered 4, Rhyzophaga humeralis numbered 5, Rhyzophaga humeralis numbered 10, Rhyzophaga humeralis numbered 12, Rhyzophaga humeralis numbered 13, Rhyzophaga humeralis numbered 14, respectively.

[0135] Results are shown in Table 1 Figure 4 (1-9 are, in order, the grain borer numbered 1, the grain borer numbered 2, the grain borer numbered 3, the grain borer numbered 4, the grain borer numbered 5, the flower borer numbered 10, the hook borer numbered 12, the hundred-legs borer numbered 13, and the white-belly-like borer numbered 14, and N is a negative control). The results show that the reaction systems of the grain borer of different geographical populations all have obvious amplification peaks, and the reaction systems of the non-grain borer do not have obvious amplification peaks.

[0136] Example 3, specificity experiment

[0137] The to-be-tested borer 1 is the grain borer numbered 1.

[0138] The to-be-tested borer 2 is the grain borer numbered 2.

[0139] The to-be-tested borer 3 is the grain borer numbered 3.

[0140] The to-be-tested borer 4 is the grain borer numbered 4.

[0141] The to-be-tested borer 5 is the grain borer numbered 5.

[0142] The to-be-tested borer 6 is the flower borer numbered 6.

[0143] The to-be-tested borer 7 is the flower borer numbered 7.

[0144] The to-be-tested borer 8 is the flower borer numbered 8.

[0145] The to-be-tested borer 9 is the flower borer numbered 9.

[0146] The to-be-tested borer 10 is the flower borer numbered 10.

[0147] The to-be-tested borer 11 is the black borer numbered 11.

[0148] The final concentration of the inner primer FIP is 0.2 μM, and the final concentration of the inner primer BIP is 1.6 μM.

[0149] The to-be-tested borer 13 is the hundred-legs borer numbered 13.

[0150] The to-be-tested borer 14 is the white-belly-like borer numbered 14.

[0151] Each to-be-tested borer is subjected to the following steps:

[0152] 1. The genomic DNA of the to-be-tested borer is extracted by using a micro-sample genomic DNA extraction kit.

[0153] 2. The genomic DNA of the to-be-tested borer extracted in step 1 is used as a template, and a primer set TG-LAMP is used for loop-mediated isothermal amplification to obtain an amplification product.

[0154] The reaction system was 25 μL, which was composed 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 each primer in the primer set TG-LAMP. In the reaction system, the final concentration of the outer primer F3 and the outer primer B3 was 0.2 μM, and the final concentration of the inner primer FIP and the inner primer BIP was 1.6 μM.

[0155] Reaction conditions: 65℃ for 50 min, 85℃ for 3 min.

[0156] 3. The amplification product was subjected to 2% agarose gel electrophoresis.

[0157] According to the above method, the template was replaced with sterile ultrapure water, and the other steps were unchanged, serving as a negative control.

[0158] According to the above method, the template was replaced with the genomic DNA of the Rhyzopertha dominica, and the other steps were unchanged, serving as a positive control.

[0159] Some of the test results are shown in Figure 5 (1-14 are the to-be-tested beetles 1-14, and N is the negative control). The results show that when the to-be-tested beetles are Rhyzopertha dominica, they all show ladder-like bands; when the to-be-tested beetles are non-Rhyzopertha dominica, they all have no bands (i.e. do not show ladder-like bands); and the negative control also has no bands.

[0160] As can be seen, the primer set TG-LAMP has good specificity for identifying Rhyzopertha dominica.

[0161] Example 4, sensitivity experiment

[0162] I. Sensitivity experiment based on agarose gel electrophoresis

[0163] 1. The genomic DNA of Rhyzopertha dominica was extracted by using a micro sample genomic DNA extraction kit.

[0164] 2. The genomic DNA of Rhyzopertha dominica was taken and gradient diluted with sterile water to obtain diluents with the genomic DNA concentration of Rhyzopertha dominica being 10.0 ng / μL, 1.0 ng / μL, 1.0×10 -1 ng / μL, 1.0×10 -2 ng / μL and 1.0×10 -3 ng / μL, respectively.

[0165] 3. The diluents obtained in step 2 were used as templates, and the primer set TG-LAMP was used for loop-mediated isothermal amplification to obtain amplification products.

[0166] The reaction system was 25 μL, which was composed of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL diluent (1 μL diluent contained 10.0 ng, 1.0 ng, 1.0 x 10 -1 -2 - 3 ng, 1.0 x 10 - ng or 1.0 x 10 1 ng of the genomic DNA of T. granarium), a primer mixture and sterile ultrapure water. The primer mixture was a mixture of each primer in the primer set TG-LAMP. In the reaction system, the final concentration of the outer primer F3 and the outer primer B3 was 0.2 Figure 6 The results show that the primer group TG-LAMP has high sensitivity and specificity in identification of the skin beetle.

[0167] The reaction condition was 65 °C for 50 min and 85 °C for 3 min.

[0168] 4. The amplification product was subjected to 2% agarose gel electrophoresis.

[0169] According to the above method, the template was replaced with sterile ultrapure water, and the other steps were unchanged, serving as a negative control.

[0170] According to the above method, the template was replaced with the genomic DNA of T. granarium, and the other steps were unchanged, serving as a positive control.

[0171] If it is ladder-like bands, it indicates that the corresponding genomic content in the reaction system can be detected. If it is not ladder-like bands (such as no bands), it indicates that the corresponding genomic content in the reaction system cannot be detected.

[0172] Part of the detection results are shown in Table 1. Figure 7 (1-5 DNA template concentrations were 10.0 ng / μL, 1.0 ng / μL, 1.0 x 10 - 1 ng / μL, 1.0 x 10 -2 ng / μL, 1.0 x 10 -3 ng / μL, and N was a negative control). The results showed that the sensitivity of the primer set TG-LAMP for identifying T. granarium was 1.0 x 10 -2 ng / μL.

[0173] It can be seen that the primer set TG-LAMP for identifying T. granarium has high sensitivity by using agarose gel electrophoresis.

[0174] II. Visual sensitivity experiment

[0175] 1. The genomic DNA of T. granarium was extracted by using a micro sample genomic DNA extraction kit.

[0176] 2. Genomic DNA was collected from the bark beetle and serially diluted with sterile water to obtain 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.

[0177] 3. Using the dilution solution obtained in step 2 as a template, loop-mediated isothermal amplification was performed using primer set TG-LAMP to obtain the amplification product.

[0178] 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 bark beetle). -1 ng, 1.0×10 -2 ng, 1.0×10 -3 ng or 1.0×10 -4 The reaction system consists of primers (ng) and a primer mixture. The primer mixture is a mixture of the primers in the TG-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.

[0179] Reaction conditions: 65℃ for 35 min, 4℃ for 3 min.

[0180] Following the above method, replace the template with sterile ultrapure water, keeping all other steps unchanged, as a negative control.

[0181] Following the method described above, the template was replaced with genomic DNA from the bark beetle, while all other steps remained unchanged, serving as a positive control.

[0182] 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.

[0183] Some test results can be found ​ (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 -4ng, N is negative control ​ ​ The sensitivity is between 1.0 ng / μL and 1.0 x 10 - 1

[0184] It can be seen that the visual mediated primer set TG-LAMP has lower sensitivity in identifying the Trogoderma granarium, and the sensitivity is lower than that of the agarose gel electrophoresis and real-time fluorescent PCR mediated method.

[0185] III. Sensitivity experiment based on real-time fluorescent PCR

[0186] 1. The genomic DNA of Trogoderma granarium was extracted by using the micro sample genomic DNA extraction kit.

[0187] 2. The genomic DNA of Trogoderma granarium was taken and gradient diluted with sterile water to obtain the dilutions with the genomic DNA concentration of Trogoderma granarium of 10.0 ng / μL, 1.0 ng / μL, 1.0 x 10 -1 ng / μL, 1.0 x 10 -2 ng / μL, 1.0 x 10 -3 ng / μL, 1.0 x 10 -4 ng / μL, 1.0 x 10 -5 ng / μL, respectively.

[0188] 3. The dilutions obtained in step 2 were used as templates to perform loop-mediated isothermal amplification by using the primer set TG-LAMP to obtain the amplification products.

[0189] The reaction system was 25 μL, which was composed of 12.5 μL WarmStart LAMP 2X Master Mix, 1 μL dilution (1 μL dilution contained 10.0 ng, 1.0 ng, 1.0 x 10 -1 ng, 1.0 x 10 -2 ng, 1.0 x 10 - 3 ng, 1.0 x 10 -4 ng or 1.0 x 10 -5 ng of genomic DNA of Trogoderma granarium), 0.5 μL LAMP Fluorescent Dye, primer mixture and sterile ultrapure water. The primer mixture was a mixture of various primers in the primer set TG-LAMP. In the reaction system, the final concentration of the outer primer F3 and the outer primer B3 was 0.2 μM, and the final concentration of the inner primer FIP and the inner primer BIP was 1.6 μM.

[0190] The reaction conditions were as follows: 65℃ for 10 min; 65℃ for 15 s, 65℃ for 45 s, 40 cycles; 85℃ for 3 min.​

[0191] According to the above method, the template is replaced with sterile ultrapure water, and the other steps are unchanged, as a negative control.

[0192] According to the above method, the template is replaced with the genomic DNA of the Rhyzopertha dominica, and the other steps are unchanged, as a positive control.

[0193] If a clear amplification peak appears, it indicates that the corresponding genome content in the reaction system can be detected. If no clear amplification peak appears, it indicates that the corresponding genome content in the reaction system cannot be detected.

[0194] Part of the test results are shown in Table 1. ​ (1-7 DNA template concentrations are: 10.0 ng / μL, 1.0 ng / μL, 1.0×10 - 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 a negative control). The results show that the sensitivity of the primer set TG-LAMP for identifying Rhyzopertha dominica is 1.0×10 -1 ng / μL.

[0195] It can be seen that the real-time fluorescence PCR mediated primer set TG-LAMP for identifying Rhyzopertha dominica has high sensitivity, but the sensitivity is slightly lower than that of the agarose gel electrophoresis mediated method.

[0196] The above only describes the preferred embodiments of the present application, and for those skilled in the art, without departing from the technical principles of the present application, a number of improvements and refinements can be made, which should also be considered as the protection scope of the present application. SEQUENCE LISTING <110> China Agricultural University <120> LAMP primer set for identifying Rhyzopertha dominica and application thereof <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <400> 1 cgacctagca atcttcagct ta 22 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <400> 2 gttgatatta cggtcggtta gaagt 25 <210> 3 <211> 43 <212> DNA <213> Artificial Sequence <400> 3 cgggagttat tcctgtggtt cgtatcacct agcaggaatc tcc 43 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <400> 4 gatcagtagc aatcaccgcc ctgatagctc ctgctaggac g 41

Claims

1. A kit of primers for identifying or assisting in identifying Trogoderma granarium, 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 granarium; b2) identifying or assisting in identifying whether a test hide beetle is Trogoderma granarium; b3) detecting or assisting in detecting whether a test sample contains Trogoderma granarium; b4) distinguishing or assisting in distinguishing Trogoderma granarium from other hide beetles; the other hide beetles are at least one of Trogoderma variabile, 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 granarium; c2) identifying or assisting in identifying whether a test hide beetle is Trogoderma granarium; c3) detecting or assisting in detecting whether a test sample contains Trogoderma granarium; c4) distinguishing or assisting in distinguishing Trogoderma granarium from other hide beetles; the other hide beetles are at least one of Trogoderma variabile, 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 together the primers in the kit of primers of claim 1 or 2 in proportion.

6. The method of claim 5, wherein: in the 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 hide beetle is Trogoderma granarium, 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 granarium 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 granarium 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 granarium; 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 granarium 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 granarium; 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 granarium 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 granarium; if no obvious amplification peaks appear, the dermestid beetle to be tested is or is a candidate for other dermestid beetles; said other dermestid beetles being at least one of the following dermestid beetles: Trogoderma variabile, 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: ​

Images