Primer combination for multiple PCR (Polymerase Chain Reaction) identification of aspongopus, hemp skin bug, tea wing bug, short-horned melon bug and large winged bug as well as application and identification method of primer combination

Abstract

The invention discloses a primer combination for multiple PCR (Polymerase Chain Reaction) identification of aspongopus, hemp skin bug, tea wing bug, short-horned melon bug and large winged bug, and further discloses application of the primer combination and an identification method based on the primer combination. The specific identification method of the aspongopus raw material is established by a multiplex PCR method, the original animal of the aspongopus and the adulterants of the aspongopus are simultaneously identified through the same amplification condition, and the aspongopus medicinal material and the adulterants of the aspongopus are simultaneously identified, namely the adulterants of the aspongopus, the adulterants of the aspongopus, the adulterants of the aspongopus, the adulterants of the aspongopus, the adulterants of the aspongopus, and the adulterants of the aspongopus are simultaneously identified. The method can be used for simultaneously identifying the aspongopus decoction pieces and adulterants such as hemp skin bugs, tea wing bugs, cucurbitaceae cucurbitaceae bugs and large winged bugs, and is high in identification efficiency and good in specificity.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine identification, and in particular to a primer combination for multiplex PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs, and an application and an identification method thereof. Background Art

[0002] The 2020 edition of the Chinese Pharmacopoeia stipulates that this product is the dried body of the nine-scented insect Aspongopus chinensis Dallas of the Pentatomidae family. Catch it from November to March of the following year, put it in a suitable container, suffocate it with a little wine, take it out and dry it in the shade; or put it in boiling water to kill it, take it out and dry it. This product is salty and warm. It enters the liver, spleen and kidney meridians. It mainly regulates qi and relieves pain, warms the middle and helps yang. It is used for stomach cold and distension, liver and stomach qi pain, kidney deficiency and impotence, and soreness of the waist and knees. Nine-scented insect medicinal materials are currently mainly derived from wild resources. The commercially available nine-scented insect medicinal materials are often easily mixed with closely related species with similar morphology and chemical composition, such as large wrinkled bugs, hemp-skinned bugs, tea-winged bugs, short-horned melon bugs, stingless bugs and fine hairy bugs. Once mixed, it is extremely difficult to identify. Although traditional identification methods can be identified through properties and microscopy, they cannot accurately identify nine-scented insect medicinal materials that are mixed with a certain proportion. Multiplex PCR technology is a technology that uses multiple pairs of primers in the same PCR reaction to simultaneously amplify multiple target DNA fragments, and distinguish species based on the size of the amplified bands. Since the current patents do not involve molecular identification research on nine-scented insects and mixed counterfeits, in order to ensure the safety and clinical efficacy of traditional Chinese medicine, this study established a method for identifying nine-scented insect medicinal materials and decoction pieces based on multiplex PCR technology. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a primer combination for multiplex PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and big wrinkled stink bugs, which has high identification efficiency and good specificity.

[0004] Another technical problem to be solved by the present invention is to provide an application of the above primer combination.

[0005] The technical problem that the present invention also aims to solve is to provide an identification method based on the above-mentioned primer combination for multiple PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and big wrinkled stink bugs.

[0006] In order to solve the above technical problems, the present invention provides a primer combination for multiplex PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and big wrinkled stink bugs, including:

[0007] Primer 1 and primer 2 for identifying the nine-scented insect, the sequence of the primer 1 is shown in SEQ ID NO: 1; the sequence of the primer 2 is shown in SEQ ID NO: 2;

[0008] Primer 3 and primer 4 for identifying the scurvy bug, wherein the sequence of primer 3 is shown in SEQ ID NO: 3; the sequence of primer 4 is shown in SEQ ID NO: 4;

[0009] Primer 5 and primer 6 for identifying brown winged stink bug, the sequence of primer 5 is shown in SEQ ID NO: 5; the sequence of primer 6 is shown in SEQ ID NO: 6;

[0010] Primer seven and primer eight for identifying short-horned citrus bugs, wherein the sequence of primer seven is shown in SEQ ID NO: 7; the sequence of primer eight is shown in SEQ ID NO: 8;

[0011] Primer nine and primer ten are used for identifying wrinkled bugs, wherein the sequence of primer nine is shown in SEQ ID NO: 9; the sequence of primer ten is shown in SEQ ID NO: 10.

[0012] Furthermore, the present invention also provides the use of a primer combination for multiplex PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and big wrinkled stink bugs in any one of (1) to (4):

[0013] (1) Identify medicinal materials of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled bugs;

[0014] (2) a kit for identifying medicinal materials of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs;

[0015] (3) Identify whether the sample to be tested contains medicinal materials of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs;

[0016] (4) A test kit for identifying whether the sample to be tested contains medicinal materials of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs.

[0017] It should be noted that the present invention can simultaneously identify the original animal of the nine-scented insect and the mixed and fake products of the hemp-skinned stink bug, the tea-winged stink bug, the short-horned cucurbit stink bug, and the big wrinkled stink bug; and simultaneously identify the medicinal materials of the nine-scented insect and the mixed and fake products of the hemp-skinned stink bug, the tea-winged stink bug, the short-horned cucurbit stink bug, and the big wrinkled stink bug; and simultaneously identify the decoction pieces of the nine-scented insect and the mixed and fake products of the hemp-skinned stink bug, the tea-winged stink bug, the short-horned cucurbit stink bug, and the big wrinkled stink bug. The present invention can simultaneously identify the nine-scented insect and the mixed and fake products of the hemp-skinned stink bug, the tea-winged stink bug, the short-horned cucurbit stink bug, and the big wrinkled stink bug through the same amplification condition, with high identification efficiency and good specificity.

[0018] Accordingly, the present invention also provides a kit, which includes the above-mentioned primer combination.

[0019] In some embodiments, the kit is a PCR kit; preferably, the kit is a multiplex PCR kit. The PCR kit simulates the DNA replication process in vitro, amplifies the target DNA fragment through continuous cycle reactions, and obtains a sufficient amount of DNA for subsequent analysis. The kit includes template DNA, primers, DNA polymerase, buffer, auxiliary reagents, etc. In some embodiments, the kit includes: primer 1 and primer 2 for identifying the nine-scented insect, the sequence of primer 1 is shown in SEQ ID NO: 1; the sequence of primer 2 is shown in SEQ ID NO: 2; primer 3 and primer 4 for identifying the hemp-skinned stink bug, the sequence of primer 3 is shown in SEQ ID NO: 3; the sequence of primer 4 is shown in SEQ ID NO: 4; primer 5 and primer 6 for identifying the tea-winged stink bug, the sequence of primer 5 is shown in SEQ ID NO: 5; the sequence of primer 6 is shown in SEQ ID NO: 6; primer 7 and primer 8 for identifying the short-horned melon bug, the sequence of primer 7 is shown in SEQ ID NO: 7; the sequence of primer 8 is shown in SEQ ID NO: 8; primer 9 and primer 10 for identifying the big wrinkled stink bug, the sequence of primer 9 is shown in SEQ ID NO: 9; the sequence of primer 10 is shown in SEQ ID NO: 10.

[0020] Furthermore, the present invention also provides an identification method based on the primer combination for multiplex PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and big wrinkled stink bugs according to claim 1, comprising the following steps:

[0021] Prepare a PCR amplification system, the PCR amplification system comprising the primer combination for PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and big wrinkled stink bugs as claimed in claim 1;

[0022] The PCR amplification system performs PCR amplification according to preset amplification conditions to obtain an amplification product;

[0023] Performing electrophoresis analysis on the amplified product and recording its electrophoresis pattern;

[0024] If the pattern contains a 301bp DNA fragment, the test product is a nine-scented insect;

[0025] If the pattern contains a 129 bp DNA fragment, the test object is a stink bug;

[0026] If the pattern contains a 175 bp DNA fragment, the sample to be tested contains the brown-winged stink bug;

[0027] If the pattern contains a 117 bp DNA fragment, the sample to be tested contains the short-horned citrus bug;

[0028] If the pattern contains a 390 bp DNA fragment, the sample to be tested contains the giant wrinkled bug.

[0029] The invention establishes a specific identification method for the raw material of the nine-scented insect by a multiplex PCR method, can simultaneously identify the original animal of the nine-scented insect and mixed and fake products such as the hemp skin stink bug, the tea wing stink bug, the short-horned cucurbit stink bug and the big wrinkled stink bug through the same amplification condition, can simultaneously identify the medicinal materials of the nine-scented insect and mixed and fake products such as the hemp skin stink bug, the tea wing stink bug, the short-horned cucurbit stink bug and the big wrinkled stink bug, can also simultaneously identify the decoction pieces of the nine-scented insect and mixed and fake products such as the hemp skin stink bug, the tea wing stink bug, the short-horned cucurbit stink bug and the big wrinkled stink bug, and has high identification efficiency and good specificity.

[0030] The PCR amplification system has a significant effect on multiplex PCR. In some embodiments, the total volume of the PCR amplification system is 22 μL to 27 μL, and the PCR amplification system includes: Multiplex PCR 5X Master Mix 4 μL to 6 μL, 0.1 μL to 0.2 μL of primer 1 and primer 2, 0.25 μL to 0.35 μL of primer 3 and primer 4, 0.25 μL to 0.35 μL of primer 5 and primer 6, 0.35 μL to 0.45 μL of primer 7 and primer 8, 0.35 μL to 0.45 μL of primer 9 and primer 10, 0.5 μL to 1.5 μL of template DNA, and the rest is sterile ultrapure water. In some embodiments, the concentration of primer one, primer two, primer three, primer four, primer five, primer six, primer seven, primer eight, primer nine or primer ten is 9 μmol / L to 11 μmol / L.

[0031] It should be noted that the concentration of template DNA is crucial to the amplification effect of PCR. Too high a concentration may lead to nonspecific amplification, while too low a concentration may not obtain enough amplification products. In multiplex PCR, multiple pairs of primers need to be added to amplify multiple target sequences simultaneously. The interaction between these primers, the ability to bind to template DNA, and the specificity of the primers themselves will affect the amplification effect. Factors such as complementarity between primers, homology with non-target amplification regions, and primer concentration all affect the amplification results. In addition, DNA polymerase is a key enzyme in the PCR reaction, and its activity and stability directly affect the amplification effect. In multiplex PCR, since multiple target sequences need to be amplified simultaneously, higher performance requirements are placed on DNA polymerase.

[0032] Taking the above factors into consideration, preferably, the total volume of the PCR amplification system is 25 μL, and the PCR amplification system includes: Multiplex PCR 5X Master Mix 5 μL, primer 1 and primer 2 each 0.15 μL, primer 3 and primer 4 each 0.3 μL, primer 5 and primer 6 each 0.3 μL, primer 7 and primer 8 each 0.4 μL, primer 9 and primer 10 each 0.4 μL, template DNA 1 μL, and the rest is sterile ultrapure water. Preferably, the concentration of primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9 or primer 10 is 10 μmol / L.

[0033] In addition, the preset amplification conditions also have a key impact on the PCR amplification results. In some embodiments, the preset amplification conditions are:

[0034] The amplification system is pre-denatured at 92-97°C for 3-7 minutes, then cycled 29-32 times under a preset program, and finally extended at 70-75°C for 3-7 minutes;

[0035] The preset program is: denaturing the amplification system at 92-97° C. for 25-35 seconds, then annealing at 59-61° C. for 25-35 seconds, and then extending at 70-75° C. for 25-35 seconds.

[0036] It should be noted that temperature control in PCR reactions is critical. In multiplex PCR, since the amplification of different target sequences may require different annealing temperatures and times, the temperature conditions need to be finely adjusted to optimize the amplification effect. In addition, the number of cycles determines the efficiency and yield of PCR amplification. Therefore, in multiplex PCR, the appropriate number of cycles needs to be determined based on the abundance of the target sequence and the amplification efficiency. Too many cycles may lead to an increase in nonspecific amplification and misincorporation rates, while too few cycles may not result in sufficient amplification products.

[0037] Preferably, the PCR amplification procedure is:

[0038] The amplification system was pre-denatured at 95°C for 5 min, then cycled 31 times under the preset program, and finally extended at 72°C for 5 min;

[0039] The preset program is: denaturing the amplification system at 95°C for 30s, then annealing at 60°C for 30s, and then extending at 72°C for 30s.

[0040] Finally, after the amplification is completed, the PCR product is analyzed by gel electrophoresis and other methods to verify the amplification effect. The position, size, brightness and other information of the bands in the electrophoresis pattern are observed to determine whether the PCR amplification is successful and the analysis of the amplified product. In some embodiments, the electrophoresis analysis includes: diluting the amplified product and spotting it on a 3% agarose gel, electrophoresing it at 150V for 40 minutes, and developing it with Gelred to obtain an electrophoresis pattern.

[0041] The implementation of the present invention has the following beneficial effects:

[0042] The invention designs a primer combination for the nine-scented insect and mixed and counterfeit products such as hemp skin stink bugs, tea wing stink bugs, short-horned cucurbit stink bugs and big wrinkled stink bugs, and establishes a specific identification method for the nine-scented insect raw material through a multiple PCR method. The original animal of the nine-scented insect and mixed and counterfeit products such as hemp skin stink bugs, tea wing stink bugs, short-horned cucurbit stink bugs and big wrinkled stink bugs can be identified at the same time under the same amplification condition, and the medicinal materials of the nine-scented insect and mixed and counterfeit products such as hemp skin stink bugs, tea wing stink bugs, short-horned cucurbit stink bugs and big wrinkled stink bugs can also be identified at the same time. The nine-scented insect decoction pieces and mixed and counterfeit products such as hemp skin stink bugs, tea wing stink bugs, short-horned cucurbit stink bugs and big wrinkled stink bugs can also be identified at the same time. The identification efficiency is high and the specificity is good, and the problem of difficulty in identification after the nine-scented insect and mixed and counterfeit products such as hemp skin stink bugs, tea wing stink bugs, short-horned cucurbit stink bugs and big wrinkled stink bugs after being mixed can be effectively solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the result of primer screening; wherein, M: DNA Maker DL1000, 1. Nine-scented insect, 2. Hemp skin stink bug, 3. Tea winged stink bug, 4. Short-horned melon bug, 5. Big wrinkled stink bug, N. Blank;

[0044] Figure 2 This is a result diagram showing the effect of Taq enzyme types on PCR amplification results; where M: DNA Maker DL1000, 1. Nine-scented insect, 2. Hemp skin stink bug, 3. Tea winged stink bug, 4. Short-horned melon bug, 5. Big wrinkled stink bug, N. Blank;

[0045] Figure 3 : is the result diagram of the effect of annealing temperature on PCR amplification results; wherein, M: DNA Maker DL1000, 1. Nine-scented insect, 2. Hemp skin stink bug, 3. Tea winged stink bug, 4. Short-horned melon bug, 5. Big wrinkled stink bug, N. Blank;

[0046] Figure 4 Result diagram of the effect of primer quantity on PCR amplification results; AB. Nine-scented insect, C. Hemp stink bug, D. Tea winged stink bug, E. Short-horned cucurbit bug, F. Big wrinkled stink bug; M: DNA Maker DL1000, 1. Nine-scented insect, 2. Hemp stink bug, 3. Tea winged stink bug, 4. Short-horned cucurbit bug, 5. Big wrinkled stink bug, N. Blank;

[0047] Figure 5: is a result diagram of the effect of the amount of DNA template on the PCR amplification result; wherein, M: DNA Maker DL1000, 1. Nine-scented insect, 2. Hemp skin stink bug, 3. Tea winged stink bug, 4. Short-horned melon bug, 5. Big wrinkled stink bug, N. Blank;

[0048] Figure 6 : is the result diagram of the effect of cycle number on PCR amplification results; wherein, M: DNA Maker DL1000, 1. Nine-scented insect, 2. Hemp skin stink bug, 3. Tea winged stink bug, 4. Short-horned melon bug, 5. Big wrinkled stink bug, N. Blank;

[0049] Figure 7 This is a result diagram showing the effect of instrument type on PCR amplification results; where M: DNA Maker DL1000, 1. Nine-scented insect, 2. Hemp-skinned stink bug, 3. Tea-winged stink bug, 4. Short-horned melon bug, 5. Big wrinkled stink bug, N. Blank;

[0050] Figure 8 is the result of the applicability investigation; where M: DNA Maker DL1000, 1. Nine-scented insect, 2. Hemp-skinned stink bug, 3. Tea-winged stink bug, 4. Short-horned melon bug, 5. Big wrinkled stink bug, N. Blank;

[0051] Fig. 9 The figure is the result of multiplex PCR method for identifying mixed samples of nine-scented insects; wherein, M: DNA Maker DL1000, 1. Nine-scented insect, 2. Hemp skin stink bug, 3. Tea winged stink bug, 4. Short-horned melon bug, 5. Big wrinkled stink bug, N. Blank. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. In addition, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0053] In the description of the present invention, it is necessary to understand that the terms "length", "width", "up", "down", "left", "right", "horizontal", "top", "bottom", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0054] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0055] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, an electrical connection, or mutual communication; it can be a direct connection, or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0056] In the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may include that the first and second features are in direct contact, or may include that the first and second features are not in direct contact but are in contact through another feature between them. Moreover, a first feature being "above", "above" and "above" a second feature includes that the first feature is directly above and obliquely above the second feature, or simply indicates that the first feature is higher in level than the second feature. A first feature being "below", "below" and "below" a second feature includes that the first feature is directly below and obliquely below the second feature, or simply indicates that the first feature is lower in level than the second feature.

[0057] The disclosure below provides many different embodiments or examples to realize different structures of the present invention. In order to simplify the disclosure of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the present invention can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplification and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed. In addition, the examples of various specific processes and materials provided by the present invention, but those of ordinary skill in the art can be aware of the application of other processes and / or the use of other materials.

[0058] In the present invention, "preferred" and "better" are only used to describe implementation methods or embodiments with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features. In the present invention, when involving numerical ranges, unless otherwise specified, the two endpoints of the numerical range are included.

[0059] 1. Primer List

[0060]

[0061]

[0062] 3. Extraction of genomic DNA

[0063] Sample DNA extraction was performed according to the instructions of Wizard SV Genomic DNA Purification System Kit (A2361, Promega).

[0064] 4. DNA Concentration Determination

[0065] The above DNA samples were taken, and the DNA concentration was determined using a BioSpec-nano micro-volume UV spectrophotometer, while OD260 / OD230 and OD260 / OD280 were recorded, and the concentration was adjusted to 50-100 ng / μL using TB buffer.

[0066] 5. Primer design and screening

[0067] According to the COI tag sequences of the nine-scented insect and its fakes obtained from NCBI, DNAMAN was used to compare the differences in sequences between different species, and the region that can clearly distinguish the target species was found. The primers for PCR amplification were designed in this region using primer premier 5 software. The specific information is shown in Table 1. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0068] Table 1 Primer information

[0069]

[0070]

[0071] According to the characteristics of the enzyme and the Tm value of the primer, the initial PCR reaction system was set: it was carried out in a 200 μL centrifuge tube with a total reaction volume of 25 μL. The reaction system included 5.0 μL of Multiplex PCR 5X Master Mix, 0.3 μL of each forward and reverse primer (10 μmol / l), 1.0 μL of template DNA, and sterile ultrapure water was added to 25 μL.

[0072] Place the centrifuge tube in a PCR instrument and set the initial PCR reaction parameters: pre-denaturation at 95°C for 5 min, 31 cycles (denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s), and extension at 72°C for 5 min.

[0073] According to the above amplification system and conditions, PCR amplification was performed on the samples of the nine-scented insect using the primers shown in Table 1.

[0074] The results are as follows Figure 1 As shown in the figure, the designed primers can successfully amplify the target bands of the corresponding species, among which, the primer Jiuxiangchong J14 can obtain a single band at 301bp, the primer Hemp skin stink bug 1 can obtain a single band at 129bp, the tea wing stink bug 1-1 can obtain a single band at 175bp, the short-horned melon bug 4 can obtain a single band at 117bp, and the big wrinkled stink bug 4 can obtain a single band at 390bp.

[0075] 6. Optimization of PCR amplification conditions

[0076] (1) Investigation of Taq enzyme types

[0077] To investigate the effects of different enzyme types on multiplex PCR amplification results, experiments were conducted using Multiplex PCR5XMaster Mix, Multiplex PCR Assay Kit Ver.2, 2×T5 Super PCR Mix, and 2×Taq PCR Mix polymerase. The results are shown in Figure 2 The results showed that the use of Multiplex PCR 5X Master Mix polymerase could obtain the correct amplified bands, and the band quality and brightness were relatively uniform, so Multiplex PCR 5X Master Mix was selected as the optimal Taq enzyme.

[0078] (2) Annealing temperature investigation

[0079] According to the Tm values ​​of the primers and the results of the previous preliminary experiments, the annealing temperatures were set to 56°C, 58°C, 60°C, and 62°C, respectively. Figure 3 The results showed that nonspecific amplification occurred at 56℃-58℃; at 62℃, no amplified bands were obtained for the stink bugs, brown-winged stink bugs and large wrinkled stink bugs. In order to ensure the accuracy and repeatability of the PCR results, the annealing temperature was selected as 60℃.

[0080] (3) Primer quantity investigation

[0081] In order to investigate the effect of different primer amounts on the results of multiplex PCR amplification, based on 0.3 μL of each forward and reverse primer in the preliminary experiment, the primer concentration was 10 μmol / L, and the forward and reverse primer amounts of each variety were 0.1 μL, 0.2 μL, 0.3 μL, and 0.4 μL, respectively. The results are shown in Figure 4 The results showed that the brightness difference was large when the primer amount of the nine-scented insect was 0.1μL and 0.2μL. Subsequent tests showed that the band brightness was appropriate when the primer amount was 0.15μL.

[0082] On this basis, the amount of primers for the hemp stink bug was investigated, and it was found that the specific band was significantly weakened at 0.1-0.2 μL, and the amount of primers for the hemp stink bug was determined to be 0.3 μL.

[0083] On this basis, the amount of primers for the brown-winged stink bug was examined, and it was found that the brightness of the specific band decreased significantly at 0.1-0.2 μL, the brightness was moderate at 0.3 μL, and non-specific amplification occurred at 0.4 μL, so the amount of primers for the brown-winged stink bug was determined to be 0.3 μL.

[0084] On this basis, the primer amount of the short-horned citrus stink bug was investigated, and it was found that the brightness of the specific band decreased significantly at 0.1-0.2 μL, and the brightness was moderate at 0.3-0.4 μL, but there was more non-specific amplification at 0.3 μL, so the primer amount of the short-horned citrus stink bug was determined to be 0.4 μL.

[0085] On this basis, the amount of primers for the giant wrinkled bug was examined, and it was found that as the amount of primers increased, the brightness of the band increased. In order to ensure the amplification effect, the amount of primers for the giant wrinkled bug was determined to be 0.4 μL.

[0086] (4) DNA template quantity investigation

[0087] In order to investigate the effect of different DNA template amounts on the results of multiplex PCR amplification, the DNA concentration was adjusted to about 70 ng / μL, and the experiments were carried out using DNA template amounts of 0.5 μL, 1.0 μL, 2.0 μL, and 4.0 μL, respectively. The results are shown in Figure 5 The results showed that when the template amount was too high or too low, the PCR amplification efficiency would be inhibited, resulting in a false negative result. When the DNA template amount was 1.0 μL, a single bright band could be amplified. In order to ensure the accuracy of the PCR amplification results, the DNA template amount was selected to be 1.0 μL.

[0088] (5) Cycle number investigation

[0089] According to the preliminary experiments and the instructions of Taq enzyme, the influence of 29, 30, 31 and 32 cycles on PCR amplification results was investigated. The results are shown in Figure 6 The results showed that at 29 to 32 cycles, the corresponding specific bands could be seen in all samples, but as the number of cycles increased, the brightness of some sample bands weakened. To ensure the stability and accuracy of the results, 31 cycles were selected for PCR reaction.

[0090] (6) Tolerance study

[0091] The optimized PCR reaction conditions were investigated with different instruments to test the tolerance of the multiplex PCR identification method for the nine-scented insect. The results are shown in Figure 7 The results showed that although there were differences in the brightness of the PCR amplification bands, the three brands of PCR instruments could obtain correct identification results, indicating that the method had good durability.

[0092] VII. Applicability Examination

[0093] According to the optimized PCR amplification conditions and procedures, 33 batches of samples of nine-scented insects were amplified, including 13 batches of nine-scented insects, 5 batches of hemp stink bugs, brown-winged stink bugs, short-horned melon bugs and big wrinkled bugs. The results are shown in Figure 8 Specific bands were amplified in all samples. Although non-specific amplification occurred in some batches, it did not affect the interpretation of the results, indicating that this method can accurately distinguish between the nine-scented insect, the hemp-skinned stink bug, the tea-winged stink bug, the short-horned melon bug and the big wrinkled stink bug.

[0094] 8. Using multiplex PCR to identify samples mixed with nine-scented insects

[0095] The same batch of DNA from the same batch of nine-scented insects was spiked with 0%, 1%, 3%, 5%, 10%, and 100% of DNA from the same batch of scutellaria scabra for multiple PCR reactions to test the detection limit of the established multiple PCR method for scutellaria scabra in nine-scented insects; the same method was used to test the detection limits of tea-winged stink bugs, short-horned melon bugs, and large wrinkled stink bugs. The results are shown in Fig. 9 , if there is more than 3% of the hemp-skinned stink bug, more than 1% of the tea-winged stink bug, more than 1% of the short-horned melon stink bug, or more than 5% of the big wrinkled stink bug in the nine-scented insect sample, it can be detected, which proves that this method can realize the identification of the origin of the nine-scented insect medicinal material, and can also semi-quantitate the origin adulteration products.

[0096] In the description of this specification, the description with reference to the terms "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiments or examples are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0097] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A primer combination for multiplex PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs, characterized in that: include: Primer 1 and primer 2 for identifying the nine-scented insect, wherein the sequence of the primer 1 is shown in SEQ ID NO: 1; The sequence of the second primer is shown in SEQ ID NO: 2; Primer 3 and primer 4 for identifying the scurvy bug, wherein the sequence of primer 3 is shown in SEQ ID NO: 3; the sequence of primer 4 is shown in SEQ ID NO: 4; Primer 5 and primer 6 for identifying brown winged stink bug, the sequence of primer 5 is shown in SEQ ID NO: 5; the sequence of primer 6 is shown in SEQ ID NO: 6; Primer seven and primer eight for identifying short-horned citrus bugs, wherein the sequence of primer seven is shown in SEQ ID NO: 7; the sequence of primer eight is shown in SEQ ID NO: 8; Primer nine and primer ten are used to identify the big wrinkled bug. The sequence of primer nine is shown in SEQ ID NO: 9; the sequence of primer ten is shown in SEQ ID NO:

10.

2. Use of the primer combination for multiplex PCR identification of nine-scented insects, hemp-skinned stink bugs, brown-winged stink bugs, short-horned melon bugs and big wrinkled stink bugs as claimed in claim 1 in any one of (1) to (4): (1) Identify medicinal materials of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled bugs; (2) a kit for identifying medicinal materials of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs; (3) Identify whether the sample to be tested contains medicinal materials of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs; (4) A test kit for identifying whether the sample to be tested contains medicinal materials of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs.

3. A kit, characterized in that: It comprises the primer combination as claimed in claim 1.

4. An identification method based on the primer combination for multiplex PCR identification of nine-scented insects, hemp-skinned stink bugs, brown-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs according to claim 1, characterized in that, The following steps are involved: Prepare a PCR amplification system, the PCR amplification system comprising the primer combination for PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and big wrinkled stink bugs as claimed in claim 1; The PCR amplification system performs PCR amplification according to preset amplification conditions to obtain an amplification product; Performing electrophoresis analysis on the amplified product and recording its electrophoresis pattern; If the pattern contains a 301bp DNA fragment, the test product is a nine-scented insect; If the pattern contains a 129 bp DNA fragment, the test object is a stink bug; If the pattern contains a 175 bp DNA fragment, the sample to be tested contains the brown-winged stink bug; If the pattern contains a 117 bp DNA fragment, the sample to be tested contains the short-horned citrus bug; If the pattern contains a 390 bp DNA fragment, the sample to be tested contains the giant wrinkled bug.

5. The identification method of the primer combination for multiple PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs as claimed in claim 4, characterized in that, The total volume of the PCR amplification system is 22 μL to 27 μL, and the PCR amplification system includes: Multiplex PCR 5X Master Mix 4 μL to 6 μL, primer one and primer two, each 0.1 μL to 0.2 μL, primer three and primer four, each 0.25 μL to 0.35 μL, primer five and primer six, each 0.25 μL to 0.35 μL, primer seven and primer eight, each 0.35 μL to 0.45 μL, primer nine and primer ten, each 0.35 μL to 0.45 μL, template DNA 0.5 μL to 1.5 μL, and the rest is sterile ultrapure water.

6. The identification method of the primer combination for multiple PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs as claimed in claim 5, characterized in that, The total volume of the PCR amplification system is 25 μL, and the PCR amplification system includes: 5 μL of Multiplex PCR 5X Master Mix, 0.15 μL of primer 1 and primer 2, 0.3 μL of primer 3 and primer 4, 0.3 μL of primer 5 and primer 6, 0.4 μL of primer 7 and primer 8, 0.4 μL of primer 9 and primer 10, 1 μL of template DNA, and the rest is sterile ultrapure water.

7. The identification method of the primer combination for multiple PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs as described in claim 5 or 6, characterized in that, The concentration of the primer one, primer two, primer three, primer four, primer five, primer six, primer seven, primer eight, primer nine or primer ten is 9 μmol / L to 11 μmol / L.

8. The identification method of the primer combination for multiple PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs as claimed in claim 4, characterized in that, The preset amplification conditions are: The amplification system is pre-denatured at 92-97°C for 3-7 minutes, then cycled 29-32 times under a preset program, and finally extended at 70-75°C for 3-7 minutes; The preset program is: denaturing the amplification system at 92-97° C. for 25-35 seconds, then annealing at 59-61° C. for 25-35 seconds, and then extending at 70-75° C. for 25-35 seconds.

9. The identification method of the primer combination for multiple PCR identification of nine-scented insects, hemp-skinned stink bugs, tea-winged stink bugs, short-horned melon bugs and large wrinkled stink bugs as claimed in claim 8, characterized in that, The PCR amplification procedure is: The amplification system was pre-denatured at 95°C for 5 min, then cycled 31 times under the preset program, and finally extended at 72°C for 5 min; The preset program is: denaturing the amplification system at 95°C for 30s, then annealing at 60°C for 30s, and then extending at 72°C for 30s.

10. The identification method of primer combination for multiple PCR identification of nine-scented insects, hemp-skinned stink bugs, brown-winged stink bugs, short-horned melon bugs and big wrinkled stink bugs as claimed in claim 4, characterized in that, The electrophoresis analysis comprises: diluting the amplified product and spotting it on a 3% agarose gel, performing electrophoresis for 40 minutes at a voltage of 150V, developing the color with Gelred, and obtaining an electrophoresis pattern.

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