A method for identifying ant species

Abstract

The present invention provides a method for identifying ant species. By comparing the gene sequences of eight local ant species in Longyou, Zhejiang, specific primers are designed so that one primer can only amplify the DNA of one ant species, and the DNA fragment is of a specific size. Morphological identification is combined with molecular identification, and the species of the ants are determined by gel electrophoresis imaging. The ant species can be identified quickly and accurately. The operation is convenient and quick, and the identification time of the collected local ants can be effectively shortened.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and relates to a method for rapidly identifying ant species by utilizing molecular biology. Background Art

[0002] There are mainly eight species of ants in Longyou, Zhejiang, namely: Flat rainbow stink ant (Iridomyrmex anceps Roger), Chinese house ant (Monomorium chinense Santsch), Hairless stink ant (Ochetellus glaberMayr), Meadow pavement ant (Tetramorium caespitum Linnaeus), Nylanderiabourbonica Forel, big-headed ant (Pheidole sp.#7), big-headed ant (Pheidole sp.#9), and Crematogaster sp.

[0003] In order to conduct in-depth research on the eight native ant species in Longyou, Zhejiang, or to meet the inspection and quarantine requirements for agricultural and forestry plants, entry and exit, etc., it is usually necessary to identify the ant species. To identify the species of the collected ants, the DNA sequence of the ant species can be obtained through cloning and sequencing, and then the sequence is compared on the NCBI website. This method requires many steps, mainly including DNA extraction, PCR amplification, electrophoresis, gel recovery, ligation conversion, etc., and the sequence similarity of the eight native ant species is high, so it is time-consuming and expensive. Moreover, due to the large number of ant species, identification based on morphology is also very time-consuming. Therefore, there is an urgent need to find a more convenient and rapid method for identifying ant species. Summary of the Invention

[0004] To solve the above problems, the present invention provides a method for identifying ant species. By comparing the sequences of eight local ant species in Longyou, Zhejiang, specific primers are designed so that one primer can only amplify the DNA of one ant, and the fragment size is specific. Morphological identification is combined with molecular identification, and gel electrophoresis imaging is used to determine the species of ants. The ant species can be identified quickly and accurately, which can effectively shorten the identification time of the collected local ants.

[0005] The sequences of the eight native ants in Longyou, Zhejiang Province are very similar. Primers designed for one species of ant can often amplify the sequences of many other ants, and the specificity is difficult to guarantee. Therefore, it is difficult to accurately identify and distinguish the eight native ants. The ant species can only be determined by cloning and sequencing.

[0006] The present invention first obtains the COI gene sequences of eight ant species through cloning and sequencing methods, and designs specific primers to identify the eight main local ant species in Longyou, Zhejiang. It can ensure that one primer can only amplify the DNA of one type of ant, thus ensuring the accuracy and reliability of the identification of the eight local ant species.

[0007] The present invention first obtains the COI gene sequences of eight ant species through cloning and sequencing. Specific primers are designed based on the COI gene sequences of the eight ant species. Based on morphological identification, PCR (polymerase chain reaction) is performed directly. The amplified DNA gene is separated by agarose gel electrophoresis, stained with ethidium bromide, and observed under ultraviolet light. The result is directly determined based on the size of the amplified product. If a band of a specific size is specifically amplified, the ant species can be identified. Compared with cloning and sequencing methods, this method is faster and more convenient, and the identification results are more accurate.

[0008] Primers for identifying flat rainbow ants:

[0009] The present invention provides a primer pair for identifying flat iridescent ants. The nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.2.

[0010] The primer pair designed for the flat iris ant of the present invention can accurately identify and distinguish the flat iris ant from eight native ant species with very high sequence similarity. Therefore, it can be understood that when the flat iris ant is found alongside other non-native ant species, whose sequences have low similarity to the flat iris ant, the primer pair designed for the flat iris ant provided by the present invention will undoubtedly facilitate accurate identification of the flat iris ant.

[0011] Furthermore, the primer pair for identifying the flat iridescent ant provided by the present invention is designed based on the COI gene sequence of the flat iridescent ant, and the nucleotide sequence of the COI gene sequence of the flat iridescent ant is shown in SEQ ID NO.3.

[0012] Furthermore, the product amplified using the primer pair for identifying the flat iridescent ant provided by the present invention is 173 bp in size and has a nucleotide sequence as shown in SEQ ID NO.4.

[0013] On the other hand, the present invention provides the use of the primer pair or a kit containing the primer pair for identifying flat rainbow stink ants, wherein the primer pair can identify whether flat rainbow stink ants are contained in ants containing any one or more of the following: flat rainbow stink ants, Chinese house ants, hairless concave stink ants, grass pavement ants, Nissima brucei, Myrmecophaga #7, Myrmecophaga #9, and Myrmecophaga; the nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.2.

[0014] Primers for identifying the Chinese house ant:

[0015] The present invention provides a primer pair for identifying the Chinese house ant. The nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.6.

[0016] The primer pair designed for the Chinese house ant (Sinensis chinensis) of the present invention can accurately identify and differentiate Sinensis chinensis from eight native ant species with very high sequence similarity. Therefore, it can be understood that when Sinensis chinensis is present alongside other non-native ants, whose sequences have relatively low similarity to that of Sinensis chinensis, the primer pair designed for Sinensis chinensis provided by the present invention will undoubtedly make it easier to accurately identify Sinensis chinensis.

[0017] Furthermore, the primer pair for identifying the Chinese house ant provided by the present invention is designed based on the COI gene sequence of the Chinese house ant, and the nucleotide sequence of the COI gene sequence of the Chinese house ant is shown in SEQ ID NO.7.

[0018] Furthermore, the product amplified using the primer pair for identifying the Chinese house ant provided by the present invention is 76 bp in size and has a nucleotide sequence as shown in SEQ ID NO.8.

[0019] On the other hand, the present invention provides the use of the primer pair or the kit of the primer pair for identifying Chinese house ants, wherein the primer pair can identify whether Chinese house ants are contained in ants containing any one or more of the following: flat rainbow ants, Chinese house ants, hairless concave ants, grass pavement ants, Nissima brucei, Myrmecophaga #7, Myrmecophaga #9, and Myrmecophaga; the nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.6.

[0020] Primers for identifying the hairless ant:

[0021] The present invention provides a primer pair for identifying the hairless pit viper ant. The nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO.9, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.10.

[0022] The primer pair designed for the hairless ant (Eriophorum spp.) of the present invention can accurately identify and distinguish the hairless ant from eight native ant species with very high sequence similarity. Therefore, it can be understood that when the hairless ant is found alongside other non-native ant species, whose sequences have low similarity to that of the hairless ant, the primer pair designed for the hairless ant provided by the present invention will undoubtedly facilitate accurate identification of the hairless ant.

[0023] Furthermore, the primer pair for identifying the hairless ant provided by the present invention is designed based on the COI gene sequence of the hairless ant, and the nucleotide sequence of the COI gene sequence of the hairless ant is shown in SEQ ID NO.11.

[0024] Furthermore, the product amplified using the primer pair for identifying the hairless ant provided by the present invention is 169 bp in size and has a nucleotide sequence as shown in SEQ ID NO.12.

[0025] In another aspect, the present invention provides the use of the primer pair or a kit containing the primer pair for identifying hairless pit ants. The primer pair can identify whether hairless pit ants are present in ants containing any one or more of the following: flat rainbow pit ants, Chinese house ants, hairless pit ants, grass pavement ants, Nissima brucei, Myrmecophaga #7, Myrmecophaga #9, and Myrmecophaga. The nucleotide sequence of the forward primer of the primer pair is shown in SEQ ID NO.9, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.10.

[0026] Primers for identifying meadow pavement ants:

[0027] The invention provides a primer pair for identifying meadow pavement ants. The nucleotide sequence of the forward primer of the primer pair is shown in SEQ ID NO.13, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.14.

[0028] The primer pair designed for the meadow pavement ant of the present invention can accurately identify and distinguish the meadow pavement ant from eight native ant species with very high sequence similarity. Therefore, it can be understood that when the meadow pavement ant is found alongside other non-native ant species, whose sequences have relatively low similarity to the meadow pavement ant, the use of the primer pair designed for the meadow pavement ant provided by the present invention will undoubtedly facilitate accurate identification of the meadow pavement ant.

[0029] Furthermore, the primer pair for identifying the meadow ant provided by the present invention is designed based on the COI gene sequence of the meadow ant, and the nucleotide sequence of the COI gene sequence of the meadow ant is shown in SEQ ID NO.15.

[0030] Furthermore, the product amplified using the primer pair for identifying the meadow pavement ant provided by the present invention is 524 bp in size and has a nucleotide sequence as shown in SEQ ID NO.16.

[0031] On the other hand, the present invention provides the use of the primer pair or a kit containing the primer pair for identifying meadow pavement ants, wherein the primer pair can identify whether meadow pavement ants are present in ants containing any one or more of the following: flat rainbow ants, Chinese house ants, hairless concave ants, meadow pavement ants, Bruton's ants, Myrmecophaga #7, Myrmecophaga #9, and Myrmecophaga; the nucleotide sequence of the forward primer of the primer pair is shown in SEQ ID NO.13, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.14.

[0032] Primers for identifying Nissima brucei:

[0033] The present invention provides a primer pair for identifying Nielsenia brouweri's ant. The nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO.17, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.18.

[0034] The primer pair designed for Nisslera brucei of the present invention can accurately identify and distinguish Nisslera brucei from eight native ant species with very high sequence similarity. Therefore, it can be understood that when Nisslera brucei is present alongside other non-native ants, whose sequences have relatively low similarity to that of Nisslera brucei, the primer pair designed for Nisslera brucei of the present invention will undoubtedly make it easier to accurately identify Nisslera brucei.

[0035] Furthermore, the primer pair for identifying Nielsen's ant provided by the present invention is designed based on the COI gene sequence of Nielsen's ant, and the nucleotide sequence of the COI gene sequence of Nielsen's ant is shown in SEQ ID NO.19.

[0036] Furthermore, the product amplified using the primer pair for identifying Niederma brucei provided by the present invention is 524 bp in size and has a nucleotide sequence as shown in SEQ ID NO.20.

[0037] In another aspect, the present invention provides the use of the primer pair or a kit containing the primer pair for identifying Nissella brucei, wherein the primer pair can identify whether Nissella brucei is present in an ant species containing any one or more of the group consisting of the flat iridescent ant, the Chinese house ant, the hairless concave ant, the grass pavement ant, Nissella brucei, Myrmecophaga #7, Myrmecophaga #9, and Myrmecophaga; the nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO.17, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.18.

[0038] Primers for identifying Myrmex spp. #7:

[0039] The present invention provides a primer pair for identifying Myrmecophaga #7. The nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO.21, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.22.

[0040] The primer pair designed for Myrmecophaga #7 according to the present invention can accurately identify and distinguish Myrmecophaga #7 from eight native ant species with very high sequence similarity. Therefore, it can be understood that when Myrmecophaga #7 is found alongside other non-native ants, whose sequences have relatively low similarity to Myrmecophaga #7, the primer pair designed for Myrmecophaga #7 according to the present invention will undoubtedly make it easier to accurately identify Myrmecophaga #7.

[0041] Furthermore, the primer pair for identifying the genus Myrmecophaga #7 provided by the present invention is designed based on the COI gene sequence of the genus Myrmecophaga #7, and the nucleotide sequence of the COI gene sequence of the genus Myrmecophaga #7 is shown in SEQ ID NO.23.

[0042] Furthermore, the product amplified using the primer pair for identifying the genus Myxocephalus #7 provided by the present invention is 524 bp in size and has a nucleotide sequence as shown in SEQ ID NO.24.

[0043] On the other hand, the present invention provides the use of the primer pair or a kit containing the primer pair for identifying Myrmecophaga #7, wherein the primer pair can identify whether Myrmecophaga #7 is contained in an ant species containing any one or more of Myrmecophaga flatulosa, Myrmecophaga sinensis, Myrmecophaga hairless, Myrmecophaga pratensis, Myrmecophaga brunneri, Myrmecophaga #7, Myrmecophaga #9, and Myrmecophaga crescentus; the nucleotide sequence of the forward primer of the primer pair is shown in SEQ ID NO.21, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.22.

[0044] Primers for identifying Myrmex spp. #9:

[0045] In another aspect, the present invention provides a primer pair for identifying Myrmecophaga #9, wherein the nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO.25, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.26.

[0046] The primer pair designed for Myrmecophaga #9 according to the present invention can accurately identify and distinguish Myrmecophaga #9 from eight native ant species with very high sequence similarity. Therefore, it can be understood that when Myrmecophaga #9 is found alongside other non-native ants, whose sequences have relatively low similarity to Myrmecophaga #9, the primer pair designed for Myrmecophaga #9 according to the present invention will undoubtedly make it easier to accurately identify Myrmecophaga #9.

[0047] Furthermore, the primer pair for identifying the genus Myrmecophaga #9 provided by the present invention is designed based on the COI gene sequence of the genus Myrmecophaga #9, and the nucleotide sequence of the COI gene sequence of the genus Myrmecophaga #9 is shown in SEQ ID NO.27.

[0048] Furthermore, the product amplified using the primer pair provided by the present invention for identifying the genus Myxocephalus #9 is 524 bp in size and has a nucleotide sequence as shown in SEQ ID NO.28.

[0049] On the other hand, the present invention provides the use of the primer pair or a kit containing the primer pair for identifying the genus Bighead Myrmecophaga #9, wherein the primer pair can identify whether Bighead Myrmecophaga #9 is contained in ants containing any one or more of the following: Flat Myrmecophaga, Chinese House Myrmecophaga, Hairless Myrmecophaga, Grassland Paving Myrmecophaga, Bruton's Myrmecophaga, Bighead Myrmecophaga #7, Bighead Myrmecophaga #9, and Cleomenon; the nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO.25, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.26.

[0050] Primers for identification of the genus Cleome:

[0051] In another aspect, the present invention provides a primer pair for identifying the genus Cleomethecus, wherein the nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO. 29, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 30.

[0052] The primer pair designed for the genus Myrmex in this invention can accurately identify and distinguish Myrmex from eight native ant species with very high sequence similarity. Therefore, it can be understood that when Myrmex is present alongside other non-native ant species, whose sequences have relatively low similarity to Myrmex, the primer pair designed for Myrmex provided by this invention will undoubtedly make it easier to accurately identify Myrmex.

[0053] Furthermore, the primer pair for identifying the genus Myrmecophaga provided by the present invention is designed based on the COI gene sequence of the genus Myrmecophaga, and the nucleotide sequence of the COI gene sequence of the genus Myrmecophaga is shown in SEQ ID NO.31.

[0054] Furthermore, the product amplified using the primer pair for identifying the genus Cleometis provided by the present invention is 524 bp in size and has a nucleotide sequence as shown in SEQ ID NO.32.

[0055] In another aspect, the present invention provides the use of the primer pair or a kit containing the primer pair for identifying the genus Myrmecophaga, wherein the primer pair can identify whether the genus Myrmecophaga is contained in an ant species containing any one or more of the following: Myrmecophaga spp., Myrmecophaga spp., Myrmecophaga spp., Myrmecophaga spp. #7, Myrmecophaga spp. #9, and Myrmecophaga spp.; the nucleotide sequence of the forward primer of the primer pair is shown in SEQ ID NO.29, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.30.

[0056] In another aspect, the present invention provides the steps of identifying the ant species:

[0057] (1) Extracting DNA from the ants to be tested;

[0058] (2) using at least one primer pair among the eight primer pairs to amplify the DNA of the ant to be tested obtained in step (1);

[0059] (3) Separating and identifying the amplified products by agarose electrophoresis;

[0060] The ants are at least one of the following: flat rainbow ants, Chinese house ants, hairless concave ants, grassland pavement ants, Nieto's ants, Myrmecophaga spp. #7, Myrmecophaga spp. #9, and Myrmecophaga spp.;

[0061] The eight primer pairs are as follows: 1. The nucleotide sequence of the forward primer of the flat rainbow ant is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; 2. The nucleotide sequence of the forward primer of the Chinese house ant is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6; 3. The nucleotide sequence of the forward primer of the hairless ant is shown in SEQ ID NO.9, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.10; 4. The nucleotide sequence of the forward primer of the grassland ant is shown in SEQ ID NO.13, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.14; 5. The nucleotide sequence of the forward primer of the brunner's ant is shown in SEQ ID NO.17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.18; 6. The nucleotide sequence of the forward primer of the genus Myrmecophaga #7 is shown in SEQ ID NO.21, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.22; 7. The nucleotide sequence of the forward primer of the genus Myrmecophaga #9 is shown in SEQ ID 8. The nucleotide sequence of the forward primer of the genus Cleometis is shown in SEQ ID NO.29, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.30.

[0062] Furthermore, before step (1), the ant species can be estimated based on the ant morphology, so as to select the primer pair to be used.

[0063] Furthermore, the amplification in step (2) is performed according to a certain PCR reaction procedure, wherein the annealing temperature is 48°C.

[0064] Furthermore, the PCR reaction procedure in step (2) was as follows: pre-denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 40 s, annealing at 48°C for 40 s, and extension at 72°C for 60 s; and extension at 72°C for 7 min. The PCR product was stored at 4°C until use.

[0065] The present invention obtains the COI gene sequences of eight ant species through cloning and sequencing. Specific primers are designed by comparing the sequences of the eight ant species, so that one primer can only amplify the DNA of one ant species. Gel electrophoresis imaging is then performed to determine the ant species. In other words, after obtaining an ant species and performing morphological identification, the corresponding primers of the present invention can be used to further accurately and reliably determine the ant species.

[0066] In some methods, the DNA of the ants to be tested can be the DNA of one, two, three, four, five, six, seven or eight species of ants, and is amplified using any one of the eight primers. If the corresponding product can be amplified, it proves that the ant species corresponding to the primer exists in the ants to be tested.

[0067] In some methods, the DNA of the ant to be tested can be any one of eight ant species. After the ant species is preliminarily estimated by morphology, a primer pair corresponding to the ant species is selected to identify whether the species of the ant to be tested is the ant species corresponding to the primer pair.

[0068] In some methods, the DNA of the ant to be tested can be any one of eight species of ants, and one, two, three, four, five, six, seven or eight primer pairs are selected for simultaneous amplification. Based on the agarose electrophoresis separation and identification results of the amplified products, it is determined which primer corresponds to the product, thereby identifying the species of the ant to be tested as the ant species corresponding to the primer.

[0069] In some methods, the DNA of the ants to be tested can be the DNA of one, two, three, four, five, six, seven or eight species of ants, and one, two, three, four, five, six, seven or eight primer pairs are selected for simultaneous amplification. If the products corresponding to one, two, three, four, five, six, seven or eight primers can be amplified, it proves that the ant species corresponding to the one, two, three, four, five, six, seven or eight primers exist in the ants to be tested.

[0070] In some methods, since the fragments amplified by the eight primers are of specific sizes, the species of the ant can be determined based on the size of the amplified products.

[0071] In another aspect, the present invention provides a primer pair combination for identifying ant species, wherein the primer pair combination is at least one of the eight primer pairs described above.

[0072] In some embodiments, the primer pair combination can be any one, two, three, four, five, six, seven, or eight of the eight primer pairs.

[0073] On the other hand, the present invention provides the use of the primer pair combination as described above or a kit containing the primer pair combination as described above for identifying ant species, wherein the primer pair combination can identify the ant species from ants containing any one or more of the following: flat rainbow ants, Chinese house ants, hairless concave ants, lawn pavement ants, Nicholas brucei, Myrmecophaga #7, Myrmecophaga #9, and Myrmecophaga.

[0074] In some embodiments, the primer pair combination for identifying ant species provided by the present invention has a sensitivity of 5 ng / μL for identifying ant species.

[0075] The present invention has the following beneficial effects:

[0076] 1. Provided primer pairs for identifying 8 species of ants native to Longyou, Zhejiang, allowing accurate identification of each ant species without cloning and sequencing;

[0077] 2. Each primer pair is very specific and can only amplify the DNA of one type of ant. It can accurately distinguish eight local ant species with very high sequence similarity.

[0078] 3. The operation is convenient and quick, which can effectively shorten the identification time of the collected local ants. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 This is an agarose electrophoresis diagram of the primer amplification products of the flat rainbow ant in Example 3;

[0080] Figure 2 This is an agarose electrophoresis diagram of the amplification product of the Chinese house ant primers in Example 3;

[0081] Figure 3 This is an agarose electrophoresis diagram of the amplification products of the primers of the hairless ant in Example 3;

[0082] Figure 4 This is an agarose electrophoresis diagram of the amplification product of the primers of the meadow pavement ant in Example 3;

[0083] Figure 5 is an agarose electrophoresis diagram of the primer amplification product of Niemann's ant in Example 3;

[0084] Figure 6 This is an agarose electrophoresis diagram of the amplification product of the primer #7 of the genus Myrmecophaga in Example 3;

[0085] Figure 7 This is an agarose electrophoresis diagram of the amplification product of the primer #9 of the genus Myrmecophaga in Example 3;

[0086] Figure 8 This is the agarose electrophoresis diagram of the amplification products of the genus Myrmex primers in Example 3. DETAILED DESCRIPTION

[0087] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings. It should be noted that the following embodiments are intended to facilitate understanding of the present invention and do not limit it in any way. All features disclosed in the embodiments of the present invention, or all steps in the methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0088] Example 1 Design of primers for molecular identification of eight ant species

[0089] Ants were collected in Longyou, Zhejiang Province, using a trapping method. The specimens were frozen and then directly stored in anhydrous ethanol. Under a dissecting microscope or stereomicroscope, the specimens were classified according to morphology and DNA was extracted using a kit. The mitochondrial COI gene of eight ant species was detected using PCR and DNA sequencing.

[0090] The universal primers used were: forward primer 5'-GGTCAACAAATCATAAAGATATTGG-3' (SEQ ID NO. 33); reverse primer 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (SEQ ID NO. 34); the mitochondrial DNA COI gene of eight ant species was amplified by polymerase chain reaction; an appropriate amount of the COI DNA gene amplified by the polymerase chain reaction was separated by agarose gel electrophoresis, stained with ethidium bromide, and observed under ultraviolet light. The amplified product was determined based on its size. If a band of about 1000 bp was specifically amplified, the product was recovered by gel extraction (the gel extraction kit used was QIAquick Gel Extraction Kit (QIAGEN, Germany)); the gel-recovered product was ligated and converted using the Promega-pGEM-T Easy vector system (purchased from Promega, catalog number A1360); the cloned product was sent for sequencing, and the sequencing results were obtained using MEGA7 software and blast website ( BLAST:Basic Local Alignment Search Tool (nih.gov) ) were compared to obtain the COI gene sequences of 8 ant species, among which the COI gene sequence of the flat iridescent ant is shown in SEQ ID NO.3, the COI gene sequence of the Chinese house ant is shown in SEQ ID NO.7, the COI gene sequence of the hairless concave ant is shown in SEQ ID NO.11, the COI gene sequence of the grassland pavement ant is shown in SEQ ID NO.15, the COI gene sequence of the brunner's ant is shown in SEQ ID NO.19, the COI gene sequence of the genus Myrmecophaga #7 is shown in SEQ ID NO.23, the COI gene sequence of the genus Myrmecophaga #9 is shown in SEQ ID NO.27, and the COI gene sequence of the genus Myrmecophaga is shown in SEQ ID NO.31.

[0091] In this example, primers were used for PCR amplification. The PCR reaction system is shown in Table 1, and the reagent used was GoTaq Green Master Mix (Promega, US).

[0092] Table 1 PCR reaction system of unknown ant mitochondrial DNA COI gene (10 μL system)

[0093]

[0094]

[0095] The PCR reaction procedure in this example was as follows: pre-denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 40 s, annealing at 48°C for 40 s, and extension at 72°C for 60 s; and an additional extension at 72°C for 7 min. The PCR product was stored at 4°C until use.

[0096] Based on the sequencing results of the eight ant species, specific primers for the eight ant species were designed. Each designed primer pair was used to amplify the fragments of each ant species. The amplified products were separated by agarose gel electrophoresis, stained with ethidium bromide, and observed under ultraviolet light to investigate whether each primer pair could amplify the COI sequence of only one ant species but not the other seven ant species. The designed primers and the investigation results are shown in Table 2 (due to the large number of primers designed and screened, this example only provides the results of several typical primer pairs):

[0097] Table 2. Different primers designed for 8 ant species

[0098]

[0099]

[0100] Since the eight native ant species in Longyou, Zhejiang Province have high sequence homology (90% to 98% homology), it is difficult to design highly specific primers that can only amplify one ant species at a time when designing primers for amplification. In this example, after several rounds of primer design and amplification comparison, eight sets of highly specific primers were finally obtained as shown in Table 3. Each primer can only amplify the sequence of the corresponding specific ant species, and the bands of the amplification products of each primer are all of a specific fragment size.

[0101] Table 3. Highly specific primers designed for eight ant species

[0102]

[0103]

[0104] Example 2 Effect of annealing temperature on gel electrophoresis results of 8 sets of primer amplification products

[0105] Annealing temperature significantly affects the specificity of PCR. Therefore, different annealing temperatures will also affect the specificity of the eight primer pairs. To ensure the specificity of the eight primer pairs when used to identify ant species, this example screened the annealing temperature in the PCR program and examined the agarose electrophoresis separation results of the eight primer pairs when performing PCR at different annealing temperatures when amplifying the eight corresponding ant species. The results are shown in Table 3.

[0106]

[0107]

[0108] Note: a means good specificity, and bands can be seen during agarose electrophoresis; b means poor specificity, and no bands can be seen during agarose electrophoresis.

[0109] As can be seen from Table 3, when the annealing temperature is 45°C, the specificity of the Chinese house ants, the hairless ant, and the ant genera are poor, and no bands can be seen during agarose electrophoresis. This may be because the Tm values ​​of the corresponding primers designed for these three ants are relatively high, and the annealing temperature of 45°C is too low for these three primers, which affects the detection results and causes no bands to be seen during agarose electrophoresis.

[0110] When the annealing temperature was 50°C, the specificity of Nissima brucei and Myrmecophaga #9 was poor, and no bands could be detected during agarose electrophoresis. This may be because the annealing temperature was too high, which affected the detection results of the two primers and resulted in no bands being detected during agarose electrophoresis.

[0111] Therefore, when using the above eight primer sets for ant species identification, the annealing temperature in the PCR program must be strictly controlled at 48°C.

[0112] Example 3 Highly specific primers for molecular identification of eight ant species

[0113] Using one of the eight sets of highly specific primers obtained in Example 1, the PCR reaction system and reaction procedure provided in Example 1 were used to simultaneously amplify the COI genes of eight ant species. The amplified products were separated by agarose gel electrophoresis, stained with ethidium bromide, and observed under ultraviolet light. The results are as follows: Figures 1 to 8 As shown, Figure 1 This is the electrophoresis diagram of the primer amplification products of the flat rainbow ant. Figure 2 This is the electrophoresis diagram of the primer amplification products of Chinese house ants. Figure 3 This is the electrophoresis diagram of the primer amplification products of the hairless ant. Figure 4 This is the electrophoresis diagram of the amplified products of the primers of the grassland ant. Figure 5 This is the electrophoresis diagram of the primer amplification products of Nieto's ant. Figure 6 This is the electrophoresis diagram of the amplified product of the primer #7 of the genus Megacephalus. Figure 7This is the electrophoresis diagram of the amplified product of the primer #9 of the genus Megacephalus. Figure 8 This is the electrophoresis diagram of the primer amplification products of the genus Cleomethecus. The two leftmost lanes are maker, DL2000 and DL500 respectively. Lane 1 is the primer amplification product of the flat iridescent ant, lane 2 is the primer amplification product of the Chinese house ant, lane 3 is the primer amplification product of the hairless concave ant, lane 4 is the primer amplification product of the grassland pavement ant, lane 5 is the primer amplification product of the brunneri ant, lane 6 is the primer amplification product of the genus Megacephalus #7, lane 7 is the primer amplification product of the genus Megacephalus #9, and lane 8 is the primer amplification product of the genus Cleomethecus.

[0114] Depend on Figures 1 to 8 It can be seen that each set of primers can only amplify the sequence of a corresponding ant species, and the amplified bands are all of specific fragment sizes. The fragment size corresponding to the amplification product of the flat rainbow ant primers is 173 bp ( Figure 1 ), the size of the fragment corresponding to the amplification product of the primers of the Chinese house ant is 76bp ( Figure 2 ), the fragment size corresponding to the primer amplification product of the hairless ant is 169bp ( Figure 3 ), the fragment size corresponding to the amplification product of the primer of the grassland ant is 523bp ( Figure 4 ), the fragment size corresponding to the amplification product of the primers of Nieto's ant was 274 bp ( Figure 5 ), the fragment size corresponding to the amplification product of the primer #7 of the genus Megacephalus is 79 bp ( Figure 6 ), the fragment size corresponding to the amplification product of the primer #9 of the genus Megacephalus is 272bp ( Figure 7 ), the fragment size corresponding to the amplification product of the primers of the genus Cleome is 405bp ( Figure 8 Therefore, the size of the amplified product can be used to determine the type of ant it belongs to. It can be seen that the eight primer sets used in this example are highly specific, and the accuracy rate for identifying any of the eight ant species is 100%.

[0115] Example 4: Specific identification of ant species using primer combinations

[0116] In this example, a mixture of eight highly specific primer pairs obtained in Example 1 (0.2 μL per pair) was used, along with the PCR reaction system and procedure provided in Example 1, to amplify the COI gene of an unknown ant species. The amplified products were separated by agarose gel electrophoresis, stained with ethidium bromide, and observed under ultraviolet light. The amplification results showed that a 273 bp fragment was amplified in channel 7, confirming that the unknown ant was Myrmecophaga #9.

[0117] After further appearance identification and gene sequencing, the result was confirmed to be Big-headed Ant Genus #9. Therefore, using a combination of 8 sets of primer pairs, it is possible to determine at one time which of the eight ant species the unknown ant belongs to.

[0118] The application of the present invention is not limited to this. It can be expanded based on its application scope in environmental protection. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope defined by the claims. Sequence Listing <110> Zhejiang University <120> A method for identifying ant species <160> 34 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <400> 1 gtctatcctc ctctagcctc a 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 aagataagag taataaaata 20 <210> 3 <211> 709 <212> DNA <213> Artificial Sequence <400> 3 ggtcaacaaa tcataaagat attggtatat tatattttat ttttgctatt tgatcaggaa 60 taattggatc ctctataaga ataattattc gtctcgaatt aggtacttgt aactctatta 120 ttatgaatga tcaagcctat aattccattg taactggtca tgcatttatt ataatttttt 180 ttatagttat accttttata attggaggat tcggaaattt tttagtccca cttatattag 240 gagccccaga tatagcttat ccacgaataa ataatataag attttgacta ctccctcctt 300 caattttact actaactatc agaaacttta ttagatcagg agtaggaaca ggatgaactg 360 tctatcctcc tctagcctca aatatattcc ataatggacc atctgtagac ttagctattt 420 tctctcttca tattgctgga atatcttcaa ttttaggagc aatcaatttt atttctacta 480 ttctaaatat acatcataaa aatcttacta tagataaaat tcctctttta gtctgatcaa 540 tcttaattac tgctatttta ttactcttat ctttacccgt actagcagga gcaattacta 600 tactactaac agatcgaaac ttaaatactt ctttcttcga tccttctgga ggaggagatc 660 ctattttata tcaacattta ttttgatttt ttggtcaccc tgaagttta 709 <210> 4 <211> 173 <212> DNA <213> Artificial Sequence <400> 4 aatatattcc ataatggacc atctgtagac ttagctattt tctctcttca tattgctgga 60 atatcttcaa ttttaggagc aatcaatttt atttctacta ttctaaatat acatcataaa 120 aatcttacta tagataaaat tcctctttta gtctgatcaa tcttaattac tgc 173 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <400> 5 cattaaccct tcttattcta gg 22 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <400> 6 ggataaatcg attgaggc 18 <210> 7 <211> 709 <212> DNA <213> Artificial Sequence <400> 7 ggtcaacaaa tcataaagat attggtattt tatattttat ccttgccatt tgatcaggta 60 taattggatc ttctataaga ataattatcc gattagaatt aggatcttgt ggccccttaa 120 tcaataatga tcaaatttat aactctttag ttacaagaca cgcatttatt ataatttttt 180 ttatagttat accatttata attgggggat ttggaaactt tttagtacca ttaatacttg 240 gttcaccaga tatagcttac cctcgtataa ataatataag attttgatta ttacccccat 300 cattaaccct tcttattcta ggaagattta ttaattcagg ggtaggaact ggatgaacaa 360 tctacccccc tctagcttct aatatttttc acagaggtgc ctcaatcgat ttatccattt 420 tttctcttca tatcgctggt atgtcctcaa ttttaggagc aattaatttt atctcaacta 480 ttataaatat acatcataaa aatatttctc tagataaaat tcctttacta gtctgatcaa 540 ttataattac tgcaattctt ttactcttat ctcttcctgt tctagcaggg gccattacta 600 tacttttaac agatcgtaat ctaaatacaa ccttctttga cccaagaggt ggtggagacc 660 ctattctata ccaacactta ttttgatttt ttggtcaccc tgaagttta 709 <210> 8 <211> 76 <212> DNA <213> Artificial Sequence <400> 8 aagatttatt aattcagggg taggaactgg atgaacaatc tacccccctc tagcttctaa 60 tatttttcac agaggt 76 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <400> 9 ggaacatgca attctattat tata 24 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <400> 10 ggttagtaat agaattgatg gtg 23 <210> 11 <211> 709 <212> DNA <213> Artificial Sequence <400> 11 ggtcaacaaa tcataaagat attggaatat tatattttat ttttgcaatt tgatcaggaa 60 taataggctc ttcaataaga ataattatcc gattagaatt aggaacatgc aattctatta 120 ttataaatga tcaaatttat aattctattg taactagtca tgcctttatt ataatttttt 180 ttatagttat accttttata attggaggat ttggaaattt tttagtacct ttaatattag 240 gggcccctga tatagcttac ccacgaataa ataacataag attttgatta ttaccaccat 300 caattctatt actaaccatt agaaatttta tcagatcagg agtaggaaca ggatgaactg 360 tttatcctcc tttagcaaga aatatatttc acaatggacc atctgtagat ttagctattt 420 tttctttaca tattgccgga atatcatcaa ttctaggagc aattaatttt atttcaacta 480 ttctaaatat acatcataaa aatttttcta tagataaaat tcctttatta gtatgatcaa 540 tcttaattac tgccgtatta ttacttctat ctttaccagt attagcagga gcaattacta 600 tacttttaac cgatcgaaat ttaaatactt ctttttttga tccatcagga ggaggagatc 660 caattttata ccaacactta ttttgatttt ttggtcaccc tgaagttta 709 <210> 12 <211> 169 <212> DNA <213> Artificial Sequence <400> 12 aatgatcaaa tttataattc tattgtaact agtcatgcct ttattataat tttttttata 60 gttatacctt ttataattgg aggatttgga aattttttag tacctttaat attaggggcc 120 cctgatatag cttacccacg aataaataac ataagatttt gattattac 169 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <400> 13 gtcaacaaat cataaagata ttg 23 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <400> 14 gaagaaggag gatagccg 18 <210> 15 <211> 708 <212> DNA <213> Artificial Sequence <400> 15 ggtcaacaaa tcataaagat attggtattc tttattttct atttgctatc tgagctggaa 60 taattggatc ttcaataagt ataattattc gacttgaatt aagatcctgt ggttcattaa 120 ttaatgacga ccaggtatat aattccttag taacaagaca tgcccttgtt ataatttttt 180 ttatagttat accttttata atcggtggat ttggaaattt tttagtccca ttaatactag 240 gaactcctga tatagcctac cctcgcataa ataatataag attctgactc ctccccccct 300 ctattagatt attattaatt agaagattta ttaatacagg agtaggaaca ggatggacca 360 tttacccccc attagcatct aacatctttc atagaggacc atcaattgac ctatcaattt 420 tttctcttca tattgcagga atatcttcta ttataggcgc aattaatttt attgctacta 480 ttataaacat acatcataaa aaattaacct tagataaaat ccccttatta gtttgatcta 540 ttttaattac ggctatcctc cttcttctat ctctacccgt attagccgga gctattacta 600 tgctcctaac agaccgtaat ttaaatacct cattctttga cccatcgggg ggggggaccc 660 aattttatat caacacttat tctgattttt tggtcaccct gaagttta 708 <210> 16 <211> 523 <212> DNA <213> Artificial Sequence <400> 16 attctttatt ttctatttgc tatctgagct ggaataattg gatcttcaat aagtataatt 60 attcgacttg aattaagatc ctgtggttca ttaattaatg acgaccaggt atataattcc 120 ttagtaacaa gacatgccct tgttataatt ttttttatag ttataccttt tataatcggt 180 ggatttggaa attttttagt cccattaata ctaggaactc ctgatatagc ctaccctcgc 240 ataaataata taagattctg actcctcccc ccctctatta gattattatt aattagaaga 300 tttattaata caggagtagg aacaggatgg accatttacc ccccattagc atctaacatc 360 tttcatagag gaccatcaat tgacctatca attttttctc ttcatattgc aggaatatct 420 tctattatag gcgcaattaa ttttattgct actattataa acatacatca taaaaaatta 480 accttagata aaatcccctt attagtttga tctattttaa tta 523 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <400> 17 gtcaacaaat cataaagata ttg 23 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <400> 18 cttaataaaa ggagagaaat tg 22 <210> 19 <211> 709 <212> DNA <213> Artificial Sequence <400> 19 ggtcaacaaa tcataaagat attggaatct tatatttttt atttgctatt tgagcgggta 60 taattggaac ttctataaga atgattattc gtttagaatt aggttcccct aatcctctaa 120 ttaataatga tcaaatctat aattctatag ttactagaca tgcttttatt ataatttttt 180 ttatagttat gccattcata attggaggct ttggaaattt tttagtgcct ttaatattag 240 gttcaccaga tatagcctat cctcgaataa ataatataag attttgatta cttccccctt 300 caatttctct ccttttatta agaaatttta ttaatgatgg tgttggtacc ggctgaacag 360 tatacccccc actagcatct aacatttttc ataatggacc ttcagttgac ttagctattt 420 tctccttaca tattgcagga atatcttcaa ttttaggagc aattaacttt atttcaacaa 480 ttttaaatat acatcaaaaa aacttctcaa ttgataaaat ccccctcctt gtttgatcca 540 tttttattac agcaattctg ctccttttat ctctacctgt tttagcaggg gctatcacca 600 tattattaac tgatcgaaat ttaaatactt cattctttga tccatcagga gggggtgatc 660 cgatcctcta tcaacattta ttttgatttt ttggtcaccc tgaagttta 709 <210> 20 <211> 274 <212> DNA <213> Artificial Sequence <400> 20 atcttatatt ttttatttgc tatttgagcg ggtataattg gaacttctat aagaatgatt 60 attcgtttag aattaggttc ccctaatcct ctaattaata atgatcaaat ctataattct 120 atagttacta gacatgcttt tattataatt ttttttatag ttatgccatt cataattgga 180 ggctttggaa attttttagt gcctttaata ttaggttcac cagatatagc ctatcctcga 240 ataaataata taagattttg attacttccc cctt 274 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <400> 21 gtcaacaaat cataaagata ttg 23 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <400> 22 gttgattagt gagttgcaag 20 <210> 23 <211> 709 <212> DNA <213> Artificial Sequence <400> 23 ggtcaacaaa tcataaagat attggaatat tatattttat tctagcaatt tgagccggaa 60 tgattggatc ttctataaga ataatcattc gattagaact tggatcttgc aactcactaa 120 tcaacaacga ccaaatttat aactctctag ttacaagaca cgcttttatc ataatttttt 180 ttatagttat accttttata attgggggat ttggaaattt tttagtcccc ttaatacttg 240 gatcaccaga catagcttac ccccgtataa ataatataag attctgactt ttacccccct 300 ctctcacttt acttcttcta ggtagattta ttaattctgg agttggaacc ggatgaacaa 360 tttaccctcc tttagcctcc aatatttttc atagaggagc atccattgat ctctctattt 420 tttctctcca tattgcaggt atatcgtcta tcttaggtgc tatcaacttc atctctacaa 480 tcattaatat acatcataaa aatttcacca tagataaaat tcccttatta gtctgatcta 540 ttttaattac cgctgtatta ctcttacttt ctctccctgt tctagcaggt gctatcacta 600 tacttctcac cgatcgaaac ttaaatactt ccttttttga tccagctggt ggtggagacc 660 caattctata ccaacattta ttttgatttt ttggtcaccc tgaagttta 709 <210> 24 <211> 79 <212> DNA <213> Artificial Sequence <400> 24 atattatatt ttattctagc aatttgagcc ggaatgattg gatcttctat aagaataatc 60 attcgattag aacttggat 79 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <400> 25 gtcaacaaat cataaagata ttg 23 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <400> 26 ataaaagaag ggaaatagaa 20 <210> 27 <211> 709 <212> DNA <213> Artificial Sequence <400> 27 ggtcaacaaa tcataaagat attggaatct tatatttttt atttgctatt tgagctggga 60 taattggaac ttcaataaga ataattattc gcttagaatt aggatctcct aatcctctta 120 ttaataatga tcaaatttat aactccatag ttactaggca cgcttttatt ataatttttt 180 ttatagttat accatttata attggtggat ttggaaattt cttagtccct ttaatattag 240 gatctcctga tatagcctat cctcgaataa ataatataag attctgatta cttcctcctt 300 ctatttccct tcttttatta agtaatttta ttaatgatgg tgtaggaaca ggctgaactg 360 tatatcctcc tctggcctct aatatttttc ataatggacc ttcggtagat ttagctattt 420 tttctttaca tattgctggt atatcttcaa ttttaggagc aattaacttc atttcaacaa 480 ttttaaatat acatcataaa aatttttcaa ttgataaaat tcctcttctt gtttgatcta 540 tttttattac agcaatccta ctacttctat ctcttcctgt cttagcagga gccattacta 600 tattattaac agatcgaaat ctaaatactt ctttctttga cccttctgga ggaggagacc 660 ctattcttta ccaacattta ttttgatttt ttggtcaccc tgaagttta 709 <210> 28 <211> 272 <212> DNA <213> Artificial Sequence <400> 28 atcttatatt ttttatttgc tatttgagct gggataattg gaacttcaat aagaataatt 60 attcgcttag aattaggatc tcctaatcct cttattaata atgatcaaat ttataactcc 120 atagtacta ggcacgcttt tattataatt ttttttatag ttataccatt tataattggt 180 ggatttggaa atttcttagt ccctttaata ttaggatctc ctgatatagc ctatcctcga 240 ataataata taagattctg attacttcct cc 272 <210> 29 <211> 17 <212> DNA <213> Artificial Sequence <400> 29 ctaggctctt gtgattc 17 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <400> 30 gaatagatca agttagtaga gag 23 <210> 31 <211> 709 <212> DNA <213> Artificial Sequence <400> 31 ggtcaacaaa tcataaagat attggaattt tatattttat tttcgctatt tgagcaggca 60 taattgggtc atccataaga ataatcattc gccttgaact aggctcttgt gattctttaa 120 ttaataatga tcaaatttat aatgttttag taactagaca cgcttttatt ataattttct 180 ttatagttat acccttcata atcggaggat ttgggaattt tcttgttcct ctaatacttg 240 gctcacctga tatagcctat cctcgtataa ataatataag tttttgactc cttcctccct 300 ctattcttct ccttctactt agaagattta taaatacagg tgtaggaact ggatgaacta 360 tttatcctcc tctagcttct aatatctttc atagaggtgc ctctattgac ctttcaattt 420 tctccctcca cattgctggt atatcttcaa ttttaggagc tattaacttc atttctacta 480 tcctaaacat acatcataaa tctattacct tagataaaat ctctctacta acttgatcta 540 ttcttattac tgcaattcta ttacttcttt ctcttcctgt tcttgccggg gcaattacca 600 tacttctaac tgatcgaaat cttaatactt ctttttttga tccttctggt ggaggagatc 660 ctattttata ccaacacctt ttttgatttt ttggtcaccc tgaagttta 709 <210> 32 <211> 405 <212> DNA <213> Artificial Sequence <400> 32 tttaattaat aatgatcaaa tttataatgt tttagtaact agacacgctt ttattataat 60 tttctttata gttataccct tcataatcgg aggatttggg aattttcttg ttcctctaat 120 acttggctca cctgatatag cctatcctcg tataaataat ataagttttt gactccttcc 180 tccctctatt cttctccttc tacttagaag atttataaat acaggtgtag gaactggatg 240 aactatttat cctcctctag cttctaatat ctttcataga ggtgcctcta ttgacctttc 300 aattttctcc ctccacattg ctggtatatc ttcaatttta ggagctatta acttcatttc 360 tactatccta aacatacatc ataaatctat taccttagat aaaat 405 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <400> 33 ggtcaacaaa tcataaagat attgg 25 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <400> 34 taaacttcag ggtgaccaaa aaatca 26

Claims

1. A method for identifying which of eight ant species an ant is, characterized in that: The following steps are involved: (1) Extract DNA from the ants to be tested; (2) amplifying the DNA of the ant to be tested obtained in step (1) using a mixture of 8 primer pairs; the annealing temperature of the amplification is 48°C; (3) The amplified products were separated and identified by agarose electrophoresis; The eight ant species are the flat rainbow ant, the Chinese house ant, the hairless concave ant, the lawn pavement ant, the Bruton ant, the big-headed ant #7, the big-headed ant #9 and the genus Cleatocystis; The mixture of the eight primer pairs is a mixture of primer pairs for identifying the flat iridescent ant, the Chinese house ant, the hairless concave ant, the lawn pavement ant, the brunner's ant, the big-headed ant #7, the big-headed ant #9 and the genus Cleome; The primer pair for identifying the flat iridescent ant is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; The primer pair for identifying the Chinese house ant is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6; The primer pair for identifying the hairless ant is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.9, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.10; The primer pair for identifying the meadow pavement ant is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.13, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.14; The primer pair for identifying Nielsenia brunneri is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.18; The primer pair for identifying the genus Myrmecophaga #7 is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.21, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.22; The primer pair for identifying the genus Myrmecophaga #9 is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO. 25, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 26; The primer pair for identifying the genus Cleometheus is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.29, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

30.

2. A primer pair combination for identifying which of eight ant species an ant is, characterized in that: The primer pair combination consists of primer pairs for identifying the flat rainbow ant, the Chinese house ant, the hairless ant, the lawn pavement ant, the brunner's ant, the big-headed ant #7, the big-headed ant #9 and the genus Cleome; The primer pair for identifying the flat iridescent ant is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; The primer pair for identifying the Chinese house ant is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6; The primer pair for identifying the hairless ant is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.9, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.10; The primer pair for identifying the meadow pavement ant is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.13, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.14; The primer pair for identifying Nielsenia brunneri is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.18; The primer pair for identifying the genus Myrmecophaga #7 is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.21, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.22; The primer pair for identifying the genus Myrmecophaga #9 is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO. 25, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 26; The primer pair for identifying the genus Cleomethecus is as follows: the nucleotide sequence of the forward primer is shown in SEQ ID NO.29, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.30; The eight ant species are flat rainbow ants, Chinese house ants, hairless concave ants, grassland pavement ants, Bruton's ants, Bighead ant genus #7, Bighead ant genus #9 and Cleatocystis genus.

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