Specific primers for identifying chrysopa nipponica species and PCR (Polymerase Chain Reaction) identification method

Abstract

The invention relates to the field of insect molecular detection, in particular to species-specific primers for identifying chrysopa nipponica and a PCR (polymerase chain reaction) identification method. The chrysopa nipponica species specific primer provided by the invention can distinguish other chrysopa natural enemy insect species occurring in the same domain as chrysopa nipponica and successfully identify the real chrysopa nipponica species, the sensitivity of the species specific primer is extremely high, the lowest detection concentration of the species specific primer to DNA is 0.002625 ng / [mu] L, and the specific primer can be used for identifying the chrysopa nipponica species. The detection concentration is far lower than the lowest detection concentration of the insect species specific primer disclosed at present, and a sample with low DNA original concentration and degraded DNA is expected to be detected.

Description

Technical Field

[0001] The present invention relates to the field of insect molecular detection, and in particular to species-specific primers and a PCR identification method for identifying Japanese lacewings. Background Art

[0002] Japanese lacewings (Chrysoperla nipponensis) belong to the genus Chrysoperla in the family Chrysopidae, order Neuroptera, class Insecta, and are common lacewings species in agricultural and forestry ecosystems. The larvae and some adults of Chrysopidae insects can prey on aphids, scale insects, spider mites, leafhoppers, psyllids, whiteflies, thrips, spider mites, and eggs and young larvae of Lepidoptera, and are an important class of natural enemy insects. Different lacewings species have different adaptability to prey. If lacewings are released into crop areas where a certain pest that is not suitable for them is the dominant population, their biological control effect may be poor. The research on the difference in morphological characteristics between Japanese lacewings and other lacewings species mainly focuses on male adults. In addition, it is a holometabolous insect with a complete life history including four stages: egg, larva, pupa and adult. The morphological differences in each growth and development stage are large, and even professional lacewings taxonomists find it difficult to accurately identify them. Therefore, there are often problems with counterfeiting of Japanese lacewings circulating in the natural enemy insect market.

[0003] DNA barcoding refers to the use of single or multiple molecular markers of uniform standards to identify species or associate different developmental stages of the same species, and is not restricted by the sex of the species. This technology achieves species division based on the differences in the sequence composition of the COI gene among species. Its main process includes DNA extraction, amplification of COI gene fragments, sequencing of amplified products and sequence processing and analysis, which takes about 3 days at the fastest, and sequencing and sequence analysis require certain economic and technical costs. The rapid identification method based on species-specific primers can effectively solve the above problems. It only requires one PCR reaction and one agarose gel electrophoresis using species-specific primers to quickly identify the target species. It is simple to operate and economical and efficient. Japanese lacewings and other lacewings species such as the big lacewings, the beautiful lacewings, the fork-shaped lacewings and some species of the genera ... Summary of the invention

[0004] The present invention overcomes the difficulty in morphological identification of Japanese lacewings, reduces the time, economic and technical costs of molecular species identification based on DNA barcode universal primers, and effectively distinguishes other natural enemy insect species of the Chrysopidae family that occur in the same area as the Japanese lacewings. Species-specific primers and a PCR identification method for identifying Japanese lacewings are provided.

[0005] The object of the present invention is to provide a species-specific primer pair for identifying Japanese lacewings.

[0006] The species-specific primer pair for identifying Japanese lacewings according to the present invention comprises the following primers:

[0007] Upstream primer CHN-F: 5'-AGTTTACCCTCCTTTATCATCA-3' (SEQ ID No: 1),

[0008] Downstream primer CHN-R: 5'-CACCAGCTAATACAGGCAAT-3' (SEQ ID No: 2).

[0009] The PCR identification method of Japanese lacewings according to the present invention comprises the following steps:

[0010] PCR amplification is performed on the sample to be tested using the upstream primer CHN-F and the downstream primer CHN-R mentioned above;

[0011] The agarose gel electrophoresis results of the PCR reaction products showed that the sample with a specific band of 233 bp was a sample of the Japanese lacewings.

[0012] Compared with the existing technical methods, the present invention has the following advantages:

[0013] First, the species-specific primers and PCR identification method for identifying Japanese lacewings provided by the present invention do not require sequencing and analysis of PCR products, do not require operators to have a knowledge background in lacewing morphology and taxonomy, are applicable to any detection unit capable of DNA extraction and amplification, can obtain identification results within 1 day, and have the advantages of being economical, efficient, and simple to operate.

[0014] Second, the method of the present invention is based on the comparison of representative haplotype sequences of species to determine species difference sites, taking into account the differences in sequence composition of the COI gene within species due to geographical isolation, and designs primers that can better distinguish different species, especially between similar species, to reduce the binding of primers to similar sequences of non-target species, thereby reducing the possibility of nonspecific amplification.

[0015] Third, the species-specific primers of the Japanese lacewings provided by the present invention can distinguish other natural enemy insect species of the Chrysopidae family that occur in the same area as the Japanese lacewings, and successfully identify the true Japanese lacewings species.

[0016] Fourth, the species-specific primers for identifying the giant lacewings provided by the present invention have extremely high sensitivity, and the minimum detection concentration of DNA is 0.002625 ng / μL, which is much lower than the minimum detection concentration of the currently disclosed insect species-specific primers. It is expected to test samples with low original DNA concentration and degraded DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The electrophoresis results of PCR products based on universal primers of DNA barcodes of Chrysopidae are shown. M is DL2000DNAMaker, 1-3 are Axolotl lacewings, 4-6 are Pseudocerma lutea, 7-8 are Pseudocerma equiloba, 9-10 are Pseudocerma lacewings, 11-13 are Pseudocerma japonica, 14-16 are Pseudocerma lutea, 17-19 are Pseudocerma lutea, 20-22 are Pseudocerma lutea;

[0018] Figure 2 The electrophoresis results of PCR products based on the species-specific primers designed by Wang Dongmei et al. for Japanese lacewings are shown. M is DL2000 DNAMaker, 1-3 are Harmonia axyridis, 4-6 are Pseudocerma lutea, 7-8 are Equifolium lacewings, 9-10 are Pseudocerma lutea, 11-13 are Japanese lacewings, 14-16 are Pseudocerma lutea, 17-19 are Pseudocerma lutea, and 20-22 are Pseudocerma lutea.

[0019] Figure 3 The distribution of interspecific difference sites of 8 lacewings species on the DNA barcode and the coverage positions of species-specific primers of Japanese lacewings are shown. The highlighted sites are interspecific difference sites, the right-pointing arrow is the position covered by the upstream primer on the DNA barcode, and the left-pointing arrow is the position covered by the downstream primer on the DNA barcode;

[0020] Figure 4 The results of 4 pairs of primers in the screening process of the specific primers for Japanese lacewings are shown, (a) is the starting and ending positions of primer CHN1-F / R on the reference sequence and its electrophoresis result, (b) is the starting and ending positions of primer CHN2-F / R on the reference sequence and its electrophoresis result, (c) is the starting and ending positions of primer CHN6-F / R on the reference sequence and its electrophoresis result, (d) is the starting and ending positions of primer CHN10-F / R on the reference sequence and its electrophoresis result; M is DL2000DNAMaker, 1-3 is leaf-colored lacewings, 4-6 is big lacewings, 7-9 is beautiful lacewings;

[0021] Figure 5 The electrophoresis results of PCR products based on the primers CHN-F / R specific to Japanese lacewings are shown. M is DL2000DNAMaker, 1-3 are Axolotl lacewings, 4-6 are Pseudocerma lacewings, 7-8 are Equifolium lacewings, 9-10 are Pseudocerma lacewings, 11-13 are Japanese lacewings, 14-16 are Pseudocerma lacewings, 17-19 are Pseudocerma lacewings, and 20-22 are Pseudocerma lacewings.

[0022] Figure 6The results of sensitivity test of primer CHN-F / R specific for Japanese lacewings are shown. The samples for each concentration test are three samples of Japanese lacewings, CH0101, CH0102 and CH0103. M is D2000DNAMaker, 1-3 are original DNA of Japanese lacewings, 4-6 are 10-fold dilution of DNA of Japanese lacewings, 7-9 are 60-fold dilution of DNA of Japanese lacewings, 10-12 are 250-fold dilution of DNA of Japanese lacewings, and 13-15 are 400-fold dilution of DNA of Japanese lacewings. DETAILED DESCRIPTION

[0023] The specific embodiments of the present invention are described in detail below. It should be noted that the following reagents, materials and instruments can be obtained from commercial sources unless otherwise specified. The DNA barcodes (sequences) described herein all refer to the 658 bp segment at the 5' end of the mitochondrial COI gene.

[0024] Example 1: Extraction of total genomic DNA and qualitative testing of its quality

[0025] 1. Extraction of total genomic DNA

[0026] The total genomic DNA of 22 specimens of 8 lacewing species (specimen information is shown in Table 1) was extracted based on the HiPure Universal DNA Kit. The specimens were stored in 95% alcohol and stored in a -20°C refrigerator before DNA extraction.

[0027] Table 1 Test specimen information

[0028]

[0029]

[0030] 2. Qualitative test of total genomic DNA quality

[0031] The DNA of the 22 specimens extracted in the previous step was amplified based on the universal primers for DNA barcodes of Chrysopidae, and the size and brightness of the bands were observed by agarose gel electrophoresis to determine whether the DNA was successfully extracted. The universal primers for DNA barcodes of Chrysopidae are COI4-F 5'-TAAACTTCTGGATGTCCAAAAAATCA-3' and COI4-R 5'-TGTAAAACGACGGCCAGTAAACTAATARCCTTCAAAG-3'.

[0032] All 22 PCR products showed a single band ( Figure 1), and the band size was 850bp of the target fragment amplification product, indicating that the total genomic DNA of the 22 specimens was successfully extracted and can be used for subsequent experiments. The bands in lanes 9, 12, 13, and 22 were relatively weak, indicating that the DNA concentrations of these samples were low.

[0033] Example 2: Specificity test of existing Japanese lacewings species-specific primers

[0034] Download the species-specific primer pair of Japanese lacewings designed by Wang Dongmei et al. in 2020 from the literature. The upstream primer is CN-10F 5'-TGGTTGAACAGTTTACCCTCCT-3', and the downstream primer is CN-8R5'-TGAAAGTAATAATAAAGC-3'. Amplify the DNA of the 22 specimens extracted in the previous step.

[0035] The PCR reaction system was 25 μL, and the components of the system included: 1 μL DNA template, 12.5 μL 2×Taq PCRMasterMix, 1 μL each of upstream and downstream primers, and ddH2O was used to make up to 25 μL.

[0036] PCR amplification program: pre-denaturation at 94°C for 1 min to completely unwind the template DNA into a single strand; 39 thermal cycles: denaturation at 94°C for 30 s to melt the template DNA, annealing at 49°C for 50 s to allow the primer to specifically bind to the single strand, and extension at 65°C for 1 min; after the thermal cycle, final extension at 65°C for 10 min and storage at 4°C.

[0037] Amplification product detection: electrophoresis results show ( Figure 2 ), lanes 1-3 are for A. axyridis, lanes 4-6 are for A. lutea, lanes 7-8 are for A. isophylla, lanes 9-10 are for A. chamaeme, lanes 11-13 are for A. japonica, lanes 14-16 are for A. lepidoptera, lanes 17-19 are for A. serrata, lanes 20-22 are for A. serrata. In addition to the target band of 233 bp amplified in lanes 11-13 of A. japonica, The target band of 233bp appeared in lanes 9-10 of the lacewings, lanes 4-6 of the yellow-spotted lacewings, lanes 7-8 of the isoleaf lacewings, and lanes 14-16 of the leaf-colored lacewings, indicating that the species-specific primers CN-10F and CN-8R of the Japanese lacewings designed by Wang Dongmei et al. in 2020 are not specific to the DNA samples of the Japanese lacewings and cannot effectively identify the Japanese lacewings species.

[0038] Example 3: Design of species-specific primers

[0039] 1. Acquisition of species representative haplotype sequences

[0040] A total of 133 DNA barcode sequences of the big lacewings, beautiful lacewings, leaf-colored lacewings, Japanese lacewings, forked lacewings, isophyllous lacewings, yellow-spotted lacewings, and aberrantly colored lacewings were obtained from the DNA barcode database Barcode of Life Data System and the DNA barcode data accumulated in our laboratory. Among them, there were 24 sequences of Japanese lacewings, 4 sequences of forked lacewings, 37 sequences of the big lacewings, 15 sequences of beautiful lacewings, 6 sequences of leaf-colored lacewings, 4 sequences of isophyllous lacewings, 23 sequences of yellow-spotted lacewings, and 20 sequences of aberrantly colored lacewings. The haplotypes of multiple aligned DNA barcodes of the same species of lacewings were obtained based on DNAsp5.0 software, and the haplotype with the longest sequence and the largest number of individuals with this haplotype was taken as the haplotype of the species.

[0041] 2. Determination of interspecific difference sites

[0042] Based on Mega XI software, the haplotype sequences were compared and the interspecific difference sites were highlighted. According to the conventional method of primer design, 8 DNA barcode sequences (one for each species) were randomly selected from the 133 DNA barcode sequences of the big lacewings, beautiful lacewings, leaf-colored lacewings, Japanese lacewings, fork-shaped lacewings, isophyllous lacewings, yellow-spotted lacewings and heterochromatic fork-shaped lacewings in the previous step, and the DNA barcode sequences of the 8 species were compared based on Mega XI software to highlight the interspecific difference sites.

[0043] 3. Primer Design

[0044] Based on Primer Premier 5.0 software, specific primers for Japanese lacewings were designed using the representative haplotype sequence of Japanese lacewings and randomly selected DNA barcode sequences as reference sequences.

[0045] (1) Using the randomly selected DNA barcode sequence of Japanese lacewings as a reference for design: the primer pair CHN1-F 5'-GTCAACCAGGTTCTTTA-3' (SEQ ID No: 3) and CHN1-R 5'-ACCAGCAAGGTGTAA-3' (SEQ ID No: 4) with the highest score given by the software was the candidate primer for Japanese lacewings species-specific primers, and the length of the target fragment amplified by it was 339 bp;

[0046] (2) Using the representative haplotype sequence of Japanese lacewings as a reference for design: Based on the primer pair with the highest score given by the software, the primer binding positions were manually adjusted multiple times so that the 3' ends of the forward and reverse primers covered the most interspecies difference sites ( Figure 3), and further screened and adjusted according to the following standards: 1) The GC content of the primer should be between 40 and 60%, the Tm should be between 50 and 70°C, and the difference between the Tm of the forward and reverse primers should be less than 5°C; 2) The 3' end of the primer: no modification should be made, no more than 3 consecutive GGG or CCC should appear, and no hairpin structure or dimer should be formed; 3) The bases should be randomly distributed, and there should not be 4 consecutive bases 1 complementary between the primer itself and the forward and reverse primers; 4) The primer length should be 20 to 30 bases. Finally, the Japanese lacewing species-specific primer pair was obtained: upstream primer CHN-F 5'-AGTTTACCCTCCTTTATCATCA-3', downstream primer CHN-R 5'-CACCAGCTAATACAGGCAAT-3', the length of the target fragment amplified by it was 233 bp, and the other three primer pairs: CHN2-F 5'-TCCTTTAATACTAGCTGCTCCTGAT-3' (SEQ ID No: 5), CHN2-R 5'-TGAAATACCAGCAAGGTGTAAACTA-3' (SEQ ID No: 6), the length of the target fragment amplified by it was 220 bp; CHN6-F 5'-TGCTTCTTCTATAGTAGAAAGAGGAG-3' (SEQ ID No: 7), CHN6-R 5'-ACCAGCAAGGTGTAAACTAAAAATA-3' (SEQ ID No: 8), the length of the target fragment amplified by it was 124 bp; CHN10-F 5'-TCCTTTAATACTAGCTGCTCCTGAT-3' (SEQ ID No: 9), CHN10-R 5'-TGAAATACCAGCAAGGTGTAAACTA-3' (SEQ ID No: 10), the length of the target fragment amplified by it was 124 bp; 5'-GATTAGTTCCTTTAATACTAGC-3' (SEQ ID No: 9), CHN10-R 5'-GATGAAATACCAGCAAGG-3' (SEQ ID No: 10), the length of the amplified target fragment is 229 bp.

[0047] Example 4: Specificity test of the species-specific primers of the Japanese lacewings provided by the present invention

[0048] In order to save the consumption of total genomic DNA of experimental samples and simplify the test steps, the specific test of the species-specific primers of Japanese lacewings is divided into two stages: initial screening and final testing. In the initial screening stage, only the DNA of samples of the genus Chrysopidae, the beautiful lacewings and the leaf-colored lacewings are tested. Only the primers that cannot amplify the target bands of the DNA of these three lacewings can be used for the final testing stage. In the final testing stage, the DNA of the 22 specimens extracted in Example 1 is tested. Five pairs of candidate primers for the species-specific primers of Japanese lacewings provided by the present invention are synthesized.

[0049] (1) Preliminary screening of species-specific primers for Japanese lacewings

[0050] The candidate primer pairs based on the five pairs of species-specific primers for the Japanese lacewings were used to amplify the DNA of the nine specimens of the big lacewings, the beautiful lacewings and the leaf-colored lacewings extracted in Example 1, respectively.

[0051] The PCR reaction system was 25 μL, and the components of the system included: 1 μL DNA template, 12.5 μL 2×Taq PCRMasterMix, 1 μL upstream and downstream primers, and 25 μL was supplemented with ddH2O. PCR amplification program: 94°C pre-denaturation for 1 min to completely unwind the template DNA into a single strand; 39 thermal cycles: 94°C denaturation for 30 s to melt the template DNA, 51°C annealing for 50 s to allow the primer to specifically bind to the single strand, and 65°C extension for 1 min; after the thermal cycle, 65°C final extension for 10 min, and storage at 4°C.

[0052] Figure 4 The results of 4 pairs of primers in the screening process of Japanese lacewing-specific primers are shown, (a) is the starting and ending positions of primer CHN1-F / R on the reference sequence and its electrophoresis result, and the length of the target fragment amplified is 339bp; (b) is the starting and ending positions of primer CHN2-F / R on the reference sequence and its electrophoresis result, and the length of the target fragment amplified is 220bp; (c) is the starting and ending positions of primer CHN6-F / R on the reference sequence and its electrophoresis result, and the length of the target fragment amplified is 124bp; (d) is the starting and ending positions of primer CHN10-F / R on the reference sequence and its electrophoresis result; M is DL2000 DNAMaker, 1-3 is leaf-colored lacewing, 4-6 is big lacewing, 7-9 is beautiful lacewing, and the length of the target fragment amplified is 229bp.

[0053] Amplification product detection: electrophoresis results show ( Figure 4 ) Primers CHN1-F / R ( Figure 4 (a) in the figure and three pairs of candidate primers designed with haplotype sequences as reference sequences ( Figure 4 (b)-(d)) in the figure can amplify the target bands for the samples of the big lacewings and the leaf-colored lacewings; when the primer coverage position is adjusted to the middle of the haplotype sequence of the reference sequence, the primer CHN6-F / R binds to the non-target region in the template DNA and amplifies a fragment that does not meet the target size ( Figure 4 (c) in the figure), the primer design failed; therefore, the primers designed by the method of designing primers based on the randomly selected DNA barcode sequence of Japanese lacewings as a reference based on the primer design software are not species-specific. The species-specific site of the DNA barcode sequence of Japanese lacewings is not at its 5' end, but is located at a position about 300-560bp from the end of the DNA barcode sequence ( Figure 3). In summary, these four candidate primer pairs are not specific to Japanese lacewings; while primers CHN-F / R are specific to Japanese lacewings samples ( Figure 5 ).

[0054] (2) Final test of the specificity of the Japanese lacewings species-specific primers

[0055] Based on primers CHN-F / R, DNA from 22 samples extracted in the previous step was amplified and tested by electrophoresis. The PCR system, amplification procedure and electrophoresis operation steps were the same as those of the primer screening in the previous step. Figure 5 ) showed that lanes 11-13 were three samples of Japanese lacewings CH0101, CH0102 and CH0103, all of which amplified a 233bp band, which was consistent with the theoretical product length of 233bp obtained during primer design. No bands were amplified from samples of other lacewings species, indicating that the Japanese lacewings species-specific primer pair CHN-F / R was specific to the DNA samples of Japanese lacewings and could successfully identify the Japanese lacewings species.

[0056] Example 5: Sensitivity test of species-specific primers for Japanese lacewings

[0057] Because the total genomic DNA amount of Japanese lacewings required in this embodiment is large, it is necessary to re-extract the total genomic DNA of three samples CH0101, CH0102 and CH0103 of Japanese lacewings according to the steps of Example 1, and the original concentrations of these three samples are 1.80ng / μL, 1.05ng / μL and 1.84ng / μL, respectively. The original genomic total DNA is diluted with DNA eluent AE at a gradient of 10 times. When the dilution multiple exceeds 100 times, the original genomic total DNA is diluted with a gradient of 50 times. Based on the species-specific primers of Japanese lacewings provided by the present invention, the diluted DNA is subjected to PCR reaction and electrophoresis test. The DNA concentration corresponding to the weak band shown by electrophoresis is the minimum detection concentration of the species-specific primers of Japanese lacewings. The PCR reaction system is 25μL, and the components of the system include: 1μL DNA template, 12.5μL of 2×Taq PCR MasterMix, 1μL of upstream and downstream primers, and 25μL is supplemented with ddH2O. PCR amplification program: pre-denaturation at 94°C for 1 min to completely unwind the template DNA into a single strand; 39 thermal cycles: denaturation at 94°C for 30 s to melt the template DNA, annealing at 51°C for 50 s to allow the primer to specifically bind to the single strand, and extension at 65°C for 1 min; after the thermal cycle, final extension at 65°C for 10 min and storage at 4°C.

[0058] The results are as follows Figure 6As shown, when diluted to 400 times, the Japanese lacewing sample CH0102 (lane 14) showed a weak band. Therefore, the minimum detection concentration of the Japanese lacewing species-specific primers was 0.002625 ng / μL.

[0059] The present invention verifies that the existing designed Japanese lacewing species-specific primers are not specific. In addition to being able to amplify the DNA of Japanese lacewings, they can also amplify the DNA of forked lacewings, yellow-spotted lacewings, equal-leaf lacewings and leaf-colored lacewings, and cannot effectively identify Japanese lacewings based on PCR reactions. The Japanese lacewing species-specific primers CHN-F / R provided by the present invention can effectively distinguish Japanese lacewings and their closely related species, and the specific primers can be used to identify the real Japanese lacewings in the circulation market of natural enemy insects based on PCR reactions. In addition, the present invention verifies that compared with using a random DNA barcode sequence of the species as a reference sequence, the design of species-specific primers using a representative haplotype sequence as a reference sequence can more effectively reduce the binding of primers to similar sequences of non-target species and reduce the possibility of non-specific amplification. The minimum detection concentration of the Japanese lacewing primers provided by the present invention is extremely low, reaching 0.002625 ng / μL, and the amplified target fragment is short, which is expected to be used to test samples with low original DNA concentration and DNA degradation, such as identifying Japanese lacewings in the intestinal contents of other insects or animals that prey on lacewings and in dried lacewing specimens that have been preserved for a long time.

[0060] The specific embodiments disclosed in the present invention are only used to explain the present invention in detail, and those skilled in the relevant art may modify, supplement or make similar substitutions to the actual operation details within the scope of the claims.

Claims

1. A species-specific primer pair for identifying Japanese lacewings, characterized in that: The specific primer pair includes the following primers: Upstream primer CHN-F: 5'-AGTTTACCCTCCTTTATCATCA-3', Downstream primer CHN-R: 5′-CACCAGCTAATACAGGCAAT-3′.

2. A PCR identification method for Japanese lacewings, characterized in that: The method comprises the following steps: Amplifying the DNA of the sample to be tested by PCR using the species-specific primers for identifying the Japanese lacewings according to claim 1; The PCR reaction product is detected. If the agarose gel electrophoresis result of the PCR reaction product shows a specific band of 233 bp, the sample to be tested is a Japanese lacewings sample.

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