Common Thrips specific COI primers, kits and applications

Abstract

The present invention provides common thrips-specific COI primers, a kit and an application. According to the conserved sequence of mtDNA of common thrips, the present invention designs a pair of common thrips-specific COI primers (the forward primer sequence is shown in SEQ ID No. 1 and the reverse primer sequence is shown in SEQ ID No. 2). After detection, the pair of primers has the ability of specific amplification only for common thrips, the amplification product size is 311 bp, the DNA concentration detection limit is 0.0469 ng / μL, the sensitivity is high, and it can be used to evaluate the predation ability of natural enemies on common thrips. The present invention utilizes PCR detection technology to obtain a specific single band by amplification of specific primers, is easy to operate, has the characteristics of being fast, cheap, and highly specific, and improves the accuracy of detection.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to common giant thrips-specific COI primers, a kit and applications. Background Art

[0002] Megalurothrips usitatus is the main pest on leguminous plants in Hainan, causing serious damage to melons and vegetables in Hainan in winter. Hainan is located in the tropics, and the occurrence and outbreak period of Megalurothrips is long, and multiple generations can be reproduced in one year. Both nymphs and adults of Megalurothrips can feed on young and tender tissues, flowers and fruits of the plants they harm, especially flower buds. At the peak, there are hundreds of Megalurothrips in a flower, which often causes the flower buds of plants to shrink, become deformed or even fall off, seriously affecting the formation of fruit pods. Chemical pesticides are still the main measure to prevent and control thrips due to their efficient toxicity. However, Megalurothrips often hide in flower buds and soil, making some chemical pesticides unable to directly contact thrips, resulting in poor control effects. Moreover, due to the long-term use of chemical pesticides, the resistance of thrips to various insecticides has continued to increase, further leading to increased dosage, causing problems such as excessive pesticide residues, and seriously threatening food safety.

[0003] Biological control, such as the use of natural enemy insects to carry out pest control, has attracted more and more attention. Evaluating and screening efficient natural enemy insects is an important task in carrying out biological control. Traditionally, direct observation or enzyme-linked immunosorbent assay methods are used to evaluate the control efficiency of natural enemies against pests. However, due to the small size of thrips and the similar morphology of many species, it is difficult to observe directly, and methods such as enzyme-linked immunosorbent assay require the preparation of antibodies, which is expensive. In recent years, with the development of molecular biology, molecular detection technology has been widely used in the field of insect ecology. Its high sensitivity, rapid response and simple operation have become an important means to evaluate the predation effect. Moreover, molecular detection often uses mitochondrial genes, which have the characteristics of many copies, high evolution rate, and easy detection as target genes, which can more accurately reflect the predation effect of natural enemy insects. At present, there have been research reports on the application of this technology in China, such as the study of the predation of the protoceratops on peanut aphids, the feeding determination of lacewings on corn aphids, the predation detection of the green stink bug on cotton field pests, and the predation study of the middle black stink bug. It is feasible to detect prey components in predators using DNA marker technology. However, there are no reports on detecting common Macrothrips in predators using molecular detection technology. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a common giant thrips-specific COI primer, a kit and an application thereof.

[0005] The technical solution of the present invention is as follows:

[0006] One of the purposes of the present invention is to provide a common thrips-specific COI primer, comprising a forward primer and a reverse primer, wherein the sequence of the forward primer is shown in SEQ ID NO.1, and the sequence of the reverse primer is shown in SEQ ID NO.2.

[0007] The second object of the present invention is to provide a kit for detecting common giant thrips, wherein the kit contains the common giant thrips-specific COI primer according to claim 1.

[0008] The third object of the present invention is to provide the use of the common giant thrips-specific COI primer or the kit in detecting common giant thrips.

[0009] The fourth object of the present invention is to provide the use of the common giant thrips-specific COI primer or the kit in evaluating the predation ability of natural enemies on the common giant thrips.

[0010] The specific detection steps are:

[0011] 1) Extracting DNA from the sample to be tested;

[0012] 2) using the DNA extracted in step 1) as a template, and performing a PCR amplification reaction using the common giant thrips-specific COI primers described in claim 1;

[0013] 3) Analyze the PCR product and determine whether the sample to be tested belongs to common Macrothrips according to the size of the amplified product.

[0014] Furthermore, step 3) is: performing agarose gel electrophoresis on the PCR amplification product, and if a DNA amplification band with a size of 311 bp appears, the sample is determined to be common giant thrips.

[0015] Further, the PCR reaction system is:

[0016] Each 25 μL contains:

[0017]

[0018] Furthermore, the PCR amplification program was as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 52°C annealing for 30 s, and 72°C extension for 1 min, for a total of 30 cycles.

[0019] Compared with the prior art, the beneficial results of the present invention are:

[0020] According to the mitochondrial COI gene sequence (Accession EF213759.1) of common giant thrips published in GenBank, a pair of specific COI primers are designed. The primers are species specific and have an amplification effect only on common giant thrips, but have no amplification ability on other species in cowpea and other nearby bean crop fields, including other thrips of closely related species and other insects. The size of the amplified product is 311 bp, the detection limit of DNA concentration is 0.0469 ng / μL, and the sensitivity is very high.

[0021] The present invention utilizes PCR (polymerase chain reaction) detection technology to obtain a specific single band by amplifying specific primers, which is easy to operate, fast, cheap, and highly specific, thereby improving the accuracy of detection.

[0022] When thrips are preyed on by natural enemies, part of the thrips tissue will be digested, and the thrips individuals are originally small, resulting in a low content of thrips in the natural enemies, so it is difficult to detect the genes of thrips in the natural enemies. The specific COI primers provided by the present invention can achieve 100% detection immediately after the ladybug preys on the common large thrips, and the detection rate is still as high as 80% after 4 hours, indicating that the specific primers can be used to evaluate the predation ability of natural enemies on the common large thrips. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The PCR detection results of the common giant thrips specific primers Mus-c1-F6 and Mus-c1-R7 in Example 1 of the present invention; wherein M is a DNA molecular weight standard 2000bp ladder; 1-26 are the amplification results of the insects corresponding to the serial numbers in Table 1, and "—" is a negative control.

[0024] Figure 2 The results of DNA concentration gradient detection of common giant thrips specific primers Mus-c1-F6 and Mus-c1-R7 in Example 2 of the present invention; wherein, M is a DNA molecular weight standard 2000bp ladder; 1-6 are concentration gradients of template DNA diluted 2 times in sequence; and "-" is a negative control.

[0025] Figure 3 This is the detection result of six-spotted ladybird beetle after predation on common giant thrips by using common giant thrips specific primers Mus-c1-F6 and Mus-c1-R7 in Example 3 of the present invention. The detection rates at 0h, 2h, 4h, and 6h are 100%, 90%, 80%, and 40%, respectively. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0027] Example 1. Amplification effect of the common thrips specific primers Mus-c1-F6 and Mus-c1-R7 on the common thrips DNA.

[0028] 1. Preparation of the genome of Thrips spp.

[0029] A single common Macrothrips was placed in a 1.5 mL centrifuge tube and ground for extraction. The obtained DNA solution was stored at -20°C for later use, and 2 μL of the solution was taken as a DNA template for PCR amplification.

[0030] 2. Synthesis of specific COI primers for detecting common thrips

[0031] The common Thrips-specific COI primer sequences are as follows:

[0032] Forward primer Mus-c1-F6: 5'-TTCAATTACATTATTATTAATAGGTCTT-3' (SEQ ID No. 1)

[0033] Reverse primer Mus-c1-R7: 5'-GATCTGTTAAAAGCATTGTAATTC-3' (SEQ ID No. 2)

[0034] 3. PCR amplification

[0035] The reaction system was 25 μL, including: 0.5 μL EasyTaq DNA polymerase, 2.5 μL 10×EasyTaq buffer, 2 μL dNTPs (2.5 mM), 2 μL DNA template, 0.5 μL each of forward and reverse primers (10 μM), and 17 μL ddH2O.

[0036] PCR amplification program: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 52°C annealing for 30 s, 72°C extension for 1 min, for a total of 30 cycles.

[0037] 4. PCR product identification

[0038] The PCR amplification products were separated by 1.4% agarose gel electrophoresis, stained with ethidium bromide, and determined by the size of the amplification products in a gel imaging system, with the D2000 standard molecular weight as a reference.

[0039] 5. Results

[0040] PCR amplification was performed using primers Mus-c1-F6 and Mus-c1-R7, with DNA of common giant thrips as template and other common thrips, predatory natural enemies of common giant thrips and other insect species (see Table 1) in cowpea fields as controls. The results are shown in Table 1. Figure 1As shown, only the common giant thrips corresponding to lanes 3 and 4 amplified the target band of 311 bp, indicating that the primers of the present invention have strong specificity. The target band of 311 bp was recovered and sequenced, and its sequence is shown in SEQ ID No.3.

[0041] Table 1 Insect species used in species-specific tests

[0042]

[0043] Example 2. Determination of the minimum detection amount of DNA of common giant thrips by using common giant thrips specific primers Mus-c1-F6 and Mus-c1-R7

[0044] The single-headed common giant thrips genomic DNA was extracted according to Example 1, and amplified according to the reaction system and PCR reaction conditions described in Example 1. The common giant thrips genomic DNA was extracted as a template, and the DNA template (original concentration was 1.5000 ng / ul) was diluted 2 times in a gradient manner, and amplified with the screened specific primers, and the DNA concentration was diluted until no band was amplified.

[0045] Primers Mus-c1-F6 and Mus-c1-R7 were used to determine the minimum detection amount. PCR amplification was performed using genomic DNA of common giant thrips at different dilutions as templates. The detection limit of DNA concentration was 0.0469 ng / μL ( Figure 2 ).

[0046] Example 3. Application of common giant thrips-specific primers to evaluate the predation ability of natural enemies on this thrips

[0047] (Take the six-spotted ladybug, a dominant natural enemy in southern farmland, as an example)

[0048] Before the test, the six-spotted ladybugs were starved and placed in a 9-cm-diameter petri dish with moistened cotton wool for 24 hours. The three-instar larvae and adults of the six-spotted ladybugs were starved to prey on common giant thrips. After the feeding was completed, the digestion time was set to 0h, 2h, 4h, and 6h. After the digestion was completed, the six-spotted ladybugs were frozen to death with liquid nitrogen, and moistened cotton wool was added during the digestion process. Ten six-spotted ladybugs were required for each treatment.

[0049] According to Example 1, the genomic DNA of the six-spotted ladybug was extracted after a single head preyed on the common giant thrips (0h, 2h, 4h, 6h), and amplified according to the reaction system and PCR reaction conditions described in Example 1. The results showed that the detection rates of the six-spotted ladybug after preying on the common giant thrips were 100%, 90%, 80%, 40% and 60% respectively at 0h, 2h, 4h and 6h. Figure 3), that is, the detection rate can reach 100% immediately after the ladybugs prey on the common giant thrips, and the detection rate is still as high as 80% after 4 hours, indicating that the specific primers can be used to evaluate the predation ability of natural enemies on common giant thrips.

[0050] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Common Thrips specific COI primers, including a forward primer and a reverse primer, characterized in that: The sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO.

2.

2. A kit for detecting common Macrothrips, characterized in that: The kit contains the common Thrips-specific COI primer according to claim 1.

3. Use of the common thrips-specific COI primer according to claim 1 or the kit according to claim 2 in detecting common thrips.

4. Use of the common giant thrips-specific COI primer according to claim 1 or the kit according to claim 2 in evaluating the predation ability of natural enemies on common giant thrips.

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