A PCR detection primer group, kit and method for distinguishing between diaspidiotus oceanicus and diaspidiotus citri

Abstract

The application discloses a PCR detection primer group, a kit and a method for distinguishing between Dialeuropora oceanica and Dialeuropora citri, and belongs to the technical field of molecular biology. The PCR detection primer group comprises a primer Pmi-PCR-F and a primer Pmi-PCR-R, and the primer sequences are specifically as follows: Pmi-PCR-F: 5'-GAAATTAATACGACTCACTATAGGGCATTATTGGAAGATTAAGAAATTGACTCTTAC-3'; and Pmi-PCR-R: 5'-ATAAAATTAATTGATCTAAAAATAGAAGAAATCCC-3'. The application has high specificity, can accurately distinguish between Dialeuropora oceanica and its similar species Dialeuropora citri and other whiteflies, the result can be visualized, does not need to be sequenced, the equipment has high popularization degree and is mature in technology, and is suitable for routine detection in a laboratory.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically to a PCR detection primer set, kit, and method for distinguishing between the oceanic mealybug and the citrus mealybug. Background Technology

[0002] With the continued expansion of global cross-border trade in agricultural products, the frequency of transporting live plants such as fruits, vegetables, and seedlings has increased significantly. The oceanic mealybug, explicitly designated as a high-risk invasive pest in my country, has become a key target for control in port quarantine and field monitoring. The citrus mealybug, a native species, is extremely similar. Both types of mealybugs can damage over 250 species of fruits, vegetables, and flowers, including citrus, grapes, and mangoes. They induce sooty mold by sucking plant sap and secreting honeydew, causing crop decline, a sharp drop in fruit quality, and in severe cases, death of the entire plant, posing a significant threat to the safety of China's fruit and vegetable industry and ecological balance. Currently, accurate identification of these mealybugs is a crucial preliminary step in preventing their invasion and spread in the field of plant quarantine, directly impacting the effectiveness of the national biosecurity defense line.

[0003] Currently, the two mainstream methods for identifying mealybugs in the industry both have significant shortcomings: First, the traditional morphological identification method. Because the morphological similarity between the Pacific mealybug and the citrus mealybug exceeds 90%, and their common characteristics such as body size, body color, and distribution of wax powder on the body surface highly overlap, they can only be distinguished by microscopic features such as the number of antennal segments and the length of the anal lobes. This not only requires operators to have extremely high classification and identification experience, but also makes it easy to misjudge if the specimens are damaged or immature specimens such as nymphs. The identification process usually takes several hours, which is completely unsuitable for the timeliness requirements of high-volume customs clearance at ports. Second, the existing general molecular detection technology mostly uses broad-spectrum insect DNA barcoding primers for amplification. Subsequent identification requires complex sequence comparison, which is cumbersome, has a long detection cycle, and lacks specificity for closely related species. It is easy to have false positive results due to the amplification of non-target fragments, making it difficult to achieve rapid and accurate differentiation between the two types of mealybugs.

[0004] Existing publicly available PCR detection technologies for mealybugs mostly design primers for a single mealybug species, making it impossible to simultaneously identify both *Mealybug squarrosa* and *Mealybug citrus* in the same reaction system. To detect the two species separately, multiple reaction systems are required, which not only doubles the detection cost but also increases the risk of sample cross-contamination. Other attempts at detection based on multiplex PCR suffer from severe primer dimer interference, blurred amplification bands, and insufficient sensitivity for low-concentration insect samples, making it impossible to accurately detect single nymphs. This results in extremely poor applicability for promotion in grassroots quarantine laboratories.

[0005] In summary, how to provide a simple, specific, and sensitive dedicated PCR detection solution that can quickly and accurately distinguish between the oceanic mealybug and the citrus mealybug in the same reaction system is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a PCR detection primer set, kit and method for distinguishing between the oceanic mealybug and the citrus mealybug, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A PCR detection primer set for distinguishing between the oceanic mealybug and the citrus mealybug includes primer Pmi-PCR-F (upstream primer) and primer Pmi-PCR-R (downstream primer). The primer sequences are as follows: Pmi-PCR-F: 5'-GAAATTAATACGACTCACTATAGGGCATTATTGGAAGATTAAGAAATTGACTCTTAC-3' (SEQ ID NO. 1); Pmi-PCR-R: 5'-ATAAAATTAATTGATCTAAAAATAGAAGAAATCCC-3' (SEQ ID NO. 2).

[0009] A PCR detection kit for distinguishing between the oceanic mealybug and the citrus mealybug includes the above-mentioned PCR detection primer set.

[0010] Furthermore, the PCR detection kit also includes PCR reaction buffer, DNA polymerase, and dNTPs.

[0011] A PCR detection method for distinguishing between the oceanic mealybug and the citrus mealybug includes the following steps: (1) Extract DNA from the sample to be tested; (2) PCR amplification was performed using primers Pmi-PCR-F and Pmi-PCR-R from the primer set as described in claim 1 to obtain PCR amplification products; (3) The PCR amplification products were detected by agarose gel electrophoresis; if a specific band appeared, it was identified as Pacific mealybug; if no band appeared, it was identified as Citrus mealybug or other mealybugs.

[0012] Furthermore, in step (2) above, the PCR amplification reaction system is as follows: 2 μL DNA template, 1 uL of 10 μmol / L primer Pmi-PCR-F, 1 uL of 10 μmol / L primer Pmi-PCR-R, 13.5 μL of 2× Phanta Max Master Mix, and water added to 25 μL.

[0013] Furthermore, in step (2) above, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing for 20 s, 72℃ extension for 18 s, for a total of 35 cycles; 72℃ final extension for 5 min.

[0014] Furthermore, in step (3) above, the voltage for agarose gel electrophoresis is 120V and the time is 30 min.

[0015] The present invention also claims protection for the use of the above-mentioned PCR detection primer set in distinguishing between the oceanic mealybug and the citrus mealybug.

[0016] The present invention also claims protection for the use of the above-mentioned PCR detection kit in distinguishing between the oceanic mealybug and the citrus mealybug.

[0017] The present invention also claims protection for the application of the above-mentioned PCR detection method in distinguishing between the oceanic mealybug and the citrus mealybug.

[0018] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: This invention is based on conventional PCR amplification technology, utilizing specific primers to amplify the target sequence, and determining the results via agarose gel electrophoresis, thereby rapidly distinguishing between the oceanic mealybug and the citrus mealybug. This invention exhibits high specificity, accurately differentiating the oceanic mealybug from its closely related species, the citrus mealybug, and other mealybugs. The results are visualized, requiring no sequencing, and the equipment is widely available and the technology is mature, making it suitable for routine laboratory testing. Attached Figure Description

[0019] Figure 1 This is a graph showing the agarose gel electrophoresis results in Example 3. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 The PCR detection primer set for distinguishing between the oceanic mealybug and the citrus mealybug includes primers Pmi-PCR-F and Pmi-PCR-R, with the specific primer sequences as follows: Pmi-PCR-F: 5'-GAAATTAATACGACTCACTATAGGGCATTATTGGAAGATTAAGAAATTGACTCTTAC-3'; Pmi-PCR-R: 5'-ATAAAATTAATTGATCTAAAAATAGAAGAAATCCC-3'.

[0022] Example 2 A PCR detection kit for distinguishing between the oceanic mealybug and the citrus mealybug includes the PCR detection primer set in Example 1, as well as PCR reaction buffer, DNA polymerase, and dNTPs.

[0023] Example 3 The PCR detection method for distinguishing between the oceanic mealybug and the citrus mealybug includes the following steps: (1) Take the mealybug sample to be tested, add 10 µL of lysis buffer (Tiosbio® Star Flash Nucleic Acid Release Agent Cat. No. BT0069), and repeatedly crush and grind it with the pipette tip to fully break the sample; blow and aspirate the lysis buffer several times with the pipette tip, and take the supernatant as a DNA template for later use; (2) PCR amplification was performed using the PCR detection primer set in Example 1 to obtain PCR amplification products; The PCR amplification reaction system (25 μL system) consisted of: 2 μL DNA template, 1 μL 10 μmol / L primer Pmi-PCR-F, 1 μL 10 μmol / L primer Pmi-PCR-R, 13.5 μL 2× Phanta Max Master Mix, and water added to a final volume of 25 μL. The PCR amplification reaction program was as follows: pre-denaturation: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing for 20 s, 72℃ extension for 18 s, for a total of 35 cycles; final extension at 72℃ for 5 min. (3) Electrophoresis detection Take 5 μL of PCR amplification product and perform electrophoresis on a 2% agarose gel at 120V for 30 min. Observe the results in a gel imaging system. If a specific amplification band appears at the expected size and position, the sample is identified as *Mealyx citrus*. If no amplification band is found, the sample is identified as *Mealyx citrus* or other mealybugs.

[0024] The agarose gel electrophoresis results in Example 3 are shown in the figure below. Figure 1As shown, the marker is a DNA molecular weight marker; the samples in each lane are: 1 for *Stachys aquaticus*, 2 for *Stachys citrus*, 3 for *Stachys nigra*, 4 for *Stachys papaya*, 5 for *Stachys lycoris*, 6 for *Stachys hibiscus*, 7 for *Stachys citrus*, and 8 for the negative control. Figure 1 It can be seen that only the oceanic mealybug lane showed a specific amplification band, while other samples did not show any bands.

[0025] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A PCR detection primer set for distinguishing between the oceanic mealybug and the citrus mealybug, characterized in that, This includes primers Pmi-PCR-F and Pmi-PCR-R, with the following primer sequences: Pmi-PCR-F: 5'-GAAATTAATACGACTCACTATAGGGCATTATTGGAAGATTAAGAAATTGACTCTTAC-3'; Pmi-PCR-R: 5'-ATAAAATTAATTGATCTAAAAATAGAAGAAATCCC-3'.

2. A PCR detection kit for distinguishing between the oceanic mealybug and the citrus mealybug, characterized in that, Includes the PCR detection primer set as described in claim 1.

3. The PCR detection kit for distinguishing between the oceanic mealybug and the citrus mealybug according to claim 2, characterized in that, It also includes PCR reaction buffer, DNA polymerase, and dNTPs.

4. A PCR detection method for distinguishing between the oceanic mealybug and the citrus mealybug, characterized in that, Specifically, the following steps are included: (1) Extract DNA from the sample to be tested; (2) PCR amplification was performed using primers Pmi-PCR-F and Pmi-PCR-R from the primer set as described in claim 1 to obtain PCR amplification products; (3) The PCR amplification products were detected by agarose gel electrophoresis; if a specific band appeared, it was identified as Pacific mealybug; if no band appeared, it was identified as Citrus mealybug or other mealybugs.

5. The PCR detection method for distinguishing between the oceanic mealybug and the citrus mealybug according to claim 4, characterized in that, In step (2), the PCR amplification reaction system is as follows: 2 μL DNA template, 1 μL 10 μmol / L primer Pmi-PCR-F, 1 μL 10 μmol / L primer Pmi-PCR-R, 13.5 μL 2× Phanta Max Master Mix, and water added to 25 μL.

6. The PCR detection method for distinguishing between the oceanic mealybug and the citrus mealybug according to claim 4, characterized in that, In step (2), the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing for 20 s, 72℃ extension for 18 s, for a total of 35 cycles; 72℃ final extension for 5 min.

7. The PCR detection method for distinguishing between the oceanic mealybug and the citrus mealybug according to claim 4, characterized in that, In step (3), the voltage for agarose gel electrophoresis is 120V and the time is 30 min.

8. The application of the PCR detection primer set as described in claim 1 in distinguishing between the oceanic mealybug and the citrus mealybug.

9. The application of a PCR detection kit as described in claim 2 or 3 in distinguishing between the oceanic mealybug and the citrus mealybug.

10. The application of a PCR detection method as described in any one of claims 4-7 in distinguishing between the oceanic mealybug and the citrus mealybug.

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