Primer pairs, kits and applications for identifying three dominant symbiotic bacteria of corn aphids

Abstract

The present invention discloses primer pairs, a kit, and their applications for identifying three dominant symbiotic bacteria of corn aphids. By designing primers for the three dominant symbiotic bacteria, Acinetobacter soli, Sphingomonas paucimobilis, and Acidovorax wautersii, a multiplex PCR reaction system was constructed for simultaneous detection of the three dominant symbiotic bacteria. This detection method can simultaneously identify the infection status of the three dominant symbiotic bacteria in corn aphid samples using a single PCR reaction system, saving time, reducing costs, and conserving precious experimental samples.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer pair, a kit and applications thereof for identifying three dominant symbiotic bacteria of corn aphids, Acinetobacter soli, Sphingomonas paucimobilis and Acidovorax wautersii. Background Art

[0002] Several aphid species can infest corn plants, with the corn aphid, Rhopalosiphum maidis, being the dominant species. Corn aphids not only damage corn but also rice and various grass weeds. Besides feeding directly on foliar sap, corn aphids secrete honeydew, which impacts crop photosynthesis. More seriously, corn aphids can transmit viruses such as maize dwarf mosaic virus, barley yellow dwarf virus, and sugarcane mosaic virus, and can even transfer viruses from corn to other grass crops such as sugarcane and rice. Corn aphid damage is increasing annually, becoming a dominant foliar pest in many corn and rice-growing regions in my country, significantly impacting crop yield and quality. Research has shown that corn aphids are resistant to various chemical pesticides, yet current control measures still primarily rely on chemical pesticides. Therefore, research and development of new, green control technologies for corn aphids are crucial.

[0003] Studies have shown that symbiotic bacteria can facilitate the transmission of plant viruses to host insects, such as Hamiltonella and Arsenophonus symbionts in whiteflies and Buchnera symbionts in aphids. Therefore, research on the diversity and function of aphid symbionts will provide new insights into the "bacteria-to-insect" control strategy. Currently, research on the diversity of corn aphid symbionts is relatively limited, and identification methods for the three dominant symbionts of corn aphids, Acinetobacter soli, Sphingomonas paucimobilis, and Acidovorax wautersii, have not yet been developed. Therefore, it is necessary to develop primer pairs, kits, and application technologies for identifying corn aphid symbionts. Summary of the Invention

[0004] In light of this, the present invention aims to provide a primer pair, kit, and application thereof for identifying the three dominant symbiotic bacteria of corn aphids, Acinetobacter soli, Sphingomonas paucimobilis, and Acidovorax wautersii. This multiplex PCR reaction allows for simultaneous identification of infection status of these three dominant symbiotic bacteria in corn aphid samples, saving time, reducing costs, and conserving valuable laboratory samples.

[0005] The first object of the present invention is to provide a primer set for identifying three dominant symbiotic bacteria of corn aphids, Acinetobactersoli, Sphingomonas paucimobilis, and Acidovorax wautersii, the primer set comprising:

[0006] Primers used to identify Acinetobacter soli:

[0007] AS-F: 5'-GGCTAGAGTGTGGGAGAGGA-3' (SEQ ID NO. 1);

[0008] AS-R: 5'-GCGCCACTAAAGCCTCAAAG-3' (SEQ ID NO. 2);

[0009] Primers used to identify Sphingomonas paucimobilis:

[0010] SP-F: 5'-CACACTGGGACTGAGACACG-3' (SEQ ID NO.3);

[0011] SP-R: 5'-ACGCCCAGTAATTCCGAACA-3' (SEQ ID NO.4);

[0012] Primers used to identify Acidovorax wautersii:

[0013] AW-F: 5'-GGTGATGTAAGACAGATGTG-3' (SEQ ID NO. 5);

[0014] AW-R: 5'-GGTGTTTCCTCCGCATATC-3' (SEQ ID NO. 6).

[0015] The second object of the present invention is to provide a kit comprising the above primer set.

[0016] Preferably, the kit further comprises 2×Taq Mix and ddH2O.

[0017] The third object of the present invention is to provide the use of the above primer set or kit in identifying the three dominant symbiotic bacteria of corn aphids, Acinetobacter soli, Sphingomonas paucimobilis, and Acidovorax wautersii.

[0018] A fourth object of the present invention is to provide a method for identifying three dominant symbiotic bacteria of corn aphids, Acinetobactersoli, Sphingomonas paucimobilis, and Acidovorax wautersii, comprising the following steps:

[0019] (1) Extracting total DNA from corn aphid samples;

[0020] (2) using the extracted DNA as a template, a multiplex PCR amplification reaction was performed using the above primer set;

[0021] (3) The multiplex PCR amplification products were analyzed by electrophoresis.

[0022] Preferably, the multiplex PCR reaction system in step (2) comprises: 12.5 μL of 2×Taq Mix, 0.5 μL each of AS-F / R, 0.5 μL each of SP-F / R, 0.5 μL each of AW-F / R, 8.5 μL of ddH2O, and 1.0 μL of DNA template, for a total of 25 μL.

[0023] Preferably, the multiplex PCR reaction program in step (2) is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 6 s, 34 cycles; extension at 72°C for 5 min.

[0024] Preferably, the electrophoresis analysis in step (3) is specifically as follows: when the amplified product contains a 211 bp fragment, it indicates that the corn aphid sample contains the symbiotic bacterium Acinetobacter soli; when the amplified product contains a 235 bp fragment, it indicates that the corn aphid sample contains the symbiotic bacterium Sphingomonas paucimobilis; when the amplified product contains a 134 bp target fragment, it indicates that the corn aphid sample contains the symbiotic bacterium Acidovorax wautersii.

[0025] Based on the detection of the diversity of corn aphid symbionts, the present invention simplifies the identification process of the dominant symbionts of corn aphids by designing primers for the three dominant symbionts of corn aphids, Acinetobacter soli, Sphingomonas paucimobilis, and Acidovorax wautersii, analyzing the primer sensitivity, and constructing a multiplex PCR reaction system for simultaneously detecting the three dominant symbionts.

[0026] This detection method can improve detection efficiency, shorten detection time, and provide more accurate and effective primer pairs for identifying the dominant symbiotic bacteria of corn aphids. It can simultaneously identify the infection status of three dominant symbiotic bacteria in corn aphid samples in a single PCR reaction system, saving time, reducing costs, and saving precious experimental samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results of the symbiotic bacteria Acinetobacter soli are shown in Figure 1. Lane M: DL2000 marker; Lanes 1-6 represent winged, wingless, 1st, 2nd, 3rd, and 4th instars of the corn aphid, respectively; Lane 7 is the negative control, H2O.

[0028] Figure 2 The results of the symbiotic bacteria Sphingomonas paucimobilis were shown. Lane M: DL2000 marker; Lanes 1-6: winged adults, wingless adults, 1st instar, 2nd instar, 3rd instar, and 4th instar of the corn aphid; Lane 7: negative control H2O.

[0029] Figure 3 The results of the symbiotic bacteria Acidovorax wautersii test are shown in Figure 1. Lane M: DL2000 marker; Lanes 1-6 represent winged, wingless, 1st, 2nd, 3rd, and 4th instar aphids, respectively; Lane 7 is the negative control, H2O.

[0030] Figure 4 The sensitivity test results of primer AS-F / R are shown in Figure 1. The DNA concentrations in lanes 1-4 are 40, 20, 2, and 0.2 μg / mL, respectively, and lane 5 is the negative control, H2O.

[0031] Figure 5 The sensitivity test results of primer SP-F / R are shown in Figure 1. The DNA concentrations in lanes 1-4 are 40, 20, 2, and 0.2 μg / mL, respectively, and lane 5 is the negative control, H2O.

[0032] Figure 6 The sensitivity test results of primers AW-F / R are shown in Figure 1. The DNA concentrations in lanes 1-4 are 40, 20, 2, and 0.2 μg / mL, respectively, and lane 5 is the negative control, H2O.

[0033] Figure 7 The results of simultaneous amplification using three primer pairs are shown. Lane M: DL2000 marker; Lanes 1-6: winged adults, wingless adults, 1st instar, 2nd instar, 3rd instar, and 4th instar of corn aphid, respectively; Lane 7: negative control H2O. DETAILED DESCRIPTION

[0034] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0035] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0036] Example 1

[0037] Winged and wingless adults of corn aphids, as well as 1st, 2nd, 3rd, and 4th instars, were selected and rinsed multiple times in 75% alcohol to remove bacterial contamination on the aphids' surface. After the alcohol was fully evaporated, genomic DNA of corn aphids was extracted using the Rapid DNA Extraction Kit EE101 (Beijing Quanshijin Biotechnology Co., Ltd.).

[0038] The DNA extraction steps are as follows:

[0039] 1) Place 30 corn aphids in a sterile 1.5 mL centrifuge tube, add 100 μL LB2 and 20 μL Proteinase K (ensure that the corn aphids are completely incorporated into the centrifuge tube), and incubate at 55° C. until completely lysed.

[0040] 2) Add 20 μL of RNAase A to the sample and incubate at room temperature for 2 minutes.

[0041] 3) Centrifuge at 12000×g for 5 minutes and transfer the supernatant to a sterile centrifuge tube.

[0042] 4) Add 500 μL of BB2, vortex immediately for 5 seconds, and incubate at room temperature for 5 minutes.

[0043] 5) Add all the solution to the centrifuge column, centrifuge at 12000×g for 30 seconds, and discard the flow-through.

[0044] 6) Add 500 μL of CB2 (containing ethanol), centrifuge at 12,000 × g for 30 seconds, and discard the flow-through.

[0045] 7) Add 500 μL WB2 (containing ethanol), centrifuge at 12,000 × g for 30 seconds, and discard the flow-through.

[0046] 8) Repeat step 7) once.

[0047] 9) Centrifuge at 12000×g for 2 minutes to completely remove residual WB2.

[0048] 10) Place the spin column in a clean centrifuge tube and add 50-200 μL of preheated EB (60°C-70°C) to the center of the column. Let stand at room temperature for 1 minute. Centrifuge at 12,000 × g for 1 minute to elute the DNA. Store the eluted DNA at -20°C until needed.

[0049] The extracted DNA was amplified by PCR using primers AS-F / R, SP-F / R and AW-F / R (Table 1).

[0050] Table 1 Primer sequences

[0051]

[0052]

[0053] The PCR reaction system is:

[0054]

[0055] The PCR reaction program is:

[0056]

[0057] Take 5 μL of PCR product and detect it by 2% agarose gel electrophoresis. Figure 1-3 shown.

[0058] Depend on Figure 1-3 As shown, primers AS-F / R, SP-F / R, and AW-F / R were used to amplify different samples of corn aphids, effectively amplifying the three dominant symbiotic bacteria of corn aphids, Acinetobacter soli, Sphingomonaspaucimobilis, and Acidovorax wautersii. This result demonstrates that all three primer pairs provided by the present invention have good specificity.

[0059] Example 2

[0060] DNA of the first instar of corn aphid No. 7 was randomly selected and diluted to 40, 20, 2.0, and 0.2 μg / mL, respectively, for primer sensitivity testing. The primers are shown in Table 1.

[0061] The PCR reaction system is:

[0062]

[0063]

[0064] The PCR reaction procedure is the same as that of Experiment 1.

[0065]

[0066] Take 5 μL of PCR product and detect it by 2% agarose gel electrophoresis. Figure 4-6 shown.

[0067] Depend on Figure 4It can be seen that primer AS-F / R can also effectively amplify the target fragment of the symbiotic bacterium Acinetobacter soli when the template DNA concentration is 0.2 μg / mL.

[0068] Depend on Figure 5 It can be seen that primer SP-F / R can also effectively amplify the target fragment of the symbiotic bacterium Sphingomonas paucimobilis when the template DNA concentration is 0.2 μg / mL.

[0069] Depend on Figure 6 It can be seen that primers AW-F / R can also effectively amplify the target fragment of the symbiotic bacterium Acidovorax wautersii when the template DNA concentration is 2 μg / mL.

[0070] The above results show that the three pairs of primers provided by the present invention all have good detection sensitivity.

[0071] Example 3

[0072] Winged adults, wingless adults, 1st, 2nd, 3rd, and 4th instars of corn aphids were selected, and three primer pairs (AS-F / R, SP-F / R, and AW-F / R) (Table 1) were added to each sample for PCR amplification.

[0073] PCR system

[0074]

[0075] The PCR reaction program is:

[0076]

[0077] Take 5 μL of PCR product and detect it by 2% agarose gel electrophoresis. Figure 7 shown.

[0078] Depend on Figure 7 It can be seen that under the above PCR amplification system and conditions, the three pairs of primers used to detect the three dominant symbiotic bacteria of corn aphids can be used to detect corn aphid symbiotic bacteria by multiplex PCR.

[0079] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer set for identifying three dominant symbiotic bacteria of corn aphids, Acinetobacter soli, Sphingomonaspaucimobilis, and Acidovoraxwautersii, characterized in that: The primer set includes: Primers used to identify Acinetobacter soli: AS-F: 5'-GGCTAGAGTGTGGGAGAGGA-3'; AS-R: 5'-GCGCCACTAAAGCCTCAAAG-3'; Primers used to identify Sphingomonas paucimobilis: SP-F: 5'-CACACTGGGACTGAGACACG-3'; SP-R: 5'-ACGCCCAGTAATTCCGAACA-3'; Primers used to identify Acidovorax wautersii: AW-F: 5'-GGTGATGTAAGACAGATGTG-3'; AW-R: 5'-GGTGTTTCCTCCGCATATC-3'.

2. A kit, characterized in that The kit comprises the primer set according to claim 1.

3. The kit according to claim 2, wherein The kit also includes 2×Taq Mix and ddH 2 O.

4. Use of the primer set according to claim 1 or the kit according to claim 2 in identifying three dominant symbiotic bacteria of corn aphids, Acinetobacter soli, Sphingomonas paucimobilis, and Acidovorax wautersii.

5. A method for identifying three dominant symbiotic bacteria of corn aphids, Acinetobacter soli, Sphingomonaspaucimobilis, and Acidovoraxwautersii, characterized in that: The following steps are involved: (1) Extracting total DNA from corn aphid samples; (2) using the extracted DNA as a template and performing a multiplex PCR amplification reaction using the primer set described in claim 1; (3) The multiplex PCR amplification products were analyzed by electrophoresis.

6. The identification method according to claim 5, characterized in that The multiplex PCR reaction system in step (2) includes: 12.5 μL of 2×Taq Mix, 0.5 μL each of AS-F / R, 0.5 μL each of SP-F / R, 0.5 μL each of AW-F / R, 8.5 μL of ddH2O, and 1.0 μL of DNA template, for a total of 25 μL.

7. The identification method according to claim 5, characterized in that The multiplex PCR reaction program in step (2) was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 6 s, for 34 cycles; and extension at 72°C for 5 min.

8. The identification method according to claim 5, characterized in that The electrophoresis analysis in step (3) is specifically as follows: when the amplified product contains a 211 bp fragment, it indicates that the corn aphid sample contains the symbiotic bacterium Acinetobacter soli; when the amplified product contains a 235 bp fragment, it indicates that the corn aphid sample contains the symbiotic bacterium Sphingomonas paucimobilis; when the amplified product contains a 134 bp target fragment, it indicates that the corn aphid sample contains the symbiotic bacterium Acidovorax wautersii.

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