Novel culex wolbachia subtype as well as detection primer, detection method and detection kit thereof

Abstract

The invention provides a culex neo-wolbachia subtype as well as a detection primer, a detection method and a detection kit thereof. The invention discloses a novel Wolbachia subtype, and designs a specific detection primer, a probe and a detection method aiming at a gene segment of the novel Wolbachia. By utilizing the detection primer and the probe disclosed by the invention, the neowolbachia culex subtype can be rapidly, efficiently and specifically detected according to the detection method disclosed by the invention. The method has the advantages of rapidness, high efficiency, simplicity and convenience in operation, high specificity, high sensitivity and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a new Wolbachia subtype of Culex pipiens and a detection primer, a detection method and a detection kit thereof. Background Art

[0002] Wolbachia, also known as Wolbachia, is an obligate intracellular symbiont that is maternally transmitted and was first discovered in the reproductive tissues of Culex pipiens. Wolbachia is widely distributed in nature, and more than 65% of insects are naturally infected with Wolbachia. After Wolbachia infects mosquitoes, it can cause cytoplasmic incompatibility in the host, thereby affecting the reproduction of mosquitoes; on the other hand, it can block the replication and proliferation of pathogens (such as dengue virus and Zika virus) in mosquitoes, thereby blocking the spread of related pathogens, and can be used for on-site prevention and control of mosquito-borne infectious diseases.

[0003] Different insects carry different subtypes of Wolbachia. The discovery of new subtypes will help to more accurately control the spread of mosquito-borne infectious diseases and can also help researchers gain a deeper understanding of the interaction mechanism between Wolbachia and insects.

[0004] To fully utilize the potential of Wolbachia, it is crucial to accurately identify and distinguish different subtypes. Therefore, it is particularly important to develop specific detection methods for different Wolbachia subtypes. Commonly used detection methods for Wolbachia include polymerase chain reaction (PCR), immunostaining and electron microscopy, and different methods have their own advantages and disadvantages. The PCR method is sensitive and simple, but it is easy to produce false positives and cannot explain the distribution of Wolbachia in mosquitoes. Several antibodies used in the immunostaining method have different degrees of non-specific staining, making it difficult to interpret the experimental results. Electron microscopy can provide direct evidence of the distribution of Wolbachia in mosquitoes, but it requires expensive equipment and complex technology, which is inconvenient to use in daily research work. Therefore, the invention of new detection methods is conducive to the rapid and accurate detection of Wolbachia. Summary of the invention

[0005] In view of the above problems existing in the prior art, the object of the present invention is to provide a new Wolbachia subtype of Culex pipiens and its detection primers, detection method and detection kit.

[0006] In order to achieve the above purpose, the scheme of the present invention is as follows:

[0007] On the one hand, the present invention provides a Culex pipiens new Wolbachia subtype gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] To achieve the detection of the genes of the new Wolbachia subtype in Culex mosquitoes, the present invention also provides a detection primer, a probe for the new Wolbachia subtype in Culex mosquitoes, and a kit containing the primer and the probe. The sequences of the detection primers are shown in SEQ ID NO.3 and SEQ ID NO.4. The sequence of the fluorescent probe is shown in SEQ ID NO.2, and a fluorescent generating group and a fluorescent quenching group are respectively bound to both ends of the probe.

[0009] Furthermore, the present invention also provides a method for detecting the new Wolbachia subtype in Culex mosquitoes for non-disease diagnosis and treatment purposes, comprising the following steps:

[0010] Extract the genomic DNA of the sample. Using the sample DNA as a template, perform fluorescence quantitative PCR amplification with the detection primers shown in SEQ ID NO.3 and SEQ ID NO.4, and the fluorescent probe shown in SEQ ID NO.2. After the amplification reaction is completed, read and record the Ct value (the number of PCR cycles) of the sample. Based on the Ct value of the sample, determine whether the sample contains the new Wolbachia subtype in Culex mosquitoes.

[0011] Furthermore, the PCR amplification system:

[0012] 1 μL of template DNA, 10 μL of 2X TaqMan Fast qPCR Master Mix buffer, 0.2 μL of 10 μmol / L probe, 0.3 μL of each 10 μmol / L detection primer, and 8.7 μL of deionized water.

[0013] Furthermore, the PCR reaction conditions:

[0014] 94°C for 3 min; 94°C for 5 s; 60°C for 30 s, 40 cycles; collect fluorescence at 60°C.

[0015] Furthermore, the method for determining whether the sample contains the new Wolbachia subtype in Culex mosquitoes based on the Ct value of the sample is as follows:

[0016] If the amplification reaction curve of the gene to be detected appears and the Ct value is less than 38, it is judged as positive; if 38 < Ct < 40, it is judged as suspicious. Increase the template amount and repeat the amplification. If the same experimental result is obtained, it is judged as positive, otherwise as negative; if the Ct value is 0 or 40, it is judged as negative.

[0017] Furthermore, the samples include Culex mosquitoes and Aedes mosquitoes.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] The present invention discloses a Culex new Wolbachia subtype, and specific detection primers and probes are designed for the gene sequence of the subtype. The fluorescent detection primers and probes of the present invention can be used to quickly, efficiently and specifically detect the Culex new Wolbachia subtype according to the detection method of the present invention. The present invention has the advantages of high specificity (only the Culex new Wolbachia subtype is detected, while the Aedes Wolbachia type A and type B are not amplified), high sensitivity (the minimum detection limit is 100 copies / ml), etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a graph of the experimental results of the sensitivity of the new Wolbachia subtype of Culex pipiens, A and B represent the detection results of two mosquitoes in Example 1, respectively;

[0021] Figure 2 1 is a graph showing the experimental results of the repeatability of the new Wolbachia subtype of Culex pipiens, wherein A and B represent the results of the fluorescent quantitative PCR of the genomic DNA of the new Wolbachia subtype of Culex pipiens in two mosquitoes in Example 2;

[0022] Figure 3 This is a graph of experimental results on the specificity of the new Wolbachia subtype in Culex pipiens, in which the six curves represent the results of fluorescent quantitative PCR of the genomic DNA of Wolbachia in six mosquitoes. DETAILED DESCRIPTION

[0023] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0024] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0025] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0026] The inventors discovered a new Wolbachia subtype of Culex pipiens and obtained its gene fragment (SEQ ID NO.1). Specific primers for the new Wolbachia were designed for the gene fragment. The following experiments verified the specificity, sensitivity and repeatability of the primers.

[0027] Example 1 Sensitivity Detection

[0028] (1) Two Culex pipiens mosquitoes (carrying the new Wolbachia subtype) were frozen at -20°C for 30 minutes and their abdomens were dissected and placed in a 1.5 mL EP tube.

[0029] (2) Add 120uL DEB solution (the formula of DEB solution is: 10% SDS, 5M NaCl, 0.5M EDTA, 1.0M Tris pH 8.0, RNase A, the solvent is water, the same below).

[0030] (3) Grind thoroughly for 1 min to 2 min, incubate at 37 °C in a water bath for 60 min, add 3 μL proteinase K, and incubate at 50 °C for 60 min;

[0031] (4) Add 60 μL of phenol and 60 μL of chloroform, vortex and centrifuge at 14,000 rpm for 15 min.

[0032] (5) Take 200 μL of the supernatant and transfer it to a new 1.5 mL Eppendorf tube. Add 300 μL of anhydrous ethanol and shake to mix.

[0033] (6) Incubate at -20°C overnight.

[0034] (7) Centrifuge at 14,000 rpm for 10 min, remove the supernatant, and incubate at 37°C for 10 min.

[0035] (8) Add 50 μL of deionized water, resuspend the DNA, and incubate at 65°C for 15 min to obtain the genomic DNA of the new Wolbachia subtype of Culex pipiens. The DNA extract was stored at -20°C.

[0036] (9) The two genomic DNA extracts containing the new Wolbachia subtype of Culex pipiens were diluted 10, 100, and 1000 times as template DNA, for a total of 8 samples.

[0037] (10) PCR amplification system: 1 μL template DNA, 10 μL 2X TaqMan Fast qPCR Master Mix buffer, 0.2 μL 10 μmol / L probe (5′-CGCGATCGCATTATTGTTAACTCCTGTTGGATCGCG-3′, SEQ ID NO. 2, with a fluorescence generating group and a fluorescence quenching group bound to both ends of the probe, respectively), 0.3 μL 10 μmol / L fluorescence detection primer wGelF (5′-GATTGACTGGTATCTTGCTATT-3′, SEQ ID NO. 3), 0.3 μL 10 μmo / L fluorescence detection primer wGelR (5′-ATCCCACAATAAATGACGAAT-3′, SEQ ID NO. 4), and 8.7 μL deionized water.

[0038] (11) Mix well and centrifuge before performing PCR amplification.

[0039] (12) PCR reaction conditions:

[0040] Pre-denaturation at 94°C for 3 min

[0041] Amplification at 94°C for 5 s

[0042] 60°C for 30 s for 40 cycles, fluorescence collected at 60°C.

[0043] (13) Observe the amplification curve after the amplification is completed.

[0044] The specific results are as Figure 1 shown. Result judgment criteria: If the amplification reaction curve of the gene to be detected appears and the Ct value is less than 38, it is judged as positive. If 38 < Ct < 40, it is judged as suspicious. For the suspicious case, increase the template amount and repeat the amplification. If the same experimental result is obtained, it is judged as positive, otherwise it is negative. If the Ct value is 0 or 40, it is judged as negative.

[0045] It can be seen from Figure 1 that the DNA extraction stock solution and the solutions diluted 10, 100, and 1000 times can all amplify the reaction curve, and their Ct values are all less than 38, which is judged as positive. The concentration in the solution diluted 1000 times is 100 copies / ml. Thus, the detection limit of the detection primer of the present invention is 100 copies / ml.

[0046] Example 2: Repeatability detection

[0047] (1) Two Culex mosquitoes (carrying a new Wolbachia subtype) were stunned at -20°C for 30 minutes, and then the abdomen was dissected and placed in a 1.5 mL EP tube.

[0048] (2) Add 120 μL of DEB solution, grind thoroughly for 1 min - 2 min, and incubate in a water bath at 37°C for 60 min.

[0049] (3) Add 3 μL of proteinase K and incubate at 50°C for 60 min.

[0050] (4) Add 60 μL each of phenol and chloroform, vortex, and centrifuge at high speed for 15 min at 14000 rpm.

[0051] (5) Take 200 μL of the supernatant and transfer it to a new 1.5 mL Ep tube, add 300 μL of absolute ethanol, and mix well by oscillation.

[0052] (6) Incubate at -20°C overnight.

[0053] (7) Centrifuge at high speed for 10 min at 14000 rpm, remove the supernatant, and incubate at 37°C for 10 min.

[0054] (8) Add 50 μL of deionized water to resuspend the DNA, and incubate at 65°C for 15 min to obtain the genomic DNA of the new Wolbachia subtype of two Culex mosquitoes.

[0055] (9) The genomic DNA of each of the above-mentioned new Wolbachia subtypes of Culex mosquitoes was used as template DNA for 7 repetitions, for a total of 16 samples.

[0056] (10) PCR amplification system: 1 μL of template DNA, 10 μL of 2X TaqMan Fast qPCR Master Mix buffer, 0.2 μL of 10 μmol / L probe (5’-CGCGATCGCATTATTGTTAACTCCTGTTGGATCGCG-3’, with a fluorescent generating group and a fluorescent quenching group respectively bound to both ends of the probe), 0.3 μL of 10 μmol / L fluorescent detection primer wGelF (5’-GATTGACTGGTATCTTGCTATT-3’), 0.3 μL of 10 μmol / L fluorescent detection primer wGelR (5’-ATCCCACAATAAATGACGAAT-3’), and 8.7 μL of deionized water.

[0057] (11) After mixing and centrifuging, PCR amplification was carried out.

[0058] (12) PCR reaction conditions:

[0059] Pre-denaturation at 94 °C for 3 min

[0060] Amplification at 94 °C for 5 s

[0061] 60 °C for 30 s for 40 cycles, and fluorescence was collected at 60 °C.

[0062] (13) After the amplification was completed, the amplification curve was observed.

[0063] Specific results are as Figure 2 (shown in A and B). Result judgment criteria: If an amplification reaction curve of the gene to be detected appears and the Ct value is less than 38, it is judged as positive; if 38 < Ct < 40, it is judged as suspicious. For the suspicious cases, increase the template amount and repeat the amplification. If the same experimental results are obtained, it is judged as positive, otherwise as negative. If the Ct value is 0 or 40, it is judged as negative.

[0064] From Figure 2 It can be seen that for the genomic DNA of 2 new Wolbachia subtypes of Culex mosquitoes, reaction curves could be amplified in each of the 7 repetitions, and their Ct values were all less than 38, which was judged as positive. Thus, it shows that the fluorescence detection method of the present invention has good detection repeatability.

[0065] Example 3: Specificity detection

[0066] (1) Six Aedes albopictus mosquitoes (carrying Wolbachia type A and B), two Aedes albopictus mosquitoes with only type A and two Aedes albopictus mosquitoes with only type B, a total of six Aedes mosquitoes, were frozen at -20℃ for 30 minutes, and their abdomens were dissected and placed in 1.5 mL EP tubes.

[0067] (2) Add 120uL DEB solution, grind thoroughly for 1min to 2min, and place in a 37℃ water bath for 60min.

[0068] (3) Add 3 μL of proteinase K and incubate at 50°C for 60 min.

[0069] (4) Add 60 μL of phenol and 60 μL of chloroform, vortex and centrifuge at 14,000 rpm for 15 min.

[0070] (5) Take 200 μL of the supernatant and transfer it to a new 1.5 mL Eppendorf tube. Add 300 μL of anhydrous ethanol and shake to mix.

[0071] (6) Incubate at -20°C overnight.

[0072] (7) Centrifuge at 14,000 rpm for 10 min, remove the supernatant, and incubate at 37°C for 10 min.

[0073] (8) Add 50 μL of deionized water, resuspend the DNA, and incubate at 65°C for 15 min to obtain one Wolbachia genomic DNA from each of the six mosquitoes.

[0074] (9) Each of the above Wolbachia genomic DNAs was used as template DNA for fluorescence quantitative PCR reaction.

[0075] (10) PCR amplification system: 1 μL template DNA, 10 μL 2X TaqMan Fast qPCR Master Mix buffer, 0.2 μL 10 μmol / L probe (5′-CGCGATCGCATTATTGTTAACTCCTGTTGGATCGCG-3′, with a fluorescence generating group and a fluorescence quenching group bound to both ends of the probe, respectively), 0.3 μL 10 μmol / L fluorescence detection primer wGelF (5′-GATTGACTGGTATCTTGCTATT-3′), 0.3 μL 10 μmo / / L fluorescence detection primer wGelR (5′-ATCCCACAATAAATGACGAAT-3′), and 8.7 μL deionized water.

[0076] (11) Mix well and centrifuge before performing PCR amplification.

[0077] (12) PCR reaction conditions:

[0078] Pre-denaturation 94℃3min

[0079] Amplification at 94°C for 5 s

[0080] 40 cycles at 60°C for 30 s, fluorescence collection at 60°C

[0081] (13) Observe the amplification curve after the amplification is completed.

[0082] The specific results are as Figure 3 shown. Result judgment criteria: If the amplification reaction curve of the gene to be detected appears and the Ct value is less than 38, it is judged as positive; if 38 < Ct < 40, it is judged as suspicious. Increase the template amount for the suspicious sample and repeat the amplification. If the same experimental results are obtained, it is judged as positive, otherwise as negative. If the Ct value is 0 or 40, it is judged as negative.

[0083] From Figure 3 it can be seen that none of the six Wolbachia samples (i.e., two samples carrying Wolbachia A and B, two samples with single Wolbachia A, and two samples with single Wolbachia B) can amplify the reaction curve. This shows that the fluorescence detection primers of the present invention cannot amplify Wolbachia A, Wolbachia B, or a mixture of Wolbachia A and B, that is, only the new subtype of Culex Wolbachia can be detected, and Aedes Wolbachia does not amplify. This shows that the fluorescence detection method of the present invention has good specificity.

[0084] Example 4: Detection kit for the new subtype of Culex Wolbachia

[0085] The kit consists of the probe described above (5’-CGCGATCGCATTATTGTTAACTCCTGTTGGATCGCG-3’, with a fluorescent generating group and a fluorescent quenching group respectively bound to both ends of the probe), the fluorescence detection primers wGelF (5’-GATTGACTGGTATCTTGCTATT-3’), wGelR (5’-ATCCCACAATAAATGACGAAT-3’), and the conventional reagents required for PCR detection, such as PCR reaction buffer, deionized water, etc.

[0086] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A new Wolbachia subtype gene of Culex pipiens, characterized in that: The nucleotide sequence of the said gene is shown as SEQ ID NO.

1.

2. A primer for detecting a new Wolbachia subtype of Culex pipiens, characterized in that: The gene sequence of the new Wolbachia subtype of Culex mosquitoes is shown as SEQ ID NO.1, and the sequences of the detection primers are shown as SEQ ID NO.3 and SEQ ID NO.

4.

3. A detection kit for a new Wolbachia subtype in Culex pipiens, characterized in that: It contains the detection primers described in claim 1.

4. The detection kit according to claim 3, characterized in that It also contains a fluorescent probe, the sequence of which is shown as SEQ ID NO.2, and a fluorescent generating group and a fluorescent quenching group are respectively bound to both ends of the probe.

5. A method for detecting a new Wolbachia subtype in Culex pipiens for purposes other than disease diagnosis and treatment, characterized in that: It includes the following steps: Extract the genomic DNA of the sample. Using this sample DNA as a template, perform fluorescence quantitative PCR amplification with the detection primers shown as SEQ ID NO.3 and SEQ ID NO.4, and the fluorescent probe shown as SEQ ID NO.

2. After the amplification reaction is completed, read and record the Ct value of the PCR cycle number of the sample. According to the Ct value of the sample, determine whether the sample contains the new Wolbachia subtype of Culex mosquitoes.

6. The detection method according to claim 5, characterized in that: PCR amplification system: Template DNA 1 μL, 2X TaqMan Fast qPCR Master Mix buffer 10 μL, 10 μmol / L probe 0.2 μL, 10 μmol / L detection primers each 0.3 μL, deionized water 8.7 μL.

7. The detection method according to claim 5, characterized in that: PCR reaction conditions: 94 °C for 3 min; 94 °C for 5 s; 60 °C for 30 s, 40 cycles; collect fluorescence at 60 °C.

8. The detection method according to claim 5, characterized in that: The method for determining whether the sample contains the new Wolbachia subtype of Culex mosquitoes according to the Ct value of the sample is as follows: If the amplification reaction curve of the gene to be detected appears and the Ct value is less than 38, it is judged as positive; if 38 < Ct < 40, it is judged as suspicious, increase the template amount, repeat the amplification, if the same experimental result is obtained, it is judged as positive, otherwise it is negative; if the Ct value is 0 or 40, it is judged as negative.

9. The detection method according to claim 5, characterized in that: The said samples include Culex mosquitoes and Aedes mosquitoes.

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