WO2016197843A1 - Fluorescence detection primer, detection method, and detection kit for culex wolbachia - Google Patents

Abstract

A fluorescence detection primer, detection method, and detection kit for Culex Wolbachia. The fluorescence detection primer is: wPipF: 5'-GTTTGTGCAGCTAATAG-3'; wPipR: 5'-GTCTGCAAGGCCTATTTCTACTG-3'; probe: 5'-CTTTCAATTGAAAAGATTCGATCAAC-3'; the two ends of the probe are respectively combined with a fluorescence generation group and a fluorescence quenching group. Culex Wolbachia can be detected by utilizing the fluorescence detection primer and the probe of the present invention on the basis of the detection method. In the method, the lowest detection limit is 100 copies/ml, and no amplification of Aedes Wolbachia occurs.

Description

Fluorescent detection primer of Culex mosquitoes, and detection method and detection kit thereof Technical field:

The invention belongs to the field of molecular biology, and particularly relates to a fluorescent detection primer of Culex mosquitoes, a detection method thereof and a detection kit.

Background technique:

Wolbachia is a symbiotic microbe that is widely distributed in arthropods and is probably the most abundant group of insect symbiotic microorganisms. It is distributed in insect species such as Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Lepidoptera. It uses vertical propagation as its basic mode of transmission between host generations. It is stably present in the germ cells of the host, is transmitted to the host progeny through the egg cells, and can regulate the reproductive activities of the host by various means such as cytoplasmic incompatibility, femaleification and male sterility. Through these regulatory actions, it promotes its widespread spread within the host population.

Cytoplasmic Incompatibility (CI) is the most common type of host reproductive behavior change caused by Wolbachia. It is manifested by the fact that when male mosquitoes infected with Wolbachia are mated with uninfected female mosquitoes or male and female mosquitoes infected with different species of Wolbachia, the embryos cannot develop after sperm and egg binding. The cytoplasmic incompatibility of Wolbachia can cause Wolbachia-infected mosquitoes to invade mosquitoes that are not infected or infected with different species of Wolbachia for population suppression and population replacement. Its application has great value for the prevention and control of mosquito-borne diseases. Recent studies have found that Wolbachia not only has the above characteristics, but also interacts with some important pathogens (such as dengue viruses, malaria parasites, etc.) carried by mosquitoes in mosquitoes and inhibits them. The proliferation and spread in mosquitoes thus blocks the transmission of these pathogens to humans. This inhibition of Wolbachia is related to its distribution in the tissues of mosquitoes. Therefore, understanding the tissue distribution of Wolbachia in mosquitoes can help to quickly screen out the most blocking effects. Good Wolbachia-infected mosquitoes also help to monitor the vertical transmission of Wolbachia in mosquitoes, and also to efficiently monitor large-scale large-area Walls. Infection of Wolbachia in mosquito tissues.

In nature, there are many types of mosquitoes, and there are two types of mosquitoes, Aedes aegypti and Culex pipiens. Aedes mosquitoes are divided into Aedes albopictus and Aedes aegypti, in which Aedes aegypti does not carry Wolbachia. Because Wolbachia can only survive in the host and cannot live freely in vitro, how to effectively and specifically detect Wolbachia is an indispensable tool for the study of this bacterium. The more commonly used detection methods are polymerase chain reaction (PCR) and immunostaining. Immunostaining is time consuming and labor intensive. With the popularity of PCR methods, great progress has been made in the detection of Wolbachia. Different mosquito species Carrying different Wolbachia, the results of the PCR can visually show the different types of Wolbachia carried by mosquitoes, which provides great convenience for our daily testing. At present, the methods for different Wolbachia PCR detection are still not perfect.

Summary of the invention:

It is an object of the present invention to provide a fluorescent detection primer for the specificity, high specificity and sensitivity of Culex pipiens.

The fluorescent detection primer of the Culex mosquitoes of the present invention is characterized in that it comprises:

wPipF: 5'-GTTTGTGCAGCTAATAG-3' (the nucleotide sequence of which is shown in SEQ ID NO. 1);

wPipR: 5'-GTCTGCAAGGCCTATTTCTACTG-3' (the nucleotide sequence thereof is shown as SEQ ID NO. 2);

Probe: 5'-CTTTCAATTGAAAAGATTCGATCAAC-3' (the nucleotide sequence of which is shown in SEQ ID NO. 3);

Both ends of the probe are bound to a fluorescence generating group and a fluorescence quenching group, respectively.

The fluorescent generating group is FAM, and the fluorescent quenching group is BHQ1.

A second object of the present invention is to provide a method for detecting Culex pipiens pallens for non-disease diagnosis and treatment purposes, which comprises extracting sample DNA, using the sample DNA as a template, and using the above-mentioned Culex The wPipF and wPipR of the fluorescence detection primer of the erbucker, and the probe are subjected to real-time PCR amplification. After the amplification reaction is completed, the sample is read and recorded according to the fluorescence signal of the fluorescent moiety of the probe label. The number of PCR cycles Ct, according to the Ct value of the sample, according to the established judgment criteria, determine whether the sample contains Coiba mosquito Wolbachia.

The fluorescent quantitative PCR amplification is carried out, and the reaction conditions are preferably: pre-denaturation 50 ° C for 5 minutes, 95 ° C for 15 minutes; amplification 94 ° C for 15 seconds, 55 ° C for 45 seconds, 40 cycles; 55 ° C when collecting fluorescence signal.

A third object of the present invention is to provide a detection kit for Culex pipa, including a fluorescent detection primer, characterized in that

The fluorescent detection primers are:

wPipF: 5'-GTTTGTGCAGCTAATAG-3';

wPipR: 5'-GTCTGCAAGGCCTATTTCTACTG-3';

Probe: 5'-CTTTCAATTGAAAAGATTCGATCAAC-3';

Both ends of the probe are bound to a fluorescence generating group and a fluorescence quenching group, respectively.

The fluorescent generating group is FAM, and the fluorescent quenching group is BHQ1.

According to the detection method of the invention, the detection method of the invention can quickly and efficiently detect the Wolveria sinensis, which has strong specificity and high specificity (only detection of Culex bacillus The austenoid, while the Aedes aegypti Wolbachia does not undergo amplification, has high sensitivity, and the minimum detection limit is 100 copies/ml.

Therefore, the invention has the advantages of rapid and high efficiency, simple operation, high specificity, high sensitivity, and simple identification.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 is a graph showing the experimental results of the sensitivity of Culex pipiens. The DNA extracts, 10×, 100×, 1000× represent DNA extracts, 10×, 100×, 1000× times dilutions, respectively. DNA extract;

2 is a graph showing experimental results of the reproducibility of Culex pipiens pallens, wherein I and II represent the results of real-time PCR of genomic DNA of Culex pipiens pallens in two mosquitoes in Example 2;

Fig. 3 is a graph showing the experimental results of the specificity of Culex pipiens. The six curves represent the results of real-time PCR of genomic DNA of Wolbachia in six mosquitoes.

detailed description:

The following examples are intended to further illustrate the invention and not to limit the invention.

Example 1: Sensitivity

1. A Guangzhou-induced Culex pipiens pallens (carrying Culex bacillus) is smoked by carbon dioxide, and the abdomen is placed in a 0.2ul EP tube;

2. Add 20 ul of DNA extract (the formula of DNA extract is: 30 mM NaOH, 0.25 mM EDTA, 15 mM Tris-HCl, the solvent is water, the same below, shaking before use, adding white flakes together);

3, fully mix;

4. After centrifugation, warm bath at 99 ° C for 10 minutes;

5. The DNA extract is stored at -20 ° C to obtain genomic DNA of Culex mosquitoes;

6. The DNA extract of the above-mentioned genomic DNA containing Culex pipiens pallens was diluted 10, 100, 1000 times as template DNA, and each gradient was two parallels, a total of 8 samples;

7. PCR amplification system:

Template DNA 2μl, 10×Taqman buffer 5μl, 5mmol/L MgCl ₂ 4μl, 2.5mol/L dNTPs 2μl, 20μmol/L probe (5′-CTTTCAATTGAAAAGATTCGATCAAC-3′) 1μl, 20μmol/L fluorescence detection primer wPipF(5 '-GTTTGTGCAGCTAATAG-3') 1 μl, 20 μmol/L fluorescence detection primer wPipR (5'-GTCTGCAAGGCCTATTTCTACTG-3') 1 μl, 0.55 U UNG enzyme 0.2 μl, 2.5 U/μl Taq polymerase 3 μl, deionized water 10.8 μl.

8. Mix and centrifuge for PCR amplification;

9, PCR reaction conditions:

10. Observe the amplification curve after the end of amplification.

The specific results are shown in Figure 1. The result is judged as follows: If the amplification reaction curve of the gene to be tested appears, and the Ct value is less than 38, it is judged as positive. If 38<Ct<40, it is judged to be suspicious. Repeat amplification, if it is the same experimental result, it is judged as positive, otherwise it is negative, if the Ct value is 0 or 40, it is judged to be negative.

It can be seen from Fig. 1 that the DNA extract, 10, 100, and 1000-fold diluted solutions can amplify the reaction curve, and the Ct is less than 38, which is judged to be positive. The DNA concentration in the 1000-fold diluted solution is 100 copies. /ml. Thus, the detection limit of the detection primer of the present invention is 100 copies/ml.

Example 2: Repeatability

1. Two Guangzhou-induced Culex pipiens pallens (carrying Culex bacillus) after being smoked by carbon dioxide, the anatomical abdomen was placed in a 0.2ul EP tube;

2. Add 20 ul of DNA extract separately (osmbrate before use, add white flakes together);

3, fully mix;

4. After centrifugation, warm bath at 99 ° C for 10 minutes;

5. The DNA extract is stored at -20 ° C, thereby obtaining genomic DNA of each of the two mosquitoes, Volkbach.

6. The genomic DNA of each of the above-mentioned Culex bacillus is used as template DNA for 10 repetitions, for a total of 20 samples;

7. PCR amplification system:

Template DNA 2 μl, 10×Taqman buffer 5 μl, 5 mmol/L MgCl ₂ 4 μl, 2.5 mol/L dNTPs 2 μl, 20 μmol/L probe 1 μl, 20 μmol/L fluorescence detection primer wPipF (5′-GTTTGTGCAGCTAATAG-3′) 1 μl, 20μmol/L fluorescence detection primer wPipR (5'-GTCTGCAAGGCCTATTTCTACTG-3') 1μl, 0.55U UNG enzyme 0.2μl, 2.5U/μl Taq polymerase 3μl, deionized water 10.8μl

8. Mix and centrifuge for PCR amplification;

9, PCR reaction conditions:

10. Observe the amplification curve after the end of amplification.

The specific results are shown in Figure 2. The result is judged as follows: If the amplification reaction curve of the gene to be tested appears, and the Ct value is less than 38, it is judged as positive. If 38<Ct<40, it is judged as suspicious and suspiciously increases the amount of template. Repeat amplification, if it is the same experimental result, it is judged as positive, otherwise it is negative, if the Ct value is 0 or 40, it is judged to be negative.

It can be seen from Fig. 2 that the genomic DNA of 2 parts of Culex bacillus can amplify the reaction curve for 10 repetitions, and the Ct is less than 38, which is judged to be positive, thereby indicating the fluorescence detection of the present invention. Primers are reproducible.

Example 3: Specificity

1. Two Aedes albopictus (carrying A-type and B-type Wolbachia), two single-type A-type Aedes albopictus and two single-B-type Aedes albopictus, a total of 6 Aedes mosquitoes smoked by carbon dioxide After halo, the anatomical abdomen was placed in a 0.2 ul EP tube;

2. Add 20 ul of DNA extract separately (osmbrate before use, add white flakes together);

3, fully mix;

4. After centrifugation, warm bath at 99 ° C for 10 minutes;

5. The DNA extract is stored at -20 ° C, thereby obtaining genomic DNA of each of the six mosquitoes of Wolbachia;

6. The genomic DNA of each of the above Wolbachia is used as a template DNA fluorescent quantitative PCR reaction;

7. PCR amplification system:

Template DNA 2 μl, 10×Taqman buffer 5 μl, 5 mmol/L MgCl ₂ 4 μl, 2.5 mol/L dNTPs 2 μl, 20 μmol/L probe 1 μl, 20 μmol/L fluorescence detection primer wPipF (5′-GTTTGTGCAGCTAATAG-3′) 1 μl, 20μmol/L fluorescence detection primer wPipR (5'-GTCTGCAAGGCCTATTTCTACTG-3') 1μl, 0.55U UNG enzyme 0.2μl, 2.5U/μl Taq polymerase 3μl, deionized water 10.8μl

8. Mix and centrifuge for PCR amplification;

9, PCR reaction conditions:

10. Observe the amplification curve after the end of amplification.

The specific results are shown in Figure 3. The result is judged as follows: If the amplification reaction curve of the gene to be tested appears, and the Ct value is less than 38, it is judged as positive. If 38<Ct<40, it is judged to be suspicious, and the amount of template is suspected to be increased. Repeat amplification, if it is the same experimental result, it is judged as positive, otherwise it is negative, if the Ct value is 0 or 40, it is judged to be negative.

As can be seen from Figure 3, six parts of Wolbachia (ie two with type A and B type Wolbachia, two single type A Wolbachia and two single B type Wolbacher) The reaction curve can not be amplified, thereby indicating that the fluorescence detecting primer of the present invention cannot amplify a mixture of type A, type B, or a mixture of type A and type B wolbachia. Only Culb buta was detected, while Aedes albob has not expanded. Thus, the fluorescence detecting primer of the present invention has good specificity.

Claims (6)

1. A fluorescent detection primer for Culex mosquitoes, which is characterized by comprising:

   wPipF: 5'-GTTTGTGCAGCTAATAG-3';

   wPipR: 5'-GTCTGCAAGGCCTATTTCTACTG-3';

   Probe: 5'-CTTTCAATTGAAAAGATTCGATCAAC-3';

   Both ends of the probe are bound to a fluorescence generating group and a fluorescence quenching group, respectively.
2. The fluorescent detection primer according to claim 1, wherein the fluorescent generating group is FAM, and the fluorescent quenching group is BHQ1.
3. A method for detecting Culex pipiens pallens for non-disease diagnosis and treatment purposes, characterized in that sample DNA is extracted, and the sample DNA is used as a template, and the Culex pipa var. The fluorescence detection primers wPipF and wPipR, and the probe are subjected to real-time PCR amplification. After the amplification reaction is completed, the PCR cycle number of the sample is read and recorded according to the fluorescent signal of the fluorescent moiety of the probe label. According to the Ct value of the sample, according to the established judgment standard, it is judged whether the sample contains Culex pipiens.
4. The detection method according to claim 3, wherein the fluorescence quantitative PCR amplification is carried out under the following conditions: pre-denaturation at 50 ° C for 5 minutes, 95 ° C for 15 minutes; amplification at 94 ° C for 15 seconds, 55 ° C. 45 seconds, 40 cycles.
5. A detection kit for Culex mosquitoes, including a fluorescent detection primer, characterized in that

   The fluorescent detection primers are:

   wPipF: 5'-GTTTGTGCAGCTAATAG-3';

   wPipR: 5'-GTCTGCAAGGCCTATTTCTACTG-3';

   Probe: 5'-CTTTCAATTGAAAAGATTCGATCAAC-3';

   Both ends of the probe are bound to a fluorescence generating group and a fluorescence quenching group, respectively.
6. The test kit according to claim 5, wherein said fluorescent generating group is FAM and said fluorescent quenching group is BHQ1.

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