WO2016197837A1

WO2016197837A1 - Fluorescence detection primers of three types of wolbachia and detection method and detection kit thereof

Info

Publication number: WO2016197837A1
Application number: PCT/CN2016/084213
Authority: WO
Inventors: 高秀洁; 杨翠; 周其伟; 李丽梅; 奚志勇; 朱俭
Original assignee: 广州威佰昆生物科技有限公司; 中山大学达安基因股份有限公司
Priority date: 2015-06-11
Filing date: 2016-05-31
Publication date: 2016-12-15
Also published as: CN104911267A

Abstract

Disclosed are fluorescence detection primers of three types of Wolbachia and a detection method and detection kit thereof. Using the fluorescence detection primers of the present invention, Aedes type A, Aedes type B and Culex Wolbachia can be detected according to the detection method of the present invention, and only Aedes Wolbachia is detected in the Aedes, while only Culex Wolbachia is detected in the Culex, and the lowest detection limit is 100 copies/ml.

Description

Three kinds of Wolbachia fluorescence detection primers, detection method and detection kit thereof

Technical field:

The invention belongs to the field of molecular biology, and particularly relates to three kinds of Wolbachia fluorescent detection primers, 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:

The object of the present invention is to provide a fluorescence detecting primer for three kinds of Wolbachia, such as Aedes type A, Aedes type B and Culex bacillus, which are highly specific, highly specific and sensitive.

Three kinds of Wolbachia fluorescence detecting primers of the present invention, characterized in that

(1) For Aedes type A Wolbachia:

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

wAlbAR: 5'-GCACCAGCTTTTACTTGACC-3' (the nucleotide sequence thereof is shown in SEQ ID NO. 2);

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

The two ends of the probe A are respectively combined with a fluorescence generating group FAM and a fluorescence quenching group BHQ1;

(2) For Aedes B type Wolbachia:

wAlbBF: 5'-AAAGGAACCGAAGTTCATGAT-3' (the nucleotide sequence of which is shown in SEQ ID NO. 4);

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

Probe B: 5'-CAACATTTGCTCCAACAACTGTTGC-3' (the nucleotide sequence of which is shown in SEQ ID NO. 6);

The two ends of the probe B are respectively combined with a fluorescence generating group HEX and a fluorescence quenching group BHQ1;

(3) For Culex mosquitoes:

wPipF: 5'-GTTTGTGCAGCTAATAG-3' (the nucleotide sequence of which is shown in SEQ ID NO. 7);

wPipR: 5'-GTCTGCAAGGCCTATTTCTACTG-3' (the nucleotide sequence of which is shown in SEQ ID NO. 8);

Probe C: 5'-CTTTCAATTGAAAAGATTCGATCAAC-3' (the nucleotide sequence of which is shown in SEQ ID NO. 9);

Both ends of the probe C are bound to a fluorescence generating group Texas Red and a fluorescence quenching group BHQ2, respectively.

A second object of the present invention is to provide a method for detecting three Wolbachia for non-disease diagnosis and treatment purposes, characterized in that sample DNA is extracted, and the sample DNA is used as a template to Fluke detection primers wAlbAF, wAlbAR, probe A, wAlbBF, wAlbBR, probe B, wPipF, wPipR and The probe C is subjected to real-time PCR amplification. After the amplification reaction is completed, the PCR cycle number Ct of the sample is read and recorded according to the fluorescence signal of the fluorescence-generating group labeled by the probe, and the Ct value of the sample is determined according to the establishment. Standard, determine whether the sample contains Aedes type A, Aedes type B and Culex pipiens.

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 test kit for three Wolbachia, comprising a fluorescent detection primer and a PCR reagent, wherein the fluorescent detection primer comprises:

(1) For Aedes type A Wolbachia:

(2) For Aedes B type Wolbachia:

(3) For Culex mosquitoes:

According to the detection method of the invention, the detection method of the invention can quickly and efficiently detect the Aedes type A, the Aedes type B and the Culex pipiens, which have specificity and high specificity. Mosquitoes detected A. sinensis Wolbachia, while Culex pipiens detected only C. sinensis, and the sensitivity was high. The minimum detection limit was 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 results of the sensitivity of Aedes A type Wolbachia, wherein I, II, III, and IV represent DNA extracts, 10, 100, and 1000-fold diluted DNA extracts, respectively;

2 is a graph showing experimental results of the sensitivity of Aedes B-type Wolbachia, wherein I, II, III, and IV represent DNA extracts, 10, 100, and 1000-fold diluted DNA extracts, respectively;

3 is a graph showing experimental results of the sensitivity of Culex pipiens, wherein I, II, III, and IV represent DNA extracts, 10, 100, and 1000-fold diluted DNA extracts, respectively;

Figure 4 is a graph showing the experimental results of the repeatability of Aedes aegypti and Culex pipiens, wherein I and II represent amplification curves of different samples, respectively;

Figure 5 is a graph showing the results of the specificity of the FAM detection channel for Aedes type A Wolbachia;

Figure 6 is a graph showing the experimental results of the specificity of the FAM detection channel for Aedes type B and wPip type Wolbachia (Culex vaccae);

Figure 7 is a graph showing the results of the specificity of the HEX detection channel for Aedes B-type Wolbachia;

Figure 8 is a graph showing the results of the specificity of the HEX detection channel for Aedes type A and wPip type Wolbachia (Culex vaccae);

Figure 9 is a graph showing the results of experiments on the specificity of the Texas Red detection channel for wPip type Wolbachia (Culex vaccae);

Figure 10 is a graph showing the experimental results of the specificity of the Texas Red detection channel for Aedes type A and B type Wolbachia.

detailed description:

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

Example 1: Sensitivity

First, the sensitivity of Aedes A type Wolbachia

1. A mosquito carrying Aedes A-type Wolbachia 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, shake it before use, and add white flakes together);

3, fully mix;

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

5. Obtaining the DNA extract and storing at -20 ° C to obtain the genomic DNA of Aedes type A Wolbachia;

6. The DNA extract is subjected to 10, 100, 1000 gradient dilution, and each gradient has two parallel, a total of 8 samples, and PCR amplification is performed as template DNA;

7. PCR amplification system:

2 μl of template DNA, 18 μl of PCR A solution and 2 μl of PCR B solution.

The PCR A solution contains 10 pmol of fluorescent detection primer wAlbAF (5'-CAGGGTTGATGTTGAAGGAG-3'), 10 pmol of fluorescent detection primer wAlbAR (5'-GCACCAGCTTTTACTTGACC-3'), and 5 pmol of probe A (5'-TATCTTCAATTGCTATATCGTAATAAACG- per 18 μl) 3', both ends of the probe are combined with a fluorescent generating group FAM and a fluorescence quenching group BHQ1), 10 pmol of a fluorescent detection primer wAlbBF (5'-AAAGGAACCGAAGTTCATGAT-3'), and 10 pmol of a fluorescent detection primer wAlbBR (5'-TTGTTTAGTTGTGAGTAAAGTCCC -3'), 5 pmol of probe B (5'-CAACATTTGCTCCAACAACTGTTGC-3', both ends of the probe are bound to the fluorescence generating group HEX and the fluorescence quenching group BHQ1, respectively), 10 pmol of the fluorescent detection primer wPipF (5'-GTTTGTGCAGCTAATAG -3'), 10pmol fluorescence detection primer wPipR (5'-GTCTGCAAGGCCTATTTCTACTG-3'), 5pmol probe C (5'-CTTTCAATTGAAAAGATTCGATCAAC-3', both ends of the probe are bound to the fluorescence generating group Texas Red and fluorescence quenching The group BHQ2), the rest is a buffer with a pH of 8.0. The buffer component was 50 mmol/L Tris-HCl, 8 mmol/L MgCl ₂ , 250 mmol/L KCl, and the solvent was water.

The PCR B solution consists of a hot-start Taq enzyme, a reverse transcriptase, and dNTPs, and each 2 μl of the PCR B solution contains a hot-start Taq enzyme 5 U, a reverse transcriptase 2.5 U, and dNTPs 10 mmol, and the rest is water.

8. After mixing, perform PCR amplification;

9, PCR reaction conditions:

Pre-denaturation 50 ° C 2 min

95 ° C 15 min

Amplification 94 ° C 15s

55 ° C 45s 40 cycles, collecting fluorescence at 55 ° C

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 DNA extracts can amplify the reaction curve, and the Ct is less than 38, which is judged to be positive, and the DNA in the 1000-fold diluted solution The concentration is 100 copies/ml. Thus, the detection limit of the fluorescent detection primer of the present invention is 100 copies/ml.

Second, the sensitivity of Aedes B type Wolbachia

The experimental procedure for the sensitivity of Aedes B-type Wolbachia is the same as that of Aedes A-type Wolbachia, except that the template DNA is replaced with the genomic DNA of Aedes B-type Wolbachia.

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

Second, the sensitivity of Culex pipa

The experimental procedure for the sensitivity of Culex mosquitoes to Wolbachia was tested with the sensitivity of Aedes type A Wolbachia, except that the template DNA was replaced with the genomic DNA of Culex pipiens.

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.

It can be seen from Fig. 3 that the DNA extract, 10, 100, and 1000-fold diluted DNA extracts can amplify the reaction curve, and the Ct is less than 38, which is judged to be positive, and the DNA in the 1000-fold diluted solution The concentration is 100 copies/ml. Thus, the detection limit of the fluorescent detection primer of the present invention is 100 copies/ml.

Example 2: Repeatability

1. Two triple-infected Aedes albopictus (carrying Aedes type A, Aedes type B and Culex mosquitoes) after being smoked by carbon dioxide, the anatomical abdomen was placed in a 0.2ul EP tube;

3, fully mix;

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

5. Obtain DNA extract and store at -20 °C to obtain genomic DNA of Aedes type A, Aedes type B and Culex pipiens.

6. Genomic DNA of the above Aedes type A, Aedes type B and Culex platicoides as template DNA 10 repetitions, for a total of 20 samples;

7. PCR amplification system:

2 μl of template DNA, 18 μl of PCR A solution and 2 μl of PCR B solution.

8. After mixing, perform PCR amplification;

9, PCR reaction conditions:

Pre-denaturation 50 ° C 2 min

95 ° C 15 min

Amplification 94 ° C 15s

55 ° C 45s 40 cycles, collecting fluorescence at 55 ° C

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

The specific results are shown in Figure 4. 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.

It can be seen from Fig. 4 that the genomic DNA samples of two Aedes type A, Aedes type B and Culex bacillus can amplify the reaction curve for 10 times, and the Ct is less than 38, which is judged as Positive, thereby indicating that the fluorescent detection primer of the present invention can simultaneously detect Aedes type A, Aedes type B and Culex pipiens, and has good repeatability.

Example 3: Specificity

1. Two Wolbachia containing a single type A, two Aedes aegypti with a single B type, and two Culex pipiens pallens (including Culex boswellii) After a total of 6 mosquitoes were smoked by carbon dioxide, the anatomical abdomen was placed in a 0.2 ul EP tube;

2. Add 20 ul of DNA extract respectively (the formula of DNA extract is: 30 mM NaOH, 0.25 mM EDTA, 15 mM Tris-HCl, the solvent is water, shake it before use, and add white flakes together);

3, fully mix;

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

5. Obtain the DNA extract and store at -20 °C, that is, obtain two genomic DNAs of A. sinensis type A Wolbachia, two genomic DNAs of Aedes B type Wolbachia and two Culex pipiens A total of six genomic DNAs of Wolbachia were used for PCR amplification using these six genomic DNAs as template DNA, respectively.

6,

PCR group 1:

2 μl of template DNA, 18 μl of PCR A solution and 2 μl of PCR B solution.

The PCR A solution contains 10 pmol of fluorescent detection primer wAlbAF (5'-CAGGGTTGATGTTGAAGGAG-3'), 10 pmol of fluorescent detection primer wAlbAR (5'-GCACCAGCTTTTACTTGACC-3'), and 5 pmol of probe A (5'-TATCTTCAATTGCTATATCGTAATAAACG- per 18 μl) 3', both ends of the probe are combined with a fluorescent generating group FAM and a fluorescent quenching group BHQ1), and the rest are buffers having a pH of 8.0. The buffer component was 50 mmol/L Tris-HCl, 8 mmol/L MgCl ₂ , 250 mmol/L KCl, and the solvent was water.

PCR group 2:

2 μl of template DNA, 18 μl of PCR A solution and 2 μl of PCR B solution.

The PCR A solution contains 10 pmol of fluorescent detection primer wAlbBF (5'-AAAGGAACCGAAGTTCATGAT-3'), 10 pmol of fluorescent detection primer wAlbBR (5'-TTGTTTAGTTGTGAGTAAAGTCCC-3'), and 5 pmol of probe B (5'-CAACATTTGCTCCAACAACTGTTGC- per 18 μl) 3', both ends of the probe are combined with a fluorescence generating group HEX and a fluorescence quenching group BHQ1), and the rest are buffers having a pH of 8.0. The buffer component was 50 mmol/L Tris-HCl, 8 mmol/L MgCl ₂ , 250 mmol/L KCl, and the solvent was water.

PCR group 3:

2 μl of template DNA, 18 μl of PCR A solution and 2 μl of PCR B solution.

The PCR A solution contains 10 pmol of fluorescent detection primer wPipF (5'-GTTTGTGCAGCTAATAG-3'), 10 pmol of fluorescent detection primer wPipR (5'-GTCTGCAAGGCCTATTTCTACTG-3'), and 5 pmol of probe C (5'-CTTTCAATTGAAAAGATTCGATCAAC- per 18 μl) 3', the two ends of the probe are respectively combined with a fluorescent generating group Texas Red and a fluorescence quenching group BHQ2), and the rest are buffers having a pH of 8.0. The buffer component was 50 mmol/L Tris-HCl, 8 mmol/L MgCl ₂ , 250 mmol/L KCl, and the solvent was water.

8. After mixing, perform PCR amplification;

9, PCR reaction conditions:

Pre-denaturation 50 ° C 2 min

95 ° C 15 min

Amplification 94 ° C 15s

55 ° C 45s 40 cycles, collecting fluorescence at 55 ° C

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

The specific results are shown in Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9 and Fig. 10, and the result judgment criterion is: if the amplification reaction curve of the gene to be detected appears, and the Ct value is less than 38, it is judged to be positive if 38<Ct<40, judged to be suspicious, suspiciously increased the amount of template, repeated 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 to be negative.

As can be seen from Fig. 5 and Fig. 6, the FAM channel (only for the Aedes type A Wolbachia fluorescent detection primers wAlbAF, wAlbAR and probe A, PCR group 1) only shows Aedes type A Wolbach The austenite amplification curve is positive, while both B-type Wolbachia and Culex pipiens have no amplification curve and are negative.

As can be seen from Figure 7 and Figure 8, the HEX channel (only for fluorescence detection primers for Aedes B-type Wolbachia) wAlbBF, wAlbBR and probe B, PCR group 2) only shows the amplification curve of Aedes B type Wolbachia, which is positive, while neither type A Wolbachia nor C. sinensis Wolbachia The amplification curve was negative.

As can be seen from Fig. 9 and Fig. 10, the Texas Red channel (only for the fluorescence detection primers for Culex pipa, wPipF, wPipR and probe C, PCR group 3) only shows the mosquitoes of Volkswagen The amplification curve was positive, while both type A Wolbachia and B-type Wolbachia had no amplification curve and were negative.

Therefore, the fluorescent detection primer of the present invention can detect Aedes A, Aedes B type Wolbachia and Culex pipiens, and can distinguish Aedes type A and Aedes type B Wall Bark's body and Culex pipiens.

Claims

A three-volume detection primer for Wolbachia, characterized in that it comprises

(1) For Aedes type A Wolbachia:

wAlbAF: 5'-CAGGGTTGATGTTGAAGGAG-3';

wAlbAR: 5'-GCACCAGCTTTTACTTGACC-3';

Probe A: 5'-TATCTTCAATTGCTATATCGTAATAAACG-3';

The two ends of the probe A are respectively combined with a fluorescence generating group FAM and a fluorescence quenching group BHQ1;

(2) For Aedes B type Wolbachia:

wAlbBF: 5'-AAAGGAACCGAAGTTCATGAT-3';

wAlbBR: 5'-TTGTTTAGTTGTGAGTAAAGTCCC-3';

Probe B: 5'-CAACATTTGCTCCAACAACTGTTGC-3';

The two ends of the probe B are respectively combined with a fluorescence generating group HEX and a fluorescence quenching group BHQ1;

(3) For Culex mosquitoes:

wPipF: 5'-GTTTGTGCAGCTAATAG-3';

wPipR: 5'-GTCTGCAAGGCCTATTTCTACTG-3';

Probe C: 5'-CTTTCAATTGAAAAGATTCGATCAAC-3';

Both ends of the probe C are bound to a fluorescence generating group Texas Red and a fluorescence quenching group BHQ2, respectively.
A method for detecting Wolbachia for non-disease diagnosis and treatment purposes, characterized in that sample DNA is extracted, the sample DNA is used as a template, and the three Wolbachias according to claim 1 The fluorescent detection primers wAlbAF, wAlbAR, probe A, wAlbBF, wAlbBR, probe B, wPipF, wPipR and probe C are subjected to real-time PCR amplification, and after completion of the amplification reaction, the fluorescent-generating group is labeled according to the probe. Fluorescence signal, read and record the number of PCR cycles of the sample Ct, according to the Ct value of the sample, according to the established judgment criteria, determine whether the sample contains Aedes type A, Aedes type B and Culex bacillus.
The detection method according to claim 2, 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; collect fluorescent signal at 55 °C.
A test kit for three Wolbachia, comprising a fluorescent detection primer and a PCR reagent, wherein the fluorescent detection primer comprises:

(1) For Aedes type A Wolbachia:

wAlbAF: 5'-CAGGGTTGATGTTGAAGGAG-3';

wAlbAR: 5'-GCACCAGCTTTTACTTGACC-3';

Probe A: 5'-TATCTTCAATTGCTATATCGTAATAAACG-3';

The two ends of the probe A are respectively combined with a fluorescence generating group FAM and a fluorescence quenching group BHQ1;

(2) For Aedes B type Wolbachia:

wAlbBF: 5'-AAAGGAACCGAAGTTCATGAT-3';

wAlbBR: 5'-TTGTTTAGTTGTGAGTAAAGTCCC-3';

Probe B: 5'-CAACATTTGCTCCAACAACTGTTGC-3';

The two ends of the probe B are respectively combined with a fluorescence generating group HEX and a fluorescence quenching group BHQ1;

(3) For Culex mosquitoes:

wPipF: 5'-GTTTGTGCAGCTAATAG-3';

wPipR: 5'-GTCTGCAAGGCCTATTTCTACTG-3';

Probe C: 5'-CTTTCAATTGAAAAGATTCGATCAAC-3';

Both ends of the probe C are bound to a fluorescence generating group Texas Red and a fluorescence quenching group BHQ2, respectively.