CN103146834A - Allglo probe-based detection method of anopheles sinensis knockdown resistance gene mutation site - Google Patents

Abstract

The invention discloses a detection method for anopheles sinensis knockdown resistance gene mutation site based on Allglo probe. The special primer provided by the present invention is made up of primer 1 and primer 2 shown in SEQ ID NO:1 and SEQ ID NO:2 respectively by nucleotide sequence; Described reagent is made up of described special primer, probe 1 (sequence SEQ ID NO: ID NO:3), probe 2 (sequence SEQ ID NO:4) and probe 3 (sequence SEQ ID NO:5); the reaction reagent is composed of the reagent and Allglo reaction buffer; the kit Include the reagents. The invention provides an efficient, quick and convenient detection method for the kdr gene mutation site of Anopheles sinensis, which has high specificity and sensitivity and can quickly and accurately detect the mutation type of the kdr gene of Anopheles sinensis.

Description

Detection method of knockdown resistance gene mutation site in Anopheles sinensis based on Allglo probe

technical field

The invention belongs to the technical field of gene mutation detection, in particular to a detection method for anopheles sinensis knockdown resistance (kdr) gene mutation site, in particular to an Anopheles sinensis knockdown resistance (kdr) gene mutation site based on Allglo probe Fluorescent quantitative PCR detection method and kit.

Background technique

Anopheles sinensis is one of the main malaria vectors in my country. Recent reports point out that the transmission ability of Anopheles sinensis in central my country has been greatly improved compared with the last century, and the enhancement of its malaria transmission ability may be the same as this century. Malaria outbreaks in central my country and the Huanghuai region are closely related. The use of insecticides to control Anopheles mosquitoes is a very important means of malaria prevention and control. Humans have used insecticides to control mosquitoes for more than 80 years. Since the early 1980s, pyrethroid insecticides have been used to treat mosquito nets and spray Mosquito eradication has gradually become one of the important antimalarial measures. Resistance of malaria vectors to insecticides has spread rapidly across the globe due to the heavy use of insecticides over the years. In recent years, the monitoring data of vector resistance in central provinces such as Hubei and Jiangsu show that the resistance of local Anopheles sinensis to deltamethrin insecticide has increased significantly, and the insecticide of Anopheles sinensis in some areas of Hubei province has The median lethal concentration (LC50) of the dose has increased by 306 times compared with 1998.

Knockdown resistance (kdr) is one of the molecular mechanisms for mosquitoes to develop resistance to pyrethroid insecticides. It is caused by mutations in the amino acid sequence encoded by the S6 segment of the second domain of the insect sodium ion channel protein, namely kdr Gene L1014 site (TTG) mutation. The study found that there are both L1014F and L1014S mutations in Anopheles gambiae. These mutations lead to changes in the binding sites of pyrethroid insecticides and sodium ion channel proteins, and the ability to bind insecticides is weakened, thereby affecting its Reduced sensitivity (D.Martinez-Torres, F.Chandre, M.S.Williamson, et al.. Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malariavector Anopheles gambiae s.s [J]. Insect Mol Biol, 1998, 7 (2) : 179-184; J.Pinto, A.Lynd, N.Elissa, et al.．Co-occurrence of East and West African kdr mutations suggests high levels of resistance to pyrethroid insecticides in Anopheles gambiae from Libreville, Gabon[J]．Med Vet Entomol, 2006, 20 (1): 27-32); in 2007, KimH et al. reported that there were two mutant types of Anopheles sinensis kdr gene, L1014F (TTT) and L1014C (TGT). Ester sensitivity was significantly reduced (H.Kim, J.H.Baek, W.-J.Lee, et al.. Frequency detection of pyrethroid resistance allele in Anophelessinensis populations by real-time PCR amplification of specific allele(rtPASA)[J]. Biochemistry and Physiology, 2007, 87(1):54-61). Therefore, the determination of the mutation frequency of genes related to mosquito knockdown resistance in different regions can provide a scientific basis for the formulation of malaria vector control measures.

At present, there have been reports on the detection of drug-resistant kdr gene mutations in Anopheles mosquitoes in China by using PCR and TaqMan method. In 2011, Wu Song et al. reported the establishment of PCR detection method for knockdown resistance mutation of Anopheles sinensis (Wu Song, Ma Jian, Liu Qian et al. Establishment of PCR detection method for knockdown resistance mutation of Anopheles sinensis[J]. Journal of Comorbid Diseases, 2011, 27(11): 1008-1010); Chinese patents CN102373280A and CN102373282A respectively disclose a kind of Anopheles sinensis drug resistance AS-PCR detection kit and PCR-RFLP detection kit, but the above PCR method Need to open the cover to test the results, which is easy to cause cross-contamination and false positives. The result needs to be judged according to the strength of the electrophoresis band, which reduces the sensitivity and specificity of the test, and is easy to misjudgment and misjudgment. It is not conducive to the health of operators; Chinese patent CN102373281A discloses a TaqMan PCR detection kit for Anopheles sinensis drug resistance, but the synthesis of TaqMan probes is complicated and expensive, and it is necessary to use different TaqMan probe combinations in two tubes to perform fluorescence quantitative amplification respectively. The operation and detection procedures are cumbersome, and the detection sensitivity and specificity need to be improved; in addition, the above methods need to pre-extract Anopheles mosquito genome DNA as the amplification template, which is time-consuming, labor-intensive, low detection efficiency, and easy to contaminate.

In field vector monitoring, there is still a lack of high sensitivity and specificity, low cost, simple and rapid detection method and corresponding reagents and kits for the mutation of Anopheles sinensis kdr gene.

Contents of the invention

In view of the above-mentioned defects in the existing Anopheles sinensis kdr gene mutation site detection method and kit, the first purpose of the present invention is to provide a special primer for detecting the Anopheles sinensis knockdown resistance gene mutation site, which is composed of nuclear The nucleotide sequence is composed of primer 1 and primer 2 shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

Another object of the present invention is to provide a reagent for detecting the mutation site of the Anopheles sinensis knockdown resistance gene, which consists of the special primers, probe 1, probe 2 and probe 3,

The nucleotide sequence of the probe 1 is shown in SEQ ID NO:3;

The nucleotide sequence of the probe 2 is shown in SEQ ID NO:4;

The nucleotide sequence of the probe 3 is shown in SEQ ID NO:5;

The 5' and 3' ends of the probe 1 are labeled with MAR groups;

Both the 5' and 3' ends of the probe 2 are marked with a JUP group;

Both the 5' and 3' ends of the probe 3 are labeled with NEP groups.

The 7th base C in the nucleotide sequence of the probe 1, the probe 2 and the probe 3 and the 12th base T in the nucleotide sequence of the probe 2 are locked nucleotides.

The third object of the present invention is to provide a reaction reagent for detecting the mutation site of Anopheles sinensis knockdown resistance gene, consisting of the reagent and Allglo reaction buffer, in the reaction reagent, the primer 1 and the primer The final concentration of 2 is 300-800nM, preferably 500nM, and the final concentration of probe 1, probe 2 and probe 3 is 200-600nM, preferably 400nM.

The fourth object of the present invention is to provide a kit for detecting the mutation site of the knockdown resistance gene of Anopheles sinensis, including the reaction reagent.

The fifth object of the present invention is to provide the special primer or the reagent or the reaction reagent or the kit in the detection of the Anopheles sinensis knockdown resistance gene mutation site and the preparation of the Anopheles sinensis knockdown resistance gene Application in mutation site detection products, wherein the mutation site is L1014F or L1014C.

The sixth object of the present invention is to provide a method for detecting the mutation site of the Anopheles sinensis knockdown resistance gene, the steps are as follows: take 10-20 μl of the reaction reagent or the reaction reagent in the kit, add One Anopheles sinensis mosquito leg was subjected to real-time fluorescence quantitative amplification with Allglo probe. After the amplification was completed, the instrument automatically analyzed the amplification curve and judged the result according to the amplification curve;

The amplification reaction conditions are as follows:

Pre-denaturation at 95°C for 3-10 minutes, denaturation at 95°C for 10-30s, annealing and extension at 60°C for 20-60s, and signal collection during the annealing and extension stage, a total of 35-45 cycles; preferably, pre-denaturation at 95°C for 5 minutes, 95°C Denaturation for 15s, annealing and extension at 60°C for 30s, signal collection during the annealing and extension stage, a total of 40 cycles;

The criteria for judging the results are as follows:

When only the FAM channel has an amplification signal, the knockdown resistance gene does not contain the mutation site and is TTG homozygous wild type;

When only the VIC/HEX channel has an amplification signal, the knockdown resistance gene has a L1014F mutation site, which is a TTT homozygous mutant type;

When only the CY5 channel has an amplification signal, the knockdown resistance gene has a L1014C mutation site, which is a TGT homozygous mutant type;

When any two of FAM, VIC/HEX and CY5 channels have amplification signals, the knockdown resistance gene is heterozygous. Wherein, the heterozygous type is wild mutation heterozygous type or mutation heterozygous type, including TTG/TTT wild mutation heterozygous type, TTG/TGT wild mutation heterozygous type or TTT/TGT mutant heterozygous type.

Beneficial technical effect of the present invention is as follows:

The invention provides a method for detecting mutation sites of Anopheles sinensis knockdown resistance gene based on Allglo probe and corresponding reagents and kits, which have high specificity and sensitivity and can detect Anopheles sinensis kdr quickly and accurately Gene mutation type, compared with the existing technology, has the following advantages:

1. Compared with ordinary PCR, AS-PCR and PCR-RFLP, the present invention is a completely closed-tube detection, which can effectively avoid cross-contamination and false positives, without electrophoresis and other post-processing, and can directly carry out mutation analysis according to the fluorescence quantitative amplification curve. Type identification, no misjudgment and misjudgment, high accuracy;

2. Compared with the TaqMan probe, the Allglo probe of the present invention uses the same fluorophore (MAR, JUP, NEP) at both ends, which are mutually a reporter group and a quencher group, which is different from a TaqMan probe with a fluorophore at one end. The structure of one end is a quenching group, which can reduce the influence of the background while increasing the Tm value, improve the detection sensitivity and specificity, and the synthesis is simple and the price is cheap;

3. The invention does not need to extract the genomic DNA of Anopheles mosquito, and can directly take the mosquito legs as the amplification template, and combine the triple probes into one tube for simultaneous detection. The operation is very simple, the detection efficiency is high, and the cost is low;

4. The present invention establishes an efficient, accurate, fast and simple method for detecting the mutation site of the kdr gene of Anopheles sinensis, which is expected to be further popularized and applied in the monitoring work of Anopheles sinensis vector, thereby providing a new tool for malaria monitoring work.

Description of drawings

Figure 1 is a schematic diagram of the fluorescent quantitative amplification primers and Allglo probe design area of the present invention, wherein, Primer-F and Primer-R: fluorescent quantitative amplification upstream and downstream primers; Probe: Allglo probe design area.

Fig. 2 is the TTG homozygous wild type Allglo fluorescence quantitative PCR amplification map of the L1014 site of the Anopheles sinensis kdr gene.

Figure 3 is the Allglo fluorescent quantitative PCR amplification map of the homozygous TTT mutation at the L1014 site of the Anopheles sinensis kdr gene.

Figure 4 is the Allglo fluorescent quantitative PCR amplification map of the homozygous TGT mutation at the L1014 site of the Anopheles sinensis kdr gene.

Figure 5 is the Allglo fluorescent quantitative PCR amplification map of the TTG/TTT wild mutation heterozygous type at the L1014 site of the Anopheles sinensis kdr gene.

Fig. 6 is an Allglo fluorescence quantitative PCR amplification map of the TTG/TGT wild mutation heterozygous type at the L1014 site of the Anopheles sinensis kdr gene.

Fig. 7 is an Allglo fluorescence quantitative PCR amplification map of TTT/TGT mutation heterozygous type L1014 site of Anopheles sinensis kdr gene.

Detailed ways

The present invention will be described in detail below through examples.

The reagents and instruments used in the following examples are as follows: Roche Probe Master (2×) was purchased from Roche; probe 1 (kdr-TTG), probe 2 (kdr-TTT) and probe 3 (kdr-TGT) were purchased from Shanghai Synthesized by Huirui Biological Co., Ltd.; LightCycler480 fluorescent quantitative eight-tube was purchased from Bioplastic Company; fluorescent quantitative PCR instrument Roche Lightcycler480II was purchased from Roche Company; Primer 1 (upstream primer Primer-F) and primer 2 (downstream primer Primer-R) were obtained from Synthesized by Nanjing KingScript Biotechnology Co., Ltd.; Fast Tissue-to-PCR Kit Anopheles sinensis gDNA Extraction Kit, Thermo Scientific Maxima Probe qPCR Master Mix (2×) were purchased from Thermo, USA; other materials or reagents, unless otherwise specified , are commercially available.

The design of embodiment 1 primer and probe

According to the Anopheles sinensis knockdown resistance (kdr) gene sequence (GenBank accession number GI84646709) and the L1014 site mutation, a pair of primers and three probes were designed with Primer Premier6.0: Primer 1 (upstream primer Primer-F ) and primer 2 (downstream primer Primer-R) nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, probe 1 (kdr-TTG), probe 2 (kdr-TTT) and probe 3 (kdr-TGT) nucleotide sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 respectively, and the 5' and 3' ends of the above three probes are respectively MAR, JUP, NEP gene markers, wherein the 7th base C and the 8th base T of the three probes are locked nucleotides. Refer to Figure 1 for the specific design regions of the above primers and probes.

The design of embodiment 2 reaction system and conditions

The invention adopts a triple probe detection method, that is, each reaction tube contains three probes kdr-TTG, kdr-TTT and kdr-TGT that can detect TTG, TTT and TGT.

Prepare the Allglo real-time fluorescence quantitative PCR reaction system: Roche Probe Master (2×) 5 μl, the final concentration of the upstream primer Primer-F and the downstream primer Primer-R is 500nM, the final concentration of the probes kdr-TTG, kdr-TTT and kdr-TGT is 400nM, Make up to 10 μl of sterilized water, one leg of Anopheles sinensis, and put it in LightCycler480 fluorescent quantitative eight-connected tube.

Determine the Allglo real-time fluorescent quantitative PCR reaction conditions: put the eight-tube tube in Roche Lightcycler480II for real-time fluorescent quantitative PCR amplification. The reaction conditions are set as follows: 95°C pre-denaturation for 5 minutes, 95°C denaturation for 15s, 60°C annealing and extension for 30s, a total of 40 cycle, the signal is collected during the annealing and extension stage, and the instrument automatically analyzes the amplification curve after the amplification is completed.

Probe kdr-TTG detection uses FAM channel (detection wavelength 465-510nm), probe kdr-TTT detection uses VIC/HEX channel (detection wavelength 533-580nm), probe kdr-TGT detection uses CY5 channel (detection wavelength 618- 680nm).

Embodiment 3 detection result judgment

Combined with the amplification curve to analyze and interpret the detection results.

Kdr genotype identification: identify specific genotypes based on the fluorescence quantitative amplification curves of different channels. When only the FAM channel has an amplification signal, the kdr gene does not contain a mutation site, and it is a TTG homozygous wild type; when only the VIC/HEX channel has an amplification signal, the kdr gene has a L1014F mutation site, which is a TTT homozygous mutation type; when only the CY5 channel has an amplification signal, the kdr gene has the L1014C mutation site, which is a TGT homozygous mutant type; when any two of the FAM, VIC/HEX and CY5 channels have amplification signals, the kdr gene is Mutants (wild mutant heterozygous or mutation heterozygous), that is, when both FAM and VIC/HEX channels have amplification signals, but CY5 channels have no amplification signals, the kdr gene is heterozygous for TTG/TTT wild mutations, When both FAM and CY5 channels have amplification signals, but VIC/HEX channels have no amplification signals, the kdr gene is heterozygous for TTG/TGT wild mutation; when both VIC/HEX and CY5 channels have amplification signals, and FAM channels When there is no amplification signal, the kdr gene is heterozygous for the TTT/TGT mutation.

Example 4 Allglo probe real-time fluorescent quantitative PCR detection of Anopheles sinensis field samples

140 samples of Anopheles sinensis were collected from adult mosquitoes in Sihong, Jiangsu and Liuhe, Nanjing. Take one mosquito leg from each Anopheles mosquito mentioned above, perform Allglo real-time fluorescent quantitative PCR amplification with the method described in Examples 1 to 3, and set a template-free negative control (NTC) at the same time. The results were analyzed and interpreted, and the typing identification results are shown in Table 1, in which, TTG wild homozygous, TTT mutation homozygous, TGT mutation homozygous, TTG/TTT wild mutation heterozygous, TTG/TGT wild mutation heterozygous The real-time quantitative PCR amplification curves of kdr gene of TTT/TGT mutation heterozygous type and TTT/TGT mutation type are shown in Fig. 2 to Fig. 7 respectively.

Comparative example 1 TaqMan probe real-time fluorescent quantitative PCR detection of Anopheles sinensis field samples

Use Fast Tissue-to-PCR Kit Anopheles sinensis gDNA Extraction Kit to extract the above 140 Anopheles sinensis genomic DNA, the extraction steps are as follows: Take 1~3 Anopheles sinensis mosquito legs and put them in a 1.5ml centrifuge tube, add 100μl protease K, make the tissue completely submerged in the liquid, incubate at 55°C for 10 minutes, incubate at 95°C for 3 minutes, add 100 μl of Neutralization solution, mix well, centrifuge at 8000g for 3 minutes, absorb the supernatant and store it in a new centrifuge tube for later use.

According to the Anopheles sinensis sodium channel protein (VGSC) gene sequence (Genbank accession number: GI84646709) and its common mutation types at the L1014 site (H Kim, JH Baek, WJ Lee, et al. Frequency detection of pyrethroid resistance allele in Anopheles sinensispopulations by real-time PCR amplification of specific allele(rtPASA)[J].PesticideBiochemistry and Physiology,2007,87(1):54-61.), design a pair of real-time fluorescence quantitative PCR amplification primers and 3 probes, The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO:6 and SEQ ID NO:7 respectively, TaqMan-MGB probe kdr-TTG (wild type), TaqMan-MGB probe kdr-TTT (L1014F mutation) And the nucleotide sequences of TaqMan-MGB probe kdr-TGT (L1014C mutation) are shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 respectively, and the 5' ends of the three probes are respectively made of FAM , HEX and NED tags, and the 3' end is modified with MGB. The primers were synthesized by Nanjing GenScript Biotechnology Company, and the TaqMan-MGB probe was synthesized by Shanghai Jikang Biotechnology Company.

TaqMan probe real-time fluorescent quantitative PCR reaction system: total reaction system 10μl, including ThermoScientific Maxima Probe qPCR Master Mix (2×) 5μl, primer concentration 500nmol/L, TaqMan-MGB probe concentration 500nmol/L, DNA template 2μl, placed in Roche Lightcycler480II was used for PCR amplification.

TaqMan probe real-time fluorescent quantitative PCR reaction conditions: two-step method, namely 50°C for 2 minutes, 95°C for 5 minutes; 95°C for 15 seconds, 60°C for 30 seconds, 40 cycles; 40°C for 10 seconds; 60°C for 10 seconds; Acquire the Signal.

The FAM channel (to detect the kdr-TTG probe) and the VIC/HEX channel (to detect the kdr-TTT and kdr-TGT probes) were used to identify the wild-type and mutant types of the L1014 site of the kdr gene, respectively. The results of typing and identification are shown in Table 1.

Another 140 legs of Anopheles mosquitoes mentioned above were taken, and the genomic DNA was extracted respectively (the extraction method is the same as above), and the kdr gene was amplified by PCR (see the following literature for specific methods: D.Zhong, X.Chang, G.Zhou, et al.. Relationship between Knockdown Resistance, Metabolic Detoxification and Organismal Resistance to Pyrethroids in Anopheles sinensis[J].PLoS One, 2013, 8(2):e55475), was submitted to Nanjing GenScript Biotechnology Co., Ltd. for sequencing, and the kdr gene analysis was carried out according to the sequencing results For type identification, the results of type identification in Example 4 and Comparative Example 1 were compared with it, and the results are shown in Table 1.

Table 1 Comparison of the identification results of TaqMan and Allglo fluorescence quantitative typing

From the data in Table 1, it can be seen that the Allglo fluorescent quantitative PCR detection result of the present invention is completely consistent with the sequencing result, and there are 3 sample errors in the TaqMan fluorescent quantitative PCR detection. It can be seen that the detection accuracy of the Allglo fluorescent quantitative PCR of the present invention is higher than that of the TaqMan fluorescent quantitative PCR detection. .

The above are only preferred implementations of the present invention, and the present invention is not limited to the above examples. It can be understood that other improvements and changes directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims (7)

1. A special primer for detecting anopheles sinensis knockdown resistance gene mutation site, characterized in that: primer 1 and primer 2 are respectively shown in SEQ ID NO:1 and SEQ ID NO:2 by nucleotide sequence composition. 2. A reagent for detecting anopheles sinensis knockdown resistance gene mutation site, characterized in that: it is composed of special primers, probe 1, probe 2 and probe 3 according to claim 1, The nucleotide sequence of the probe 1 is shown in SEQ ID NO:3; The nucleotide sequence of the probe 2 is shown in SEQ ID NO:4; The nucleotide sequence of the probe 3 is shown in SEQ ID NO:5; The 5' and 3' ends of the probe 1 are labeled with MAR groups; Both the 5' and 3' ends of the probe 2 are marked with a JUP group; Both the 5' and 3' ends of the probe 3 are labeled with NEP groups. The 7th base C in the nucleotide sequence of the probe 1, the probe 2 and the probe 3 and the 12th base T in the nucleotide sequence of the probe 2 are locked nucleotides. 3. A reaction reagent for detecting Anopheles sinensis knockdown resistance gene mutation site, characterized in that: it is made up of reagent and Allglo reaction buffer according to claim 2, and in said reaction reagent, said primer 1 and primer The final concentrations of 2 are all 300-800 nM, and the final concentrations of probe 1, probe 2 and probe 3 are all 200-600 nM. 4. A kit for detecting the mutation site of anopheles sinensis knockdown resistance gene, characterized in that it comprises the reaction reagent according to claim 3. 5. the special primer of claim 1 or the reagent of claim 2 or the reaction reagent of claim 3 or the kit of claim 4 in detecting Anopheles sinensis knockdown resistance gene mutation site and preparing Anopheles sinensis Knockdown resistance gene mutation site detection product, the mutation site is L1014F or L1014C. 6. A detection method for anopheles sinensis knockdown resistance gene mutation site, characterized in that the steps are as follows: take 10-20 μl of the reaction reagent according to claim 3 or the reaction reagent in the kit according to claim 4 , add 1 Anopheles sinensis mosquito leg for real-time fluorescence quantitative amplification of Allglo probe, after the amplification is completed, the instrument will automatically analyze the amplification curve, and judge the result according to the amplification curve; The amplification reaction conditions are as follows: Pre-denaturation at 95°C for 3-10 minutes, denaturation at 95°C for 10-30s, annealing and extension at 60°C for 20-60s, signal collection during the annealing and extension stage, a total of 35-45 cycles; The criteria for judging the results are as follows: When only the FAM channel has an amplification signal, the knockdown resistance gene does not contain the mutation site and is TTG homozygous wild type; When only the VIC/HEX channel has an amplification signal, the knockdown resistance gene has a L1014F mutation site, which is a TTT homozygous mutant type; When only the CY5 channel has an amplification signal, the knockdown resistance gene has a L1014C mutation site, which is a TGT homozygous mutant type; When any two of FAM, VIC/HEX and CY5 channels have amplification signals, the knockdown resistance gene is heterozygous. 7. according to the detection method of the described Anopheles sinensis knockdown resistance gene mutation site of claim 6, it is characterized in that: described heterozygous type is wild mutation heterozygous type or mutant heterozygous type, comprises TTG/TTT wild mutation Heterozygous, TTG/TGT wild mutation heterozygous, or TTT/TGT mutant heterozygous.

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