WO2016197837A1 - Amorces de détection fluorimétrique de trois types de wolbachia et procédé de détection et kit de détection associés - Google Patents

Amorces de détection fluorimétrique de trois types de wolbachia et procédé de détection et kit de détection associés Download PDF

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WO2016197837A1
WO2016197837A1 PCT/CN2016/084213 CN2016084213W WO2016197837A1 WO 2016197837 A1 WO2016197837 A1 WO 2016197837A1 CN 2016084213 W CN2016084213 W CN 2016084213W WO 2016197837 A1 WO2016197837 A1 WO 2016197837A1
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probe
wolbachia
fluorescence
aedes
type
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高秀洁
杨翠
周其伟
李丽梅
奚志勇
朱俭
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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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • 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.
  • 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 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.
  • Aedes aegypti and Culex pipiens 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.
  • PCR polymerase chain reaction
  • 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.
  • 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;
  • 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;
  • 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:
  • 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;
  • 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;
  • 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.
  • 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
  • Culex pipiens detected only C. sinensis, and the sensitivity was high.
  • the minimum detection limit was 100 copies/ml.
  • the invention has the advantages of rapid and high efficiency, simple operation, high specificity, high sensitivity, and simple identification.
  • 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;
  • FIG. 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;
  • FIG. 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.
  • a mosquito carrying Aedes A-type Wolbachia is smoked by carbon dioxide, and the abdomen is placed in a 0.2ul EP tube;
  • 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);
  • 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;
  • 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 ⁇ l) 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
  • the PCR B solution consists of a hot-start Taq enzyme, a reverse transcriptase, and dNTPs, and each 2 ⁇ l 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.
  • 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.
  • the detection limit of the fluorescent detection primer of the present invention is 100 copies/ml.
  • 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 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.
  • the detection limit of the fluorescent detection primer of the present invention is 100 copies/ml.
  • 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.
  • the detection limit of the fluorescent detection primer of the present invention is 100 copies/ml.
  • 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;
  • 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);
  • 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 ⁇ l) 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
  • the PCR B solution consists of a hot-start Taq enzyme, a reverse transcriptase, and dNTPs, and each 2 ⁇ l 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.
  • 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.
  • 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);
  • 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 ⁇ l) 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.
  • the PCR B solution consists of a hot-start Taq enzyme, a reverse transcriptase, and dNTPs, and each 2 ⁇ l 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.
  • 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 ⁇ l) 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.
  • the PCR B solution consists of a hot-start Taq enzyme, a reverse transcriptase, and dNTPs, and each 2 ⁇ l 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.
  • 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 ⁇ l) 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.
  • the PCR B solution consists of a hot-start Taq enzyme, a reverse transcriptase, and dNTPs, and each 2 ⁇ l 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne des amorces de détection fluorimétrique de trois types de Wolbachia et un procédé de détection et un kit de détection associés. En utilisant lesdites amorces de détection fluorimétrique de la présente invention, le procédé de détection peut être utilisé pour détecter Aedes de type A, Aedes de type B et Culex Wolbachia, et seule Aedes Wolbachia est détectée dans la famille Aedes, tandis que seule Culex Wolbachia est détectée dans la famille Culex, et la limite inférieure de détection est de 100 copies/mL.
PCT/CN2016/084213 2015-06-11 2016-05-31 Amorces de détection fluorimétrique de trois types de wolbachia et procédé de détection et kit de détection associés WO2016197837A1 (fr)

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CN201510320913.6A CN104911267A (zh) 2015-06-11 2015-06-11 三种沃尔巴克氏体的荧光检测引物及其检测方法和检测试剂盒
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CN102382898A (zh) * 2011-12-07 2012-03-21 广州沃巴克生物科技有限公司 快速定量检测蚊子组织中沃尔巴克氏体（Wolbachia）的引物及其试剂盒和方法

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CN102382898A (zh) * 2011-12-07 2012-03-21 广州沃巴克生物科技有限公司 快速定量检测蚊子组织中沃尔巴克氏体（Wolbachia）的引物及其试剂盒和方法

Non-Patent Citations (1)

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Title
CALVITTI, M. ET AL.: "Wolbachia Density and Cytoplasmic Incompatibility in Aedes Albopictus: Concerns with Using Artificial Wolbachia Infection as a Vector Suppression Tool", PLOS ONE, vol. 10, no. 3, 26 March 2015 (2015-03-26), XP055333462 *

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