CN104878108A - Fluorescence detection primer, detection method and detection kit of aedes A type and culex wolbachia - Google Patents

Abstract

The invention discloses a fluorescence detection primer, a detection method and a detection kit of aedes A type and culex wolbachia. The aedes A type and culex wolbachia can be quickly, efficiently and singly detected by utilizing the fluorescence detection primer according to the detection method, the singleness is strong, the specificity is high (only the aedes A type and culex wolbachia is detected, but aedes B type wolbachia is not amplified), the sensitivity is high, and the lowest detection limit is 100 copies/ml.

Description

The fluorescent detector primers of a kind of yellow-fever mosquito A type and culex Wolbachia and detection method thereof and detection kit

Technical field:

The invention belongs to biology field, be specifically related to the fluorescent detector primers of a kind of yellow-fever mosquito A type and culex Wolbachia and detection method thereof and detection kit.

Background technology:

Wolbachia (Wolbachia) is distributed widely in the symbiotic microorganism in arthropods body, and it may be monoid the abundantest in insect symbiotic microorganism.It is distributed in the castes such as Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera, lepidopteran.It is using vertical transmission as its basic model at host's intergenerational transmission.It is stably present in the sexual cell of host, passes to host's filial generation by ovum, and as not affine in tenuigenin by various ways, feminize and the reproduction activity of the modulate host such as gametocide.Its wide-scale distribution in host population is promoted by these regulating and controlling effects.

In host's reproductive behavior change that Wolbachia (Wolbachia) causes, tenuigenin not affine (CytoplasmicIncompatibility, CI) is a modal type.It shows as when the male mosquito infected by Wolbachia (Wolbachia) is not with when infecting female mosquito or infects the male and female mosquito mating of different sorts Wolbachia, and sperm and ovum combine embryo afterwards and cannot grow.The mosquito that the incompatible characteristic of kytoplasm of Wolbachia (Wolbachia) can cause Wolbachia to infect can invade the mosquito group not infecting or infect Wolbachia not of the same race carry out population compacting and population alternative.Its application has huge value to the control of mosquito matchmaker disease.Recent studies have found that, Wolbachia (Wolbachia) not only has outside above-mentioned characteristic, simultaneously its some important causal organism that can also carry with mosquito in mosquito body is (as dengue virus, plasmodium etc.) interact, suppress their increments in mosquito body and diffusion thus propagate in the mankind at these causal organisms of blocking-up.This restraining effect of Wolbachia (Wolbachia) is relevant in the distribution of mosquito in-vivo tissue to it, so, understand Wolbachia (Wolbachia) to contribute to rapid screening at mosquito distribution and go out the mosquito that the best Wolbachia of transmission blockage effect (Wolbachia) infects, also contribute to monitoring Wolbachia (Wolbachia) vertical transmission in mosquito group, also be conducive to Wolbachia (Wolbachia) infection conditions in mosquito tissue of monitoring extensive large territorial scope expeditiously simultaneously.

At occurring in nature, the kind of mosquito has a lot, common are yellow-fever mosquito and culex two kinds.Yellow-fever mosquito is divided into Aedes albopictus and Aedes aegypti, and wherein Aedes aegypti does not carry Wolbachia.Because Wolbach Salmonella can only survive in host, can not free living in vitro, thus how detecting Wolbach Salmonella effectively is specifically indispensable instrument to this bacterial studies.Relatively more conventional detection means has polymerase chain reaction method (PCR method) and immunostaining.Immunostaining takes time and effort, and specificity is bad.Along with popularizing of PCR method, the detection for Wolbachia makes great progress.Different mosquito kind carries different Wolbachias, can find out the different Wolbachia types that mosquito is carried intuitively, like this for our routine testing provides great convenience by PCR result.At present for the method also imperfection that different Wolbachia PCR detects.

Summary of the invention:

The object of this invention is to provide the fluorescent detector primers of a species specificity is strong, specificity is high and highly sensitive yellow-fever mosquito A type and culex Wolbachia.

The fluorescent detector primers of yellow-fever mosquito A type of the present invention and culex Wolbachia, is characterized in that, comprise

(1) for yellow-fever mosquito A type Wolbachia:

WAlbAF:5 '-GACATCAGGGTTGATGTTGA-3 ' (its nucleotide sequence is as shown in SEQ ID NO.1);

WAlbAR:5 '-GGAGTGATAGGCATATCTTCAAT-3 ' (its nucleotide sequence is as shown in SEQ ID NO.2);

Probe A:5 '-ATTTACCCCAGCAGATACTATTGCG-3 ' (its nucleotide sequence is as shown in SEQ ID NO.3);

The two ends of probe A are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively;

(2) for culex Wolbachia:

WPipF:5 '-GCAGGGCTATATTTTGGG-3 ' (its nucleotide sequence is as shown in SEQ ID NO.4);

WPipR:5 '-ATCAGTTTCTAGGTCATT-3 ' (its nucleotide sequence is as shown in SEQ ID NO.5);

Probe B:5 '-TCATCTATTAATAAAATAGGAGTATTACTAT-3 ' (its nucleotide sequence is as shown in SEQ IDNO.6);

The two ends of probe B are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively.

Second object of the present invention is to provide a kind of yellow-fever mosquito A type of Diagnosis and Treat object of non-diseases and the detection method of culex Wolbachia, it is characterized in that, extract sample DNA, with this sample DNA for template, use the fluorescent detector primers wAlbAF of above-mentioned yellow-fever mosquito A type and culex Wolbachia, wAlbAR, probe A, wPipF, wPipR and probe B, carry out fluorescent quantitative PCR, after amplified reaction completes, according to the fluorescent signal of the fluorescence generation group of probe mark, read and record the PCR cycle index Ct of sample, Ct value per sample, according to the judging criterion set up, whether judgement sample is containing yellow-fever mosquito A type and culex Wolbachia.

Described carries out fluorescent quantitative PCR, and its reaction conditions is preferably: denaturation 50 DEG C 5 minutes, 95 DEG C 15 minutes; Increase 94 DEG C 15 seconds, 55 DEG C 45 seconds, 40 circulations; Fluorescent signal is collected when 55 DEG C.

3rd object of the present invention is to provide the detection kit of a kind of yellow-fever mosquito A type and culex Wolbachia, and comprise fluorescent detector primers and PCR reagent, it is characterized in that, described fluorescent detector primers comprises

(1) for yellow-fever mosquito A type Wolbachia:

wAlbAF：5’-GACATCAGGGTTGATGTTGA-3’；

wAlbAR：5’-GGAGTGATAGGCATATCTTCAAT-3’；

Probe A:5 '-ATTTACCCCAGCAGATACTATTGCG-3 ';

The two ends of probe A are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively;

(2) for culex Wolbachia:

wPipF：5’-GCAGGGCTATATTTTGGG-3’；

wPipR：5’-ATCAGTTTCTAGGTCATT-3’；

Probe B:5 '-TCATCTATTAATAAAATAGGAGTATTACTAT-3 ';

The two ends of probe B are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively.

Utilize fluorescent detector primers of the present invention can the rapidly and efficiently single-minded yellow-fever mosquito A type that detects and culex Wolbachia according to detection method of the present invention, there is specificity strong, specificity is high (only detects yellow-fever mosquito A type and culex Wolbachia, and yellow-fever mosquito Type B Wolbachia does not increase), highly sensitive, its lowest detection is limited to 100copies/ml.

Therefore, the advantage such as the present invention has rapidly and efficiently, easy and simple to handle, high specific, highly sensitive, qualification are easy.

Accompanying drawing illustrates:

Fig. 1 is the experimental result picture of the sensitivity of yellow-fever mosquito A type Wolbachia, wherein I, II, III, IV difference representation DNA extract, the DNA extracts of 10,100,1000 times of dilutions;

Fig. 2 is the experimental result picture of the sensitivity of culex Wolbachia, wherein I, II, III, IV difference representation DNA extract, the DNA extracts of 10,100,1000 times of dilutions;

Fig. 3 is the experimental result picture of the repeatability of yellow-fever mosquito A type and culex Wolbachia, wherein I, II amplification curve representing different samples respectively;

Fig. 4 is the specific experimental result picture of FAM sense channel to A type Wolbachia;

Fig. 5 is FAM sense channel to the specific experimental result picture of Type B and wPip type Wolbachia (culex Wolbachia);

Fig. 6 is the specific experimental result picture of HEX sense channel to wPip type Wolbachia (culex Wolbachia);

Fig. 7 is HEX sense channel to the specific experimental result picture of A type and Type B Wolbachia.

Embodiment:

Following examples further illustrate of the present invention, instead of limitation of the present invention.

Embodiment 1: sensitivity

One, the sensitivity of yellow-fever mosquito A type Wolbachia

1, mosquito of carrying yellow-fever mosquito A type Wolbachia through carbonic acid gas smoked dizzy after, dissect belly and be placed in 0.2ul EP pipe;

2,20ulDNA extracting solution (formula of DNA extraction liquid is: 30mM NaOH, 0.25mM EDTA, 15mMTris-HCl, and solvent is water, needs vibration, white plates together added before using) is added;

3, fully mix;

4, after brief centrifugation, 99 DEG C of temperature are bathed 10 minutes;

5, obtain DNA extract ,-20 DEG C of preservations, namely obtain the genomic dna of yellow-fever mosquito A type Wolbachia;

6, DNA extract is carried out 10,100,1000 gradient dilutions, each gradient have two parallel, totally 8 samples, carry out pcr amplification as template DNA;

7, PCR amplification system:

Template DNA 2 μ l, PCR A liquid 18 μ l and PCR B liquid 2 μ l.

Described PCR A liquid, every 18 μ l contain 10pmol fluorescent detector primers wAlbAF (5 '-GACATCAGGGTTGATGTTGA-3 '), 10pmol fluorescent detector primers wAlbAR (5 '-GGAGTGATAGGCATATCTTCAAT-3 '), 5pmol probe A (5 '-ATTTACCCCAGCAGATACTATTGCG-3 ', the two ends of probe are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively), 10pmol fluorescent detector primers wPipF (5 '-GCAGGGCTATATTTTGGG-3 '), 10pmol fluorescent detector primers wPipR (5 '-ATCAGTTTCTAGGTCATT-3 '), 5pmol probe B (5 '-TCATCTATTAATAAAATAGGAGTATTACTAT-3 ', the two ends of probe are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively), all the other for pH value be the damping fluid of 8.0.Described buffer components is 50mmol/L Tris-HCl, 8mmol/L MgCl2,250mmol/L KCl, and solvent is water.

Described PCR B liquid is made up of hot start Taq polymerase, reversed transcriptive enzyme, dNTPs, and containing hot start Taq polymerase 5U, reversed transcriptive enzyme 2.5U and dNTPs 10mmol in every 2 μ l PCR B liquid, all the other are water.

8, the amplification of laggard performing PCR is mixed;

9, PCR reaction conditions:

Denaturation 50 DEG C of 2min

95℃ 15min

Increase 94 DEG C of 15s

55 DEG C of 45s, 40 circulations, collect fluorescence when 55 DEG C

10, amplification terminates rear observation amplification curve.

Concrete outcome as shown in Figure 1, result judging criterion: if there is the amplified reaction curve of testing gene, and Ct value is less than 38, then be judged as the positive, if 38<Ct<40, be judged as suspicious, suspiciously add large form amount, repeat amplification protcol, if obtain identical experimental result, be then judged as the positive, otherwise be negative, if Ct value is 0 or 40, be then judged as feminine gender.

As can be seen from Figure 1, the DNA extract of DNA extract, 10,100,1000 times of dilutions can amplify response curve, and its Ct is less than 38, is judged as the positive, and the DNA concentration in the solution of 1000 times of dilutions is 100copies/ml.Thus, the Monitoring lower-cut of fluorescent detector primers of the present invention is 100copies/ml.

Two, the sensitivity of culex Wolbachia

The experiment flow of the sensitivity of culex Wolbachia is with the sensitivity test of A type Wolbachia, and just template DNA changes the genomic dna of culex Wolbachia into.

Concrete outcome as shown in Figure 2, result judging criterion: if there is the amplified reaction curve of testing gene, and Ct value is less than 38, then be judged as the positive, if 38<Ct<40, be judged as suspicious, suspiciously add large form amount, repeat amplification protcol, if obtain identical experimental result, be then judged as the positive, otherwise be negative, if Ct value is 0 or 40, be then judged as feminine gender.

As can be seen from Figure 2, the DNA extract of DNA extract, 10,100,1000 times of dilutions can amplify response curve, and its Ct is less than 38, is judged as the positive, and the DNA concentration in the solution of 1000 times of dilutions is 100copies/ml.Thus, the Monitoring lower-cut of fluorescent detector primers of the present invention is 100copies/ml.

Embodiment 2: repeatability

1, two mosquitoes of carrying yellow-fever mosquito A type and culex Wolbachia through carbonic acid gas smoked dizzy after, dissect belly and be placed in 0.2ul EP pipe respectively;

2,20ul DNA extraction liquid (formula of DNA extraction liquid is: 30mM NaOH, 0.25mM EDTA, 15mMTris-HCl, and solvent is water, needs vibration, white plates together added before using) is added;

3, fully mix;

4, after brief centrifugation, 99 DEG C of temperature are bathed 10 minutes;

5, obtain DNA extract ,-20 DEG C of preservations, namely obtain the genomic dna of yellow-fever mosquito A type and culex Wolbachia;

6, the genomic dna of above-mentioned yellow-fever mosquito A type and culex Wolbachia carries out 10 repetitions as template DNA, amounts to 20 samples;

7, PCR amplification system:

Template DNA 2 μ l, PCR A liquid 18 μ l and PCR B liquid 2 μ l.

Described PCR A liquid, every 18 μ l contain 10pmol fluorescent detector primers wAlbAF (5 '-GACATCAGGGTTGATGTTGA-3 '), 10pmol fluorescent detector primers wAlbAR (5 '-GGAGTGATAGGCATATCTTCAAT-3 '), 5pmol probe A (5 '-ATTTACCCCAGCAGATACTATTGCG-3 ', the two ends of probe are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively), 10pmol fluorescent detector primers wPipF (5 '-GCAGGGCTATATTTTGGG-3 '), 10pmol fluorescent detector primers wPipR (5 '-ATCAGTTTCTAGGTCATT-3 '), 5pmol probe B (5 '-TCATCTATTAATAAAATAGGAGTATTACTAT-3 ', the two ends of probe are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively), all the other for pH value be the damping fluid of 8.0.Described buffer components is 50mmol/L Tris-HCl, 8mmol/L MgCl2,250mmol/L KCl, and solvent is water.

Described PCR B liquid is made up of hot start Taq polymerase, reversed transcriptive enzyme, dNTPs, and containing hot start Taq polymerase 5U, reversed transcriptive enzyme 2.5U and dNTPs 10mmol in every 2 μ l PCR B liquid, all the other are water.

8, the amplification of laggard performing PCR is mixed;

9, PCR reaction conditions:

Denaturation 50 DEG C of 2min

95℃ 15min

Increase 94 DEG C of 15s

55 DEG C of 45s, 40 circulations, collect fluorescence when 55 DEG C

10, amplification terminates rear observation amplification curve.

Concrete outcome as shown in Figure 3, result judging criterion: if there is the amplified reaction curve of testing gene, and Ct value is less than 38, then be judged as the positive, if 38<Ct<40, be judged as suspicious, suspiciously add large form amount, repeat amplification protcol, if obtain identical experimental result, be then judged as the positive, otherwise be negative, if Ct value is 0 or 40, be then judged as feminine gender.

As can be seen from Figure 3, each 10 repetitions of genome DNA sample of 2 parts of yellow-fever mosquito A and culex Wolbachia can amplify response curve, and its Ct is less than 38, is judged as the positive, illustrates thus, and fluorescent detector primers of the present invention is reproducible.

Embodiment 3: specificity

1, two Wolbachias containing single A type, two Aedes albopictus containing the Wolbachia of single Type B and two Guangzhou Culex quinquefasciatus (containing culex Wolbachia), totally 6 mosquitoes through carbonic acid gas smoked dizzy after, dissect belly and be placed in 0.2ulEP pipe respectively;

2,20ul DNA extraction liquid (formula of DNA extraction liquid is: 30mM NaOH, 0.25mM EDTA, 15mMTris-HCl, and solvent is water, needs vibration, white plates together added before using) is added respectively;

3, fully mix;

4, after brief centrifugation, 99 DEG C of temperature are bathed 10 minutes;

5, DNA extract is obtained,-20 DEG C of preservations, namely obtain the genomic dna totally six parts of the genomic dna of two parts of yellow-fever mosquito A type Wolbachias, the genomic dna of two parts of yellow-fever mosquito Type B Wolbachias and two parts of culex Wolbachias respectively, carry out pcr amplification using these six parts of genomic dnas as template DNA.

6、

PCR group 1:

Template DNA 2 μ l, PCR A liquid 18 μ l and PCR B liquid 2 μ l.

Described PCR A liquid, every 18 μ l contain 10pmol fluorescent detector primers wAlbAF (5 '-GACATCAGGGTTGATGTTGA-3 '), 10pmol fluorescent detector primers wAlbAR (5 '-GGAGTGATAGGCATATCTTCAAT-3 '), 5pmol probe A (5 '-ATTTACCCCAGCAGATACTATTGCG-3 ', the two ends of probe are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively), all the other for pH value be the damping fluid of 8.0.Described buffer components is 50mmol/L Tris-HCl, 8mmol/L MgCl2,250mmol/L KCl, and solvent is water.

Described PCR B liquid is made up of hot start Taq polymerase, reversed transcriptive enzyme, dNTPs, and containing hot start Taq polymerase 5U, reversed transcriptive enzyme 2.5U and dNTPs 10mmol in every 2 μ l PCR B liquid, all the other are water.

PCR group 2:

Template DNA 2 μ l, PCR A liquid 18 μ l and PCR B liquid 2 μ l.

Described PCR A liquid, every 18 μ l contain 10pmol fluorescent detector primers wPipF (5 '-GCAGGGCTATATTTTGGG-3 '), 10pmol fluorescent detector primers wPipR (5 '-ATCAGTTTCTAGGTCATT-3 '), 5pmol probe B (5 '-TCATCTATTAATAAAATAGGAGTATTACTAT-3 ', the two ends of probe are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively), all the other for pH value be the damping fluid of 8.0.Described buffer components is 50mmol/L Tris-HCl, 8mmol/L MgCl2,250mmol/L KCl, and solvent is water.

Described PCR B liquid is made up of hot start Taq polymerase, reversed transcriptive enzyme, dNTPs, and containing hot start Taq polymerase 5U, reversed transcriptive enzyme 2.5U and dNTPs 10mmol in every 2 μ l PCR B liquid, all the other are water.

8, the amplification of laggard performing PCR is mixed;

9, PCR reaction conditions:

Denaturation 50 DEG C of 2min

95℃ 15min

Increase 94 DEG C of 15s

55 DEG C of 45s, 40 circulations, collect fluorescence when 55 DEG C

10, amplification terminates rear observation amplification curve.

Concrete outcome as shown in Figure 4, Figure 5, Figure 6 and Figure 7, result judging criterion: if there is the amplified reaction curve of testing gene, and Ct value is less than 38, then be judged as the positive, if 38<Ct<40, be judged as suspicious, suspiciously add large form amount, repeat amplification protcol, if obtain identical experimental result, be then judged as the positive, otherwise be negative, if Ct value is 0 or 40, be then judged as feminine gender.

As can be seen from Figure 4 and Figure 5, FAM passage is (only with for fluorescent detector primers wAlbAF, wAlbAR of yellow-fever mosquito A type Wolbachia and probe A, PCR group 1) amplification curve of only display yellow-fever mosquito A type Wolbachia, it is positive, and Type B Wolbachia and culex Wolbachia all do not have amplification curve, it is feminine gender.

As can be seen from Figures 6 and 7, HEX passage is (only with for fluorescent detector primers wPipF, wPipR of culex Wolbachia and probe B, PCR group 2) amplification curve of only display culex Wolbachia, it is positive, and A type Wolbachia and Type B Wolbachia all do not have amplification curve, it is feminine gender.

Illustrate thus, fluorescent detector primers of the present invention only detects yellow-fever mosquito A type Wolbachia and culex Wolbachia, and Type B Wolbachia does not increase, and can tell yellow-fever mosquito A type Wolbachia and culex Wolbachia.

Claims (4)

1. a fluorescent detector primers for yellow-fever mosquito A type and culex Wolbachia, is characterized in that, comprises

(1) for yellow-fever mosquito A type Wolbachia:

wAlbAF：5’-GACATCAGGGTTGATGTTGA-3’；

wAlbAR：5’-GGAGTGATAGGCATATCTTCAAT-3’；

Probe A:5 '-ATTTACCCCAGCAGATACTATTGCG-3 ';

The two ends of probe A are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively;

(2) for culex Wolbachia:

wPipF：5’-GCAGGGCTATATTTTGGG-3’；

wPipR：5’-ATCAGTTTCTAGGTCATT-3’；

Probe B:5 '-TCATCTATTAATAAAATAGGAGTATTACTAT-3 ';

The two ends of probe B are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively.

2. the yellow-fever mosquito A type of the Diagnosis and Treat object of a non-diseases and the detection method of culex Wolbachia, it is characterized in that, extract sample DNA, with this sample DNA for template, the yellow-fever mosquito A type of use described in claim 1 and the fluorescent detector primers wAlbAF of culex Wolbachia, wAlbAR, probe A, wPipF, wPipR and probe B, carry out fluorescent quantitative PCR, after amplified reaction completes, according to the fluorescent signal of the fluorescence generation group of probe mark, read and record the PCR cycle index Ct of sample, Ct value per sample, according to the judging criterion set up, whether judgement sample is containing yellow-fever mosquito A type and culex Wolbachia.

3. detection method according to claim 2, is characterized in that, described carries out fluorescent quantitative PCR, and its reaction conditions is: denaturation 50 DEG C 5 minutes, 95 DEG C 15 minutes; Increase 94 DEG C 15 seconds, 55 DEG C 45 seconds, 40 circulations; Fluorescent signal is collected when 55 DEG C.

4. a detection kit for yellow-fever mosquito A type and culex Wolbachia, comprises fluorescent detector primers and PCR reagent, it is characterized in that, described fluorescent detector primers comprises

(1) for yellow-fever mosquito A type Wolbachia:

wAlbAF：5’-GACATCAGGGTTGATGTTGA-3’；

wAlbAR：5’-GGAGTGATAGGCATATCTTCAAT-3’；

Probe A:5 '-ATTTACCCCAGCAGATACTATTGCG-3 ';

The two ends of probe A are combined with fluorescence generation group FAM and fluorescent quenching group B HQ1 respectively;

(2) for culex Wolbachia:

wPipF：5’-GCAGGGCTATATTTTGGG-3’；

wPipR：5’-ATCAGTTTCTAGGTCATT-3’；

Probe B:5 '-TCATCTATTAATAAAATAGGAGTATTACTAT-3 ';

The two ends of probe B are combined with fluorescence generation group HEX and fluorescent quenching group B HQ1 respectively.