CN102373280B - Anopheles sinensis drug resistance AS-PCR (Polymerase Chain Reaction) detection kit and special primers thereof - Google Patents
Anopheles sinensis drug resistance AS-PCR (Polymerase Chain Reaction) detection kit and special primers thereof Download PDFInfo
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Abstract
The invention discloses an Anopheles sinensis drug resistance AS-PCR (Polymerase Chain Reaction) detection kit and special primers thereof. Primers for detecting kdr gene mutational sites in Anopheles sinensis provided by the invention consist of a primer group A, a primer group B and a primer group C, wherein the primer group A comprises a primer 2 and a primer 3, and the nucleotide sequences of the primer 2 and the primer 3 are respectively a sequence 2 and a sequence 3 in a sequence table; the primer group B comprises a primer 2 and a primer 4, and the nucleotide sequences of the primer 2 and the primer 4 are respectively a sequence 2 and a sequence 4 in the sequence table; and the primer group C comprises a primer 2 and a primer 5, and the nucleotide sequences of the primer 2 and the primer 5 are respectively a sequence 2 and a sequence 5 in the sequence table. An experiment of the invention proves that the invention provides a quick, easy and flexible Anopheles sinensis kdr mutational allele detection kit.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of Anopheles sinensis resistance AS-PCR detection kit and primer special thereof.
Background technology
Anopheles sinensis (Anopheles sinensis) is distributed in China all provinces and cities except Qinghai and Xinjiang, it is the dominant mosquito in the network of rivers, China Plain and Rice Cropping area, family is wild amphibious, perch that crop around the village is planted daytime more and reed clump, thicket in, part is perched at the cowhouse animal house, partially have a liking for poultry blood (take draught animals such as ox, horse, pigs as main), hold concurrently and inhale human blood, survive the winter with adult mosquito.Anopheles sinensis is the important communication media of China's malaria, the area to the north of 34 ° of north latitude especially, and it is main or unique communication media.At present, malaria there is no effective vaccine and can use, and the population quantity of malaria control medium is the important means of preventing and treating malaria.Chemical prevention is easy to operate because of it, can be rapidly, Control pests population effectively, in the anti-system planning of insect vector, occupy critical role.Come in the past few decades, for the life habit of Anopheles sinensis, use pyrethroid insecticides, adopt the anti-malaria measure of the kill mosquitos such as residual spray and Impregnated Mosquito Nets, obtained unusual effect.Yet chemical prevention has also brought counter productive when giving full play to its advantage, has caused the drug-fast generation of Anopheles sinensis.Since the nineties, the Anopheles sinensis of domestic various places report is more and more obvious to the resistance of pyrethroid insecticides.The Chongqing Anopheles sinensis reached elementary resistance to Deltamethrin and DDT in 1993,10 Anopheles sinensis populations that gathered in Yunnan in 1998 have 5 to DDT generation resistance, the Anopheles sinensis population on the ground such as Wenzhou District of Zhejiang Province in 1999, Ningbo reaches high resistance population (R/S=15-20), 2009, the Anopheles sinensis of some areas, Jiangsu produced highly resistant to Deltamethrin.
Insect produces after the resistance sterilant, and the effect of chemical prevention will descend along with drug-fast rising, and the control of insect also can be seriously influenced.In order to tackle the resistance of pest population, reach the prevention effect of expection in order to overcome resistance, the Utilization of pesticides amount, the formulation rate and the spraying times that comprise unit surface, just have to continue to increase, this is tantamount to continuing to increase the selective pressure of sterilant to the insect vector resistance, and this resistance also can be to remote diffusion, indirectly also may cause the generation of some medium disease and popular.To the monitoring of pest resistance to insecticide, the speed that resistance increases is much larger than the speed of novel pesticide, novel form research and development from these years.Therefore, how to strengthen insect vector resistance management, to delay drug-fast generation and development, existing Utilization of pesticides life-span of prolongation be the important content during insect is administered.
Insect mainly is knock down resistance (knockdown resistance, kdr) to the resistance of pyrethroid insecticides, and the kdr gene has vital role in the resistance of Anopheles sinensis to pyrethroid.The action target of pyrethroid mainly is the sodium-ion channel on the insect voltage-dependent neuron membrane, action target spot on the ionic channel has reduced susceptibility and has produced knock down resistance sterilant, therefore knock down resistance is different from metabolic resistance, can not be reduced by the inhibitor of esterase or mixed-functional oxidase.By part sodium channel sequence relatively responsive, the resistance Anopheles sinensis, the L1014F (leucine is to phenylalanine) that finds sodium channel gene is that the sudden change of TTG → TTT and sudden change that L1014C (leucine is to halfcystine) is TTG-TGT are relevant with knock down resistance.This is that China is about the reported first of Anopheles sinensis knock down resistance and transgenation thereof.
The kdr gene can be used as insect to a molecule marker of pyrethroid generation resistance, by monitoring kdr allelotrope or genotype frequency, understands the state of resistant gene in Genetic Constitution of Population.Tan Weilong etc. (2011) use the specific alleles PCR method, and 11 Anopheles sinensis populations adopting Jiangsu, Anhui are carried out the mensuration of median lethal concentration(LC﹠-{50}) (LC50) and kdr gene frequency, and find LC
50For the representative the effective cypermethrin resistance and Anopheles sinensis kdr gene frequency between have positive correlation.Research explanation Kdr gene can be used as Anopheles sinensis to a molecule marker of pyrethroid generation resistance, by monitoring kdr allelotrope or genotype frequency, understands the state of resistant gene in Genetic Constitution of Population, can predict generation and the development of resistance.
China does not also have kdr resistance molecular detecting method and the test kit for Anopheles sinensis of moulding at present.
Summary of the invention
An object of the present invention is to provide a kind of primer that detects kdr gene mutation site in the Anopheles sinensis.
Primer provided by the invention is comprised of primer sets A, primer sets B and primer sets C:
Described primer sets A comprises primer 2 and primer 3, and the nucleotide sequence of described primer 2 and primer 3 is respectively in the sequence table sequence 3 in the sequence 2 and sequence table;
Described primer sets B comprises primer 2 and primer 4, and the nucleotide sequence of described primer 2 and primer 4 is respectively in the sequence table sequence 4 in the sequence 2 and sequence table;
Described primer sets C comprises primer 2 and primer 5, and the nucleotide sequence of described primer 2 and primer 5 is respectively in the sequence table sequence 5 in the sequence 2 and sequence table.
Described primer sets A also comprises primer 1, and the nucleotides sequence of described primer 1 is classified sequence 1 in the sequence table as;
Described primer sets B also comprises primer 1, and the nucleotides sequence of described primer 1 is classified sequence 1 in the sequence table as;
Described primer sets C also comprises primer 1, and the nucleotides sequence of described primer 1 is classified sequence 1 in the sequence table as.
Described primer sets A is comprised of primer 1-3;
Described primer sets B is comprised of primer 1,2 and 4;
Described primer sets C is comprised of primer 1,2 and 5;
Described primer sets A, described primer sets B and described primer sets C are independent packaging;
Described mutational site is L1014F or L1014C.
Second purpose of the present invention provides a kind of PCR reagent that detects kdr gene mutation site in the Anopheles sinensis.
PCR reagent provided by the invention is comprised of reagent A, reagent B and reagent C;
Described reagent A is comprised of described primer sets A and PCR damping fluid;
Described reagent B is comprised of described primer sets B and PCR damping fluid;
Described reagent C is comprised of described primer sets C and PCR damping fluid;
The final concentration of described primer 1 in the described reagent of its correspondence is 1.33ng/ μ l;
The final concentration of described primer 2 in the described reagent of its correspondence is 6ng/ μ l;
The final concentration of described primer 3-5 in the described reagent of its correspondence is 6.67ng/ μ l;
Described mutational site is L1014F or L1014C.
The 3rd purpose of the present invention provides a kind of method that detects the reagent of kdr gene mutation site in the Anopheles sinensis for preparing.
Method provided by the invention comprises the steps:
1) respectively the described primer sets A in the described primer, described primer sets B and described primer sets C are carried out independent packaging, obtain independent packaging primer sets A, independent packaging primer sets B and independent packaging primer sets C;
2) again with step 1) the independent packaging primer sets A, the step 1 that obtain) the independent packaging primer sets B and the step 1 that obtain) the independent packaging primer sets C that obtains packs, and obtains reagent;
Described mutational site is L1014F or L1014C.
Reagent by described method preparation also is the scope of protection of the invention.
The 4th purpose of the present invention provides a kind of test kit that detects kdr gene mutation site in the Anopheles sinensis.
Test kit provided by the invention is following 1) or 2):
1) test kit shown in is comprised of test kit A, test kit B and test kit C;
Described test kit A is the test kit that contains the reagent A in the described PCR reagent;
Described test kit B is the test kit that contains the reagent B in the described PCR reagent;
Described test kit C is the test kit that contains the reagent C in the described PCR reagent;
2) test kit shown in is the test kit that contains described reagent;
Described mutational site is L1014F or L1014C.
The application that kdr gene mutation site, detection Anopheles sinensis resistance or preparation detect in the Anopheles sinensis resistance product in detecting Anopheles sinensis of described primer or described reagent or described test kit also is the scope of protection of the invention; Described mutational site is specially L1014F or L1014C, and described resistance is anti-chrysanthemum esters medicine; Described chrysanthemum esters medicine is specially Deltamethrin, permethrin or effective cypermethrin.
The 5th purpose of the present invention provides the method in the mutational site of kdr gene in a kind of detection or the auxiliary detection Anopheles sinensis.
Method provided by the invention comprises the steps:
Respectively Anopheles sinensis to be measured is carried out pcr amplification with described primer sets A, primer sets B and primer sets C in described primer or described reagent or the described test kit, obtain pcr amplification product;
Detect and respectively organize the product that primer amplification obtains,
If described primer sets B amplification obtains the 169bp product, and described primer C do not increase and obtains the 169bp product, and then the mutational site of described kdr gene is or the candidate is L1014F;
If described primer sets C amplification obtains the 169bp product, and described primer B do not increase and obtains the 169bp product, and then the mutational site of described kdr gene is or the candidate is L1014C;
If described primer sets A amplification obtains the 169bp product, and described primer sets B and described primer sets C all amplification obtain the 169bp product, then described kdr gene does not contain or the candidate is not contained described mutational site.
The 6th purpose of the present invention provides the genotypic method of kdr gene in a kind of detection or the auxiliary detection Anopheles sinensis.
Method provided by the invention comprises the steps:
Respectively Anopheles sinensis to be measured is carried out pcr amplification with the described primer sets A primer sets in described primer or described reagent or the described test kit, primer sets B primer sets and primer sets C primer sets, obtain pcr amplification product;
Detect and respectively organize the product that primer amplification obtains,
If described primer sets B amplification obtains the 169bp product, and described primer A and described primer C all amplification obtain the 169bp product, then described kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014F;
If described primer sets C amplification obtains the 169bp product, and described primer A and described primer B do not increase and obtain the 169bp product, and then described kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014C;
Obtain the 169bp product if described primer sets B and described primer sets C all increase, and described primer A do not increase and obtain the 169bp product, then described kdr gene is to contain or the candidate is contained the heterozygous gene of mutational site L1014F and L1014C;
If described primer sets A amplification obtains the 169bp product, and described primer sets B and described primer sets C all amplification obtain the 169bp product, then described kdr gene is not for containing or the candidate is not contained the homozygous gene of mutational site L1014F and L1014C;
In the described pcr amplification, take the genomic dna of Anopheles sinensis to be measured as template;
The annealing temperature of described pcr amplification is 55 ℃-60 ℃, and annealing temperature is specially 55 ℃;
The method of described detection pcr amplification product is agarose gel electrophoresis or order-checking.
Know-why of the present invention: on the basis of Anopheles sinensis Kdr transgenation, take the base in mutational site as 3 ' end, design three specificity inner primers (2 kinds of mutants and a kind of not mutated type), can only be combined with target DNA and the nonmutationed target DNA of 2 kinds of sudden changes respectively.Simultaneously, at two non-specific outer primers of mutating alkali yl upstream and downstream place design, utilize the characteristics of Taq enzymatic defect 3 ' → 5 ' 5 prime excision enzyme activity, with mutant primer amplification normal DNA template the time, extension can not carry out because the phosphoric acid ester bond is difficult to form, also just can not get the band of special length, thereby show that template DNA suddenlys change without this.Otherwise showing has sudden change to produce.Mutant and not mutated type primer are respectively applied to the amplification of same dna profiling, judge the specific sudden change that has or not the specific site gene.
Of the present inventionly experiment showed, a kind of quick, easy, sensitive Anopheles sinensis kdr mutation allele detection kit provided by the invention, be used for grasping the Anopheles sinensis kdr allelic situation relevant with the pyrethroid insecticides resistance.Kdr mutation allele detection kit of the present invention has designed respectively 3 group-specific primerses for the allelic genotype of Anopheles sinensis kdr, can detect fast the kdr genotype of other Anopheles sinensis.Adopt kdr mutation allele detection kit of the present invention, only need to extract the DNA of single Anopheles sinensis, therefore the quantity of Anopheles sinensis to be measured do not had special requirement, also need not in addition raising farm and the facility of Anopheles sinensis, do not need to drop into long manpower and materials yet and observe.Tested sample can accurate and effective, directly detect point mutation, can identify idiotype.Judge that the relevant genotype of kdr lays the foundation for the resistance of the German little sickle of research to pyrethroid insecticides.
Description of drawings
Fig. 1 is AS-PCR principle of design figure
Fig. 2 is the allelic detected result of genome 1014 site kdr of Anopheles sinensis
Fig. 3 is the dependency of range gene type frequency and biotic resistance phenotype
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Foundation--the AS-PCR method of embodiment 1, detection Anopheles sinensis sodium channel gene point mutation process
1, AS-PCR detects principle and design of primers
The principle of AS-PCR is: according to mosquito sodium channel gene sudden change characteristics, 3 ' end take the base in mutational site as primer, design two Auele Specific Primers (mutant and not mutated type), be combined with allelic dna sequence and the nonmutationed allelic dna sequence of sudden change respectively.Simultaneously, design respectively two non-specific outer primers in the mutational site upstream and downstream, utilize the characteristics of Taq enzymatic defect 3 ' → 5 ' 5 prime excision enzyme activity, with mutant primer amplification normal DNA template the time, PCR reaction meeting can not be extended because the phosphoric acid ester bond is difficult to form, also just can not get the band of special length, thereby show that the template DNA sequence suddenlys change without this.Otherwise showing has sudden change to produce.Mutant and not mutated type primer are respectively applied to the amplification of same sample allelotrope sequence, judge the allelotrope sequence that has or not the specific site transgenation.
According to Anopheles sinensis sodium channel gene II S4--II S6 partial sequence (kdr sensitive gene cDNA sequence, its partial nucleotide sequence is sequence 10 (L1014 classifies 293-295 position TTG as at the nucleotides sequence of this sequence), its aminoacid sequence is sequence 11 (L1014 is the 107th at the aminoacid sequence of this sequence), accession number at GENBANK is JN002364, and resistant gene CDNA sequence (L1014F, L1014C), design a pair of non-specific outer primer CD1 and CD2 (amplification contrast band).1014 sites of L1014F are 3 ' end, design respectively three specificity inner primer Cgd3, Cgd4 and Cgd5 (amplifying specific band).Wherein, Cgd3 is used for increasing not mutated allelotrope, the 1014th the amino acid that Cgd4 is used for amplification sudden change F allelotrope (TTT) sports phenylalanine by leucine, the 1014th the amino acid that Cgd5 is used for amplification sudden change C allelotrope (TGT) sports halfcystine by leucine, concrete primer sequence such as following table 1:
Table 1 is AS-PCR primer sequence table
Synthetic above-mentioned AS-PCR primer, the PAGE purifying.
2, AS-PCR reaction
Extract respectively the genomic dna of the open-air Anopheles sinensis that is numbered 1-6 that catches, for subsequent use, obtain being numbered the DNA of the sample to be tested of 1-6, respectively 6 kinds of samples are carried out following detection:
AS-PCR is designed to parallel three pipe methods: three parallel PCR reaction systems are for the identification of same idiotype, a reaction system (A pipe) primer is CD1, CD2, Cgd3, another reaction system (B pipe) primer is CD1, CD2, Cgd4, and the 3rd reaction system (C pipe) primer is CD1, CD2, Cgd5.B, C pipe only have a band if the A pipe amplifies two specific bands, and then idiotype is responsive homozygote (SS); A, C pipe only have a band if the B pipe amplifies two specific bands, then idiotype is resistance homozygote (RR-F/F), A, B pipe only have a band if the C pipe amplifies two specific bands, and then idiotype is resistance homozygote (RR-C/C); The 3rd pipe only has a band if any two Guan Jun amplify two specific bands, and then idiotype is resistance heterozygote (RR-F/C, RS-F, RS-C); If three pipes amplify two specific bands simultaneously, then test is polluted.Its principle as shown in Figure 1.
A tube reaction cumulative volume 30 μ l, comprise Taq DNA Polymerase (TaKaRa, D7205) (0.08U/ μ l) 0.5 μ l, 10 * PCR Buffer (TaKaRa), 3.0 μ l, 4 * dNTP (0.17mM), 2.0 μ l, template DNA 1.0 μ l, CD1 (1.33ng/ μ l) 0.2 μ l, CD2 (6ng/ μ l) 0.9 μ l, Cgd3 (6.67ng/ μ l) 1.0 μ l, ddH2O 20.5 μ l;
B tube reaction cumulative volume 30 μ l, comprise Taq DNA Polymerase (TaKaRa, D7205) (0.08U/ μ l) 0.5 μ l, 10 * PCR Buffer, 3.0 μ l, 4 * dNTP (0.17mM), 2.0 μ l, template DNA 1.0 μ l, CD1 (1.33ng/ μ l) 0.2 μ l, CD2 (6ng/ μ l) 0.9 μ l, Cgd4 (6.67ng/ μ l) 1.0 μ l, ddH2O 20.5 μ l;
C tube reaction cumulative volume 30 μ l, comprise Taq DNA Polymerase (TaKaRa, D7205) (0.08U/ μ l) 0.5 μ l, 10 * PCR Buffer, 3.0 μ l, 4 * dNTP (0.17mM), 2.0 μ l, template DNA 1.0 μ l, CD1 (1.33ng/ μ l) 0.2 μ l, CD2 (6ng/ μ l) 0.9 μ l, Cgd5 (6.67ng/ μ l) 1.0 μ l, ddH2O 20.5 μ l;
Reaction conditions is 94 ℃, 8min; 94 ℃, 1min, 55 ℃, 2min, 72 ℃, 2min, 40cycles; 72 ℃, 10min.
If B amplification obtains the 169bp product, and A and C all amplification obtain the 169bp product, then the mutational site of described kdr gene is L1014F, the genotype of described kdr gene is RR; RR represents the kdr gene of homozygous sudden change.
If C amplification obtains the 169bp product, and A and B all amplification obtain the 169bp product, then the mutational site of described kdr gene is L1014C, the genotype of described kdr gene is RR; RR represents the kdr gene of homozygous sudden change.
Obtain the 169bp product if B and A all increase, and C do not increase and obtain the 169bp product, then the mutational site of described kdr gene is L1014F, and the genotype of described kdr gene is RS; RS represents the kdr gene of heterozygous mutation.
Obtain the 169bp product if C and A all increase, and B do not increase and obtain the 169bp product, then the mutational site of described kdr gene is L1014C, and the genotype of described kdr gene is RS;
If A amplification obtains the 169bp product, and B and C all amplification obtain the 169bp product, then there is not the mutational site in described kdr gene, the genotype of described kdr gene is SS; SS represents the homozygous not kdr gene of sudden change.
The result as shown in Figure 2, wherein, 1-6 is numbered respectively the Anopheles sinensis of 1-6, and 3 swimming lanes of each numbering from left to right are A, B and C pipe, Marker is DNA Marker.263bp is the amplified band (obtain contrasting the PCR product by CD1 and CD2 amplification, checking order is sequence 6) of non-specific outer primer, and 169bp is the amplified band (being obtained by CD2 and Cgd3, Cgd4 or Cgd5 amplification respectively) of specificity inner primer.
Being numbered 1 Anopheles sinensis, length is only arranged in the A pipe is that the fragment 1 of 169bp is (through order-checking, be sequence 7, wherein 20-22 position Nucleotide is TTG), illustrate that there is not the kdr mutator gene in this sample, be the responsive homozygote SS of kdr, have the homozygous not kdr gene of sudden change;
Being numbered 2 Anopheles sinensis, only length to be arranged in the B pipe be the fragment (through order-checking, being sequence 8, is TTT from 5 ' terminal 20-22 position Nucleotide) of 169bp, illustrates that this sample is the homozygote of kdr mutator gene; The kdr gene that has homozygous sudden change is described, the mutational site of kdr gene is L1014F.
Being numbered 3 Anopheles sinensis, only length to be arranged in the C pipe be the fragment (through order-checking, being sequence 9, is TGT from 5 ' terminal 20-22 position Nucleotide) of 169bp, illustrates that this sample is the homozygote of kdr mutator gene; The kdr gene that has homozygous sudden change is described, the mutational site of kdr gene is L1014C.
Be numbered 4 Anopheles sinensis and the fragment that length is 169bp in B pipe and C pipe, occur simultaneously (through checking order, the B pipe is sequence 9 for sequence 8, C pipe), illustrate that this sample is that the sudden change of F type and the sudden change of C type are present in the one by one resistance heterozygote of body simultaneously for this sample of explanation.
Be numbered 5 Anopheles sinensis and the fragment that length is 169bp (through order-checking, the A pipe is sequence 9 for sequence 7, C pipe) occur simultaneously at A pipe and C pipe, illustrate that this sample is the heterozygote of kdr mutator gene; Illustrate that these are the kdr gene of heterozygous mutation, the genotype of kdr gene is RS, and the mutational site of kdr gene is L1014C.
Be numbered 6 Anopheles sinensis and the fragment that length is 169bp (through order-checking, the B pipe is sequence 7 for sequence 8, A pipe) in A pipe and B pipe, occurs, illustrate that this sample is the heterozygote of kdr mutator gene; Illustrate that these are the kdr gene of heterozygous mutation, the genotype of kdr gene is RS, and the mutational site of kdr gene is L1014F.
If occur the phenomenon that two bands appear in 3 pipes simultaneously, be abnormal condition, may there be other new sudden change or be judged to error.
All samples are errorless by gene sequencing proof experimental result.
Anopheles sinensis laboratory sensitive strain ((susceptible (Ss) mosquito) strain, be documented in Song, F.L., X.M.Cao, T.Y.Zhao, Y.D.Dong and B.L.Lu.2007.Pyrethroid resistance and distribution of kdr allele in Culex pipiens pallens in north China.Int J Pest Manag 53:25-34., the public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL): from Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C insectarium, isolation cultivated more than 10 years, never contacted any sterilant.
The Anopheles sinensis Natural Population comprises: Anopheles Sinensis Larvae in Paddy Fields resistance group, pick up from 5 areas such as Xuzhou, Huaiyin, Changshu, Nanjing, Suzhou in Jiangsu Province; When the larva quantity of collection in worksite is more, directly carries out larva and give birth to survey, quantity is inadequate takes back laboratory breeding, utilizes first brood of larvae to carry out bioassay.
1, the anopheles adult mosquito that is used for the test of larva median lethal concentration(LC﹠-{50})
5 Natural Population Anopheles sinensis strains and numbering are respectively: Xuzhou strain (XZ), Huaiyin, Jiangsu Province strain (HY), Nanjing strain (NJ), Law Firm Suzhou Jiangsu strain (SZ) and Changshu, Jiangsu Province strain (CS) (table 2).These 5 Natural Population Anopheles sinensises are picked up from July, 2009 to September and in July, 2010 to September.The Anopheles sinensis adult mosquito is taken back the insectarium, laboratory and raises behind collection in worksite, the mosquito cultivation is all raised under the condition of relatively stable temperature (26 ± 1 ℃), relative humidity 80 ± 5%, illumination 12h/d.
Laboratory sensitive strain and 5 local larvas that gather the Natural Population northern house are done toxicity test.Adopt pickling process, with preparing protodrug (Deltamethrin (95%, Yangnong Chemical Co., Ltd., Jiangsu); Es-EXTHIN (93%, French Russell-excellent Ke Fu company); Permethrin (92%, military medical research institute of Nanjing Military Command); Effective cypermethrin (97%, Tianjin agricultural chemicals limited-liability company) is mixed with 5-7 concentration with acetone, adds the 199ml clear water that spends the night in the container, put into 25 4 age Initial instar larvae, add the liquid of the different gradient concentrations of 1ml.1ml acetone compares, and repeats 3 times, and the 24h observations can not be escaped mechanical stimulus as dead take larva.
This research uses effective cypermethrin that the Anopheles sinensis of 5 Natural Populations and the median lethal concentration(LC﹠-{50}) (LC50) of laboratory sensitive strain have been carried out measuring (table 3).Take sensitive strain as reference, Wild population LC50 is resistance index (R/S) divided by sensitive population LC50.
Table 2 is the distribution of the relevant resistance group Anopheles sinensis population spot sampling point of larva
Use log dosage-mortality ratio machine value tracing analysis (Raymond et al.1993 after the data gathering; Finney et al.1971).Each Natural Population Anopheles Sinensis Larvae in Paddy Fields all has in various degree resistance (table 3) to effective cypermethrin, from 677 to 2076 times of resistance indexs do not wait, and the Changshu, Jiangsu Province population that resistance is the highest reaches 2076 times, what resistance was minimum is the Law Firm Suzhou Jiangsu population, and resistance index is 677 times.
Table 3 is that 5 Anopheles sinensis populations are measured and resistance index the median lethal concentration(LC﹠-{50}) of effective cypermethrin
2, the relationship analysis of Anopheles sinensis LC50 resistance index and kdr gene frequency and genotype frequency
Exponential relationship figure such as Fig. 3 that Anopheles sinensis LC50 resistance index and kdr gene frequency make up, the total mutation frequency dependency of resistance index and F, C is obvious, R=0.981, with the relative index of F mutation frequency be 0.9548, with the relation conefficient of C sudden change be 0.9548 (table 4).Can find out from following correlation figure, detect the resistant phenotype that Anopheles sinensis point mutation frequency also can be derived population indirectly.
The dependency of table 4 effective cypermethrin LC50 resistance index and kdr gene frequency, genotype frequency
Claims (10)
1. primer sets compound that detects kdr gene mutation site in the Anopheles sinensis is comprised of primer sets A, primer sets B and primer sets C:
Described primer sets A comprises primer 2 and primer 3, and the nucleotide sequence of described primer 2 and primer 3 is respectively in the sequence table sequence 3 in the sequence 2 and sequence table;
Described primer sets B comprises primer 2 and primer 4, and the nucleotide sequence of described primer 2 and primer 4 is respectively in the sequence table sequence 4 in the sequence 2 and sequence table;
Described primer sets C comprises primer 2 and primer 5, and the nucleotide sequence of described primer 2 and primer 5 is respectively in the sequence table sequence 5 in the sequence 2 and sequence table.
2. primer sets compound according to claim 1 is characterized in that:
Described primer sets A also comprises primer 1, and the nucleotides sequence of described primer 1 is classified sequence 1 in the sequence table as;
Described primer sets B also comprises primer 1, and the nucleotides sequence of described primer 1 is classified sequence 1 in the sequence table as;
Described primer sets C also comprises primer 1, and the nucleotides sequence of described primer 1 is classified sequence 1 in the sequence table as.
3. primer sets compound according to claim 1 and 2 is characterized in that:
Described primer sets A is comprised of primer 1,2 and 3;
Described primer sets B is comprised of primer 1,2 and 4;
Described primer sets C is comprised of primer 1,2 and 5;
Described primer sets A, described primer sets B and described primer sets C are independent packaging;
Described mutational site is L1014F or L1014C.
4. a PCR reagent that detects kdr gene mutation site in the Anopheles sinensis is comprised of reagent A, reagent B and reagent C;
Described reagent A is comprised of described primer sets A and PCR damping fluid;
Described reagent B is comprised of described primer sets B and PCR damping fluid;
Described reagent C is comprised of described primer sets C and PCR damping fluid;
Described primer sets A is comprised of primer 1,2 and 3;
Described primer sets B is comprised of primer 1,2 and 4;
Described primer sets C is comprised of primer 1,2 and 5;
The nucleotide sequence of described primer 1, primer 2, primer 3, primer 4 and primer 5 is respectively sequence 1 in the sequence table, sequence 2, sequence 3, sequence 4 and sequence 5;
The final concentration of described primer 1 in the described reagent of its correspondence is 1.33ng/ μ l;
The final concentration of described primer 2 in the described reagent of its correspondence is 6ng/ μ l;
The final concentration of described primer 3-5 in the described reagent of its correspondence is 6.67ng/ μ l;
Described mutational site is L1014F or L1014C.
5. one kind prepares the method that detects the reagent of kdr gene mutation site in the Anopheles sinensis, comprises the steps:
1) respectively described primer sets A, described primer sets B and described primer sets C in arbitrary described primer sets compound among the claim 1-3 are carried out independent packaging, obtain independent packaging primer sets A, independent packaging primer sets B and independent packaging primer sets C;
2) the independent packaging primer sets C that the independent packaging primer sets A that again step 1) is obtained, the independent packaging primer sets B that step 1) obtains and step 1) obtain packs, and obtains reagent;
Described mutational site is L1014F or L1014C.
6. the reagent that is prepared by method claimed in claim 5.
7. a test kit that detects kdr gene mutation site in the Anopheles sinensis is following 1) or 2):
1) test kit shown in is comprised of test kit A, test kit B and test kit C;
Described test kit A is the test kit that contains the reagent A in the described PCR reagent of claim 4;
Described test kit B is the test kit that contains the reagent B in the described PCR reagent of claim 4;
Described test kit C is the test kit that contains the reagent C in the described PCR reagent of claim 4;
2) test kit shown in is the test kit that contains reagent claimed in claim 6;
Described mutational site is L1014F or L1014C.
8. arbitrary described primer sets compound or claim 4 or 6 described reagent or the described test kit of claim 7 application in kdr gene mutation site, detection Anopheles sinensis resistance or the preparation detection Anopheles sinensis resistance product in detecting Anopheles sinensis among the claim 1-3; Described mutational site is L1014F or L1014C, and described resistance is anti-chrysanthemum esters medicine; Described chrysanthemum esters medicine is Deltamethrin, permethrin or effective cypermethrin.
One kind detect or the auxiliary detection Anopheles sinensis in the method in mutational site of kdr gene, comprise the steps:
Respectively Anopheles sinensis to be measured is carried out pcr amplification with described primer sets A, primer sets B and primer sets C in arbitrary described primer sets compound or claim 4 or 6 described reagent or the described test kit of claim 7 among the claim 1-3, obtain pcr amplification product;
Detect and respectively organize the product that primer amplification obtains,
If described primer sets B amplification obtains the 169bp product, and described primer C do not increase and obtains the 169bp product, and then the mutational site of described kdr gene is or the candidate is L1014F;
If described primer sets C amplification obtains the 169bp product, and described primer B do not increase and obtains the 169bp product, and then the mutational site of described kdr gene is or the candidate is L1014C;
If described primer sets A amplification obtains the 169bp product, and described primer sets B and described primer sets C all amplification obtain the 169bp product, then described kdr gene does not contain or the candidate is not contained described mutational site.
One kind detect or the auxiliary detection Anopheles sinensis in the genotypic method of kdr gene, comprise the steps:
Respectively Anopheles sinensis to be measured is carried out pcr amplification with described primer sets A, primer sets B and primer sets C in arbitrary described primer sets compound or claim 4 or 6 described reagent or the described test kit of claim 7 among the claim 1-3, obtain pcr amplification product;
Detect and respectively organize the product that primer amplification obtains,
If described primer sets B amplification obtains the 169bp product, and described primer A and described primer C all amplification obtain the 169bp product, then described kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014F;
If described primer sets C amplification obtains the 169bp product, and described primer A and described primer B do not increase and obtain the 169bp product, and then described kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014C;
Obtain the 169bp product if described primer sets B and described primer sets C all increase, and described primer A do not increase and obtain the 169bp product, then described kdr gene is to contain or the candidate is contained the heterozygous gene of mutational site L1014F and L1014C;
If described primer sets A amplification obtains the 169bp product, and described primer sets B and described primer sets C all amplification obtain the 169bp product, then described kdr gene is not for containing or the candidate is not contained the homozygous gene of mutational site L1014F and L1014C;
In the described pcr amplification, take the genomic dna of Anopheles sinensis to be measured as template;
The annealing temperature of described pcr amplification is 55 ℃-60 ℃;
The method of described detection pcr amplification product is agarose gel electrophoresis or order-checking.
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| CN109337989A (en) * | 2018-11-01 | 2019-02-15 | 宁波国际旅行卫生保健中心 | A kind of primer, probe and the method for the mutation of detection Culex pipiens pallens drug resistance gene |
| CN113528673A (en) * | 2021-06-07 | 2021-10-22 | 南方医科大学 | Aedes albopictus molecular marker related to resistance of deltamethrin insecticide, primer and application |
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