CN102373280A - 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 except that Qinghai and all provinces and cities the Xinjiang; Be that the network of rivers, China Plain and paddy rice are planted geographic dominant mosquito, family is wild amphibious, perch daytime more crop around the village plant with reed clump, thicket in; Part is perched at the cowhouse animal house; Have a liking for poultry blood (is main with draught animals such as ox, horse, pigs) partially, 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 does not still have effective vaccine and can use, and the population quantity of control malaria media is the important means of preventing and treating malaria.Chemical prevention is easy to operate because of it, can be rapidly, Pest Control population effectively, in the anti-system planning of insect vector, occupy critical role.Come in the past few decades,, use pyrethroid insecticides, adopt the anti-malaria measures of kill mosquito such as residual spray and immersion mosquito net, obtained unusual effect to the life habit of Anopheles sinensis.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 of gathering in Yunnan in 1998 have 5 to DDT generation resistance; The Anopheles sinensis population on ground such as Wenzhou, Zhejiang in 1999, Ningbo reaches high anti-population (R/S=15-20); 2009, the Anopheles sinensis of some areas, Jiangsu produced the height resistance 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; In order to overcome the control effect that resistance reaches expection, the Utilization of pesticides amount comprises the formulation rate and the spraying times of 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, also possibly cause the generation of some media disease and popular indirectly.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, the existing Utilization of pesticides life-span of resistance management, suspension of pesticide resistance generation and development, prolongation how to strengthen insect vector is the important content during insect is administered.
Insect mainly is that (knockdown resistance, kdr), the kdr gene has vital role to knock down resistance(kdr) in the resistance of Anopheles sinensis to pyrethroid to the resistance of pyrethroid insecticides.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(kdr) sterilant; Therefore knock down resistance(kdr) is different from metabolic resistance, can not be reduced by the suppressor factor of esterase or mixed-functional oxidase.Through part sodium channel sequence relatively more responsive, the resistance Anopheles sinensis, the L1014F (leucine is to phenylalanine(Phe)) 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(kdr).This is the reported first of China about Anopheles sinensis knock down resistance(kdr) and transgenation thereof.
The kdr gene can be used as the molecule marker of insect to pyrethroid generation resistance, through 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
50There is positive correlation between beta_cypermethrin resistance and the Anopheles sinensis kdr gene frequency for representative.Research explanation Kdr gene can be used as the molecule marker of Anopheles sinensis to pyrethroid generation resistance, through monitoring kdr allelotrope or genotype frequency, understands the state of resistant gene in Genetic Constitution of Population, can predict the generation and the development of resistance.
China does not also have the kdr resistance molecular detecting method and the test kit to 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 made up of primer sets A, primer sets B and primer sets C:
Said primer sets A comprises primer 2 and primer 3, and the nucleotide sequence of said primer 2 and primer 3 is respectively in the sequence table sequence 3 in sequence 2 and the sequence table;
Said primer sets B comprises primer 2 and primer 4, and the nucleotide sequence of said primer 2 and primer 4 is respectively in the sequence table sequence 4 in sequence 2 and the sequence table;
Said primer sets C comprises primer 2 and primer 5, and the nucleotide sequence of said primer 2 and primer 5 is respectively in the sequence table sequence 5 in sequence 2 and the sequence table.
Said primer sets A also comprises primer 1, and the nucleotides sequence of said primer 1 is classified sequence 1 in the sequence table as;
Said primer sets B also comprises primer 1, and the nucleotides sequence of said primer 1 is classified sequence 1 in the sequence table as;
Said primer sets C also comprises primer 1, and the nucleotides sequence of said primer 1 is classified sequence 1 in the sequence table as.
Said primer sets A is made up of primer 1-3;
Said primer sets B is made up of primer 1,2 and 4;
Said primer sets C is made up of primer 1,2 and 5;
Said primer sets A, said primer sets B and said primer sets C are independent packaging;
Said 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 made up of reagent A, reagent B and reagent C;
Said reagent A is made up of said primer sets A and PCR damping fluid;
Said reagent B is made up of said primer sets B and PCR damping fluid;
Said reagent C is made up of said primer sets C and PCR damping fluid;
The final concentration of said primer 1 in its corresponding said reagent is 1.33ng/ μ l;
The final concentration of said primer 2 in its corresponding said reagent is 6ng/ μ l;
The final concentration of said primer 3-5 in its corresponding said reagent is 6.67ng/ μ l;
Said mutational site is L1014F or L1014C.
The 3rd purpose of the present invention provides a kind of compositions and methods that detects kdr gene mutation site in the Anopheles sinensis for preparing.
Method provided by the invention comprises the steps:
1) respectively the said primer sets A in the described primer, said primer sets B and said 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) 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;
Said mutational site is L1014F or L1014C.
Reagent by described method preparation also is the scope that the present invention protects.
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 made up of test kit A, test kit B and test kit C;
Said test kit A is the test kit that contains the reagent A in the said PCR reagent;
Said test kit B is the test kit that contains the reagent B in the said PCR reagent;
Said test kit C is the test kit that contains the reagent C in the said PCR reagent;
2) test kit shown in is the test kit that contains described reagent;
Said 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 said primer or said reagent or said test kit also is the scope that the present invention protects; Said mutational site is specially L1014F or L1014C, and said resistance is anti-chrysanthemum esters medicine; Said chrysanthemum esters medicine is specially Deltamethrin, WL 43479 or beta_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:
Said primer sets A, primer sets B and primer sets C with in said primer or said reagent or the said test kit carry out pcr amplification to Anopheles sinensis to be measured respectively, obtain pcr amplification product;
Detect and respectively organize the product that primer amplification obtains,
If said primer sets B amplification obtains the 169bp product, and said primer C do not increase and obtains the 169bp product, and the mutational site of then said kdr gene is or the candidate is L1014F;
If said primer sets C amplification obtains the 169bp product, and said primer B do not increase and obtains the 169bp product, and the mutational site of then said kdr gene is or the candidate is L1014C;
If said primer sets A amplification obtains the 169bp product, and said primer sets B and said primer sets C all amplification obtain the 169bp product, then said kdr gene does not contain or the candidate is not contained said mutational site.
The 6th purpose of the present invention provides the method for kdr gene genotype 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 said primer sets A primer sets in said primer or said reagent or the said 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 said primer sets B amplification obtains the 169bp product, and said primer A and said primer C all amplification obtain the 169bp product, then said kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014F;
If said primer sets C amplification obtains the 169bp product, and said primer A and said primer B do not increase and obtain the 169bp product, and then said kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014C;
If said primer sets B and said primer sets C all increase and obtain the 169bp product, and said primer A do not increase and obtains the 169bp product, and then said kdr gene is to contain or the candidate is contained the heterozygous gene of mutational site L1014F and L1014C;
If said primer sets A amplification obtains the 169bp product, and said primer sets B and said primer sets C all amplification obtain the 169bp product, then said kdr gene is not for containing or the candidate is not contained the homozygous gene of mutational site L1014F and L1014C;
In the said pcr amplification, be template with the genomic dna of Anopheles sinensis to be measured;
The annealing temperature of said pcr amplification is 55 ℃-60 ℃, and annealing temperature is specially 55 ℃;
The method of said 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; Base with the mutational site is 3 ' end; Design three specificity inner primers (2 kinds of mutants and a kind of not mutated type), can only combine with the target DNA and the nonmutationed target DNA of 2 kinds of sudden changes respectively.Simultaneously; Two non-specific outer primers of design at mutating alkali yl upstream and downstream place; 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 of the phosphoric acid ester bond is difficult to form; Also just can not get the band of special length, thereby show that template DNA does not have this sudden change.Otherwise showing has sudden change to produce.Mutant and not mutated type primer are respectively applied for 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 3 group-specific primerses respectively to the allelic genotype of Anopheles sinensis kdr, can detect the kdr genotype of other Anopheles sinensis fast.Adopt kdr mutation allele detection kit of the present invention; Only need to extract the DNA of single Anopheles sinensis; Therefore the quantity to Anopheles sinensis to be measured does not have special requirement, also need not raising farm and the facility of Anopheles sinensis in addition, need not drop into long manpower and materials yet and observe.Seized 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 the AS-PCR principle chart
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 like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources 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; Base with the mutational site is 3 ' end of primer; Design two Auele Specific Primers (mutant and not mutated type), allelic dna sequence and the nonmutationed allelic dna sequence with sudden change combines respectively.Simultaneously; Upstream and downstream designs two non-specific outer primers respectively in the mutational site; 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 of 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 do not have this sudden change.Otherwise showing has sudden change to produce.Mutant and not mutated type primer are respectively applied for 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), designs a pair of non-specific outer primer CD1 and CD2 (amplification contrast band).1014 sites of L1014F are 3 ' end, design three specificity inner primer Cgd3, Cgd4 and Cgd5 (amplifying specific band) respectively.Wherein, The Cgd3 not mutated allelotrope that is used to increase; The 1014th the amino acid that Cgd4 is used for amplification sudden change F allelotrope (TTT) sports phenylalanine(Phe) 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 an AS-PCR primer sequence table
Synthetic above-mentioned AS-PCR primer, the PAGE purifying.
2, AS-PCR reaction
Extract the genomic dna of the open-air Anopheles sinensis that is numbered 1-6 that catches respectively, subsequent use, obtain being numbered the DNA of the sample to be tested of 1-6, respectively 6 kinds of samples are detected as follows:
AS-PCR is designed to parallel three pipe methods: three parallel PCR reaction systems are used to identify 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 have only a band if the A pipe amplifies two specific bands, and then idiotype is responsive homozygote (SS); A, C pipe have only a band if the B pipe amplifies two specific bands, and then idiotype is resistance homozygote (RR-F/F), and A, B pipe have only a band if the C pipe amplifies two specific bands, and then idiotype is resistance homozygote (RR-C/C); The 3rd pipe has only a band if any two Guan Jun amplify two specific bands, then idiotype be the resistance heterozygote (RR-F/C, RS-F, RS-C); If three pipes amplify two specific bands simultaneously, then test is polluted.Its principle is as shown in Figure 1.
A tube reaction TV 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 TV 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 TV 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 all increases with C and obtains the 169bp product, and the mutational site of then said kdr gene is L1014F, and said kdr gene genotype is RR; RR representes the kdr gene of homozygous sudden change.
If C amplification obtains the 169bp product, and A all increases with B and obtains the 169bp product, and the mutational site of then said kdr gene is L1014C, and said kdr gene genotype is RR; RR representes the kdr gene of homozygous sudden change.
If B and A all increase and obtain the 169bp product, and C do not increase and obtains the 169bp product, and the mutational site of then said kdr gene is L1014F, and said kdr gene genotype is RS; RS representes the kdr gene of heterozygous mutation.
If C and A all increase and obtain the 169bp product, and B do not increase and obtains the 169bp product, and the mutational site of then said kdr gene is L1014C, and said kdr gene genotype is RS;
If A amplification obtains the 169bp product, and B all increases with C and obtains the 169bp product, and there is not the mutational site in then said kdr gene, and said kdr gene genotype is SS; SS representes the homozygous not kdr gene of sudden change.
The result is as shown in Figure 2, and wherein, 1-6 is numbered the Anopheles sinensis of 1-6 respectively, and 3 swimming lanes of each numbering from left to right are A, B and C pipe, and 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), explain 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, explains 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, explains 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 in B pipe and C pipe, occur the fragment that length is 169bp simultaneously (through checking order; The B pipe is that sequence 8, C pipe are sequence 9), explain that this sample suddenlys change for the F type for this sample of explanation and the sudden change of C type is present in the resistance heterozygote of body one by one simultaneously.
Be numbered 5 Anopheles sinensis and occur the fragment that length is 169bp (through order-checking, the A pipe is a sequence 9 for sequence 7, C pipe) simultaneously, explain that this sample is the heterozygote of kdr mutator gene at A pipe and C pipe; Explain that these are the kdr gene of heterozygous mutation, the kdr gene genotype is RS, and the mutational site of kdr gene is L1014C.
Be numbered 6 Anopheles sinensis and in A pipe and B pipe, the fragment that length is 169bp (through order-checking, the B pipe is a sequence 7 for sequence 8, A pipe) occur, explain that this sample is the heterozygote of kdr mutator gene; Explain that these are the kdr gene of heterozygous mutation, the kdr gene genotype is RS, and the mutational site of kdr gene is L1014F.
The phenomenon that two bands appear in 3 pipes simultaneously occurs if having, be abnormal condition, possibly have other new sudden change or be judged to error.
All samples are errorless through 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 Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C of the Chinese People's Liberation Army): from Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C insectarium, isolate and culture more than 10 years, never contacted any sterilant.
Population comprises for Anopheles sinensis nature: Anopheles sinensis larva resistance group, pick up from 5 areas such as Xuzhou, Huaiyin, Changshu, Nanjing, Suzhou in Jiangsu Province; The larva quantity of collection in worksite is more directly carried out larva and give birth to be surveyed, and quantity is not enough 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 strain (HY), Nanjing strain (NJ), Suzhou, Jiangsu strain (SZ) and Changshu, Jiangsu 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, mosquito is cultured and all under the condition of relatively stable temperature (26 ± 1 ℃), relative humidity 80 ± 5%, illumination 12h/d, raises.
Laboratory sensitive strain and 5 local larvas of gathering nature population northern house are done toxicity test.Adopt pickling process, with raw insecticide medicine (Deltamethrin (95%, Yangnong Chemical Co., Ltd., Jiangsu); Es-EXTHIN (93%, French Russell-excellent Ke Fu company); WL 43479 (92%, military medicine institute of Nanjing Military Command); Beta_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 the initial stage in age larva, add the soup of the different gradient concentrations of 1ml.1ml acetone compares, repeats 3 times, and the 24h observations, can not escape mechanical stimulus with larva is death.
This research uses beta_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).With the sensitive strain is reference, and open-air 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
After the data gathering with log dosage-mortality ratio machine value tracing analysis (Raymond et al.1993; Finney et al.1971).Each natural population Anopheles sinensis larva all has resistance (table 3) in various degree to beta_cypermethrin; Resistance index does not wait for from 677 to 2076 times, and Changshu, Jiangsu population that resistance is the highest reaches 2076 times; What resistance was minimum is 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 beta_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 dependency 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 beta_cypermethrin LC50 resistance index and kdr gene frequency, genotype frequency
Claims (10)
1. primer that detects kdr gene mutation site in the Anopheles sinensis, form by primer sets A, primer sets B and primer sets C:
Said primer sets A comprises primer 2 and primer 3, and the nucleotide sequence of said primer 2 and primer 3 is respectively in the sequence table sequence 3 in sequence 2 and the sequence table;
Said primer sets B comprises primer 2 and primer 4, and the nucleotide sequence of said primer 2 and primer 4 is respectively in the sequence table sequence 4 in sequence 2 and the sequence table;
Said primer sets C comprises primer 2 and primer 5, and the nucleotide sequence of said primer 2 and primer 5 is respectively in the sequence table sequence 5 in sequence 2 and the sequence table.
2. primer according to claim 1 is characterized in that:
Said primer sets A also comprises primer 1, and the nucleotides sequence of said primer 1 is classified sequence 1 in the sequence table as;
Said primer sets B also comprises primer 1, and the nucleotides sequence of said primer 1 is classified sequence 1 in the sequence table as;
Said primer sets C also comprises primer 1, and the nucleotides sequence of said primer 1 is classified sequence 1 in the sequence table as.
3. primer according to claim 1 and 2 is characterized in that:
Said primer sets A is made up of primer 1,2 and 3;
Said primer sets B is made up of primer 1,2 and 4;
Said primer sets C is made up of primer 1,2 and 5;
Said primer sets A, said primer sets B and said primer sets C are independent packaging;
Said mutational site is L1014F or L1014C.
4. a PCR reagent that detects kdr gene mutation site in the Anopheles sinensis is made up of reagent A, reagent B and reagent C;
Said reagent A is made up of said primer sets A and PCR damping fluid;
Said reagent B is made up of said primer sets B and PCR damping fluid;
Said reagent C is made up of said primer sets C and PCR damping fluid;
The final concentration of said primer 1 in its corresponding said reagent is 1.33ng/ μ l;
The final concentration of said primer 2 in its corresponding said reagent is 6ng/ μ l;
The final concentration of said primer 3-5 in its corresponding said reagent is 6.67ng/ μ l;
Said mutational site is L1014F or L1014C.
5. one kind prepares the compositions and methods that detects kdr gene mutation site in the Anopheles sinensis, comprises the steps:
1) respectively the said primer sets A in arbitrary described primer among the claim 1-3, said primer sets B and said 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) 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;
Said mutational site is L1014F or L1014C.
6. the reagent for preparing by the described method of 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 made up of test kit A, test kit B and test kit C;
Said test kit A is the test kit that contains the reagent A in the said PCR reagent of claim 4;
Said test kit B is the test kit that contains the reagent B in the said PCR reagent of claim 4;
Said test kit C is the test kit that contains the reagent C in the said PCR reagent of claim 4;
2) test kit shown in is the test kit that contains the described reagent of claim 6;
Said mutational site is L1014F or L1014C.
8. arbitrary said primer or claim 4 or 6 said reagent or the said test kit of claim 7 kdr gene mutation site, detection Anopheles sinensis resistance or preparation in detecting Anopheles sinensis detect the application in the Anopheles sinensis resistance product among the claim 1-3; Said mutational site is specially L1014F or L1014C, and said resistance is anti-chrysanthemum esters medicine; Said chrysanthemum esters medicine is specially Deltamethrin, WL 43479 or beta_cypermethrin.
One kind detect or the auxiliary detection Anopheles sinensis in the method in mutational site of kdr gene, comprise the steps:
Said primer sets A, primer sets B and primer sets C with in arbitrary said primer or claim 4 among the claim 1-3 or 6 said reagent or the said test kit of claim 7 carry out pcr amplification to Anopheles sinensis to be measured respectively, obtain pcr amplification product;
Detect and respectively organize the product that primer amplification obtains,
If said primer sets B amplification obtains the 169bp product, and said primer C do not increase and obtains the 169bp product, and the mutational site of then said kdr gene is or the candidate is L1014F;
If said primer sets C amplification obtains the 169bp product, and said primer B do not increase and obtains the 169bp product, and the mutational site of then said kdr gene is or the candidate is L1014C;
If said primer sets A amplification obtains the 169bp product, and said primer sets B and said primer sets C all amplification obtain the 169bp product, then said kdr gene does not contain or the candidate is not contained said mutational site.
One kind detect or the auxiliary detection Anopheles sinensis in the method for kdr gene genotype, comprise the steps:
Said primer sets A, primer sets B and primer sets C with in arbitrary said primer or claim 4 among the claim 1-3 or 6 said reagent or the said test kit of claim 7 carry out pcr amplification to Anopheles sinensis to be measured respectively, obtain pcr amplification product;
Detect and respectively organize the product that primer amplification obtains,
If said primer sets B amplification obtains the 169bp product, and said primer A and said primer C all amplification obtain the 169bp product, then said kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014F;
If said primer sets C amplification obtains the 169bp product, and said primer A and said primer B do not increase and obtain the 169bp product, and then said kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014C;
If said primer sets B and said primer sets C all increase and obtain the 169bp product, and said primer A do not increase and obtains the 169bp product, and then said kdr gene is to contain or the candidate is contained the heterozygous gene of mutational site L1014F and L1014C;
If said primer sets A amplification obtains the 169bp product, and said primer sets B and said primer sets C all amplification obtain the 169bp product, then said kdr gene is not for containing or the candidate is not contained the homozygous gene of mutational site L1014F and L1014C;
In the said pcr amplification, be template with the genomic dna of Anopheles sinensis to be measured;
The annealing temperature of said pcr amplification is 55 ℃-60 ℃, and annealing temperature is specially 55 ℃;
The method of said detection pcr amplification product is agarose gel electrophoresis or order-checking.
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