CN102242215A - Kit for detecting drug resistance of culex pipiens and special primer thereof - Google Patents
Kit for detecting drug resistance of culex pipiens and special primer thereof Download PDFInfo
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Abstract
The invention discloses a kit for detecting the drug resistance of culex pipiens and a special primer thereof. The invention provides a primer for detecting kdr (knock-down resistance) gene mutation sites in culex pipiens, which is composed of a primer group A, a primer group B and a primer group C, wherein the primer group A is composed of a primer 1, a primer 2 and a primer 3; the primer group B is composed of the primer 1, the primer 2 and a primer 4; and the primer group C is composed of the primer 1, the primer 2 and a primer 5. In the invention, the experiment proves that by the quick, simple, convenient and sensitive method for detecting kdr mutation in culex pipiens, the site mutation of the detected sample can be accurately, effectively and directly detected, thus resistant individuals and sensitive individuals can be distinguished, and the genotypes of the individuals can be identified.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of northern house resistance detection kit and primer special thereof.
Background technology
Northern house complex group (Culex pipiens complex) belongs to Culex culex subgenus, this kind distribution on global.It is generally acknowledged that this complex group comprises northern house (Cx.p.pipiens), Culex quinquefasciatus (Cx.p.quinquefasciatus), culex pipiens pollens (Cx.p.pallens) and Culex molestus (Cx.p.molestus).Above-mentioned 4 subspecies all have distribution in China.This complex group kind not only distributes extensively, and has very big medical importance.It is one of main object of China's diseases prevention kill mosquito.Chemical prevention will remain the important means of mosquito control in present and foreseeable future.Yet the generation of resistance seriously has influence on the control of media mosquito.In the northern house complex group multiple mosquito to the pyrethroid insecticides resistance in the common existence of China.And resistance detects and monitoring is the prerequisite that the prevention resistance takes place and develops, and is the basis of resistance management work.
At present, the drug-fast detection method of mosquito class is continued to use the bioassay method of 1970 world health organisation recommendations always, measures responsive virulence (LD-p) baseline and the LD of insect to medicament
50Or LC
50Value.Gather pest population of the same race from testing the area again, adopt and identical living survey method and the control condition of mensuration sensitive strain, draw LD-p line and the LD of population to be measured
50Or LC
50Value is with population to be measured and the responsive LD that plants strain
50Or LC
50Ratio between the value (being the resistant multiple) is represented resistance level.But this method has strict statistical requirements to institute's acquisition data, and necessary strict control test condition needs repeated tests, and also demonstrates significantly limitation in practice.Traditional bioassay method is more loaded down with trivial details, from the worm source, raise mensuration and be difficult to accomplish real stdn, and the LD that obtains by the LD-p line
50Or LC
50The repeatability and the accuracy of value are lower, are difficult to accurately measure when low and resistant population is various when population resistance, and therefore the resistance level that records often has hysteresis quality, is not suitable for early stage resistance and detects, and is unfavorable for formulating corresponding Preventing Countermeasures.
In view of present many antagonism games depend on the early detection of resistance, so the research of resistance monitoring and detection technique shows important especially.Along with the diversification of resistance monitoring and testing goal, resistance monitoring means and method also develop towards the diversification direction.
The pest resistance to insecticide molecular detection technology is based on what set up on the machine-processed basis of understanding of pest resistance to insecticide, promptly utilizes Protocols in Molecular Biology to detect the resistance site of sterilant action target or the enhancing expression of metabolic detoxification enzyme gene.Based on the reason of operability, practicality and economic dispatch aspect, at present nearly all resistance detects research and all concentrates on the target resistance aspect, promptly detects the sudden change of target gene.
The action target of pyrethroid insecticides is a sodium-ion channel on the insect neuron membrane, and the point mutation in that sodium channel II S6 sections takes place is called as knock down resistance (kdr), receives much attention because of it is relevant with the pyrethroid insecticides resistance.At present, carried out a large amount of molecular biology experiments for knock down resistance mechanism.L1014F (Ile-Phe, Phe/kdr) sudden change is because of its extensive existence, and classical sudden change is otherwise known as.And also have two other sudden change in same site, be respectively that leucine sports the point mutation L1014S (Ser/kdr) that Histidine (His/kdr) and leucine sport Serine.The report that only has two kinds of sudden changes in the Culex mosquito is respectively L1014F and L1014S sudden change.Along with the knock down resistance Study on Molecular Mechanism deeply and the development of Protocols in Molecular Biology, kdr gene specific allelotrope polymerase chain reaction (PASA) Fast Detection Technique produces thereupon.
The PASA technology is to detect a kind of new round pcr of point mutation, and the ultimate principle of this technology is that wherein PCR product primer a 3 ' end is arranged at the resistant mutation site.Utilize these primers to carry out pcr amplification, can the increase gene fragment of responsive insect of S primer, and the gene fragment of the resistant insect that can not increase; The R primer is then opposite.The use of PASA technology need design special primer at mutating alkali yl, therefore requires all base mutations that cause resistance are perfectly clear.In northern house complex group mosquito, there are two types of L1014F and L1014S simultaneously in the kdr transgenation in bibliographical information has been arranged culex pipiens pollens and the Culex quinquefasciatus.When multiple resistant mutation appears in same base site, need a plurality of primers of design, carry out repeatedly PCR, could determine the character of sudden change.Though the AS-PCR technology has been used the mosquito resistance and detected, be mostly the method for setting up at the sudden change of kdr gene a single point.At present also there be not a kind of molecular detecting method and test kit that can detect a plurality of mutational sites simultaneously for northern house complex group mosquito.
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 northern house.
Primer provided by the invention, form by 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, described primer sets B and described primer sets C are independent packaging;
The mass ratio of primer 1 described in the described primer sets A, primer 2 and primer 3 is (2-5): 10: (8-5), mass ratio was specially 3: 10: 7;
The mass ratio of primer 1 described in the described primer sets B, primer 2 and primer 4 is (2-5): 10: (8-5), mass ratio was specially 3: 10: 7;
The mass ratio of primer 1 described in the described primer sets C, primer 2 and primer 5 is (2-5): 10: (8-5), mass ratio was specially 3: 10: 7;
Described mutational site is L1014F or L1014S.
Second purpose of the present invention provides a kind of PCR reagent that detects kdr gene mutation site in the northern house.
The invention provides PCR reagent is made up of reagent A, reagent B and reagent C;
Described reagent A is made up of described primer sets A and PCR damping fluid;
Described reagent B is made up of described primer sets B and PCR damping fluid;
Described reagent C is made up of described primer sets C and PCR damping fluid;
The concentration of each primer in the described PCR reagent of correspondence is respectively in described each primer sets: the final concentration of described primer 1 is 0.08-0.4uM, and the final concentration of described primer 1 is specially 0.12uM; The final concentration of described primer 2 is 0.08-0.4uM, and the final concentration of described primer 2 is specially 0.4uM; The final concentration of described primer 3,4,5 is 0.32-0.4uM, and the final concentration of described primer 3,4,5 is specially 0.28uM.
Described mutational site is L1014F or L1014S.
The PCR damping fluid is reagent T, available from precious biotechnology company limited, production number DR011.
The 3rd purpose of the present invention provides a kind of compositions and methods that detects kdr gene mutation site in the northern house 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) 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 L1014S.
The reagent that is prepared by described method 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 northern house.
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;
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 L1014S.
The application that kdr gene mutation site, detection northern house resistance or preparation detect in the northern house resistance product in detecting northern house of described primer or described reagent or described test kit also is a scope of protection of the invention, described mutational site is L1014F or L1014S, and described resistance is anti-chrysanthemum esters medicine; Described chrysanthemum esters medicine is specially Deltamethrin, Es-EXTHIN, 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 northern house.
Method provided by the invention comprises the steps:
With the described primer 2 among the described primer sets A and described primer 3 as primer to A, as primer C is carried out pcr amplification to northern house to be measured respectively to B with the described primer 2 among the described primer sets C, described primer 5 as primer with the described primer 2 among the described primer sets B, described primer 4, obtain pcr amplification product;
Detect and respectively organize the product that primer amplification obtains,
If described primer obtains the 366-378bp product to B amplification, and described primer C do not increase and obtains the 366-378bp product, and the mutational site of then described kdr gene is or the candidate is L1014F;
If described primer obtains the 366-378bp product to C amplification, and described primer B do not increase and obtains the 366-378bp product, and the mutational site of then described kdr gene is or the candidate is L1014S;
If described primer obtains the 366-378bp product to A amplification, and described primer to B and described primer to C all amplification obtain the 366-378bp 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 method for kdr gene genotype in a kind of detection or the auxiliary detection northern house.
Method provided by the invention comprises the steps:
With the described primer 2 among the described primer sets A and described primer 3 as primer to A, as primer C is carried out pcr amplification to northern house to be measured respectively to B with the described primer 2 among the described primer sets C, described primer 5 as primer with the described primer 2 among the described primer sets B, described primer 4, obtain pcr amplification product;
Detect and respectively organize the product that primer amplification obtains,
Described primer obtains the 366-378bp product to B amplification, and described primer A and described primer C all amplification obtain the 366-378bp product, then described kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014F;
If described primer obtains the 366-378bp product to C amplification, and described primer A and described primer B do not increase and obtain the 366-378bp product, and then described kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014S;
If described primer all increases to C to B and described primer and obtains the 366-378bp product, and described primer A do not increase and obtains the 366-378bp product, and then described kdr gene is to contain or the candidate is contained the heterozygous gene of mutational site L1014F and L1014S;
If described primer obtains the 366-378bp product to A amplification, and described primer to B and described primer to C all amplification obtain the 366-378bp product, then described kdr gene is not for containing or the candidate is not contained the homozygous gene of mutational site L1014F and L1014S;
In the described pcr amplification, be template with the genomic dna of northern house to be measured;
The annealing temperature of described pcr amplification is (60-65) ℃, and annealing temperature is specially 60 ℃;
The method of described detection pcr amplification product is agarose gel electrophoresis or order-checking.
In the above pcr amplification, primer 1 and primer 2 among described primer sets A or B or the C carry out pcr amplification, obtain the amplification size and are the 487-499bp product; Whether this product can be used for detecting the PCR reaction system correct, if the 487-499bp product is arranged, illustrates that this system is correct.
Of the present inventionly experiment showed, a kind of quick, easy, sensitive northern house resistance detection kit provided by the invention, be used for grasping the resistance situation of northern house pyrethroid insecticides.Resistance detection kit of the present invention is at the genotype relevant with northern house kdr, the resistance detection kit is at two kinds of genotype relevant with resistance of northern house complex group mosquito kdr sudden change, 3 group-specific primerses have been designed respectively, synthetic through certain proportioning, can detect two kinds of sudden changes of northern house complex group simultaneously.Adopt resistance detection kit of the present invention, only need to extract the DNA of single mosquito, therefore the quantity to mosquito to be measured does not have special requirement, also need not raising farm and the facility of mosquito in addition, also do not need to drop into long manpower and materials and observe, independent 1 people can finish the check originally of 20 increments in 2 hours, and tested sample can accurate and effective, directly detect point mutation, resistance individuality and sensitive individual not only can be distinguished, and idiotype can be identified.Reach more than 95% at the laboratory inspection coincidence rate, try out, quite be subjected to favorable comment on China institute of inspection section and Jiangsu Prov. Disease Preventing and Controlling Center and other places.Judge that the relevant genotype of kdr lays the foundation for the resistance of research northern house to pyrethroid insecticides.
Description of drawings
Fig. 1 is resistance qualification result figure
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.
Embodiment 1, northern house resistance detect
One, pcr amplification
1, primer design
At the relevant kdr of the anti-medicine of northern house and pyrethrins insecticide (Accession number:AY573288) gene design Auele Specific Primer:
CD1:5 ' AAC TTC ACC GAC TTC ATG CAC 3 ' (sequence 1, downstream primer)
CD2:5 ' CAA GGC TAA GAA AAG GTT AAG AAC 3 ' (sequence 2, upstream primer)
CD3:5 ' CCA CCG TAG TGA TAG GAA ATT TA 3 ' (sequence 3, downstream primer)
Auele Specific Primer according to the design of the mutant kdr L1014F type mutator gene (sequence 6) of kdr:
CD1:5 ' AAC TTC ACC GAC TTC ATG CAC 3 ' (sequence 1, downstream primer)
CD2:5 ' CAA GGC TAA GAA AAG GTT AAG AAC 3 ' (sequence 2, upstream primer)
CD4:5 ' CCA CCG TAG TGA TAG GAA ATT TA 3 ' (sequence 4, downstream primer)
Auele Specific Primer according to the design of the mutant kdr L1014S type mutator gene (sequence 7) of kdr:
CD1:5 ' AAC TTC ACC GAC TTC ATG CAC 3 ' (sequence 1, downstream primer)
CD2:5 ' CAA GGC TAA GAA AAG GTT AAG AAC 3 ' (sequence 2, upstream primer)
CD5:5 ' CCA CCG TAG TGA TAG GAA ATT TA 3 ' (sequence 5, downstream primer)
2, genome DAN obtains
Extract the genomic dna of open-air 6 northern houses that are numbered 1-6 that catch respectively, standby, obtain being numbered the DNA of 6 samples to be tested of 1-6.
3, the pcr amplification of the DNA of sample to be tested (amplification system such as table 1):
(1) gets 12uL reagent T (precious biotechnology company limited, production number DR011) respectively and join in 3 PCR pipes, be labeled as pipe A, pipe B and C pipe.
(2) in pipe A, add 1uL reagent P
A, in pipe B, add 1uL reagent P
B, in pipe C, add 1uL reagent P
C
(3) get 11.5uLddH
2O is to managing among A, pipe B and the pipe C.
(during a plurality of pattern detection, according to above proportioning, divide A, B and C pipe with reagent mix after packing, better effects if)
(4) getting 0.5uL DNA respectively joins among pipe A, pipe B and the pipe C.
(5) after C builds with above pipe A, pipe B and pipe, put into the PCR instrument, by following condition setting reaction conditions (in order to make the reagent mixing, centrifugal a little better effects if).
Table 1 is the pcr amplification system
Table 2 is the prescription of T reagent
Described reagent P
AFor at the responsive mosquito kdr of northern house complex group gene-specific primer mixture, composed as follows:
CD1:5 ' AAC TTC ACC GAC TTC ATG CAC 3 ' (final concentration in the PCR system is 0.12uM)
CD2:5 ' CAA GGC TAA GAA AAG GTT AAG AAC 3 ' (final concentration is 0.4uM)
CD3:5 ' CCA CCG TAG TGA TAG GAA ATT TA 3 ' (final concentration is 0.28uM)
All primer dissolved dilutions become 20uM,
P
A: by CD1, CD2 and CD3 mix, and the dose volume ratio is: CD1: CD2: CD3=3: 10: 7 (mass ratio in the A pipe is CD1: CD2: CD4=3: 10: 7).
Described reagent P
BBe Auele Specific Primer mixture at the design of northern house complex group mosquito kdr L1014F type mutator gene, composed as follows:
CD1：5’AAC?TTC?ACC?GAC?TTC?ATG?CAC 3’
CD2：5’CAA?GGC?TAA?GAA?AAG?GTT?AAG?AAC 3’
CD4:5 ' CCA CCG TAG TGA TAG GAA ATT TA 3 ' (final concentration is 0.28uM)
All primer dissolved dilutions become 20uM,
P
B: by CD1, CD2 and CD4 mix, and the dose volume ratio is: CD1: CD2: CD4=3: 10: 7 (mass ratio in the B pipe is CD1: CD2: CD4=3: 10: 7).
Described reagent P
CBe Auele Specific Primer mixture at the design of northern house complex group mosquito kdr L1014S type mutator gene, composed as follows:
CD1：5’AAC?TTC?ACC?GAC?TTC?ATG?CAC 3’
CD2：5’CAA?GGC?TAA?GAA?AAG?GTT?AAG?AAC 3’
CD5:5 ' CCA CCG TAG TGA TAG GAA ATT TA 3 ' (final concentration is 0.28uM)
All primer dissolved dilutions become 20uM,
P
C: by CD1, CD2 and CD5 mix, and the dose volume ratio is: CD1: CD2: CD5=3: 10: 7 (mass ratio in the C pipe is CD1: CD2: CD4=3: 10: 7).
Described ddH
2O is a sterile purified water.
3, pcr amplification condition
Two, detect
(1) takes by weighing the 3g agarose, add 200mL TAE Buffer.
(2) with microwave oven the glue of above preparation is dissolved (2-5min) fully.
(3) after the glue cooling, add 10uLEB, fully behind the mixing, pour in the glue groove of inserting good comb in advance, treat that gelling is solid after, take out comb, put into electrophoresis chamber.
(4) add 5uLDNA Marker in the well in first road, add the PCR product 5uL of sample in the duct of back, the A of each product manages product, B pipe product is preferably adjacent with C pipe product, so that differentiate.
(5) add TAE Buffer to flooding whole glue face, open electrophoresis apparatus, voltage is transferred to 120V, electrophoresis 25 minutes.
(6) after electrophoresis finishes, take pictures and differentiate with gel imaging system.
If the fragment that length is 487-499bp does not appear in any pipe product, represent that all results of this sample are invalid, it is the fragment of 487-499bp that length is all arranged in this experiment.
If B amplification obtains the 366-378bp product, and A and C all amplification obtain the 366-378bp product, the mutational site of then described kdr gene is L1014F, described kdr gene genotype is RR; RR represents the kdr gene of homozygous sudden change.
If C amplification obtains the 366-378bp product, and A and B all amplification obtain the 366-378bp product, the mutational site of then described kdr gene is L1014S, described kdr gene genotype is RR;
If B and A all increase and obtain the 366-378bp product, and C do not increase and obtains the 366-378bp product, and the mutational site of then described kdr gene is L1014F, and described kdr gene genotype is RS; RS represents the kdr gene of heterozygous mutation.
If C and A all increase and obtain the 366-378bp product, and B do not increase and obtains the 366-378bp product, and the mutational site of then described kdr gene is L1014S, and described kdr gene genotype is RS;
If A amplification obtains the 366-378bp product, and B and C all amplification obtain the 366-378bp product, there is not the mutational site in then described kdr gene, described kdr gene genotype is SS; SS represents the homozygous not kdr gene of sudden change.
The result as shown in Figure 1, wherein, 1-6 represents to be numbered the northern house of 1-6.
Being numbered 1 northern house, only length to be arranged in the A pipe be the fragment (through order-checking, being sequence 9) of 377bp, illustrates that this sample is the responsive homozygote of kdr.
Being numbered 2 northern house, only length to be arranged in the B pipe be the fragment (through order-checking, being sequence 10) of 371bp, illustrates that this sample is a F type sudden change resistance homozygote.
Being numbered 3 northern house, only length to be arranged in the C pipe be the fragment (through order-checking, being sequence 11) of 368bp, illustrates that this sample is a s type sudden change resistance homozygote.
Be numbered 4 northern house and in A pipe and B pipe, occur the fragment that length is 376bp (through order-checking, the A pipe is sequence 13 for sequence 12, B pipe) simultaneously, illustrate that this sample is the F type resistance heterozygote that suddenlys change.
Be numbered 5 northern house and occur the fragment that length is 377bp (through order-checking, the A pipe is sequence 15 for sequence 14, C pipe) simultaneously, illustrate that this sample is the S type resistance heterozygote that suddenlys change at A pipe and C pipe.
Be numbered 6 northern house and the fragment that length is 370bp (through order-checking, the B pipe is sequence 17 for sequence 16, C pipe) in B pipe and C pipe, occurs, illustrate that this sample is present in the resistance heterozygote of body one by one simultaneously for the sudden change of F type and S type suddenly change.
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.
60 northern house complex group mosquito samples adopt present method to carry out the kdr genotype detection, and pcr amplification product is carried out sequencing.Wherein the detected result of 55 pipes is consistent with sequencing result.
With 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) and 11 the local larvas of gathering nature population northern houses in Pekinese do 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); Permethrin (92%, military medicine 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 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.
Use the median lethal concentration (LC of four kinds of pyrethroid insecticideses (Deltamethrin, Es-EXTHIN, permethrin, effective cypermethrin) to 11 natural populations (yellow pasture, loudspeaker Gou Men, length whistle battalion, Tang Hekou, Hou Shanpu, Beizhen City, bosom, cattle pen mountain, Sun He, Fengtai, Tongzhou, Zhangjiakou) and laboratory sensitive strain
50) measure, result such as following table 3,
Table 3 median lethal concentration (LC
50)
Can be rated as from table 3, is reference with the sensitive strain, natural population LC
50Divided by sensitive population LC
50(resistance index thinks that more than 5 times this nature population has resistance to be resistance index.)。To from 2.82 to 19.01 times of deltamethrin resistance indexes, to from 2.16 to 8.77 times of Es-EXTHIN resistance indexs, to from 2.57 to 28.07 times of permethrin resistance indexs, to from 2.46 to 26.32 times of effective cypermethrin resistance indexs.To deltamethrin resistance the highest be grandson river population (27.26%), minimum is Zhangjiakou population (4.02%); To Es-EXTHIN resistance resistance the highest be cattle pen mountain population (8.77%), minimum is Zhangjiakou population; What santochlor ether chrysanthemum ester resistance was the highest is grandson river population (28.07%), minimum population (2.57%); To the effective cypermethrin resistance the highest be cattle pen mountain population (26.32%), minimum is Tongzhou population (2.46%).All in all higher cattle pen mountain and the grandson river population of resistance, what resistance was lower is yellow pasture and Tongzhou population.
The detected result of kdr gene frequency and genotype frequency sees Table 4, and detection method is with above-mentioned Molecular Detection.
Table 4kdr gene frequency and genotype frequency
Laboratory population is randomly drawed 10 individualities, is responsive homozygote (SS), and the kdr gene frequency is 0.Nature population kdr gene frequency height does not wait cattle pen mountain population the highest (37.80%), yellow pasture population minimum (7.89%).
Northern house is to the resistance of 4 kinds of pyrethroid insecticideses (Deltamethrin, Es-EXTHIN, permethrin, effective cypermethrin) and the relation between the kdr gene frequency, analysis revealed by statistics, dependency is preferably all arranged between the two, and relation conefficient is as follows: and esbiothrin (r=0.947, P=0.0001), Deltamethrin (r=0.747, P=0.006), and permethrin (r=0.565, P=0.05), effective cypermethrin (r=0.867, P=0.006).These results show that northern house is closely related with the sudden change of sodium channel related locus (kdr) to the resistance (particularly knock down resistance) of pyrethroid.
Claims (10)
1. primer that detects kdr gene mutation site in the northern house, form by 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 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 according to claim 1 and 2 is characterized in that:
Described primer sets A, described primer sets B and described primer sets C are independent packaging;
The mass ratio of primer 1 described in the described primer sets A, primer 2 and primer 3 is (2-5): 10: (8-5), the mass ratio of primer 1 described in the described primer sets A, primer 2 and primer 3 was specially 3: 10: 7;
The mass ratio of primer 1 described in the described primer sets B, primer 2 and primer 4 is (2-5): 10: (8-5), the mass ratio of primer 1 described in the described primer sets B, primer 2 and primer 4 was specially 3: 10: 7;
The mass ratio of primer 1 described in the described primer sets C, primer 2 and primer 5 is (2-5): 10: (8-5), the mass ratio of primer 1 described in the described primer sets C, primer 2 and primer 5 was specially 3: 10: 7;
Described mutational site is L1014F or L1014S.
4. a PCR reagent that detects kdr gene mutation site in the northern house is made up of reagent A, reagent B and reagent C;
Described reagent A is made up of described primer sets A and PCR damping fluid;
Described reagent B is made up of described primer sets B and PCR damping fluid;
Described reagent C is made up of described primer sets C and PCR damping fluid;
The concentration of each primer in the described PCR reagent of correspondence is respectively in described each primer sets: the final concentration of described primer 1 is 0.08-0.4uM, and the final concentration of described primer 1 is specially 0.12uM; The final concentration of described primer 2 is 0.08-0.4uM, and the final concentration of described primer 2 is specially 0.4uM; The final concentration of described primer 3,4,5 is 0.32-0.4uM, and the final concentration of described primer 3,4,5 is specially 0.28uM;
Described mutational site is L1014F or L1014S.
5. one kind prepares the compositions and methods that detects kdr gene mutation site in the northern house, comprises the steps:
1) respectively the described primer sets A in arbitrary described primer among the claim 1-3, 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) 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 L1014S.
6. by the reagent of the described method of claim 5 preparation.
7. a test kit that detects kdr gene mutation site in the northern house is following 1) or 2):
1) test kit shown in is made up 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 the described reagent of claim 6;
Described mutational site is L1014F or L1014S.
8. arbitrary described primer or claim 4 or 6 described reagent or the described test kit of claim 7 application in kdr gene mutation site, detection northern house resistance or the preparation detection northern house resistance product in detecting northern house among the claim 1-3; Described mutational site is specially L1014F or L1014S, and described resistance is anti-chrysanthemum esters medicine; Described chrysanthemum esters medicine is specially Deltamethrin, Es-EXTHIN, permethrin or effective cypermethrin.
One kind detect or the auxiliary detection northern house in the method in mutational site of kdr gene, comprise the steps:
With the described primer 2 among the described primer sets A in arbitrary described primer or claim 4 among the claim 1-3 or 6 described reagent or the described test kit of claim 7 and described primer 3 as primer to A, as primer C is carried out pcr amplification to northern house to be measured respectively to B with the described primer 2 among the described primer sets C, described primer 5 as primer with the described primer 2 among the described primer sets B, described primer 4, obtain pcr amplification product;
Detect and respectively organize the product that primer amplification obtains,
If described primer obtains the 366-378bp product to B amplification, and described primer C do not increase and obtains the 366-378bp product, and the mutational site of then described kdr gene is or the candidate is L1014F;
If described primer obtains the 366-378bp product to C amplification, and described primer B do not increase and obtains the 366-378bp product, and the mutational site of then described kdr gene is or the candidate is L1014S;
If described primer obtains the 366-378bp product to A amplification, and described primer to B and described primer to C all amplification obtain the 366-378bp product, then described kdr gene does not contain or the candidate is not contained described mutational site.
One kind detect or the auxiliary detection northern house in the method for kdr gene genotype, comprise the steps:
With the described primer 2 among the described primer sets A in arbitrary described primer or claim 4 among the claim 1-3 or 6 described reagent or the described test kit of claim 7 and described primer 3 as primer to A, as primer C is carried out pcr amplification to northern house to be measured respectively to B with the described primer 2 among the described primer sets C, described primer 5 as primer with the described primer 2 among the described primer sets B, described primer 4, obtain pcr amplification product;
Detect and respectively organize the product that primer amplification obtains,
If described primer obtains the 366-378bp product to B amplification, and described primer A and described primer C all amplification obtain the 366-378bp product, then described kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014F;
If described primer obtains the 366-378bp product to C amplification, and described primer A and described primer B do not increase and obtain the 366-378bp product, and then described kdr gene is to contain or the candidate is contained the homozygous gene of mutational site L1014S;
If described primer all increases to C to B and described primer and obtains the 366-378bp product, and described primer A do not increase and obtains the 366-378bp product, and then described kdr gene is to contain or the candidate is contained the heterozygous gene of mutational site L1014F and L1014S;
If described primer obtains the 366-378bp product to A amplification, and described primer to B and described primer to C all amplification obtain the 366-378bp product, then described kdr gene is not for containing or the candidate is not contained the homozygous gene of mutational site L1014F and L1014S;
In the described pcr amplification, be template with the genomic dna of northern house to be measured;
The annealing temperature of described pcr amplification is (60-65) ℃, and annealing temperature is specially 60 ℃;
The method of described detection pcr amplification product is agarose gel electrophoresis or order-checking.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011101955177A CN102242215A (en) | 2011-07-13 | 2011-07-13 | Kit for detecting drug resistance of culex pipiens and special primer thereof |
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| CN2011101955177A CN102242215A (en) | 2011-07-13 | 2011-07-13 | Kit for detecting drug resistance of culex pipiens and special primer thereof |
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| CN103146834A (en) * | 2013-03-25 | 2013-06-12 | 江苏省血吸虫病防治研究所 | Allglo probe-based detection method of anopheles sinensis knockdown resistance gene mutation site |
| CN109234408A (en) * | 2018-11-01 | 2019-01-18 | 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 aedes albopictus drug resistance gene |
| 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 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103146834A (en) * | 2013-03-25 | 2013-06-12 | 江苏省血吸虫病防治研究所 | Allglo probe-based detection method of anopheles sinensis knockdown resistance gene mutation site |
| CN109234408A (en) * | 2018-11-01 | 2019-01-18 | 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 aedes albopictus drug resistance gene |
| 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 |
| CN109234408B (en) * | 2018-11-01 | 2021-08-20 | 宁波国际旅行卫生保健中心 | Primer, probe and method for detecting drug resistance gene mutation of aedes albopictus |
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Application publication date: 20111116 |