CN108588243A - Quickly differentiate specific primer, multiple PCR detection kit and its detection method of adventive Pomacea canaliculata - Google Patents
Quickly differentiate specific primer, multiple PCR detection kit and its detection method of adventive Pomacea canaliculata Download PDFInfo
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Abstract
The invention discloses quick specific primer, multiple PCR detection kit and its detection methods for differentiating adventive Pomacea canaliculata.Specific primer provided by the present invention includes specific primer sequence shown in SEQ ID NO.1~SEQ ID NO.6.The method is that the genomic DNA of extraction Pomacea canaliculata first, using special primer to carrying out multi-PRC reaction under regular-PCR reagent system and program, by detected through gel electrophoresis, determines the hybrid of Pomacea canaliculata as template.It is demonstrated experimentally that this method is quick and accurate, a PCR reaction is only needed, can differentiate three kinds of different Pomacea canaliculatas, there is stronger repeatability and stability.Compared with sequence analysis method, this method is simple and economical, and without complicated technical step and expensive reagent, those skilled in the art are operable, is the ideal method for differentiating Pomacea canaliculata type.
Description
Technical field
The invention belongs to biotechnology, it is related to the specific primer, more that a clock quickly differentiates adventive Pomacea canaliculata
Weight PCR detection kit and its detection method.
Background technology
Pomacea canaliculata (apple snail), is under the jurisdiction of Gastropoda, newly into abdominal foot mesh, Ampullariidae, and Pomacea canaliculata category (Pomacea),
It is a kind of large-scale Freshwater Snails for originating in South America Amazon Basin.A variety of Pomacea canaliculatas are introduced into Asia, North America, Europe and Australia
The torrid zone, subtropical zone and the temperate zone region in continent cause to seriously threaten to local agricultural production and ecological environment.In addition, Pomacea canaliculata
It is the vector of multiple hazard parasite, can such as causes human body eosinophilic meningoencephalitis's angiostrongylus cantonensis, diffusion speed
Spend and break out the Discase intensity that range directly affects parasitic disease.2000, invasive species expert group of World Conservation Union
Pomacea canaliculata is classified as most threatening one of the invasive species in 100 kinds of the world；2003, Pomacea canaliculata was included in the first batch by Chinese environmental protection general bureau
16 kinds of very harmful Invasive Alien Species " blacklist ".
In China, Pomacea canaliculata introduced inland from 1981 through Taiwan, has invaded more than 30 years of diffusion so far, has become south rice
One big calamity of growing area.According to statistics, China has up to a million hectares of rice field by the different degrees of harm of Pomacea canaliculata, water every year
Rice per unit area yield reduces up to 70%.At present the spiral shell be rapidly spread to Zhejiang, Fujian, Guangdong, Guangxi, Hainan, Yunnan, Guizhou,
Provinces such as Jiangxi, Sichuan, Chongqing, Hunan and Anhui and causing harm are caused disaster.Meanwhile climate variation, seedling allocation and transportation and domain contact
Deng influence, the diffusion tendency in China of Pomacea canaliculata is aggravated.Trend shown by investigational data shows that Pomacea canaliculata will be China
One of great harmful organism that must cope with from now on.China is widely distributed and seriously endangers the invasive Fu Shou that simultaneously large area is broken out
Spiral shell, in addition to including tubule Pomacea canaliculata and spot Pomacea canaliculata and newfound the third invasive Pomacea canaliculata (Pomacea
sp.).These three Pomacea canaliculatas are similar in morphological feature height, influenced in addition by factors such as food source, external environment and puberties,
Morphological feature is changeable in Pomacea canaliculata kind.Therefore, differentiate Pomacea canaliculata in the presence of very big difficult and uncertain by traditional morphological feature
Property, bring very big difficulty to the formulation for decision of quarantining and remove the evil.
Protocols in Molecular Biology is fast-developing in recent years, by the comparative analysis of molecular sequences, it can be achieved that type it is quick,
Precise Identification greatly makes up the defect of traditional form identification.Studies have reported that based on mitochondrial COI sequence design one
Group special primer, although tubule Pomacea canaliculata and spot Pomacea canaliculata can be distinguished, cannot effectively differentiate spot Pomacea canaliculata and
Pomacea sp., thus it is unsuitable for the quick detection of three kinds of Pomacea canaliculatas of China's distribution.It has no at present and differentiates three kinds of invasive good fortune
The molecular engineering of longevity spiral shell is reported.
Invention content
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of quick discriminating adventive Pomacea canaliculatas
Specific primer, multiple PCR detection kit and its detection method.
The quick specific primer for differentiating adventive Pomacea canaliculata of the present invention, including three pairs of specific primers pair, point
It is not：
(1) primer pair 1 that primer sequence forms shown in SEQ ID NO.1 and SEQ ID NO.2；
(2) primer pair 2 that primer sequence forms shown in SEQ ID NO.3 and SEQ ID NO.4；
(3) primer pair 3 that primer sequence forms shown in SEQ ID NO.5 and SEQ ID NO.6.
Wherein, sequence 1 is made of 20 nucleotide；Sequence 2 is made of 21 nucleotide；Sequence 3 is by 20 nucleotide groups
At；Sequence 4 is made of 25 nucleotide；Sequence 5 is made of 19 nucleotide；Sequence 6 is made of 19 nucleotide.Draw described
Object centering, the primer pair 1 are specific to tubule Pomacea canaliculata, and the primer pair 2 is specific to spot Pomacea canaliculata, and the primer pair 3 is special
Different from Pomacea sp..
Preferably, the molal quantity of six primers of three primer pairs is equal.
The adventive Pomacea canaliculata is at least one of following：Tubule Pomacea canaliculata, spot Pomacea canaliculata, newfound
Three kinds of invasive Pomacea canaliculata Pomacea sp..
The invention also discloses a kind of multiple PCR detection kit of quick discriminating adventive Pomacea canaliculata, including it is described
The quick specific primer for differentiating adventive Pomacea canaliculata.
Preferably, the multiple PCR detection kit of the quick discriminating adventive Pomacea canaliculata further includes that PCR reactions are slow
Fliud flushing, archaeal dna polymerase and 4 kinds of dNTP.By 4 institute of the primer pair 1 being made of sequence in sequence table 1 and sequence 2, sequence 3 and sequence
It is and following after each single stranded DNA for the primer pair 3 that the primer pair 2 and sequence 5 and sequence 6 of composition are formed individually is packed
At least one of substance is packaged in same reagent box：PCR reaction buffers, archaeal dna polymerase and 4 kinds of dNTP.
The invention also discloses a kind of multi-PCR detection methods identifying or assisting in discriminating adventive Pomacea canaliculata, including
Following steps：
(a) using the genomic DNA of Pomacea canaliculata to be measured as template, with quick discriminating adventive good fortune described in claim 1
The specific primer of longevity spiral shell carries out PCR amplification；
(b) clip size for the PCR product that detecting step (a) obtains, determined as follows according to PCR product described in
The type of Pomacea canaliculata to be measured：If the PCR product is the DNA fragmentation of a 1238bp, the Pomacea canaliculata to be measured is tubule good fortune
Longevity spiral shell；If the PCR product is the DNA fragmentation of a 901bp, the Pomacea canaliculata to be measured is spot Pomacea canaliculata；If the PCR
Product is the DNA fragmentation of a 571bp, then the Pomacea canaliculata to be measured is Pomacea sp..
Preferably, the annealing temperature that the step (a) carries out PCR amplification is 60 DEG C.
Preferably, six primers that the primer pair 1, the primer pair 2 and the primer pair 3 form are in PCR reactants
Molar ratio in system is 1:1:1:1:1:1.In the present invention, six single stranded DNA rubbing in PCR reaction systems of two primer pairs
Your concentration is 0.4 μM.
Preferably, in step (b), the nucleotide sequence of the DNA fragmentation of the 1238bp is as shown in SEQ ID NO.7；Institute
The nucleotide sequence of the DNA fragmentation of 901bp is stated as shown in SEQ ID NO.8；The nucleotide sequence of the DNA fragmentation of the 571bp
As shown in SEQ ID NO.9.
The present invention is entered what tubule Pomacea canaliculata, spot Pomacea canaliculata and Pomacea sp. these three China's large area were broken out
On the basis of the mitochondria whole genome sequence of invading property Pomacea canaliculata, the special primer of three kinds of Pomacea canaliculatas of identification is designed and filtered out
It is right, the multiplex PCR identification reagent box based on special primer method is formed, realizes the accurate discriminating of three kinds of invasive Pomacea canaliculatas.This
The method and kit for being differentiated adventive Pomacea canaliculata based on multiplex PCR provided, not only high specificity, sensitivity are provided
Height, and be the ideal method for differentiating Pomacea canaliculata with higher repeatability and stability.
Description of the drawings
Fig. 1 is multiplex PCR system amplification under different annealing temperature；
Wherein, M：DL2000marker；1~4：Annealing temperature is 56 DEG C；5~8：Annealing temperature is 58 DEG C；9~12：It moves back
Fiery temperature is 60 DEG C；1、5、9：Tubule Pomacea canaliculata；2、6、10：Spot Pomacea canaliculata；3、7、11：Newfound invasive Pomacea canaliculata；
4、8、12：ddH2O。
Fig. 2 is the specificity for 3 kinds of Pomacea canaliculatas that multiplex PCR detects different geographic populations；
Wherein, M：DL2000marker；1~7：Tubule Pomacea canaliculata；8~14：Spot Pomacea canaliculata；15~21：It is newfound
Invasive Pomacea canaliculata；22：ddH2O。
Fig. 3 multiplex PCRs detect the sensitivity of 3 kinds of Pomacea canaliculatas；
Wherein, M：DL2000marker；1~5：Tubule Pomacea canaliculata；6~10：Spot Pomacea canaliculata；11~15：It is newfound
Invasive Pomacea canaliculata；1~5,6~10,11~15：DNA profiling amount is followed successively by 10,1,0.1,0.01,0.001ng；16：ddH2O。
The testing result for the Pomacea canaliculata sample that Fig. 4 multiplex PCRs acquire the Chongqing City temples Hua Yan；
Wherein, M：DL2000marker；1~2：At spiral shell；3~11：Simple grain ovum；12：ddH2O。
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Three kinds of exotic invasive Pomacea canaliculatas：Tubule Pomacea canaliculata, spot Pomacea canaliculata and the third newfound invasive Pomacea canaliculata.
Wherein, tubule Pomacea canaliculata is related to following geographical population：Dali population, Zhejiang Hangzhou population, Jiangxi Shangrao population, Jiangxi
State population, Fujian Foochow population, Law Firm Suzhou Jiangsu population, Guizhou Kweiyang population；Spot Pomacea canaliculata is related to following geographical population：Chongqing
Hechuan population, Sichuan Chengdu population, Sichuan Suining population, Zhejiang Hangzhou population；The third newfound invasive Pomacea canaliculata relates to
And following geographical population：Xiamen of Fujian Province population, Zhejiang Yuyao population, Nanning population, Guangzhou Guangdong population, Haikou kind
Group, Changsha population.Three of the above Pomacea canaliculata title is documented in " translated name in Zhou Xiaonong, Zhang Yi, Lv Shan Pomacea canaliculata scientific names
Inquire into China's parasitology and parasitic disease magazine .2009,27 (1):62-64. " and " Lv S, Zhou Y, Lou MZ, Hu L,
Liu Q,Wei FR,Guo YH,Steinmann P,Hu W,Zhou XN,Utzinger J.Phylogenetic evidence
for multiple and secondary introductions of invasive snails:Pomacea species
in the People's Republic of China.Diversity&Distributions,2013,19(2):147–
In 156. ".
Embodiment 1, for three kinds of Pomacea canaliculatas special primer design and synthesis
One, the acquisition of three kinds of Pomacea canaliculata mitochondrial genomes sequences
The mitochondrial genome complete sequence of three kinds of Pomacea canaliculatas is downloaded from GenBank, wherein tubule Pomacea canaliculata mitochondria is complete
Genome sequence row number is KJ739609；Spot Pomacea canaliculata mitochondria whole genome sequence number is MF401379；Newfound third
Kind invasive Pomacea canaliculata mitochondria whole genome sequence number is KR350466.
Two, for the special primer design and synthesis of three kinds of Pomacea canaliculatas
1, special primer designs
By the mitochondria whole genome sequence of tubule Pomacea canaliculata, spot Pomacea canaliculata and the third newfound Pomacea canaliculata, profit
The multiple alignment of sequence is carried out with the ClustalW Alignment programs built in Geneious R7 softwares.In sequence variations area
Section separately designs specific primer, and design of primers follows principle：1) 20~25bp of sequence length；2) G/C content is 40~50%；
3) single primer includes at least three specific site；4) the 3 ' of primer are held as 1~2 specific site G/C；5) primer amplification length
For 500~1500bp；6) expanding fragment length of three pairs of primers differs 100bp or more.
According to the above, design and synthesize to obtain for tubule Pomacea canaliculata, spot Pomacea canaliculata and it is newfound the third
The special primer of Pomacea canaliculata is following (table 1)：
The special primer that 1 present invention of table designs
The assessment of embodiment 2, multiplex PCR system annealing temperature
One, experiment material
Experiment material：It is related to three kinds of Pomacea canaliculatas, specially tubule Pomacea canaliculata, spot Pomacea canaliculata and newfound the third enters
Invading property Pomacea canaliculata, wherein tubule Pomacea canaliculata relate to Dali geographical population；Spot Pomacea canaliculata is related to Sichuan Chengdu geographical population：Newly
It was found that the third invasive Pomacea canaliculata be related to Guangzhou Guangdong's geographical population.
Two, experimental method
1, the extraction of genomic DNA
Using the blood/tissue/cellular genome extracts kit of TIANGEN Biotech (Beijing) Co., Ltd., it is extracted into
The total DNA of spiral shell abdominal foot portion musculature.It is carried out with reference to kit specification, concrete operation step is as follows：
(1) 75% alcohol washes surface and wash with distilled water twice of spiral shell musculature (10mg) is taken into, in clean
Filter paper on dry.
(2) by treated, tissue is transferred to the PCR pipe of 1.5mL to centrifuge tube, and liquid nitrogen grinding is added to pulverize tissue, is added
180 μ L GA buffer solutions (subsidiary with kit), centrifuge the several seconds.
(3) Proteinase K (subsidiary with kit) of 20 μ L is added, shakes mixing, 56 DEG C of water-bath 1h centrifuge the several seconds.
(4) 200 μ L buffer solutions GB (subsidiary with kit), fully reverse mixing, 70 DEG C of water-bath 10min, solution change is added
It is limpid, centrifuge 30s.
(5) 200 μ L absolute ethyl alcohols are added, shake 20s, centrifuges 1min, solution is fully transferred to adsorption column CB3 (with examination
Agent box is subsidiary) in, adsorption column is put into collecting pipe, and 12000rpm/s centrifuges 30s, outwells waste liquid, adsorption column puts back to collecting pipe.
(7) 500 μ L buffer solutions GD (subsidiary with kit) are added into adsorption column CB3,12000rpm/s centrifuges 30s,
Fall waste liquid, adsorption column puts back to collecting pipe.
(8) 600 μ L buffer solutions PW (subsidiary with kit) are added into adsorption column CB3,12000rpm/s centrifuges 30s,
Fall waste liquid, adsorption column puts back to collecting pipe.
(9) step (8) is repeated.
(10) adsorption column CB3 is centrifuged into 2min with 12000rpm/s, outwells waste liquid, adsorption column CB3 is placed in and is placed at room temperature for
5min。
(11) adsorption column CB3 is transferred in the centrifuge tube of a clean 1.5mL, and it is bis- that 150 μ L are vacantly added dropwise to adsorption column
Water is steamed, it is placed at room temperature for 2~after five minutes, 12000rpm/s centrifuges 2min, obtains Genomic DNA solution
(12) DNA solution will be obtained and is stored in -20 DEG C for subsequent research.
3, the assessment of primer annealing temperature
Using gained genomic DNA as template, using the chondriogen sequence for three kinds of Pomacea canaliculatas designed by embodiment 1
Primer pair PcF/PcR (SEQ ID NO.1 and SEQ ID NO.2), PmF/PmR (SEQ ID in row special primer (table 1)
NO.3 and SEQ ID NO.4) and NDF/NDR (SEQ ID NO.5 and SEQ ID NO.6) be combined into multiplexed PCR amplification, to examine
Survey the specificity of the primer (table 1) designed by embodiment 1.
(1) amplification system：It is 25 μ L, wherein Premix ExTaq that PCR, which reacts total volume,TM(precious bioengineering is big by 12.5 μ L
Even Co., Ltd), ddH28.5 μ L of O, 1 μ L of DNA profiling (20ng/ μ L), each 0.5 μ L of 6 primers (a concentration of 10 μM).Draw upstream
The final concentration of object and downstream primer in PCR reaction systems is 0.4 μM.
Setting replaces the negative control of template with water simultaneously.
(2) amplification condition：
Annealing temperature gradient is respectively set and is followed successively by 56 DEG C, 58 DEG C, 60 DEG C, actual conditions are as follows：
(3) amplified production detects：After reaction, 5 μ L point samples of PCR product is taken to carry out electrophoresis in 1% Ago-Gel
35min is observed and is imaged in gel imaging system after being dyed using ethidium bromide (EB).
Three, experimental result
Multiplex PCR testing result such as Fig. 1 utilizes what designed, designed obtained to draw when annealing temperature is set as 58 DEG C and 60 DEG C
Object forms PcF/PcR (sequence 1 and sequence 2), PmF/PmR (sequence 3 and sequence 4) and NDF/NDR (sequence 5 and sequence 6)
Multiplex PCR system can amplify the purpose band of a 1238bp from tubule Pomacea canaliculata, amplify one from spot Pomacea canaliculata
The purpose band of 901bp, amplifies the target fragment of a 571bp from the third newfound Pomacea canaliculata, and negative control does not have
There is amplified reaction；When annealing temperature is 56 DEG C, multiplex PCR system is from tubule Pomacea canaliculata except the mesh for amplifying a 1238bp
Band, also amplify the non-specific band of one~1000bp.Show that annealing temperature is possible to generate at 56 DEG C and when following
Non-specific amplification, when improving annealing temperature to 58~60 DEG C, the specific binding of primer and purpose type simultaneously amplifies bright
Purpose band.To ensure the specificity of multiplex PCR system, with 60 DEG C for annealing temperature in detecting below.
Embodiment 3, primer specificity detection
One, experiment material
Experiment material：It is related to three kinds of Pomacea canaliculatas, specially tubule Pomacea canaliculata, spot Pomacea canaliculata and newfound the third enters
Invading property Pomacea canaliculata, wherein tubule Pomacea canaliculata are related to following geographical population：Dali population, Zhejiang Hangzhou population, Jiangxi Shangrao
Population, Ganzhou population, Fujian Foochow population, Law Firm Suzhou Jiangsu population, Guizhou Kweiyang population；Spot Pomacea canaliculata is related to as follows
Manage population：Chongqing Hechuan population, Sichuan Chengdu population, Sichuan Suining population, Zhejiang Hangzhou population；The third newfound invasion
Property Pomacea canaliculata is related to following geographical population：Xiamen of Fujian Province population, Zhejiang Yuyao population, Nanning population, Guangzhou Guangdong population,
Haikou population, Changsha population.
Two, experimental method
Using the blood/tissue/cellular genome extracts kit of TIANGEN Biotech (Beijing) Co., Ltd., it is extracted into
The total DNA of spiral shell abdominal foot portion musculature.Concrete operations are referring to kit specification.Using gained genomic DNA as template, using reality
Apply the primer pair PcF/PcR (SEQ in the Mitochondrial gene sequence special primer (table 1) for three kinds of Pomacea canaliculatas designed by example 1
ID NO.1 and SEQ ID NO.2), PmF/PmR (SEQ ID NO.3 and SEQ ID NO.4) and NDF/NDR (SEQ ID NO.5
With SEQ ID NO.6) it is combined into multiplexed PCR amplification, to detect the specificity of the primer (table 1) designed by embodiment 1.
1, amplification system：It is 25 μ L, wherein Premix ExTaq that PCR, which reacts total volume,TM(the precious bioengineering Dalian 12.5 μ L
Co., Ltd), ddH28.5 μ L of O, 1 μ L of DNA profiling (20ng/ μ L), each 0.5 μ L of 6 primers (a concentration of 10 μM).Sense primer
It it is 0.4 μM with final concentration of the downstream primer in PCR reaction systems.
Setting replaces the negative control of template with water simultaneously.
2, amplification condition：
3, amplified production detects：After reaction, 5 μ L point samples of PCR product is taken to carry out electrophoresis in 1% Ago-Gel
35min is observed and is imaged in gel imaging system after being dyed using ethidium bromide (EB).
Three, experimental result
Multiplex PCR testing result such as Fig. 2, primer pair PcF/PcR (SEQ the ID NO.1 and SEQ obtained using designed, designed
ID NO.2), PmF/PmR (SEQ ID NO.3 and SEQ ID NO.4) and NDF/NDR (SEQ ID NO.5 and SEQ ID NO.6)
Detect the specificity of designed primer pair under multiple PCR method.Multiplex PCR is from Dali population, Zhejiang Hangzhou population, Jiangxi
Shangrao population, Ganzhou population, Fujian Foochow population, Law Firm Suzhou Jiangsu population, Guizhou Kweiyang population tubule Pomacea canaliculata in only
Amplify the purpose band of a 1238bp；From Chongqing Hechuan population, Sichuan Chengdu population, Sichuan Suining population, Zhejiang Hangzhou
The purpose band of a 901bp is only amplified in the spot Pomacea canaliculata of population；From Xiamen of Fujian Province population, Zhejiang Yuyao population, wide
The third newfound invasive Pomacea canaliculata of southwestern peaceful population, Guangzhou Guangdong population, Haikou population, Changsha population
In only amplify the purpose band of a 571bp, negative control does not have amplified reaction.
In summary experimental result, it is seen that the Mitochondrial gene sequence for three kinds of Pomacea canaliculatas designed by embodiment 1 is special
Different primer has higher specificity.
Embodiment 4, primer sensitivity technique
One, experiment material
It is related to three kinds of Pomacea canaliculatas, specially tubule Pomacea canaliculata, spot Pomacea canaliculata and the third newfound invasive Fu Shou
Spiral shell, wherein tubule Pomacea canaliculata relate to Dali geographical population；Spot Pomacea canaliculata is related to Sichuan Chengdu geographical population：Newfound
Three kinds of invasive Pomacea canaliculatas are related to Guangzhou Guangdong's geographical population.
Two, experimental method
Using the blood/tissue/cellular genome extracts kit of TIANGEN Biotech (Beijing) Co., Ltd., it is extracted into
The total DNA of spiral shell abdominal foot portion musculature.Concrete operations are referring to kit specification.Using gained genomic DNA as template, using reality
Apply the primer pair PcF/PcR (SEQ in the Mitochondrial gene sequence special primer (table 1) for three kinds of Pomacea canaliculatas designed by example 1
ID NO.1 and SEQ ID NO.2), PmF/PmR (SEQ ID NO.3 and SEQ ID NO.4) and NDF/NDR (SEQ ID NO.5
With SEQ ID NO.6) it is combined into multiplexed PCR amplification, to detect the sensitivity of the primer (table 1) designed by embodiment 1.
1, amplification system：It is 25 μ L, wherein Premix ExTaq that PCR, which reacts total volume,TM(the precious bioengineering Dalian 12.5 μ L
Co., Ltd), ddH28.5 μ L of O, 1 μ L of DNA profiling (20ng/ μ L), each 0.5 μ L of 6 primers (a concentration of 10 μM).Sense primer
It it is 0.4 μM with final concentration of the downstream primer in PCR reaction systems.Wherein, as the Pomacea canaliculata genomic DNA to be measured of masterplate
Gradient is arranged in dosage in the reaction system：10ng, 1ng, 0.1ng, 0.01ng and 0.001ng.
Setting replaces the negative control of template with water simultaneously.
2, amplification condition：
3, amplified production detects：After reaction, PCR product μ L point samples is taken to carry out electrophoresis in 1% Ago-Gel
3min is observed and is imaged in gel imaging system after being dyed using ethidium bromide (EB).
Three, experimental result
1, tubule Pomacea canaliculata
Using three species-specific primers of designed, designed to PcF/PcR (SEQ ID NO.1 and SEQ ID NO.2), PmF/
PmR (SEQ ID NO.3 and SEQ ID NO.4) and NDF/NDR (SEQ ID NO.5 and SEQ ID NO.6) detects multiplex PCR
The sensitivity of primer pair under method.The tubule Pomacea canaliculata of different DNA concentrations (10ng, 1ng, 0.1ng, 0.01ng and 0.001ng)
The multiple PCR products Gel electrophoresis results of sample are as shown in Figure 3.When template DNA content is reduced to 0.001ng, without special
The amplified band of property product；When template DNA content be 0.01ng or more when, it is amplifiable go out size be 1238bp specificity
Purpose band.The multiplex PCR of the spot Pomacea canaliculata sample of different DNA concentrations (10ng, 1ng, 0.1ng, 0.01ng and 0.001ng)
Product gel electrophoresis result is as shown in Figure 3.When template DNA content is reduced to 0.01ng, the not no amplification item of specific product
Band；When template DNA content be 0.1ng or more when, it is amplifiable go out size be 901bp specificity purpose band.Different DNA
The multiplex PCR of the third newfound invasive Pomacea canaliculata sample of concentration (10ng, 1ng, 0.1ng, 0.01ng and 0.001ng)
Product gel electrophoresis result is as shown in Figure 3.When template DNA content is reduced to 0.1ng, the not no amplification item of specific product
Band；When template DNA content be 1ng or more when, it is amplifiable go out size be 571bp specificity purpose band.
In summary experimental result, it is seen that the special primer (table 1) for three kinds of Pomacea canaliculatas designed by embodiment 1 has
Higher sensitivity.
Embodiment 5, for 3 kinds of exotic invasive Pomacea canaliculatas special primer identification application
The special primer of the chondriogen for 3 kinds of exotic invasive Pomacea canaliculatas designed using embodiment 1 builds more
Weight PCR system, 11 unknown species Pomacea canaliculatas that detection the present inventor acquires in September, 2014 in the free life pond of Chongqing City's Huayan Temple
Sample is extracted into spiral shell using the blood/tissue/cellular genome extracts kit of TIANGEN Biotech (Beijing) Co., Ltd.
The total DNA of abdominal foot portion musculature.Using gained genomic DNA as template, three kinds of Pomacea canaliculatas are directed to using designed by embodiment 1
Mitochondrial gene sequence special primer (table 1), carry out multiplexed PCR amplification, detected using agarose gel electrophoresis.Relevant operation
Detection such as embodiment 1-4.
As a result display such as Fig. 4, wherein the bright band of 1238bp is amplified with the DNA profiling of 5 unknown species Pomacea canaliculatas, it should
In the band sequence such as sequence table of 1238bp shown in sequence 7；The bright of 901bp is amplified with the DNA profiling of 4 unknown species Pomacea canaliculatas
Bright wisp band, in the band sequence such as sequence table of the 901bp shown in sequence 8；It is amplified with the DNA profiling of 2 unknown species Pomacea canaliculatas
The bright band of 571bp, in the band sequence such as sequence table of the 571bp shown in sequence 9.Negative control does not amplify band.
The above result shows that specific primer shown in table 1 by using embodiment 1 is detected, 5 unknown species Pomacea canaliculatas are tubule
Pomacea canaliculata, 4 unknown species Pomacea canaliculatas are spot Pomacea canaliculata, and 2 unknown species Pomacea canaliculatas are the third newfound invasive Fu Shou
Spiral shell.
Meanwhile the present inventor using existing DNA bar code sequence analysis identification method (identification method referring to
" the flat .2016. of Yang Qianqian, Liu Suwen, Ru Wei Dong, Liu Guangfu, Yu Xiao are based on DNA bar code technology to Zhejiang Province's exotic invasive good fortune
Longevity spiral shell carries out Molecular Identification bio-diversities, 24 (3):341-350 " text), as a result, it was confirmed that 11 unknown species Pomacea canaliculata samples
In, 5 Pomacea canaliculatas for being accredited as tubule Pomacea canaliculata are detected through specific primer shown in the above-mentioned table 1 using embodiment 1
Really it is tubule Pomacea canaliculata, is detected through specific primer shown in the above-mentioned table 1 using embodiment 1 and is accredited as spot Fu Shou
4 Pomacea canaliculatas of spiral shell are spot Pomacea canaliculata really, are detected through specific primer shown in the above-mentioned table 1 using embodiment 1
2 Pomacea canaliculatas for being accredited as the third newfound invasive Pomacea canaliculata are the third newfound invasive Pomacea canaliculata really.
The beneficial effects are mainly as follows：The method that the present invention establishes is set by the conserved positions of multiple specificity
Specific primer is counted to distinguish the chondriogen segment of three kinds of existing Pomacea canaliculatas of China.It is fast by establishing multiple PCR technique
Speed differentiate invasion China tubule Pomacea canaliculata, spot Pomacea canaliculata and Pomacea sp., when detecting between and reduce the cost aspect
It is superior to traditional sequencing approach.
In conclusion the present invention can differentiate three kinds of Pomacea canaliculatas in invasion China, i.e. tubule Pomacea canaliculata, spot with fast accurate
Pomacea canaliculata and Pomacea sp..Specific primer design is from the mitochondria genome sequencing analysis of three kinds of Pomacea canaliculatas, institute
The multiplex PCR system of structure can expand well to Pomacea canaliculata ovum grain, young spiral shell and at spiral shell genomic DNA, widely applicable；This hair
Precisely, in technology solving Pomacea canaliculata type differentiates difficult, the costly and time consuming length of testing cost for bright easy to operate, at low cost, detection
The problem of, it can be used for port quarantine and the detections application such as plant protection department and university institute.
Sequence table
<110>Metering university of China
<120>Quickly differentiate specific primer, multiple PCR detection kit and its detection method of adventive Pomacea canaliculata
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ccttgttgtg tctgtattgg 20
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ggatggagga gcatgaaata g 21
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ctgattgtta ccgccttctt 20
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
cccacttaga agtggaaatc agtag 25
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
tacttgcgta gcagaaacc 19
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gcctgccaat aagcatatc 19
<210> 7
<211> 1238
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
ccttgttgtg tctgtattgg ttttgttttt aaccccattt ttgcataaag gagtctttcg 60
ttcattatgc ttttatcctt taagtcaaat tatattttga atttttgtta ggatttttat 120
tttgttaact tggatcggaa gatgcccagt agaggctcct tatgaacaaa tcggtcaaat 180
tttaactatt ttttattttt tatatttttt actgttacca gtaacccaaa aagtatgaga 240
tactatttta gatttttaat ttaaagtcag tggctgtcca ggggaatatt tgttttgaaa 300
acaaatttta tgtgttcaat tcacttagtc acttatactg ttttaaatta taacagcaat 360
agaggtatat aatacctttt cttcgattta caagaccaat gctacaacat agcttcaatt 420
gcacataaga tatgaggagc tattatttat ttataattgc cataactggc attagaaccg 480
gatttatttc tttaatatta caatttaaac acttacttaa ccttcttcta aggttagaag 540
ctattactat aaacattttt attttattgt atagaagaag aaatataatt ggctcttctg 600
ggttttctgc tttaatttta attactttaa gagtatgtga agctagttta ggtatatcca 660
tcttagtatc tatagttcaa agacatggaa atgattacgt ttctagattt tctagccaga 720
aatgttaaga ttgatttttg ccagaattat attatttatt attcagaatt taaatatatc 780
ttgatacata aggttctgaa gattactact gttaagactc ctgtcaatta ttcagttata 840
tataccttta tttgaatttt caatacaaaa tacatttttt ataaatgata atattagttc 900
cactttaatt tcacttactt tttgaatttc ttctatgatg cttttagcta gacagtttga 960
tattaaaatt tctataaatt caagaacaaa attttccttg tgtatcttaa tccttaactt 1020
tatattaatt atagcttttt gtataacaaa caccttgata ttttatttta tattcgaggc 1080
atcattaatc ccaacattaa tattaatttt aggctgagga taccaaccag aacggctgca 1140
agcaggaata tatataatat tatatactgt tagagcttca cttcctcttt tattaatcct 1200
tctttttgct aataaatcta tttcatgctc ctccatcc 1238
<210> 8
<211> 901
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
ctgattgtta ccgccttctt tattactttt attgtcatct gctgctgttg aaagcggtgc 60
cggaacggga tggacagtat accctccttt agctggtaat ttagctcatg caggtggttc 120
tgttgattta gcaatttttt ctctacactt agcgggagct tcttctattt taggagcagt 180
aaattttatt acaacagtaa ttaatatacg atggcgaggt atacaatttg aacgacttcc 240
tttgtttgta tgatcggtta aaattacagc tattttatta cttttatcat taccagttct 300
tgcaggtgcc attactatat tattaactga tcgaaacttt aatacatctt tttttgaccc 360
agcaggagga ggagatccta ttttatatca gcatttattt tggttttttg gtcatcctga 420
ggtatatatt ttaattcttc ctggctttgg aataatttct catattgtga gtcatcattc 480
ctctaaaaaa gaaatttttg gtactttagg aataatttat gctatattag ctattggtct 540
tttaggtttt attgtttgag ctcatcacat gtttacggtt ggtatagatg tggatactcg 600
agcttatttt acagctgcta ctatgattat tgctgtaccg actggtatta aagttttcag 660
ctgacttgct actatttatg gaagtaaaat taaatatgaa accccaatgc tttgagcttt 720
agggtttatt tttttattta cagtgggtgg attgacaggt attgttctat caaattcttc 780
tttagatatt atattacatg atacttatta tgtagtggct cattttcatt atgtattatc 840
tataggagct gtatttgcct tatttggggc ttttaactac tgatttccac ttctaagtgg 900
g 901
<210> 9
<211> 571
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
tacttgcgta gcagaaacca atcgtgcacc ttttgatttt gctgagggag agtctgaatt 60
agtatcagga tttaacattg aatatagctc agcaggtttt gctcttattt ttcttgccga 120
atatgctaat attttagtta taagattatt tagtgccatt ttgttttttg gaggaaattt 180
tttatttcaa tatgggcttg attttttact tattataaaa gttttattct tttctttttt 240
gtttatttgg gtacgtgcta gttatccacg ttttcgctat gatttgttaa taagattaac 300
ttgaaaaaaa tttttaccat ttagattatc agctctctta attattttaa gttttttatt 360
tttattagtt atcaatatct cggaattata gtttaaatag aatattggct ttgggagcta 420
aagatcaaaa tattttgtgt tccgaatgag tatattaatc ctacttagtt tttctttaac 480
tataactttt atattacccc ttataactca accattaagg ctaggtttaa ttattatgtt 540
atcaacttta ttgatatgct tattggcagg c 571
Claims (9)
1. quickly differentiating the specific primer of adventive Pomacea canaliculata, it is characterised in that including three pairs of specific primers pair, point
It is not：
(1) primer pair 1 that primer sequence forms shown in SEQ ID NO.1 and SEQ ID NO.2；
(2) primer pair 2 that primer sequence forms shown in SEQ ID NO.3 and SEQ ID NO.4；
(3) primer pair 3 that primer sequence forms shown in SEQ ID NO.5 and SEQ ID NO.6.
2. the specific primer of quick discriminating adventive Pomacea canaliculata according to claim 1, it is characterised in that described
The molal quantity of six primers of three primer pairs is equal.
3. the specific primer of quick discriminating adventive Pomacea canaliculata according to claim 1, it is characterised in that：It is described outer
It is at least one of following to carry out biological Pomacea canaliculata：Tubule Pomacea canaliculata, spot Pomacea canaliculata, Pomacea sp..
4. a kind of multiple PCR detection kit of quick discriminating adventive Pomacea canaliculata, it is characterised in that including claim 1 institute
The quick specific primer for differentiating adventive Pomacea canaliculata stated.
5. the multiple PCR detection kit of quick discriminating adventive Pomacea canaliculata according to claim 4, it is characterised in that
Further include PCR reaction buffers, archaeal dna polymerase and 4 kinds of dNTP.
6. a kind of identifying or assisting in the multi-PCR detection method for differentiating adventive Pomacea canaliculata, it is characterised in that including walking as follows
Suddenly：
(a) using the genomic DNA of Pomacea canaliculata to be measured as template, with quick discriminating adventive Pomacea canaliculata described in claim 1
Specific primer carry out PCR amplification；
(b) clip size for the PCR product that detecting step (a) obtains is determined according to PCR product described to be measured as follows
The type of Pomacea canaliculata：If the PCR product is the DNA fragmentation of a 1238bp, the Pomacea canaliculata to be measured is tubule Pomacea canaliculata；
If the PCR product is the DNA fragmentation of a 901bp, the Pomacea canaliculata to be measured is spot Pomacea canaliculata；If the PCR product
For the DNA fragmentation of a 571bp, then the Pomacea canaliculata to be measured is Pomacea sp..
7. multi-PCR detection method according to claim 6, it is characterised in that：The step (a) carries out PCR amplification
Annealing temperature be 60 DEG C.
8. the method described according to claim 6 or 7, it is characterised in that：The primer pair 1, the primer pair 2 and the primer
Molar ratio of six primers formed to 3 in PCR reaction systems is 1:1:1:1:1:1.
9. according to the method described in claim 6, it is characterized in that：In step (b), the nucleosides of the DNA fragmentation of the 1238bp
Acid sequence is as shown in SEQ ID NO.7；The nucleotide sequence of the DNA fragmentation of the 901bp is as shown in SEQ ID NO.8；It is described
The nucleotide sequence of the DNA fragmentation of 571bp is as shown in SEQ ID NO.9.
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