CN105483121B - One group of primer and its application for identifying storage booklice - Google Patents
One group of primer and its application for identifying storage booklice Download PDFInfo
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Abstract
The primer for being used to identify storage booklice the invention discloses one group and its application.The present invention discloses one group of primer, and at least a pair in the primer pair shown in following (1)-(8) forms: (1) primer pair that DNA molecular shown in SEQ ID No.1 and SEQ ID No.2 forms；(2) primer pair of the composition of DNA molecular shown in SEQ ID No.3 and SEQ ID No.4；(3) primer pair of the composition of DNA molecular shown in SEQ ID No.5 and SEQ ID No.6；(4) primer pair of the composition of DNA molecular shown in SEQ ID No.7 and SEQ ID No.8.The features of the present invention is as follows: (1) carrying out Identification of Species rapidly and efficiently using primer pair disclosed by the invention storage booklice, qualification result is accurate and reliable, detection sensitivity is high, and high-throughput detection may be implemented.(2) compared with traditional form identification method, identification object of the invention is not influenced by type gender, stage of development and Morphological Identification feature integrated degree.(3) qualification process is easy to operate, economy and facility.
Description
Technical field
The primer for being used to identify storage booklice the present invention relates to one group and its application, belong to field of biotechnology.
Background technique
Booklice (booklice) also known as paper lice (paperlice), rice lice (ricelice), are under the jurisdiction of Corrodentia
Psocoptera, booklice section Liposcelididae, booklice belong to Liposcelis Motsch μ lsky 1852.Booklice category insect
Figure is small, the long 0.6-1.5mm of body, in world's distribution in extensive range；Most of its moves in interior, and minority moves in outdoor,
It is the common insect pests in storage protection.The class pest has mobility strong, and foraging territories are wide, and growth cycle is short, modes of reproduction
Multiplicity, seriously causes harm and stores in a warehouse article and threaten human health at the features such as carrying a variety of germs and resistance and big prevention and control difficulty.
Booklice class pest can be with stored goods long-distance communications, as increasingly frequent, class pest propagation is allocated and transported in international trade and grain storage
Spread risk increasingly increases.In recent years, with increasingly frequent, inspection and quarantining for import/export department of China intercepting and capturing booklice of international trade
Batch gradually increase.
Cause harm total 10 kinds of the common storage booklice in the world of grain storage, including Leposcelis entomophia L.entomophila
(Enderlein), l.bostrychophila L.bostrychophila Badonnel, colourless booklice L.decolor (Pearman), ommatidium
Booklice L.paeta Pearman is nibbled booklice L.corrodens (Heymons), dun booklice L.brunnea Motsch μ Lsky,
L. mendax L.mendax Pearman, Pi Shi booklice L.pearmani Lienhard, three color booklice L.tricolor
Badonnel, red booklice L.rufa Broadhead.
For traditional booklice Identification of Species mostly using female adult adult morphological feature as foundation, it is high, time-consuming that there are professional skill requirements
The disadvantages of long is especially non-adult form insect, such as the precise Identification of ovum, larva and pupa is difficult to realize, and is caused to quarantine
Very big difficulty.Insect identification research is carried out using Protocols in Molecular Biology, not by sample ontogeny state and sample integrity
Influence, it can be achieved that efficient, convenient, accurate species identification.
Summary of the invention
The primer for being used to identify storage booklice the object of the present invention is to provide one group and its application.
The present invention provides one group of primer, and at least a pair in the primer pair shown in following (1)-(8) forms:
(1) primer pair of the composition of DNA molecular shown in SEQ ID No.1 and SEQ ID No.2；
(2) primer pair of the composition of DNA molecular shown in SEQ ID No.3 and SEQ ID No.4；
(3) primer pair of the composition of DNA molecular shown in SEQ ID No.5 and SEQ ID No.6；
(4) primer pair of the composition of DNA molecular shown in SEQ ID No.7 and SEQ ID No.8；
(5) primer pair of the composition of DNA molecular shown in SEQ ID No.9 and SEQ ID No.10；
(6) primer pair of the composition of DNA molecular shown in SEQ ID No.11 and SEQ ID No.12；
(7) primer pair of the composition of DNA molecular shown in SEQ ID No.13 and SEQ ID No.14；
(8) primer pair of the composition of DNA molecular shown in SEQ ID No.15 and SEQ ID No.16.
In above-mentioned primer, primer primer pair shown in following (1)-(8) is formed:
(1) primer pair of the composition of DNA molecular shown in SEQ ID No.1 and SEQ ID No.2；
(2) primer pair of the composition of DNA molecular shown in SEQ ID No.3 and SEQ ID No.4；
(3) primer pair of the composition of DNA molecular shown in SEQ ID No.5 and SEQ ID No.6；
(4) primer pair of the composition of DNA molecular shown in SEQ ID No.7 and SEQ ID No.8；
(5) primer pair of the composition of DNA molecular shown in SEQ ID No.9 and SEQ ID No.10；
(6) primer pair of the composition of DNA molecular shown in SEQ ID No.11 and SEQ ID No.12；
(7) primer pair of the composition of DNA molecular shown in SEQ ID No.13 and SEQ ID No.14；
(8) primer pair of the composition of DNA molecular shown in SEQ ID No.15 and SEQ ID No.16.
A kind of to identify or assist to identify that the kit of storage booklice also belongs to protection scope of the present invention, which includes
Any of the above-described primer；
The storage booklice is Leposcelis entomophia, colourless booklice, nibbles booklice, dun booklice, L. mendax, Pi Shi booklice, three
Color booklice and red booklice.
It is a kind of to identify or assist the method for identifying storage booklice to also belong to protection scope of the present invention, include the following steps:
Using the genomic DNA of storage booklice to be identified as template, respectively using following (1)-(8) it is any shown in primer pair as primer,
PCR amplification is carried out, pcr amplification product is obtained；If the pcr amplification product obtained using primer pair shown in following (1) as primer
For 369bp, then storage booklice candidate to be identified is Leposcelis entomophia, if obtained using primer pair shown in following (2) as primer
Pcr amplification product be 300bp, then storage booklice candidate to be identified is colourless booklice, if with primer shown in following (3)
It is 200bp to the pcr amplification product obtained for primer, then storage booklice candidate to be identified is to nibble booklice, if with following (4)
Shown in primer pair be the pcr amplification product that primer obtains be 450bp, then storage booklice candidate to be identified is dun booklice,
If the pcr amplification product obtained using primer pair shown in following (5) as primer is 506bp, storage booklice to be identified is waited
Be selected as L. mendax, if using the pcr amplification product that primer pair shown in following (6) is obtained as primer be 293bp, it is to be identified
Storage booklice candidate be Pi Shi booklice, if being using the pcr amplification product that primer pair shown in following (7) is obtained as primer
257bp, then storage booklice candidate to be identified is three color booklices, if obtained using primer pair shown in following (8) as primer
Pcr amplification product is 335bp, then storage booklice candidate to be identified is red booklice:
(1) primer pair of the composition of DNA molecular shown in SEQ ID No.1 and SEQ ID No.2；
(2) primer pair of the composition of DNA molecular shown in SEQ ID No.3 and SEQ ID No.4；
(3) primer pair of the composition of DNA molecular shown in SEQ ID No.5 and SEQ ID No.6；
(4) primer pair of the composition of DNA molecular shown in SEQ ID No.7 and SEQ ID No.8；
(5) primer pair of the composition of DNA molecular shown in SEQ ID No.9 and SEQ ID No.10；
(6) primer pair of the composition of DNA molecular shown in SEQ ID No.11 and SEQ ID No.12；
(7) primer pair of the composition of DNA molecular shown in SEQ ID No.13 and SEQ ID No.14；
(8) primer pair of the composition of DNA molecular shown in SEQ ID No.15 and SEQ ID No.16.
In the above method, the concentration ratio of primer is 1:1 in the system of the PCR amplification；
Annealing temperature is 50 DEG C in the program of the PCR amplification；
The system of the PCR amplification is specific as follows: 2 × PCR amplification buffer 12.5 μ L, ddH2O9.5 μ L, 1 μ of template
L, each 1 μ L of primer that concentration is 10 μM；
The product name of 2 × PCR amplification buffer is specially 2 × Taq PCR MasterMix, purchased from raw work biology
Engineering (Shanghai) limited liability company；
The program of the PCR amplification is specific as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72
DEG C extend 30s, 30 circulation；72 DEG C of extension 5min.
In any of the above-described method, quality >=5ng of template in the system of the PCR amplification.
Application of any of the above-described primer in the product of preparation identification or auxiliary identification storage booklice also belongs to this
The protection scope of invention.
Application of the mentioned reagent box in the product of preparation identification or auxiliary identification storage booklice also belongs to guarantor of the invention
Protect range.
In any of the above-described application, the storage booklice be Leposcelis entomophia, colourless booklice, nibble booklice, dun booklice,
L. mendax, Pi Shi booklice, three color booklices and/or red booklice.
The features of the present invention is as follows:
(1) Identification of Species is carried out rapidly and efficiently using primer pair provided by the invention storage booklice, qualification result accurately may be used
It leans on, detection sensitivity height, and high-throughput detection may be implemented.
(2) compared with traditional form identification method, identification object of the invention is not by type gender, stage of development and form
The influence of identification mark integrated degree.Morphological feature of the traditional form identification method generally just for female adult adult, needs slide
Preparation of specimen's technical ability requires sample integrity etc. high.
(3) qualification process is easy to operate, economy and facility, can standardize, and easy to operate and required cost is lower, convenient for without specially
The staff's operation and utilization of industry taxology knowledge background.
Detailed description of the invention
Fig. 1 is the specific detection result for identifying the primer pair entoF3 and entoR2 of Leposcelis entomophia.
Fig. 2 is the specific detection result for identifying the primer pair decF2 and decR4 of colourless booklice.
Fig. 3 is for identifying the specific detection result for nibbling the primer pair LcF2 and LcR3 of booklice.
Fig. 4 is the specific detection result for identifying the primer pair bruF1 and bruR2 of dun booklice.
Fig. 5 is the specific detection result for identifying the primer pair mendF1 and mendR2 of L. mendax.
Fig. 6 is the specific detection result for identifying the primer pair pearF1 and pearR2 of Pi Shi booklice.
Fig. 7 is the specific detection result for identifying the primer pair triF1 and triR2 of three color booklices.
Fig. 8 is the specific detection result for identifying the primer pair rufaF1 and rufaR1 of red booklice.
Fig. 9 is the sensitivity testing result for identifying the primer pair entoF3 and entoR2 of Leposcelis entomophia.
Figure 10 is the sensitivity testing result for identifying the primer pair decF2 and decR4 of colourless booklice.
Figure 11 is for identifying the sensitivity testing result for nibbling the primer pair LcF2 and LcR3 of booklice.
Figure 12 is the sensitivity testing result for identifying the primer pair bruF1 and bruR2 of dun booklice.
Figure 13 is the sensitivity testing result for identifying the primer pair mendF1 and mendR2 of L. mendax.
Figure 14 is the sensitivity testing result for identifying the primer pair pearF1 and pearR2 of Pi Shi booklice.
Figure 15 is the sensitivity testing result for identifying the primer pair triF1 and triR2 of three color booklices.
Figure 16 is the sensitivity testing result for identifying the primer pair rufaF1 and rufaR1 of red booklice.
Specific embodiment
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.
Following embodiment further illustrates the content of present invention, but does not limit the present invention.Experiment side in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples, unless otherwise specified,
It is to be commercially available from routine biochemistry reagent shop.Quantitative test in following embodiment, is respectively provided with and weighs three times
Multiple experiment, results are averaged.
Leposcelis entomophia [Liposcelis entomophila (Enderlein)] (sample 1: is acquired from Guangxi China Huang fine strain of millet
The Leposcelis entomophia of dream；Sample 2: the Leposcelis entomophia of acquisition Bohemia from Czech；Sample 3: it acquires from the thermophilic of kansas, U.S.A
Worm booklice), l.bostrychophila (L.bostrychophila Badonnel) (sample 4: acquires from the l.bostrychophila of Chongqing in China；Sample
Sheet 5: the l.bostrychophila of acquisition Bohemia from Czech；Sample 6: acquire from the l.bostrychophila of kansas, U.S.A), colourless booklice
[L.decolor (Pearman)] (sample 7: acquires the colourless booklice from Chongqing in China；Sample 8: it acquires from Czech's medium wave sago
Sub- colourless booklice；Sample 9: acquire from the colourless booklice of kansas, U.S.A), L. paeta (L.paeta Pearman) (sample
10: acquiring the L. paeta from Zhejiang Province, China；Sample 11: the L. paeta of acquisition Bohemia from Czech；Sample 12: acquisition
From the L. paeta of kansas, U.S.A), nibbling booklice [L.corrodens (Heymons)], (sample 13: acquisition is from Czech medium wave west
Meter Ya's nibbles booklice；Sample 14: acquisition nibbles booklice from kansas, U.S.A；Sample 15: acquisition from Portuguese nibbles booklice), it is dark
Brown booklice (L.brunnea Motschulsky) (sample 16: the dun booklice of acquisition Bohemia from Czech；Sample 17: it adopts
Collect from the dun booklice of kansas, U.S.A), (sample 18: acquisition is from Jiangsu Province, China salt for L. mendax (L.mendax Pearman)
The L. mendax in city), Pi Shi booklice (L.pearmani Lienhard) (sample 19: acquires from the Pi Shi book of kansas, U.S.A
Lice), three color booklices (L.tricolor Badonnel) (sample 20: acquiring from three color booklices of Shandong Province of China Heze), red booklice
(L.rufa Broadhead) (sample 21: acquires the red booklice from kansas, U.S.A)；
The above Leposcelis entomophia, colourless booklice, L. paeta, nibbles booklice, dun booklice, L. mendax in text at l.bostrychophila
Offer " Zhao Shuo, Li Zhihong, Qin Meng booklice and its Progress on Molecular Biology plant protection, 2009,35 (6): in 17-21. "
It is disclosed, the public can obtain from China Agricultural University.
The above three colors booklice is in document " the wsp base of tri- color booklice parachorium microorganism Wolbachia of Dong Peng, Wang Jinjun
The Molecular Detection zoological research of cause, 2004,25 (5): being disclosed in 456-459. ", and the public can obtain from China Agricultural University
?.
" Beijing mesh will: Science Press, 2002:91- is nibbled by Li Fasheng China in document for the above red booklice and Pi Shi booklice
It is disclosed in 94. ", the public can obtain from China Agricultural University.
Embodiment 1, the design of primer pair
The present invention passes through on the basis of obtaining ten various common psocids mitochondrial COI gene DNA bar code sections
The multiple alignment of sequence is analyzed, and designs primer pair for eight kinds of common storage booklices, as shown in table 1.
The common 8 kinds of storages booklice special primer list in 1 world of table
Embodiment 2, the specific detection of primer
One, the specific detection of entoF3 and entoR2
(1) genome of the single head (or bull) of the storage booklice of sample 1- sample 21 is extracted respectively using CTAB method
DNA。
(2) each genomic DNA obtained using step (1) carries out PCR by primer of entoF3 and entoR2 as template
Amplification, obtains each pcr amplification product, while with ddH2O replaces template as negative control.
PCR amplification system (total volume is 25 μ L): 2 × Taq PCR MasterMix is (purchased from raw work bioengineering (Shanghai)
Limited liability company) 12.5 μ L, ddH29.5 μ L of O, concentration are the 1 μ L of template of 100-300ng/ μ L, the primer that concentration is 10 μM
Each 1 μ L.
PCR amplification program: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30sec, 50 DEG C of annealing 30sec, 72 DEG C of extension 30sec,
30 circulations；72 DEG C of extension 5min.
(3) each pcr amplification product is subjected to agarose gel electrophoresis, as a result as shown in Figure 1.
In Fig. 1, M:DNA Relative molecular weight markers (D2000)；1 to 21 PCR amplification for being followed successively by sample 1 to sample 21 produces
Object, 22 be negative control.
Fig. 1 shows that acquisition shows a 369bp size from the Leposcelis entomophia (sample 1 to sample 3) of different geographic populations
Specific band, and other sample standard deviations do not show any band.
Two, the specific detection of decF2 and decR4
Experimentation such as step 1, only replaces with decF2 and decR4 for primer, as a result as shown in Figure 2.
It is consistent in the sample and Fig. 1 of each swimming lane in Fig. 2.
Fig. 2 shows that acquisition shows a 300bp size from the colourless booklice (sample 7 to sample 9) of different geographic populations
Specific band, and other sample standard deviations do not show any band.
Three, the specific detection of LcF2 and LcR3
Experimentation such as step 1, only replaces with LcF2 and LcR3 for primer, as a result as shown in Figure 3.
It is consistent in the sample and Fig. 1 of each swimming lane in Fig. 3.
Fig. 3 shows that acquisition shows a 200bp size from the booklice (sample 13 to sample 15) that nibbles of different geographic populations
Specific band, and other sample standard deviations do not show any band.
Four, the specific detection of bruF1 and bruR2
Experimentation such as step 1, only replaces with bruF1 and bruR2 for primer, as a result as shown in Figure 4.
It is consistent in the sample and Fig. 1 of each swimming lane in Fig. 4.
Fig. 4 shows that acquisition shows that a 450bp is big from the dun booklice (sample 16 to sample 17) of different geographic populations
Small specific band, and other sample standard deviations do not show any band.
Five, the specific detection of mendF1 and mendR2
Experimentation such as step 1, only replaces with mendF1 and mendR2 for primer, as a result as shown in Figure 5.
It is consistent in the sample and Fig. 1 of each swimming lane in Fig. 5.
Fig. 5 shows that L. mendax (sample 18) shows the specific band of a 506bp size, and other sample standard deviations are not shown
Show any band.
Six, the specific detection of pearF1 and pearR2
Experimentation such as step 1, only replaces with pearF1 and pearR2 for primer, as a result as shown in Figure 6.
It is consistent in the sample and Fig. 1 of each swimming lane in Fig. 6.
Fig. 6 shows that Pi Shi booklice (sample 19) shows the specific band of a 293bp size, and other sample standard deviations are not shown
Show any band.
Seven, the specific detection of triF1 and triR2
Experimentation such as step 1, only replaces with triF1 and triR2 for primer, as a result as shown in Figure 7.
It is consistent in the sample and Fig. 1 of each swimming lane in Fig. 7.
Fig. 7 shows that three color booklices (sample 20) show the specific band of a 257bp size, and other sample standard deviations are not
Show any band.
Eight, the specific detection of rufaF1 and rufaR1
Experimentation such as step 1, only replaces with rufaF1 and rufaR1 for primer, as a result as shown in Figure 8.
It is consistent in the sample and Fig. 1 of each swimming lane in Fig. 8.
Fig. 8 shows that red booklice (sample 21) shows the specific band of a 335bp size, and other sample standard deviations are not shown
Any band.
The above result shows that different primer pairs (Leposcelis entomophia-entoF3 and entoR2, the colourless book of eight kinds of booklices
Lice-decF2 and decR4, nibbles booklice-LcF2 and LcR3, dun booklice-bruF1 and bruR2, L. mendax-mendF1 and
MendR2, Pi Shi booklice-pearF1 and pearR2, three color booklice-triF1 and triR2, red booklice-rufaF1 and rufaR1) point
The PCR product of specific length segment can not be amplified from corresponding booklice, and to other kinds without purpose band, it is as a result clear,
It is easy to differentiate.
Embodiment 3, the detection of the sensitivity of primer
One, the sensitivity detection of entoF3 and entoR2
(1) genomic DNA of sample 2 is extracted using CTAB method, and is diluted to 0.01ng/ μ L, 0.1ng/ μ L,
The concentration of 1ng/ μ L, 5ng/ μ L, 10ng/ μ L, 25ng/ μ L, 50ng/ μ L, 75ng/ μ L and 100ng/ μ L.
(2) genomic DNA of each dilution obtained using step (1) is to draw with entoF3 and entoR2 as template
Object carries out PCR amplification, obtains each pcr amplification product, while replacing template as negative control using ddH2O.
PCR amplification system such as embodiment 2.
PCR amplification program such as embodiment 2.
(3) each pcr amplification product is subjected to agarose gel electrophoresis, as a result as shown in Figure 9.
In Fig. 9, M:DNA Relative molecular weight markers (D2000)；1 to 9 is followed successively by with 0.01ng/ μ L, 0.1ng/ μ L, 1ng/ μ
The DNA of L, 5ng/ μ L, 10ng/ μ L, 25ng/ μ L, 50ng/ μ L, 75ng/ μ L and 100ng/ μ 9 kinds of various concentrations of L is template
Pcr amplification product；10 be negative control.
Fig. 9 shows that faint purpose item can be amplified when in 25 μ LPCR systems containing 1ng Leposcelis entomophia DNA profiling
Band, containing bright purpose band can be amplified when having more than 5ng DNA profiling.
Two, the sensitivity detection of decF2 and decR4
Sample 2 is only replaced with sample 7 by experimentation such as step 1, and primer replaces with decF2 and decR4, as a result as schemed
Shown in 10.
It is consistent in the amount and Fig. 9 of the template of each swimming lane in Figure 10.
Figure 10 shows that faint purpose can be amplified when booklice DNA profiling colourless containing 1ng in 25 μ L PCR systems
Band, containing bright purpose band can be amplified when having more than 5ng DNA profiling.
Three, the sensitivity detection of LcF2 and LcR3
Sample 2 is only replaced with sample 13 by experimentation such as step 1, and primer replaces with LcF2 and LcR3, as a result as schemed
Shown in 11.
It is consistent in the amount and Fig. 9 of the template of each swimming lane in Figure 11.
Figure 11 shows that faint purpose item can be amplified when nibbling booklice DNA profiling containing 1ng in 25 μ L PCR systems
Band, containing bright purpose band can be amplified when having more than 5ng DNA profiling.
Four, the sensitivity detection of bruF1 and bruR2
Sample 2 is only replaced with sample 16 by experimentation such as step 1, and primer replaces with bruF1 and bruR2, as a result such as
Shown in Figure 12.
It is consistent in the amount and Fig. 9 of the template of each swimming lane in Figure 12.
Figure 12 shows that faint mesh can be amplified when booklice DNA profiling dun containing 0.1ng in 25 μ L PCR systems
Band, containing bright purpose band can be amplified when having more than 1ng DNA profiling.
Five, the sensitivity detection of mendF1 and mendR2
Sample 2 is only replaced with sample 18 by experimentation such as step 1, and primer replaces with mendF1 and mendR2, as a result
As shown in figure 13.
It is consistent in the amount and Fig. 9 of the template of each swimming lane in Figure 13.
Figure 13 shows that faint purpose can be amplified when in 25 μ L PCR systems containing 1ng L. mendax DNA profiling
Band, containing bright purpose band can be amplified when having more than 5ng DNA profiling.
Six, the sensitivity detection of pearF1 and pearR2
Sample 2 is only replaced with sample 19 by experimentation such as step 1, and primer replaces with pearF1 and pearR2, as a result
As shown in figure 14.
It is consistent in the amount and Fig. 9 of the template of each swimming lane in Figure 14.
Figure 14 shows that faint purpose can be amplified when in 25 μ L PCR systems containing 1ng Pi Shi booklice DNA profiling
Band, containing bright purpose band can be amplified when having more than 5ng DNA profiling.
Seven, the sensitivity detection of triF1 and triR2
Sample 2 is only replaced with sample 20 by experimentation such as step 1, and primer replaces with triF1 and triR2, as a result such as
Shown in Figure 15.
It is consistent in the amount and Fig. 9 of the template of each swimming lane in Figure 15.
Figure 15 shows that faint mesh can be amplified when in 25 μ L PCR systems containing tri- color booklice DNA profiling of 0.1ng
Band, containing bright purpose band can be amplified when having more than 1ng DNA profiling.
Eight, the sensitivity detection of rufaF1 and rufaR1
Sample 2 is only replaced with sample 21 by experimentation such as step 1, and primer replaces with rufaF1 and rufaR1, as a result
As shown in figure 16.
It is consistent in the amount and Fig. 9 of the template of each swimming lane in Figure 16.
Figure 16 shows that faint purpose item can be amplified when booklice DNA profiling red containing 1ng in 25 μ L PCR systems
Band, containing bright purpose band can be amplified when having more than 5ng DNA profiling.
The above result shows that different primer pairs (Leposcelis entomophia-entoF3 and entoR2, the colourless book of eight kinds of booklices
Lice-decF2 and decR4, nibbles booklice-LcF2 and LcR3, dun booklice-bruF1 and bruR2, L. mendax-mendF1 and
MendR2, Pi Shi booklice-pearF1 and pearR2, three color booklice-triF1 and triR2, red booklice-rufaF1 and rufaR1) point
The other mitochondrial COI gene sequence to 8 kinds of booklices of storing in a warehouse has very high sensitivity, in 25 μ L PCR reaction systems,
When DNA profiling is more than 5ng, specific amplification goes out purpose bright wisp band.
It is accurate and reliable that synthesis result shows that 8 pairs of special primers of the invention have in detecting 8 kinds of common storage booklices,
The characteristics of high sensitivity.
Claims (5)
1. one group of primer, the primer pair shown in following (1)-(8) is formed:
(1) primer pair of the composition of DNA molecular shown in SEQ ID No.1 and SEQ ID No.2；
(2) primer pair of the composition of DNA molecular shown in SEQ ID No.3 and SEQ ID No.4；
(3) primer pair of the composition of DNA molecular shown in SEQ ID No.5 and SEQ ID No.6；
(4) primer pair of the composition of DNA molecular shown in SEQ ID No.7 and SEQ ID No.8；
(5) primer pair of the composition of DNA molecular shown in SEQ ID No.9 and SEQ ID No.10；
(6) primer pair of the composition of DNA molecular shown in SEQ ID No.11 and SEQ ID No.12；
(7) primer pair of the composition of DNA molecular shown in SEQ ID No.13 and SEQ ID No.14；
(8) primer pair of the composition of DNA molecular shown in SEQ ID No.15 and SEQ ID No.16.
2. the kit of a kind of identification or auxiliary identification storage booklice, which includes primer described in claim 1.
3. application of the primer described in claim 1 in the product of preparation identification or auxiliary identification storage booklice.
4. application of the kit as claimed in claim 2 in the product of preparation identification or auxiliary identification storage booklice.
5. application according to claim 3 or 4, it is characterised in that: the storage booklice be Leposcelis entomophia, colourless booklice,
Nibble booklice, dun booklice, L. mendax, Pi Shi booklice, three color booklices and/or red booklice.
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| CN201410478720.9A CN105483121B (en) | 2014-09-18 | 2014-09-18 | One group of primer and its application for identifying storage booklice |
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| CN201410478720.9A CN105483121B (en) | 2014-09-18 | 2014-09-18 | One group of primer and its application for identifying storage booklice |
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| CN105483121A CN105483121A (en) | 2016-04-13 |
| CN105483121B true CN105483121B (en) | 2019-01-01 |
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| CN102367482A (en) * | 2011-11-07 | 2012-03-07 | 中国农业大学 | Specific primer pair for assisting booklice identification and application thereof |
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| CN102367482A (en) * | 2011-11-07 | 2012-03-07 | 中国农业大学 | Specific primer pair for assisting booklice identification and application thereof |
Non-Patent Citations (2)
| Title |
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| Rapid molecular diagnosis of the stored-product psocid Liposcelis corrodens (Psocodea: Liposcelididae): Species-specific PCR primers of 16S rDNA and COI;Qianqian Yang et al.;《Journal of Stored Products Research》;20131231;摘要，正文第2.3部分以及附图1b * |
| 书虱分子鉴定、生殖进化及比较线粒体基因组学研究;杨倩倩;《中国博士论文全文数据库》;20160715;D046-1 * |
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