CN102936622A - Non-diagnostic method for detecting rickettsia sibirica through loop-mediated isothermal amplification technology - Google Patents
Non-diagnostic method for detecting rickettsia sibirica through loop-mediated isothermal amplification technology Download PDFInfo
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- CN102936622A CN102936622A CN2012103670294A CN201210367029A CN102936622A CN 102936622 A CN102936622 A CN 102936622A CN 2012103670294 A CN2012103670294 A CN 2012103670294A CN 201210367029 A CN201210367029 A CN 201210367029A CN 102936622 A CN102936622 A CN 102936622A
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Abstract
The present invention discloses a non-diagnostic method for detecting rickettsia sibirica through a loop-mediated isothermal amplification (LAMP) technology. According to the method, a rickettsia sibirica GltA gene sequence is adopted as a target sequence, a conserved region is selected after comparison, a set of primers is designed to carry out amplification, and various reaction conditions are optimized. According to the present invention, the LAMP is adopted as a molecular biology detection non-diagnostic method, a transmissometer, a fluorescence quantitative PCR instrument or a GenieII instrument can be adopted to detect a fluorescence signal to determine a result, and gel imaging is not required to determine the result so as to provide unique advantages of rapidness, convenience, short reaction time, specificity, sensitivity and the like.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of improved loop-mediated isothermal amplification method and the application in detecting dermacetor sibericus thereof.
Background technology
Dermacetor sibericus (Rickettsia sibirica) has another name called the north Asia tick and passes rickettsia, propagate by hard tick and to cause that the north Asia tick passes rickettsia spotted fever (North Asian tick-borne rickettsiosis) or is North Asian tick borne typhus (North Asian tick typhus), be polymorphic in the karyon of host cell productive set in blocks.When tick is bitten human body, pathogenic agent is injected human body first in regional nodes's breeding, be released into later on blood and form rickettsemia, cause endothelial cell inflammation in the blood, cause the dysfunction of each internal organs.Clinical characters has heating, first sore, regional glandular enlargement and fash etc.Rickettsia is kept lasting circulation between nature animal host and medium.
Summary of the invention
Exist for prior art: must just can observations through the running gel imaging, or the low problem of as a result signal to noise ratio that shows of turbidimeter, the object of the present invention is to provide a kind ofly to have loop-mediated isothermal amplification techniques simple to operate, the advantages such as reaction is quick, weak point consuming time and detect the non-diagnostic methods of dermacetor sibericus.
For achieving the above object, the present invention relates to the non-diagnostic methods that loop-mediated isothermal amplification technique detects dermacetor sibericus, comprising:
With dermacetor sibericus GltA gene order as target sequence, choose conservative region after the comparison, design of primers comprises the design of two outer primers and two inner primers, inner primer FIP (upper end inner primer) comprises Flc sequence and F2 (complementary sequence in F2c zone), namely 5 '-Flc-F2; Inner primer BIP (lower end inner primer) comprises Blc (complementary sequence in Bl zone) and B2 sequence, namely 5 '-Blc-B2.Outer primer is F3 and B3 sequence, 6 special zones on the energy specific combination target sequence.
The conserved sequence of choosing is as follows:
5′-GTATTAGCACGGGTATTGCCTCACTTTGGGGACCTGCTCACGG
CGGGGCTAATGAAGCGGTAATAAATATGCTTAAAGAAATCGGTAG
TTCTGAGTATATTCCTAAATATATAGCTA
F3
F2
ATGACCC
GTGCAAAGAAGTA
F1c
TTAAA
CAATCCGCTCTTACAAATA
B1c
B2
AGAGAAAATTAT
GTATTATCTAT
B3
AAAGCTATGGGTATACCGTCGCAAATGTTCACTGTACTTTTTGCA
ATAGCAAGAACCGTAGGCTGGAT-3′
Design outer primer F3, B3 and inner primer FIP, BIP are following sequence or its whole corresponding complementary sequences:
First group:
5′-GTATTAGCACGGGTATTGCCTCACTTTGGGGACCTGCTCACGG
CGGGGCTAATGAAGCGGTAATAAATATGCTTAAAGAAATCGGTA
GTTCTGAGTATATTCCTAAATATATAGCTAA
F3
F2
CTATGACCCGCG
TGCAAAGAAG
F1c
B1c
ATAGCAATAGAACTTGAA
TTT
B2
ATTGAGAGAAAATTAT
GTATTA
B3
TCTATAAAGCTATGGGTATACCGTCGCAAATGTTCACTGTACTTT
TTGCAATAGCAAGAACCGTAGGCTGGAT-3′。
Second group:
Further, described outer primer, inner primer are preferred first group.
Further, described outer primer: inner primer=proportional range is 1:4 ~ 1:12.
Further, described outer primer: inner primer=1:8.
Further, described ring mediated isothermal amplification temperature is 58 ℃ ~ 65 ℃.
Further, described ring mediated isothermal amplification temperature is 60 ℃.
Further, the described ring mediated isothermal amplification time is 15 minutes ~ 60 minutes.
Further, the described ring mediated isothermal amplification time is 60 minutes.
Further, described new detection method comprises step:
1. extract sample DNA: use genome DNA extracting reagent kit to carry out the extraction of DNA;
2. utilize described outer primer F3, B3 and inner primer FIP, BIP, sample DNA, enzyme, damping fluid to prepare 25 μ L reaction systems;
3. ring mediated isothermal amplification program;
4. electrophoresis detection analytical results or use the genieII instrument or use the quantitative real time PCR Instrument analytical results.
Further, described 25 μ L reaction system components are as follows:
Outer primer can be used as independent pcr amplification reaction primer and detects dermacetor sibericus in addition.
Beneficial effect of the present invention is: loop-mediated isothermal amplification technique (loop-mediated isothermal amlication of DNA, be called for short LAMP) only need a thermostat water bath to finish as a kind of molecular biology for detection reaction, naked eyes are judged result roughly, running gel imaging judged result is accurate, have fast and convenient, reaction times is short, the unique advantages such as special sensitivity, or use improved non-diagnostic methods to use instrument GenieII, or can direct judged result in the quantitative fluorescent PCR instrument reaction short period of time, do not need to carry out running gel imaging judged result.Having wide practical use aspect mechanism of basic unit and the field quick detection, for the detection of dermacetor sibericus provides new developing direction, for the LAMP amplification technique does not re-use the gel electrophoresis result, a kind of method of new analytical results is provided, be expected to become easy conventional sense means, for improving health level, guaranteeing that the development of human life security and promotion sanitary inspection cause has great importance.
Description of drawings
Fig. 1 is that inside and outside primer concentration is than different LA320C isothermal duplication instrument detection figure;
Fig. 2 is that LA320C isothermal duplication instrument detects turbidity speed of reaction figure, and CH1 is blank among the figure, and outer inner primer concentration ratio is CH2:1:2 respectively, CH3:1:4, CH4:1:8, CH5:1:12;
Fig. 3 a is the experimental result picture of 58 ℃ of lower LAMP augmentation detection dermacetor sibericuses;
Fig. 3 b is the experimental result picture of 60 ℃ of lower LAMP augmentation detection dermacetor sibericuses;
Fig. 3 c is the experimental result picture of 63 ℃ of lower LAMP augmentation detection dermacetor sibericuses;
Fig. 3 d is the experimental result picture of 65 ℃ of lower LAMP augmentation detection dermacetor sibericuses;
Fig. 4 a-4d is specificity experimental result (LAMP method) figure, 1 blank, 2 Saint Louis' encephalitis viruses, 3 japanese encephalitis viruses, 4 dengue fever viruss, 5 Borrelia burgdorferis, 6 Chikungunya virus, the hot rickettsia of 7Q, 8 dermacetor sibericuses among the figure;
Fig. 5 a and 5b are that LA320C isothermal duplication instrument detects the dermacetor sibericus sensitivity map;
Fig. 6 a and 6b are that LA320C isothermal duplication instrument detection dermacetor sibericus is added with the fluorescence dye sensitivity map;
Fig. 7 is the dermacetor sibericus sensitivity map that the GenieII instrument detects;
Fig. 8 is the dermacetor sibericus sensitivity map of fluorescence quantitative PCR detection.
Annotate: black region is expressed as background values among Fig. 1, Fig. 4 a-4d, Fig. 5 a, Fig. 6 a, and white portion is expressed as the amount of amplified production, and fringe area is expressed as the detection positive findings.
Embodiment
The design of primers of LAMP is the program of a complexity, and 4 primers of minimum needs are respectively forward inner primer FIP, forward outer primer F3, reverse inner primer BIP, reverse outer primer B3.Forward inner primer FIP comprises F1C section (the F1C section with target gene is identical), TTTT connexon and F2 section (with the F2C section complete complementary of target gene); Forward outer primer F3 is with the F3C section complete complementary of target gene; Oppositely inner primer BIP comprises B1C section (the B1C section with target gene is identical), TTTT connexon and B2 section (with the complete complementary of the B2C section of target gene); Oppositely outer primer B3 is with the B3C section complete complementary of target gene.
Its design of primers is mainly followed following principle:
1) the base number apart between F2 and the B2 between primer is about 120～180; Base number between F2 and F3 or B2 and B3 is in 20; The base number is 40～60 between F2 and F1 or B2 and the B1;
2) the Tm value of primer generally speaking the Tm value be 55～65 ℃;
3) GC content and secondary structure GC content are about 50%～60%, avoid the generation of secondary structure as far as possible, and especially 3 ' end in addition, is also noted that rich AT structure can not appear in 3 ' end;
4) 5 of the stable F1C of primer end and B1C ' end, the dG value of 6 bases of 3 of F2/B2, F3/B3 ' end is less than-4kalmol
-1
Dermacetor sibericus GltA gene order is extracted for the applicant uses the test kit operation, or all can be obtained by the upper announcement of GenBank.After the dermacetor sibericus GltA gene order comparison that obtains, choose conservative region, with PrimerExplorer V4 design outer primer F3, B3 and inner primer FIP, BIP.
Loop-mediated isothermal amplification technique is for 4 special primers of 6 zone design of target gene, utilize a kind of archaeal dna polymerase (Bst archaeal dna polymerase) with strand displacement activity, hatched about 60 minutes at constant temperature (about 65 ℃), can finish nucleic acid amplification reaction, produce macroscopic byproduct of reaction-white magnesium pyrophosphate precipitation.By reversed transcriptive enzyme, also can carry out RT-LAMP and finish amplification for RNA.Amplification is available fluorescence dye fluorexon dyeing in conjunction with double-stranded DNA also, judges by naked eyes that under ultraviolet lamp or daylight if contain amplified production, it is green that reaction mixture turns; Otherwise, then keep the orange constant of fluorescence dye.
Concrete experimental implementation step is as follows:
(1) sample DNA extracts
1. bacterium liquid is managed in EP after getting cultivation, and is centrifugal, after the collection, abandons supernatant;
2. use dH
2The resuspended washing of O bacterium, centrifugal, abandon supernatant, repeat once, use at last dH
2O is resuspended;
3. boiling water bath is processed the cracking bacterium, and is centrifugal, makes the contamination precipitation such as bacterioprotein in the bottom, get supernatant to manage in EP, supernatant dna solution ,-20 ℃ of refrigerators are placed for subsequent use or are adopted genome DNA extracting reagent kit to extract.
(2) ring mediated isothermal amplification (LAMP)
Adding following reagent in Reagent Tube, is 25 μ L with the distilled water constant volume:
(3) LAMP amplification program:
Available LA320C isothermal duplication instrument increases, or thermostat water bath, or carries out constant-temperature amplification in the PCR instrument, or uses the genieII instrument to increase, and the amplification temperature is 60 ℃, and proliferation time is 60 minutes; When using the amplification of quantitative fluorescent PCR instrument, amplification program adopts 59 ℃, 10s, and 60 ℃, 50s circulates 60 times, collects fluorescent signal at 60 ℃.Annotate: when using genieII instrument or quantitative real time PCR Instrument, in above-mentioned (2), add SYBR Green I dyestuff; When using quantitative real time PCR Instrument, in above-mentioned (2), add fluorexon, select the FAM passage to detect fluorescent signal.
(4) electrophoretic examinations
The LAMP product carries out dyeing with Ethidum Eremide behind the electrophoresis with 2.0% sepharose, and electrophoresis result is observed under uv analyzer, and positive findings should be the band that scalariform distributes.
(5) optimization experiment of the right ratio reaction conditions of interior outer primer
Most of products of LAMP are by FIP and BIP amplification, inner primer plays an important role in the reaction process of guiding DNA, and outer primer mainly works in the formation of ring texture DNA, inner primer is larger on the impact of reaction, the fixing amount of outer primer (0.2uM), select outer primer: inner primer carries out condition optimizing in the scope of 1:2 ~ 1:12.
Select the outer inner primer (1:2 of different concns ratio according to DNA Amplification Kit, 1:4,1:8,1:12), the differential responses time (15 ~ 60min) and (58 ℃ of temperature, 60 ℃, 63 ℃, 65 ℃) carry out LAM P reaction, find out from the result of LA320C isothermal duplication instrument amplification: outer inner primer concentration ratio is 1:4,1:8, all can amplify product during 1:12, but from rate of amplification Fig. 1 and Fig. 2 consider select concentration ratio be 1:8 be best outside inner primer concentration-response condition.
The selection of LAMP amplification temperature can be found out 58 ℃ from Fig. 3 a ~ 3d, 60 ℃, 63 ℃, 65 ℃ of these four temperature all can amplified production, but can find out have at first product to increase out in the time of 60 ℃ from spreading rate, so select 60 ℃ of the most optimal reaction temperatures.
(6) specificity experiment
The LAMP method of setting up according to the present invention, to Saint Louis' encephalitis virus, japanese encephalitis virus, dengue fever virus, Borrelia burgdorferi, Chikungunya virus, the hot rickettsia of Q, dermacetor sibericus detects, six kinds of virus amplification are all negative sees Fig. 4, in addition to escherichia coli, Vibrio parahemolyticus, Corynebacterium pseudotuberculosis, the glanders burkholderia, ox type Bacillus brucellae S19 strain, Escherichia coli O 157: H7, francisella tularensis, Yersinia intermedia, the Bai Shi Yersinia, Burkholderia Pseudomallei, the template DNA of Yersinia enterocolitica detects, the amplification all primer specificity of negative explanation institute establishment method is strong, does not have non-specific amplification.
(7) sensitivity experiment
With synthetic 10 times of doubling dilutions to 10 of plasmid
-1~ 10
-6, the plasmid of getting each weaker concn carries out the LAMP amplification as template, the test detection sensitivity, and amplified production carried out 1.5% agarose gel electrophoresis, simultaneously, will under same template concentrations, add the fluorescence dye fluorexon and not add the dyestuff fluorexon and do contrast, the results are shown in Figure 5, Fig. 6.
The little gradient of as a result sensitivity that the conclusion that LA320C isothermal duplication instrument draws is run out of than electrophorogram, if single perceptible conclusion of result that obtains from instrument obtains false-negative result easily, we can find out that also we should singly also should not come judged result from agarose gel electrophoresis figure from relying on LA320C isothermal duplication instrument judged result from figure, can find out that from electrophorogram it is 0.35fg/ μ L that the detection sensitivity that does not add fluorescence dye reaches the detection sensitivity that 0.035fg/ μ L is added with fluorescence dye, macroscopic result can reach 3.5fg/ μ L.
(8) the GenieII instrument detects
When using the GenieII instrument to detect, do not need to carry out the running gel imaging and show the result, can directly detect in real time the fluorescent signal of synthetic product, judged result by the GenieII instrument, in reaction system, need to add SYBR Green I, by the 10 times of doubling dilutions to 10 of plasmid to synthesizing
-1~ 10
-6, the plasmid of getting each weaker concn carries out the LAMP amplification as template, tests its detection sensitivity and detects, and the results are shown in Figure 7, and we can find out that detection sensitivity reaches 0.035fg/ μ L from figure.
(9) quantitative fluorescent PCR is to detect
When using quantitative fluorescent PCR to detect, can add fluorexon in reaction system, the FAM that selects is as sense channel, or adds SYBR Green I in reaction system, and the passage of selecting is SYBR, by to synthetic 10 times of doubling dilutions to 10 of plasmid
-1~ 10
-6, the plasmid of getting each weaker concn carries out the LAMP amplification as template, tests its detection sensitivity and detects, and the results are shown in Figure 8, and we can find out that detection sensitivity reaches 0.035fg/ μ L from figure.
Utilize fluorescence dye SYBR Green I can with the double-stranded DNA minor groove binding, not when double-stranded DNA is combined, its fluorescent signal is very weak, when strengthening also greatly, its fluorescent signal after double-stranded DNA is combined to be detected by instrument, the product that we utilize this application of principle to amplify in detection LAMP method, the result shows that amplification efficiency is better, amplification can directly show, the result that will increase again carries out electrophoresis, and electrophoresis result shows and the coming to the same thing of instrument display.
Embodiment
The detection of mouse nephridial tissue sample, tick sample
(1) gets respectively 39 parts of mouse nephridial tissue samples and 10 parts of tick samples, smash respectively and be treated to cell suspension, then extract DNA according to tissue gene group DNA extraction test kit;
(2) dna solution of getting part extraction carries out the regular-PCR amplification;
(3) surplus DNA solution is carried out dermacetor sibericus LAMP amplification, utilize described outer primer F3, B3 and inner primer FIP, BIP, the genomic dna of extraction, enzyme, damping fluid to prepare 25 μ L reaction systems;
(4) ring mediated isothermal amplification program;
(5) electrophoresis detection analytical results, more common dermacetor sibericus pcr amplification and dermacetor sibericus LAMP amplification.
Check result shows that 2 duplicate samples are positive, and this is consistent with the result that regular-PCR obtains, and illustrates that institute's establishment method and regular-PCR method do not have difference.
SEQUENCE LISTING
＜110〉China Inst. of Quarantine Inspection Sciences
＜120〉loop-mediated isothermal amplification technique detects the non-diagnostic methods of dermacetor sibericus
＜130〉invention
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> GltA-F3
<400> 1
AAGCTAAGGATAAAAATGATCCA 22
<210> 2
<211> 20
<212> DNA
<213> GltA-B3
<400> 2
<210> 3
<211> 23
<212> DNA
<213> GltA-FIP
<400> 3
GTTTCTTTAAGTACTGCGGCACGTTTAGATTAATGGGTTTTGGTCA 46
<210> 4
<211> 20
<212> DNA
<213> GltA-BIP
<400> 4
GAACTCGGGCAGCTAGACAA-ATATTCATCTTTAAGAGCGATAGC 44
Claims (10)
1. loop-mediated isothermal amplification technique detects the non-diagnostic methods of dermacetor sibericus, it is characterized in that the method comprises:
As target sequence, choose conservative region with dermacetor sibericus GltA gene order after the comparison, design outer primer F3, B3 and inner primer FIP, BIP are following sequence or its whole corresponding complementary sequences:
2. non-diagnostic methods as claimed in claim 1 is characterized in that, described outer primer: inner primer=proportional range is 1:4 ~ 1:12.
3. non-diagnostic methods as claimed in claim 2 is characterized in that, described outer primer: inner primer=1:8.
4. non-diagnostic methods as claimed in claim 1 is characterized in that, described ring mediated isothermal amplification temperature is 58 ℃ ~ 65 ℃.
5. non-diagnostic methods as claimed in claim 4 is characterized in that, described ring mediated isothermal amplification temperature is 60 ℃.
6. non-diagnostic methods as claimed in claim 1 is characterized in that, the described ring mediated isothermal amplification time is 15 minutes ~ 60 minutes.
7. non-diagnostic methods as claimed in claim 6 is characterized in that, the described ring mediated isothermal amplification time is 60 minutes.
8. non-diagnostic methods as claimed in claim 1 is characterized in that, described non-diagnostic methods comprises step:
1. extract sample DNA: use genome DNA extracting reagent kit to carry out the extraction of DNA;
2. utilize described outer primer F3, B3 and inner primer FIP, BIP, sample DNA, enzyme, damping fluid to prepare 25 μ L reaction systems;
3. ring mediated isothermal amplification program;
4. electrophoresis detection analytical results or use quantitative real time PCR Instrument, GenieII direct analysis result.
9. non-diagnostic methods as claimed in claim 8 is characterized in that, described 25 μ L reaction system components are as follows:
10. non-diagnostic methods as claimed in claim 8, it is characterized in that, described isothermal duplication adopts water bath with thermostatic control, PCR, Real-time PCR, LC320 turbidimeter, GenieII, when using instrument GenieII to increase, adds SYBR Green I dyestuff in the reagent and increases; When using described quantitative real time PCR Instrument to increase, add SYBR Green I dyestuff in the reagent, select the SYBR passage to carry out fluorescent signal when increasing and collect; Adding in the reagent when fluorexon increases selects the FAM passage to carry out the fluorescent signal collection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110468222A (en) * | 2019-09-16 | 2019-11-19 | 中国人民解放军军事科学院军事医学研究院 | The special primer for checking of one group of China's Rana amurensis |
| CN117327822A (en) * | 2023-11-16 | 2024-01-02 | 河南中检食安生物科技有限公司 | Primer group and kit for detecting hansaibatong bodies |
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| CN102251039A (en) * | 2011-07-19 | 2011-11-23 | 中国疾病预防控制中心传染病预防控制所 | Kit and method for detecting anaplasmosis and rickettsioses based on loop-mediated isothermal amplification (LAMP) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110468222A (en) * | 2019-09-16 | 2019-11-19 | 中国人民解放军军事科学院军事医学研究院 | The special primer for checking of one group of China's Rana amurensis |
| CN110468222B (en) * | 2019-09-16 | 2022-08-30 | 中国人民解放军军事科学院军事医学研究院 | Special detection primers for rickettsiae in China Heilongjiang |
| CN117327822A (en) * | 2023-11-16 | 2024-01-02 | 河南中检食安生物科技有限公司 | Primer group and kit for detecting hansaibatong bodies |
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Application publication date: 20130220 |