CN106755455A - A kind of method for improving extremely low copy number target dna detection sensitivity - Google Patents
A kind of method for improving extremely low copy number target dna detection sensitivity Download PDFInfo
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Abstract
The present invention relates to a kind of method for improving extremely low copy number target dna detection sensitivity, key step is as follows：To addition external source background genes group DNA in detected sample DNA；Digestion is carried out to mixed sample DNA using the restriction enzyme that generation randomized bases cohesive end can be specifically cut to target dna；It is attached according to cohesive end design produced after target dna cutting joint complementary therewith and therewith；Enter performing PCR to connection product using the universal primer contained in joint to react；With PCR primer as template, specific nucleic acid augmentation detection is carried out using the specific primer designed for target dna.This method applicability is wide, can effectively improve the detection sensitivity of the target dnas such as the extremely low virus of quantity, fungi, bacterium, and method flexible design, cost easy to operate is relatively low, with significant practical application meaning.
Description
Technical field
The invention belongs to nucleic acid detection technique field, and in particular to one kind improves existing nucleic acid detection technique to extremely low copy
The method of number target dna detection sensitivity.
Background technology
Nucleic acid detection method based on external DNA cloning technology has the advantages that high sensitivity and high specific, compares tool
Representational is PCR (alternating temperature reaction) and ring mediated isothermal amplification (LAMP), in biology, medical science, environmental science, legal medical expert
The fields such as, Food Science play extremely important effect.In field of detection of food safety, traditional microculture identification
Method needs a couple of days detection cycle, and nucleic acid detection method just can be completed within a few houres, and sensitivity is above passing with specificity
System method；It is widely used in quick and precisely detection and medicament research and development of various diseases etc. in medical domain nucleic acid detection method
In various projects；DNA detections are the important means of all kinds of criminal cases of investigating and prosecuting in medical jurisprudence.
But the acquisition of nucleic acid detection method high sensitivity result is generally required under conditions of using ideal sample
Obtain, the sample of (or denier target dna mixes in a large amount of background nucleics) extremely low relative to some target dna quantity, core
The sensitivity of sour detection method may be reduced so as to cause the result of false negative or false positive, such as in medical science detection with specificity
" analysis " sensitivity and the inconsistent situation of " clinic " sensitivity.Therefore, being purified for this class sample a large amount of to eliminate
The influence of background nucleic turns into one of method for improving this kind of sample detection efficiency, such as United States Patent (USP) US 13/933,919
(Richmond et al, 2013) discloses that a kind of selective enrichment is special from a large amount of background nucleics using anionexchangetechnique
The method of stator segment length DNA, but the method be applied to relatively large sample purifying and need to prepare specific anion exchange
Microballon, the target dna of extremely low quantity is easily lost in absorption and elution process；Zhou etc. reports species specificity removal people
So as to the method for improving Salmeterol fluticasone propionate sensitivity in blood sample, it first uses fel bovis and microballoon sclerotium to genomic DNA
Sour enzyme individually extracts salmonella DNA and is detected again after human gene group DNA is degraded, but the method is suitable only for whole blood sample
Treatment and comparatively cumbersome (Zhou L, the Pollard A J.A novel method of selective of operating process
removal of human DNA improves PCR sensitivity for detection of Salmonella
Typhi in blood samples[J].BMC infectious diseases,2012,12(1):164.).For without the back of the body
The pure dna sample of scape nucleic acid, although PCR methods can reach the detection sensitivity of 1 copy in theory, but extremely strict due to needing
Experimental situation and optimal experiment condition (primer, reagent, parameter etc.), therefore because being extremely difficult to this mesh in practical operation
Mark, the detection threshold value of PCR is about 100 copies even more many (Wang Yiqian, leaf rue foundation detection aerogenesis long in many document reports
The TaqMan quantitative fluorescent PCRs and regular-PCR method [J] disease surveillance of enterobacteria, 2016,31 (2):153-158；
Sritunyalucksana K,Srisala J,McColl K,et al.Comparison of PCR testing methods
for white spot syndrome virus(WSSV)infections in penaeid shrimp[J]
.Aquaculture,2006,255(1):95-104.), though increasing amplification cycles number can to a certain extent improve detection sensitivity
But false positive risk is increased simultaneously.
Obviously, if research and development it is a kind of it is with low cost, easy to operate, low method can improve pole to be required to experimental facilities etc.
The detection sensitivity of low abundance (being less than hundred a ten thousandths) DNA, while ensureing the high specific of detection, will effectively improve nucleic acid inspection
The efficiency that survey method is detected in the case of various actual complex, has a extensive future.
The content of the invention
Regarding to the issue above, the present invention is intended to provide it is a kind of efficiently, easy can improve existing nucleic acid detecting sensitivity
Method, make extremely low quantity target dna can by high sensitivity, high specific detect.
A kind of method for improving extremely low copy number target dna detection sensitivity, key step is as follows：To detected sample
External source background genes group DNA is added in DNA；Randomized bases cohesive end is produced using can specifically be cut to target dna
Restriction enzyme carries out digestion to mixed sample DNA；According to produced cohesive end design after target dna cutting with
Complementation joint and be attached therewith；Enter performing PCR to connection product using the universal primer contained in joint to react；With
PCR primer is template, and specific nucleic acid augmentation detection is carried out using the specific primer designed for target dna.
More specifically step is：
A) to the external source background genes group DNA that final concentration of 10-500ng/ μ L are added in detected sample DNA；
B) step a) is obtained using the restriction enzyme that generation randomized bases cohesive end can be cut to target dna
Sample carry out digestion, endonuclease reaction parameter is determined by selected restriction endonuclease；
C) to the double-stranded DNA joint added in digestion products for the design of genes of interest endonuclease bamhi, and in DNA ligase
In the presence of be attached reaction, the two ends of endonuclease bamhi is all connected with top connection, on the joint carry universal primer sequence
Row, joint ultimate density is 50-400nM, and coupled reaction parameter ligase according to selected by determines；
D) with connection product as template, enter performing PCR using universal primer and react, response parameter is according to final goal piece segment length
Degree determines；
E) above One_step PCR product is template, and performing PCR or other nucleic acid are entered using the specific primer of target dna
Amplification method realizes the final detection to target gene, and reaction condition and the parameter method for detecting specificity according to selected by determine.
The extremely low copy number refers generally to copy number less than 50, when higher than the copy number, experiment of the Standard PCR in optimization
Under the conditions of can detect target dna, detection is then difficult during less than the copy number, and be also easy to produce false positive.
Wherein, step a) the external source background genes group DNA include all Eukaryotic genomic DNAs, preferably salmon
The industrialized productions such as smart DNA are more ripe, can largely prepare, the genomic DNA that purity is high and price is more cheap.
Restriction enzyme described in step b) is that a class can produce randomized bases cohesive end to target dna cutting
The cohesive end of the fragment produced by restriction endonuclease, i.e. digestion is made up of randomized bases.Selection recognition site is 4-6 bases, produces
Cohesive end length carry out endonuclease reaction for 4-9 bases restriction endonuclease, and selected restriction endonuclease at least should contain in target gene
Two restriction enzyme sites or using two kinds in target gene respectively containing a restriction endonuclease for restriction enzyme site, to produce two ends to have
There is the endonuclease bamhi of randomized bases cohesive end, two distances of restriction enzyme site are 70-2000bp on the target gene.It is described
Restriction endonuclease includes but is not limited to Alw26I, BbvI, FokI, HgaI, TspRI etc..
Target dna (known to sequence) and background genes group DNA by after the abundant digestion of restriction endonuclease, generate it is a large amount of have with
The sequence and internal sequence of the fragment of machine base cohesive ends, the wherein cohesive end of target gene endonuclease bamhi as joint and
The basis of specific primer design；Background genes group DNA is had and target gene in the number of fragments after the same enzyme digestion
The fragment ratio of the identical cohesive end of endonuclease bamhi is about 1/42n(n is the base of random sequence in a cohesive end
Number), it is 1/4 with either end base identical ratio in two cohesive ends of target gene endonuclease bamhin.By what is added
Background genes group DNA generates hundreds of millions of number of fragments after digestion, therefore wherein containing identical with target endonuclease bamhi
The segment number of cohesive end be up to more than thousands of.
" for the double-stranded DNA joint of genes of interest endonuclease bamhi design " by the different list of two length described in step c)
Chain DNA is constituted, and wherein one section of universal primer sequence is contained at the end of long-chain moiety 5 ', and the end of short chain moieties 5 ' is contained can be with target gene enzyme
" linking " sequence of section section cohesive end complete complementary pairing.Then contain the complementary pairing of >=8 bases between long-chain and short chain
Part, therefore the partially double stranded structure of stabilization can be formed.All joint long-chain all sames, and short chain then has different linking sequences
Row are (according to the different and different of target endonuclease bamhi cohesive end).
The DNA ligase mainly includes that T3, T4, T7DNA ligase and E.coli DNA ligases and Taq DNA connect
Enzyme is connect, T3, T4, T7DNA ligase that preferably joint efficiency is high, reaction condition is gentle are attached reaction.Connection Time 0.5-
2.5h, the long Connection Time will cause the probability of mispairing between joint and endonuclease bamhi to increase, and the too short Connection Time may
Target fragment is caused to fail to be attached with joint completely.
After the double-stranded DNA joint and DNA ligase for the design of genes of interest endonuclease bamhi is added, joint and purpose
There is coupled reaction in genetic fragment, the two ends of target endonuclease bamhi is contained a joint design；Meanwhile, background genes group
The endonuclease bamhi containing cohesive end identical with target endonuclease bamhi is also connected with joint simultaneously in DNA, i.e.,：By coupled reaction
Afterwards, the fragment being connected with joint in endonuclease bamhi includes target gene and a part of background genes group DNA fragmentation, following
PCR courses of reaction in, this Partial Fragment in background genes group DNA can be expanded by universal primer simultaneously with target dna, be improved
The starting template quantity of PCR reactions, reaches on hundreds thousand of copies, and meeting PCR will to the minimum of amplification initial profiling number
Ask, therefore can improve the efficiency of reaction and sensitivity.
Universal primer described in step d) refers to one section of oligonucleotide sequence of designed, designed, while being also all double-strands
The long-chain moiety of DNA joints, length is 18-26 base, and whether fusing point is 60 DEG C ± 5 DEG C, can be formed according between target dna
It is unfavorable for the secondary structure of amplification and changes.Concentration of the universal primer in PCR system is 0.1-1 μM.In the embodiment of the present invention
The sequence of universal primer used is：TCGTCACTGCACGGTGACTCAGCA.
Specific primer described in step e) is that amplified production is inside target fragment for designed by target endonuclease bamhi
Section of DNA sequence, method for detecting specificity includes but is not limited to the conventional detection of nucleic acids means such as PCR, LAMP, RCA, finally makes
Testing and appraisal is carried out to product with means such as gel electrophoresis or sequencings.
Compared to the deficiencies in the prior art, the invention has the advantages that：
(1) detection sensitivity of existing nucleic acid detection method can be improved.Nucleic acid detection method is to initial mould under normal circumstances
Plate quantity there are certain requirements (copy number is higher than 50), then cannot effectively be expanded less than the threshold value, and the present invention is by addition and target
The incoherent external source background dna of gene simultaneously makes a part of background dna act as initial amplification template by steps such as digestion connections,
Final amplification initial profiling number is set to have exceeded amplification threshold value, therefore can be finally special with reference to other with effectively start amplified reaction
Different in nature detection method can be realized to the high sensitivity less than threshold targets gene, high specific detection.
(2) relative to conventional nucleic acid detection method, improve the specificity of detection.Because digestion and joint connection procedure are equal
Specificity there are certain requirements, play a part of double control, therefore the specificity of final detection has obtained certain raising.
(3) method is easy to operate, it is not necessary to instrument and reagent special, costly.Instrument needed for of the invention is mainly
Regular thermocyclers, required reagent is common toolenzyme in molecular biology, and final detection means is conventional electrophoretic or sequencing,
Therefore it is relatively low to experiment condition requirement, the requirement in most of laboratories can be met.
(4) method applicability is strong, and final specific detection also may be selected LAMP and RCA etc. in addition to usable PCR method
Constant-temperature amplification method.
Brief description of the drawings
The techniqueflow chart of Fig. 1 embodiment of the present invention 1；
Fig. 2 embodiments 1 are to the testing result (A) of Aspergillus flavus in mixed congee and the testing result (B) of Standard PCR method.M：
DNA ladder；N：Negative control.Swimming lane 1-5 represents the Aspergillus flavus of different dilution factors, swimming lane 1 respectively：Dilution factor is 104,
Swimming lane 2：Dilution factor is 105, swimming lane 3：Dilution factor is 106, swimming lane 4：Dilution factor is 107, swimming lane 5：Dilution factor is 108；
Fig. 3 embodiments 2 are to the testing result (A) of comma bacillus in drinking water and the testing result (B) of routine LAMP methods.
Swimming lane 1-5 represents the comma bacillus of different dilution factors, swimming lane 1 respectively：Dilution factor is 104, swimming lane 2：Dilution factor is 105, swimming lane 3：
Dilution factor is 106, swimming lane 4：Dilution factor is 107, swimming lane 5：Dilution factor is 108；
Fig. 4 embodiments 3 are to the testing result (A) of salmonella in catsup and the testing result (B) of routine LAMP methods.
Swimming lane 1-5 represents the salmonella of different dilution factors, swimming lane 1 respectively：Dilution factor is 104, swimming lane 2：Dilution factor is 105, swimming lane 3：
Dilution factor is 106, swimming lane 4：Dilution factor is 107, swimming lane 5：Dilution factor is 108；
Fig. 5 use the testing result (A) of present invention bacteriophage p13 primary to Cray and the testing result (B) of Standard PCR method.
M：DNA ladder；N：Negative control.Swimming lane 1-5 represents the target dna of different copy numbers, swimming lane 1 respectively：5000 copies, swimming
Road 2：500 copies, swimming lane 3：50 copies, swimming lane 4：5 copies, swimming lane 5：0.5 copy；
Testing results (A) and the detection of Standard PCR method of Fig. 6 using the present invention to comma bacillus in actual oyster sample
As a result (B).Sample number into spectrum is marked in swimming lane top.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that specific embodiment described herein is only used to explain the present invention, not
For limiting the present invention.
Portion of reagent used is as follows in embodiment, and non-specified otherwise is conventional commercial commodity：
Genome extracts kit：Tiangeng, #DP350；
Bacterial genomes extracts kit：Tiangeng, #DP302；
Marine animal tissue gene group extracts kit：Tiangeng, #DP324；
Salmon sperm dna：Sigma-Aldrich, #D1626；
FastDigest BbvI restriction endonucleases：Thermo Scientific, #FD2074；
FastDigest HgaI restriction endonucleases：Thermo Scientific, #FD1904；
T3 DNA ligases：New England Biolabs, #M0317S；
T4 DNA ligases：New England Biolabs, #M0202S；
T7 DNA ligases：New England Biolabs, #M0318S；
Pfu archaeal dna polymerases：Thermo Scientific, #EP0502.
Embodiment 1. is detected using the present invention to Aspergillus flavus in mixed congee
1st, 100 μ L are added to pass through the aspergillus flavus bacterium solution being serially diluted to 5g mixed congees sample (Wahaha) are middle under aseptic condition
(dilution factor is respectively 104、105、106、107、108), (4C, 1792 × g) is centrifuged to mixed sample and is collected supernatant afterwards,
(4 DEG C, 16128 × g) are centrifuged to supernatant and are collected bacteria residue, the bacteria residue being collected into is carried out using genome extracts kit
DNA is extracted.The Aspergillus flavus to each dilution factor carries out colony counting to calculate its general amount simultaneously.
2nd, to salmon sperm dna is added in each DNA sample extracted in step 1, final salmon sperm dna is made in each sample
Concentration be 20ng/ μ L.
3rd, the selection of target DNA fragments：To aspergillus flavus genome (GenBank：EQ963478.1) enzyme is carried out using BbvI
Cutting is analysed, and the wherein one section endonuclease bamhi of 441bp is selected as target to be detected, in the endonuclease bamhi sequence information such as sequence table
Shown in SEQ ID No.1：
4th, sample digestion (step 1 in Fig. 1)：Endonuclease reaction, 10 μ L digestions are carried out to flavus dna using BbvI restriction endonucleases
Contain 1 μ 10 × FastDigest of L buffer, sample in 1 μ L steps 2,0.5 μ L FastDigest BbvI inscribes in system
Enzyme, uses ddH2O complements to 65 DEG C of enzyme 5min that go out after 10 μ L are incubated 5min after 37 DEG C.
5th, joint coupled reaction (step 2 in Fig. 1)：BbvI endonuclease bamhis are directed to 0.5 μ L are sequentially added in digestion system
The double-stranded DNA joint (final concentration 50nM, table 1) of design, 0.5 μ L ATP (10mM) and 1 μ L T4DNA ligases, in 25 DEG C of insulations
2h。
The joint sequence used in the embodiment 1 of table 1.
Note：Chain joint long is simultaneously as the universal primer in all embodiment PCR reactions in the present invention；Underscore part is
" be connected " sequence complementary with target fragment cohesive end, similarly hereinafter.
6th, (step 3 in Fig. 1) is reacted using the PCR of universal primer：To being directly added into 5 10 × pfu of μ L in connection product
Archaeal dna polymerase buffer, 1 μ L 10mM dNTPs, 1.5 μ L universal primers (10 μM, table 1), 0.5 μ L pfu archaeal dna polymerases
(2.5U/ μ L), uses ddH2Total system is complemented to 50 μ L by O, and response parameter is：72 DEG C of 5min, 94 DEG C of 3min, 25 cyclic amplifications
Reaction (94 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 75s) is incubated 5min for 72 DEG C afterwards.
7th, specific PCR detection (step 4 in Fig. 1)：Specific primer is designed for Aspergillus flavus DNA targets endonuclease bamhi
(table 2) enters performing PCR reaction, and product, 2 μ L pfu buffer (10 ×), 0.4 μ L10mM in 1 μ L steps 6 are contained in 20 μ L systems
DNTPs, each 0.4 μ L specificity upstream and downstream primer (table 2).Response parameter is：94 DEG C of 3min, 25 (94 DEG C of cyclic amplification reactions
30s, 63 DEG C of 30s, 72 DEG C of 45s) afterwards 72 DEG C insulation 5min.
The Specific PCR primers used in the embodiment 1 of table 2.
Standard PCR control experiment is set simultaneously, and the DNA for directly being obtained using step 1 carries out the specificity of step 7 as template
PCR detections (except period is that the 50 outer other conditions of circulation are identical), final product is detected using 2% agarose gel electrophoresis.
Fig. 2 results are shown, 10 are reached to Aspergillus flavus DNA detection sensitivities using the method for the present invention7Dilution factor (figure
2A), understand that Aspergillus flavus quantity now is 20 copies by colony counting result；Standard PCR method can detect to 106Dilution factor
(Fig. 2 B), equivalent to about 300 copies, comparing result shows that the present invention can make PCR methods improve Aspergillus flavus detection sensitivity
About 15 times.Can cause a certain amount of loss due to adding Aspergillus flavus to carry out extraction again, therefore speculate of the invention actually detected
Sensitivity should be higher than that the present embodiment result, but the comparing result display present invention by using same sample can improve PCR methods
Detection sensitivity.
Embodiment 2. is detected using the present invention to comma bacillus in drinking water
1st, 100 μ L are added to pass through the culture logarithmic phase being serially diluted to 1mL drinking pure waters (ice dew) are middle under aseptic condition
Comma bacillus (dilution factor is respectively 104、105、106、107、108), supernatant is abandoned after centrifugation (4 DEG C, 16128 × g), use bacterium
Genome extracts kit carries out DNA extractions to the bacteria residue being collected into.The comma bacillus to each dilution factor carries out bacterium colony meter simultaneously
Count to calculate its general amount.
2nd, to salmon sperm dna (Sigma-Aldrich) is added in each DNA sample extracted in step 1, final salmon is made
Concentration of the smart DNA in each sample is 50ng/ μ L.
3rd, the selection of target DNA fragments：To comma bacillus 16S rRNA genes (GenBank：U10955.1 HgaI) is used
Restriction analysis are carried out, the wherein one section endonuclease bamhi of 444bp is selected as target to be detected, the endonuclease bamhi sequence information such as sequence
In list shown in SEQ ID No.7：
4th, endonuclease reaction is carried out to the sample prepared in step 2 using HgaI restriction endonucleases, 1 μ is contained in 10 μ L digestion systems
L10 × FastDigest buffer, sample in 1 μ L steps 2,0.5 μ L FastDigest HgaI restriction endonucleases use ddH2O is supplied
65 DEG C of enzyme 5min that go out after being incubated 5min after 37 DEG C to 10 μ L.
5th, it is attached reaction (table using the double-stranded DNA joint and T3DNA ligases that are designed for HgaI endonuclease bamhis
3), the final concentration of 100nM of system center tap, other reaction conditions are identical with step 5 in embodiment 1 with parameter.
The joint sequence used in the embodiment 2 of table 3.
6th, it is template with the connection product in step 5, is reacted using the PCR of universal primer, experiment condition and response parameter
It is identical with step 6 in embodiment 1.
7th, specificity LAMP detections：To sequentially adding 8 μ L ddH in light-wall pipe2O, 2.5 10 × isothermal of μ L
Amplification buffer, 3.5 μ L dNTPs (10mM), 2 μ L MgSO4(25mM), 5 μ L betaine (5M), 1 μ L moulds
Plate (product in step 6), (each primer concentration in mix primer is respectively 2 μ L mix primers：Inner primer FIP and BIP are 10
μM, outer primer F3 and B3 are 2.5 μM, are shown in Table 4), are placed in after mixing in the thermal cycler that hot lid temperature is 95 DEG C and keep 2min
After take out and be immediately inserted into 5min in ice, then operate on ice to added in pipe 1 μ L Bst 2.0DNA polymerases (8U/ μ L,
NEB), and by reaction tube it is immediately placed in the thermal cycler being preheated, 95 DEG C of enzyme 2min that go out are to terminate after 65 DEG C of reaction 45min
Reaction.Final product is detected using 0.8% agarose gel electrophoresis.
The LAMP primer used in the embodiment 2 of table 4.
Setting the DNA obtained with step 1 simultaneously directly carries out the control group of LAMP detections as template, except use template not
Outside, other reaction conditions are completely the same with step 7 with parameter.
Fig. 3 results are shown, 10 are reached to the detection sensitivity of comma bacillus in drinking water using the method for the present invention7Dilution
Degree (Fig. 3 A), understands that comma bacillus quantity now is 16 copies by colony counting result；Conventional LAMP methods can detect to 106
Dilution factor (Fig. 3 B), equivalent to about 280 copies, comparing result shows that the present invention can make LAMP methods detect sensitive to comma bacillus
Degree improves about ten several times.Relative to the BbvI restriction endonucleases that embodiment 1 is used, the present embodiment has used HgaI restriction endonucleases, has shown
The present invention has certain flexibility in restriction endonuclease selection.
Embodiment 3. is detected using the present invention to salmonella in catsup
1st, to the sramana that culture logarithmic phases of the 100 μ L by being serially diluted is added in 1g catsup (Heng Shi) under aseptic condition
(dilution factor is respectively 10 to Salmonella4、105、106、107、108), (4 DEG C, 4032 × g) are centrifuged to mixed sample and are collected afterwards
Supernatant, (4 DEG C, 16128 × g) is centrifuged to supernatant and is collected bacteria residue, using bacterial genomes extracts kit to being collected into
Bacteria residue carry out DNA extractions.The salmonella to each dilution factor carries out colony counting to calculate its general amount simultaneously.
2nd, to salmon sperm dna (Sigma-Aldrich) is added in each DNA sample extracted in step 1, final salmon is made
Concentration of the smart DNA in each sample is 150ng/ μ L.
3rd, the selection of target DNA fragments：To salmonella SodC2 genes (GenBank：DQ023313.1) entered using HgaI
Row restriction analysis, select the wherein one section endonuclease bamhi of 346bp as target to be detected, in the endonuclease bamhi sequence such as sequence table
Shown in SEQ ID No.14：
4th, endonuclease reaction is carried out to the sample in step 2 using HgaI restriction endonucleases, in 10 μ L digestion systems containing 1 μ L10 ×
FastDigest buffer, sample in 1 μ L steps 2,0.5 μ L FastDigest HgaI restriction endonucleases use ddH2O complements to 10 μ
L is after 65 DEG C of enzyme 5min that go out after 37 DEG C of insulation 5min.
5th, it is attached reaction (table using the double-stranded DNA joint and T7DNA ligases that are designed for salmonella DNA
5), the final concentration of 200nM of system center tap, other reaction conditions are identical with step 5 in embodiment 1 with parameter.
The joint sequence used in the embodiment 3 of table 5.
6th, using connection product in step 5 as template, enter performing PCR using universal primer and react, other experiment conditions and anti-
Answer parameter identical with step 6 in embodiment 1.
7th, specificity LAMP detections：Product is detected (table 6) as LAMP reaction templates using in step 6, using being directed to
The LAMP primer of salmonella DNA designs carries out amplified reaction, and other reaction conditions and parameter are complete with step 7 in embodiment 2
Unanimously.
The LAMP primer used in the embodiment 3 of table 6.
Setting the DNA obtained with step 1 simultaneously directly carries out the control group of LAMP detections as template, except use template not
Outside, other reaction conditions are completely the same with step 7 with parameter.
Fig. 4 results are shown, 10 are reached to the detection sensitivity of salmonella in catsup using the method for the present invention7Dilution
Degree (Fig. 4 A), understands that salmonella quantity now is 12 copies by colony counting result；Conventional LAMP methods can detect to 106
Dilution factor (Fig. 4 B), equivalent to about 180 copies, comparing result shows that the present invention can make LAMP methods sensitive to Salmeterol fluticasone propionate
Degree improves ten several times.
The application present invention of embodiment 4. bacteriophage p13 primary to Cray is detected
In order to more intuitively carry out quantitatively evaluating to detection sensitivity of the invention, the purifying of known exact magnitude is used
Phage DNA afterwards is detected as target to be detected, using the inventive method and Standard PCR method and is carried out results contrast
Evaluate.
1st, the phage DNA of gradient dilution concentration known, makes its concentration be followed successively by 5000,500,50,5,0.5 copy/μ L,
Then to salmon sperm dna (Sigma-Aldrich) is added in each sample, make concentration of the final salmon sperm dna in each sample equal
It is 400ng/ μ L.
2nd, the selection of target DNA fragments：To phage genome (GenBank：SRX270599) digestion is carried out using BbvI
Analysis, selects the wherein one section endonuclease bamhi of 511bp as target to be detected, SEQ ID in the endonuclease bamhi sequence such as sequence table
Shown in No.21：
3rd, endonuclease reaction is carried out using BbvI restriction endonucleases, 1 10 × FastDigest of μ L is contained in 10 μ L digestion systems
Buffer, 1 μ L samples, 0.5 μ L FastDigest BbvI restriction endonucleases use ddH2O complements to 10 μ L and is incubated 5min after 37 DEG C
65 DEG C of enzyme 5min that go out afterwards.
4th, it is (whole to double-stranded DNA joints of the 0.5 μ L for the design of phage DNA endonuclease bamhi is sequentially added in digestion system
Concentration 300nM, table 7), 0.5 μ L ATP (10mM) and 1 μ L T4DNA ligases, be incubated 0.5h in 25 DEG C.
The joint sequence used in the embodiment 4 of table 7.
5th, using connection product in step 4 as template, enter performing PCR using universal primer and react, other experiment conditions and anti-
Answer parameter identical with step 6 in embodiment 1.
6th, enter performing PCR reaction using for target endonuclease bamhi design specific primer, 1 μ L steps are contained in 20 μ L systems
Product, 2 μ L pfu buffer (10 ×), 0.4 μ L 10mM dNTPs, each 0.4 μ L specificity upstream and downstream primer (table 8) in 5.Instead
The parameter is answered to be：94 DEG C of 3min, 25 cyclic amplification reactions (94 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 45s) are incubated 5min for 72 DEG C afterwards.
Standard PCR control experiment is set simultaneously, directly using not plus the bacteriophage p13DNA of salmon sperm dna is carried out as template
The specific PCR detection (except period is that the 50 outer other conditions of circulation are identical) of step 6, final product is solidifying using 2% agarose
Gel electrophoresis are detected.
The Specific PCR primers used in the embodiment 4 of table 8.
Fig. 5 results show that using the method for the present invention detection sensitivity can be made to reach 5 copies (Fig. 5 A), Standard PCR method is examined
It is 50 copies (Fig. 5 B) to survey sensitivity, and the present invention makes detection sensitivity improve 10 times.
Embodiment 5. is detected using the present invention to comma bacillus in actual oyster sample
1st, collection is extracted from June to 23 parts of oyster samples of in the market purchase between August using marine animal tissue gene group
Kit is extracted to the full DNA of Oyster Tissue.
2nd, the selection of target DNA fragments：With embodiment 2.
3rd, endonuclease reaction is carried out using BbvI restriction endonucleases, 1 10 × FastDigest of μ L is contained in 10 μ L digestion systems
Buffer, the oyster DNA sample extracted in 1 μ L steps 1,0.5 μ L FastDigest BbvI restriction endonucleases use ddH2O is complemented to
10 μ L are after 65 DEG C of enzyme 5min that go out after 37 DEG C of insulation 5min.
4th, to sequentially added in digestion system 0.5 μ L for endonuclease bamhi design double-stranded DNA joint (final concentration 400nM,
Table 3), 0.5 μ L ATP (10mM) and 1 μ L T4DNA ligases, be incubated 1h in 25 DEG C.
5th, be template with the connection product in step 4, using universal primer PCR react, other reaction conditions with implement
Step 6 is identical in example 1.
6th, using the product in step 5 as template, carried out using specific primer is designed for comma bacillus endonuclease bamhi
PCR reacts (table 9), and outside removing template is different from primer, other reaction conditions are identical with step 7 in parameter and embodiment 1.
The Specific PCR primers used in the embodiment 5 of table 9.
Standard PCR control experiment is set simultaneously, step 6 is directly carried out as template using the oyster DNA that extracts in step 1
Specific PCR detection (except period be the 50 outer other conditions of circulation it is identical), final product use 2% agarose gel electrophoresis
Detection.
Fig. 6 results show that finally detect 3 comma bacillus positives (Fig. 6 A) altogether using the method for the present invention, routine is high
Period PCR methods detect 2 positives (Fig. 6 B) altogether, in showing the detection present invention could apply to actual sample, and
Improve detection sensitivity.Due to inevitably introducing substantial amounts of background genes group DNA in actual sample detection, so in enzyme
Need not additionally add background genes group DNA during cutting, therefore it is especially applicable to denier target in actual sample
The detection of DNA, with significant practical application meaning.
The result of the above embodiments shows that the method for the present invention can effectively improve existing conventional nucleic acid detection method to pole
The detection sensitivity of low predicted quantitative objectives DNA, the inventive method has been separately verified to different type microorganism by multiple embodiments
The Detection results of (including virus, fungi, bacterium)；Demonstrate various different restriction endonucleases to be applicable, show the choosing of the inventive method
Select flexibility；Show that this method has fairly obvious actual application value by the testing result to actual sample, and it is overall
On requirement to instrument and reagent it is relatively low, therefore with wide actual application prospect.
It should be appreciated that for those of ordinary skills, can according to the above description be improved or converted,
And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
SEQUENCE LISTING
<110>The tall and erect molecular biosciences Science and Technology Ltd. in Qingdao thousand
<120>A kind of method for improving extremely low copy number target dna detection sensitivity
<130> 2017
<160> 27
<170> PatentIn version 3.5
<210> 1
<211> 441
<212> DNA
<213> Aspergillus flavus
<400> 1
atcaattacc tcgctgccaa gaggaaagaa ctcggtctcg atagctcgac ggtcgtcctt 60
caacaaagct cactgggatt cgacatggga cttgcacaga ctctcaatgc catcatgaac 120
ggaggaaagc ttgtcattgt gccacaagaa ttacgaggag actcgattga aattgcaaga 180
attatccgcg atcagaaggt gactttcacg ctagccactc cgtccgaata cctcgtcatg 240
ctccaacatg gtcgtgaata cctcaacaat tacaccggat ggcggcacgc ctgccttggg 300
ggtgagccat tcacagacca gctcaagagg gagtttgtga ggctgggaaa gaactgccct 360
gtggtgcagg actcctacgg tgtgaccgag atttccgcct gcacaacctt tgagacgatg 420
tctgcctcgc agctggaaga c 441
<210> 2
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> A-1
<400> 2
gcactgctga gtcac 15
<210> 3
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> A-2
<400> 3
tgattgctga gtcac 15
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223>Chain joint long（Universal primer）
<400> 4
tcgtcactgc acggtgactc agca 24
<210> 5
<211> 21
<212> DNA
<213> Aspergillus flavus
<400> 5
cgctgccaag aggaaagaac t 21
<210> 6
<211> 24
<212> DNA
<213> Aspergillus flavus
<400> 6
cttgtggcac aatgacaagc tttc 24
<210> 7
<211> 444
<212> DNA
<213> Vibrio cholerae
<400> 7
tgccttcggg agctctgaga caggtgctgc atggctgtcg tcagctcgtg ttgtgaaatg 60
ttgggttaag tcccgcaacg agcgcaaccc ttatccttgt ttgccagcac gtaatggtgg 120
gaactccagg gagactgccg gtgataaacc ggaggaaggt ggggacgacg tcaagtcatc 180
atggccctta cgagtagggc tacacacgtg ctacaatggc gtatacagag ggcagcgatc 240
ccgcgaggtg gagcgaatct cacaaagtac gtcgtagtcc ggattggagt ctgcaactcg 300
actccatgaa gtcggaatcg ctagtaatcg caaatcagaa tgttgcggtg aatacgttcc 360
cgggccttgt acacaccgcc cgtcacacca tgggagtggg ctgcaaaaga agcaggtagt 420
ttaaccttcg ggaggacgct tgcc 444
<210> 8
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> A-3
<400> 8
aggcatgctg agtcac 16
<210> 9
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> A-4
<400> 9
acttttgctg agtcac 16
<210> 10
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> FIP
<400> 10
gctgccctct gtatacgcca ttgtcaagtc atcatggccc tt 42
<210> 11
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> BIP
<400> 11
cgcgaggtgg agcgaatctc atggagtcga gttgcagact 40
<210> 12
<211> 20
<212> DNA
<213> Vibrio cholerae
<400> 12
gataaaccgg aggaaggtgg 20
<210> 13
<211> 20
<212> DNA
<213> Vibrio cholerae
<400> 13
cgcaacattc tgatttgcga 20
<210> 14
<211> 346
<212> DNA
<213> Salmonella typhimurium
<400> 14
gcctgtgcgg gtgcgcaggc cgccagcgag aaagtagaga tgaatctggt gacggcgcaa 60
ggcgtaggac agtctatcgg caccgtcgtc atcgatgaaa ccgaaggcgg cttaaaattt 120
accccacacc ttaaagcgtt gccgccgggc gagcatggtt ttcacattca tgccaacggt 180
agctgccagc ccgcgattaa agacggcaaa gcggttgccg cagaagccgc tggtggtcat 240
ctggacccac aaaataccgg caagcatgaa ggaccggaag gccaggggca tctgggcgac 300
ctcccggtgt tagtcgttaa taatgatggt atcgccagcg aaccgg 346
<210> 15
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> A-5
<400> 15
caggctgctg agtcac 16
<210> 16
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> A-6
<400> 16
ttacttgctg agtcac 16
<210> 17
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> FIP
<400> 17
tgaaaaccat gctcgcccgg gaaaccgaag gcggctta 38
<210> 18
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> BIP
<400> 18
atgccaacgg tagctgccag gggtccagat gaccacca 38
<210> 19
<211> 18
<212> DNA
<213> Salmonella typhimurium
<400> 19
ctatcggcac cgtcgtca 18
<210> 20
<211> 18
<212> DNA
<213> Salmonella typhimurium
<400> 20
ttcatgcttg ccggtatt 18
<210> 21
<211> 511
<212> DNA
<213> Klebsilla Phage P13
<400> 21
aacctgttaa cttgggctgg tgcaagtgac tactccatct ctcgcatggg tactaccatt 60
gtcatatcca gccttagtgg tgcaagcttc acggtggaca cggaggatgg ctctaaaggt 120
aaagaccttg tagccatcca gtacaaggtg acttcaactg acctgcttcc aagcaaggca 180
cctgttggtt acctggtcca ggtatggcct accggaagca agcctgagtc tcgctactgg 240
ctcaaggcgg aggcggcgga tggcaacctt gttacctggc aggaaaccct tggggcagat 300
gaggtgctgg gttttaatgg gacgactatg ccatatatca ttgagcgaac caatattgtt 360
ggtggtatcg ctcagttcac cattaagcag ggctattggg atgaccgggc ggtgggtgat 420
gagctaacca accctatgcc ttcctttatt gaccaaagcc tcagtgatat cttcatggtg 480
caaaacaggt tatgcctggc agctggagaa t 511
<210> 22
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> A-7
<400> 22
ggtttgctga gtcac 15
<210> 23
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> A-8
<400> 23
cttgtgctga gtcac 15
<210> 24
<211> 21
<212> DNA
<213> Klebsilla Phage P13
<400> 24
tgggctggtg caagtgacta c 21
<210> 25
<211> 22
<212> DNA
<213> Klebsilla Phage P13
<400> 25
gcccggtcat cccaatagcc ct 22
<210> 26
<211> 20
<212> DNA
<213> Vibrio cholerae
<400> 26
ctctgagaca ggtgctgcat 20
<210> 27
<211> 18
<212> DNA
<213> Vibrio cholerae
<400> 27
tgcttctttt gcagccca 18
Claims (10)
1. a kind of method for improving extremely low copy number target dna detection sensitivity, it is characterised in that key step is as follows：To treating
External source background genes group DNA is added in detection sample DNA；Randomized bases are produced to glue using can specifically be cut to target dna
The restriction enzyme of property end carries out digestion to mixed sample DNA；According to viscosity end produced after target dna cutting
Design joint complementary therewith in end is simultaneously attached therewith；Performing PCR is entered to connection product using the universal primer contained in joint
Reaction；With PCR primer as template, specific nucleic acid augmentation detection is carried out using the specific primer designed for target dna.
2. method according to claim 1, it is characterised in that concretely comprise the following steps：
A) to the external source background genes group DNA that final concentration of 10-500ng/ μ L are added in detected sample DNA；
B) sample that the restriction enzyme of randomized bases cohesive end can be produced to obtain step a) to target dna cutting is used
Product carry out digestion, and endonuclease reaction parameter is determined by selected restriction endonuclease；
C) to the double-stranded DNA joint added in digestion products for the design of genes of interest endonuclease bamhi, and in the work of DNA ligase
Reaction is attached under, the two ends of endonuclease bamhi is all connected with top connection, universal primer sequence is carried on the joint, connect
Head ultimate density is 50-400nM, and coupled reaction parameter ligase according to selected by determines；
D) with connection product as template, enter performing PCR using universal primer and react, response parameter is true according to final goal fragment length
It is fixed；
E) above One_step PCR product is template, and performing PCR, LAMP or RCA are entered to mesh using the specific primer of target dna
The final detection of gene is marked, reaction condition and the parameter method for detecting specificity according to selected by determine.
3. method according to claim 1 and 2, it is characterised in that step a) the external source background genes group DNA include institute
There is Eukaryotic genomic DNA.
4. method according to claim 1 and 2, it is characterised in that step a) the external source background genes group DNA are salmon
Smart DNA.
5. method according to claim 1 and 2, it is characterised in that restriction enzyme described in step b) is that a class can
To produce the restriction endonuclease of randomized bases cohesive end to target dna cutting, selection recognition site is 4-6 bases, the viscosity for producing
Tip length is 4-9 base restriction endonucleases, and selected restriction endonuclease should be at least in target gene containing two restriction enzyme sites or using
Two kinds respectively contain a restriction endonuclease for restriction enzyme site in target gene.
6. method according to claim 1 and 2, it is characterised in that joint described in step c) is different by two length
Single stranded DNA is constituted, and wherein one section of universal primer sequence is contained at the end of long-chain moiety 5 ', and the end of short chain moieties 5 ' is contained can be with target gene
" linking " sequence of endonuclease bamhi cohesive end complete complementary pairing, the complementary pairing containing >=8 bases between long-chain and short chain
Part.
7. method according to claim 1 and 2, it is characterised in that the restriction enzyme includes Alw26I, BbvI,
FokI, HgaI, TspRI.
8. method according to claim 2, it is characterised in that DNA ligase described in step c) mainly include T3, T4,
T7 DNA ligases and E.coli DNA ligases and Taq DNA ligases, the Connection Time of the DNA ligase is 0.5-
2.5h。
9. method according to claim 1 and 2, it is characterised in that universal primer described in step d) refers to one section of widow
Nucleotide sequence, while being also the long-chain moiety of all double-stranded DNA joints, length is 18-26 base, and fusing point is 60 DEG C ± 5
DEG C, concentration of the universal primer in PCR system is 0.1-1 μM.
10. method according to claim 1 and 2, it is characterised in that specific primer is for target described in step e)
Designed by endonuclease bamhi, amplified production is the section of DNA sequence inside target fragment.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108588197A (en) * | 2018-06-14 | 2018-09-28 | 珠海市人民医院 | A method of detection trace Single-stranded DNA fragments |
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| CN103866025A (en) * | 2014-03-18 | 2014-06-18 | 中国海洋大学 | Pre-amplification method for nucleic acid and application thereof |
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