CN106755443A

CN106755443A - A kind of method and reagent of the super sensitivity detection trace amount DNA from various hybrid dnas

Info

Publication number: CN106755443A
Application number: CN201611260467.5A
Authority: CN
Inventors: 王永利; 宋卓; 袁梦兮
Original assignee: Human And Future Biotechnology (changsha) Co Ltd
Current assignee: Human And Future Biotechnology (changsha) Co Ltd
Priority date: 2016-12-30
Filing date: 2016-12-30
Publication date: 2017-05-31
Anticipated expiration: 2036-12-30
Also published as: CN106755443B

Abstract

The invention discloses a kind of method and reagent of the super sensitivity detection trace amount DNA from various hybrid dnas, methods described includes：A () carries out probe preenrichment using probe to the joint connection product of the target dna containing site to be detected or region to be detected, wherein described probe is double chain oligonucleotide, the joint binding site including universal primer at the two ends of the joint connection product；B () carries out specific enrichment using the upstream specific primer and downstream specific primer for the site to be detected or region to be detected, and the universal primer at two ends to probe preenrichment product, then carry out universal primer amplification to specific enrichment product.Present method solves existing detection technique for the low technical problem of the complex samples detection sensitivity containing the pollution of a large amount of exogenous DNAs.

Description

A kind of method and reagent of the super sensitivity detection trace amount DNA from various hybrid dnas

Technical field

The present invention relates to DNA detection technique fields, more particularly to one kind super sensitivity detection trace from various hybrid dnas The method and reagent of DNA.

Background technology

It is frequently necessary to carry out nondestructive sampling when carrying out genetic test in the fields such as scientific research, clinical or health examination, It is i.e. (de- containing oral cavity by obtaining the hair of people, nail, excrement (containing Colonic exfoliative cells), urine, saliva, swill Fall cell), vomitus (containing alimentary canal cast-off cells), in the complex samples such as the tissue or body fluid of the raw bacterium that goes mouldy to a certain species Detected in the specific region of DNA domain of (typically people)；In fields such as legal medical expert, criminal investigations, the Sample preservation that also happens occasionally is not good at or specific Situation about being sampled under environment.These sample components are often and its complicated.The DNA extracted from these samples is often containing substantial amounts of Non-targeted species DNA.For example, the DNA extracted from fecal sample is the mixture of a variety of DNA.Wherein about 60% DNA From enteric bacteria, remaining most DNA derive from animal or plant swill, can be used for the colonic mucosa for detecting The one thousandth that the DNA of cast-off cells accounts for STb gene is even less.Current laboratory conventional DNA detection schemes such as QPCR, time Flight mass spectrum, generation sequencing, high-flux sequence etc. are all based on PCR (PCR) technology.And in substantial amounts of non-targeted In the presence of species DNA interference, the round pcr specificity based on primer combination template and extension can be very poor.So as to cause Conventional DNA detection schemes can be influenced to different extents above.Especially high throughput sequencing technologies are being used to mixed with a large amount of , it is necessary to put into substantial amounts of DNA sample when the trace target dna of mixing species DNA carries out target area sequencing, to ensure trace mesh Mark DNA has enough copy numbers with to its accurate detection.At this moment useless other species DNA is also more, and interference effect is also got over By force.Therefore how effectively to remove the interference of substantial amounts of non-targeted species DNA and carry out sensitive and accurate specific DNA target areas richness Collection detection turns into the scientific research technical staff technical barrier to be solved.

Current existing technical solution is prokaryotic and the eukaryotic such as directed toward bacteria during DNA is extracted Characteristic difference carry out a certain degree of differentiation.The prokaryotics such as bacterium are not easy cracking compared with eukaryotic, so in extracting DNA When do not carry out heating cracking, and the content of DNA of bacteria in STb gene can be just reduced using comparatively gentle cracking scheme.But it is real Bacterium itself will crack dead and released dna under the natural environment of border, and this technical scheme is directed to other eukaryotic cell dnas Without screening removal effect, so program effect is frequently not fine.

The content of the invention

The present invention provides a kind of method and reagent of the super sensitivity detection trace amount DNA from various hybrid dnas, solves existing Detection technique is for the low technical problem of the complex samples detection sensitivity containing the pollution of a large amount of exogenous DNAs.

According to the first aspect of the invention, the present invention provides a kind of super sensitivity detection trace amount DNA from various hybrid dnas Method, including：

A () is visited using probe to the joint connection product of the target dna containing site to be detected or region to be detected Pin preenrichment, wherein above-mentioned probe is double chain oligonucleotide, the joint at the two ends of above-mentioned joint connection product includes general drawing The binding site of thing；

B () is drawn using the upstream specific primer and downstream specificity for above-mentioned site to be detected or region to be detected Thing, and the universal primer at two ends carries out specific enrichment to probe preenrichment product, then specific enrichment product is led to Expanded with primer.

Further, above-mentioned probe is to expand the one or more pairs of coverings site to be detected or to be detected for obtaining by PCR The double chain oligonucleotide in region.

Further, above-mentioned probe carries affinity labeling；Preferably, above-mentioned affinity labeling is to be located at two chains of above-mentioned probe 5 ' end biotin labelings.

Further, above-mentioned steps (b) are constituted using a plurality of above-mentioned upstream specific primer primer sets and a plurality of above-mentioned The primer sets of downstream specific primer composition.

Further, above-mentioned probe length is 150-200bp.

Further, each above-mentioned upstream specific primer and downstream specific primer apart from above-mentioned site to be detected or The distance in region to be detected is 1-20 base, preferably 1-5 base.

Further, above-mentioned site to be detected or region to be detected are including in point mutation, insertion, missing and Gene Fusion It is at least one.

Further, the above method also includes：

C () is sequenced to the amplified production of above-mentioned steps (b), to detect above-mentioned site to be detected or region to be detected.

According to the second aspect of the invention, the present invention provides a kind of super sensitivity detection trace amount DNA from various hybrid dnas Reagent, including：

A () probe, above-mentioned probe is double chain oligonucleotide, for the target containing site to be detected or region to be detected The joint connection product of DNA carries out probe preenrichment, wherein, the joint at the two ends of above-mentioned joint connection product includes general drawing The binding site of thing；

(b) upstream specific primer and downstream specific primer, and universal primer, for entering to probe preenrichment product Row specific enrichment, and universal primer amplification is carried out to specific enrichment product.

The method of the present invention carries out preenrichment using target area probe to DNA, eliminates most exogenous DNAs and non- Target area domain dna.The method allows to add STb gene as much as possible, and then increases the copy number of target trace amount DNA.The present invention Two chains of target area to be detected are all captured using double-chain probe, increased the utilization rate of trace amount DNA.The present invention is also Probe preenrichment product is carried out specifically using the universal primer of upstream specific primer and downstream specific primer, and two ends Property enrichment, then universal primer amplification is carried out to specific enrichment product, reduce further exogenous DNA and non-target area domain dna Content, improve the content of trace amount DNA, solve existing detection technique for containing a large amount of exogenous DNAs pollution complicated sample The low technical problem of this detection sensitivity.

Brief description of the drawings

Fig. 1 is that the principle schematic of probe manufacturing is carried out using the method for PCR in the embodiment of the present invention；

Fig. 2 be the embodiment of the present invention in using double chain DNA probe carry out hybridize preenrichment principle schematic；

Fig. 3 is the mistake for carrying out specific PCR enrichment and whole library construction in the embodiment of the present invention to probe preenrichment product Journey.

Fig. 4 is the electrophoresis detection result figure in the library of structure in the embodiment of the present invention, and wherein Library represents library electrophoresis As a result, M represents Takara 100bp Marker.

Specific embodiment

The present invention is described in further detail below by specific embodiment combination accompanying drawing.

Commercialization probe manufacturing traditional at present is method for synthesizing gene, and the method is restricted to probe length (general The probe synthesis degree of accuracy of more than 100nt is poor), and it is expensive.And the present invention carries out probe manufacturing using PCR method, Not only making probe cost greatly reduces, and can make longer specificity preferably probe.Carried out using double chain DNA probe Hybridization preenrichment, compared to traditional single-stranded probe for, can with the two of Sync enrichment target area chains, make bioaccumulation efficiency and Sensitivity is higher.

The principle of probe manufacturing is carried out using the method for PCR, as shown in figure 1, probe length is generally 150-200bp.Visit There is biotin labeling at the 5 ' ends of two chains of pin, to be captured using Avidin.Probe is double chain oligonucleotide, is covered to be checked Location point or region to be detected.In a particular application, for site to be detected or the number in region to be detected, it is possible to use a pair Or multipair such Double stranded oligonucleotide acid probe.

Using double chain DNA probe hybridize the principle of preenrichment, as shown in Fig. 2 containing a large amount of external source species in sample DNA and other non-target area domain dnas, and trace target area domain dna (i.e. containing site to be detected or region to be detected DNA).Probe preenrichment is carried out using joint connection product of the probe comprising target area domain dna, you can improve target area domain dna Relative amount in the sample.Because probe may still have non-specific binding, therefore non-specific preenrichment product can be produced.This Be accomplished by next step carries out specific amplification using specific primer.

Specific PCR enrichment and whole library construction are carried out to probe preenrichment product, as shown in figure 3, using for be checked Location point or the upstream specific primer and downstream specific primer in region to be detected, and the universal primer at two ends are pre- to probe Enriched product carries out specific enrichment, then carries out universal primer amplification to specific enrichment product, that is, obtain whole library.To whole text Storehouse is sequenced, you can detect the catastrophe in site to be detected or region to be detected, in the present invention, site to be detected or to be checked Surveying region mutagenesis situation includes at least one in point mutation, insertion, missing and Gene Fusion.The non-specificity of probe preenrichment Product is due to only having universal primer in combination, and it is in combination to have no specific primer, so performing PCR enrichment cannot be entered.

As shown in figure 3, upstream specific primer and downstream specific primer can be respectively the primers of a plurality of primer composition Group, i.e. upstream specific primer group and downstream specific primer group, that is, multiple sites to be detected or region to be detected it is special The mixture of property primer.Every primer in primer sets can respectively for a kind of specific site to be detected or area to be detected Domain.Specific primer component be upstream group and downstream group, respectively two chains to DNA to be detected expand.

In a preferred embodiment of the invention, each upstream specific primer and downstream specific primer are apart from location to be checked The distance in point or region to be detected is generally 1-20 base, preferably 1-5 base.

Corresponding to the method for the embodiment of the present invention, the embodiment of the present invention also provides one kind hypersensitive from various hybrid dnas The reagent of trace amount DNA is detected, including：

Describe technical scheme in detail by the following examples, it will be appreciated that embodiment be only it is exemplary, no It is understood that to be limiting the scope of the invention.The reagent used in embodiment, in case of no particular description, is Commercially available conventional reagent.

Embodiment

The present embodiment carries out library construction to sample and 4 the 7 of gene sites of NRAS, KRAS, PI3KA, EGFR is entered Row detection, it is specific as follows：

1. the joint containing random unimolecule label is designed

IDX1-S：

CAAGCAGAAGACGGCATACGAGATNNNNNNNNggaattaGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(SEQ ID NO：1)；

IDX2-S：

CAAGCAGAAGACGGCATACGAGATNNNNNNNNatccggcGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(SEQ ID NO：2)；

IDX3-S：

CAAGCAGAAGACGGCATACGAGATNNNNNNNNcaggccgGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(SEQ ID NO：3)；

ADT-AS：pGATCGGAAGAGC(SEQ ID NO：4)；The phosphate group modification of the end of ADT-AS sequences 5 '.

IDX1-S, IDX2-S, IDX3-S (being the first chain of joint) respectively with ADT-AS sequence anneals into double-strand, constitute Three joints ADT1, ADT2, ADT3.

2. the present embodiment is detected for common 7 catastrophe points of NRAS, KRAS, PIK3CA, EGFR this 4 genes.

Q61K for NRAS is designed for making the primer of probe：

NRAS-Q61K-F:CCTTACCCTCCACACCCCCA(SEQ ID NO：5)；

NRAS-Q61K-R:GTTAATATCCGCAAAT(SEQ ID NO：6).

G12D for KRAS is designed for making the primer of probe：

KRAS-G12D-F:GTGACATGTTCTAATATAGTC(SEQ ID NO：7)；

KRAS-G12D-R:TAATATGCATATTAAAACAAG(SEQ ID NO：8).

E545K for PIK3CA is designed for making the primer of probe：

PIK3CA-E545K-F:TATTTTACAGAGTAACAGAC(SEQ ID NO：9)；

PIK3CA-E545K-R:AGATCAGCCAAATTCAGTTA(SEQ ID NO：10).

E746-A750 for EGFR is designed for making the primer of probe：

EGFR-E746-A750-F:ACAATTGCCAGTTAACGTCT(SEQ ID NO：11)；

EGFR-E746-A750-R:CAGACATGAGAAAAGGTGGG(SEQ ID NO：12).

V769_D770insASV for EGFR is designed for making the primer of probe：

EGFR-V769_D770insASV-F:AGCTTTTCCTCCATGAGTAC(SEQ ID NO：13)；

EGFR-V769_D770insASV-R:GATGCCCAGCAGGCGGCA(SEQ ID NO：14).

T790M for EGFR is designed for making the primer of probe：

EGFR-T790M-F:TCCACCGTGCAGCTCATC(SEQ ID NO：15)；

EGFR-T790M-R:ATCTCCCCTCCCCGTATCTC(SEQ ID NO：16).

L858R for EGFR is designed for making the primer of probe：

EGFR-L858R-F:ACTTGGAGGACCGTCGCTTG(SEQ ID NO：17)；

EGFR-L858R-R:AGTGGGAAGGCAGCCTGGT(SEQ ID NO：18).

All primers 5 ' for making probe are held and are modified using biotin labeling.

It is the genomic DNA of Healthy People for making the DNA profiling of probe.

Probe manufacturing experimental system (table 1) and PCR programs are as follows：

Table 1

PCR programs are as follows：

A) 98 DEG C 1 minute；

B) 15 cyclic programs are as follows：

98 DEG C 30 seconds

60 DEG C 30 seconds

72 DEG C 30 seconds

C) 72 DEG C 1 minute

D) 4 DEG C of preservations.

The amount for carrying out the material such as all probes after purification using 60 μ l Ampure XP beads mixes.

3. the present embodiment is on common 7 catastrophe points design PCR of this 4 genes of NRAS, KRAS, PIK3CA, EGFR Downstream specific enrichment primer.

Q61K for NRAS designs PCR specific enrichment primers：

Upstream U-NRAS-Q61K-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCTCATGGCACTGTACTC(SEQ ID NO：19).

Downstream D-NRAS-Q61K-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGACATACTGGATACAGCT(SEQ ID NO：20).

G12D for KRAS designs PCR specific enrichment primers：

Upstream U-KRAS-G12D-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTATCGTCAAGGCACTCTTGCC(SEQ ID NO：21).

Downstream D-KRAS-G12D-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGAACTTGTGGTAGTTGGAG(SEQ ID NO：22).

E545K for PIK3CA designs PCR specific enrichment primers,

Upstream U-PIK3CA-E545K-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGTGACTCCATAGAAAATCT(SEQ ID NO：23).

Downstream D-PIK3CA-E545K-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTTCTACACGAGATCCT(SEQ ID NO：24).

E746-A750 for EGFR designs PCR specific enrichment primers,

Upstream U-EGFR-E746-A750-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTCCTTGTTGGCTTTCGGAG(SEQ ID NO：25).

Downstream D-EGFR-E746-A750-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAGTTAAAATTCCCGTCGC(SEQ ID NO：26).

V769_D770insASV for EGFR designs PCR specific enrichment primers：

Upstream U-EGFR-V769_D770insASV-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTCCAGGAAGCCTACGTG(SEQ ID NO：27).

Downstream D-EGFR-V769_D770insASV-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGATGCCCAGCAGGCGGCA(SEQ ID NO：28).

T790M for EGFR designs PCR specific enrichment primers：

Upstream U-EGFR-T790M-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAGGCAGCCGAAGG GCATG(SEQ ID NO：29).

Downstream D-EGFR-T790M-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGGAAGCCTACGTGATGGC(SEQ ID NO：30).

L858R for EGFR designs PCR specific enrichment primers：

Upstream U-EGFR-L858R-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTATTCTTTCTCTTCCGCAC(SEQ ID NO：31).

Downstream D-EGFR-L858R-TN:

CGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTCAAGATCACAGATTTTGG(SEQ ID NO：32).

The amount mixing of the materials such as the PCR upstreams specific enrichment primer in each site to be detected constitutes PCR upstreams specificity Enriching primer group.The amount mixing of the materials such as PCR downstreams specific enrichment primer constitutes PCR downstreams specific enrichment primer sets.

4. final PCR primer sequence is as follows：

UQ-P1:CAAGCAGAAGACGGCATACGA(SEQ ID NO：33)；

UQ-P2:

AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG(SEQ ID NO：34).

5. it is that the hybrid dna extracted in a healthy human faecal mass (is carried in human faecal mass to be used for the background sample of the present embodiment The DNA of taking-up is hybrid dna, wherein about 60% DNA derives from enteric bacteria, remaining most DNA are from dynamic Thing or vegetation foodstuff residue, can be used for the colonic mucosa cast-off cells for detecting DNA account for STb gene one thousandth it is even less. Use STRATEC Molecular brandsPSP Spin Stool DNA Plus Kit(Catalogue#:1038110300) Carry out fecal sample DNA extractings.

For the mutation blending commercialization standard items of the sample from HORIZON DISCOVERY companies of the present embodiment MultiplexI cfDNA ReferenceStandardSet(Catalogue#:HD780), product 5%MultiplexI is taken The DNA sample of (each actual frequency of mutation in site to be checked see the table below 2) is with healthy background dna fragment according to 1：999 are admixed.

Sample after blending is divided into two parts, and portion is used for carrying out experiment of the invention, and another carries out digital droplet PCR Detection (control experiment).

The actual frequency of mutation in each sites of 5%Multiplex I：

Table 2

NRAS-Q61K	6.3%
		KRAS-G12D	6.3%
PIK3CA-E545K	6.3%
		EGFR-E746-A750	5%
EGFR-V769_D770insASV	5%
		EGFR-T790M	5%
EGFR-L858R	5%

6. excrement genomic DNA fragment

Excrement genomic DNA takes 50ug, using Covaris S220, the broken instrument fragmentations of ultrasonic wave DNA to average 250bp Size.

7. fragmentation DNA ends are repaired

Reaction system such as table 3 below：

Table 3

20 DEG C of warm bath 30 minutes on metal bath.

Purified using 120 μ l Ampure XP beads, 100 μ l elution buffers wash-out.

8.DNA admixes

5%MultiplexI DNA and fragmentation DNA is according to 1：999 are admixed.Taking 20ug blendings DNA carries out the present invention Following experiment.Residue blending DNA carries out digital droplet PCR experiment (control experiment).

Add poly- adenine tail in 9.3 ' ends

Reaction system such as table 4 below：

Table 4

The DNA solution that end is repaired	20ug
		Klenow enzyme buffer liquids	5μl
dATP	10μl
		Klenow exo- enzymes	3μl
Cumulative volume	50μl

37 DEG C of warm bath 30 minutes on metal bath.

Purified using 60 μ l Ampure XP beads, 100 μ l elution buffers wash-out.

10. jointing

Reaction system such as table 5 below：

Table 5

The DNA solution of 3 ' plus A	10ug
		T4DNA ligase buffer solutions	25μl
2 μM of DNA joints	1μl
		T4DNA ligases	5μl
Cumulative volume	50μl

DNA joints are ADT1 or ADT2 or ADT3.

20 DEG C of warm bath 15 minutes on metal bath.

Purified using 60 μ l Ampure XP beads, 34.8 μ l deionized waters wash-out.

11. probes hybridize preenrichment

It is formulated as follows the reaction of table 61：

Table 6

It is concentrated by evaporation to 7ul.

95 DEG C 5 minutes in PCR instrument, 66 DEG C 5 minutes.

Reaction system such as table 7 below：

Table 7

66 DEG C hybridize 16 hours in PCR instrument.

Dynabeads is used after rinsing^TMM-270Streptavidin (Catalog nos.65305, invitrogen) magnetic Pearl is screened.98 DEG C of 10 minutes heating wash-outs in PCR instrument.

12.PCR specific enrichments

The product of step 11 is divided equally two parts, is carried out using upstream specific primer group and downstream specific primer group respectively PCR specific enrichments.

Reaction system such as table 8 below：

Table 8

Probe hybridizes preenrichment product	100ng
		Amplitaq Gold360Master Mix	25μl
PCR specific enrichments primer sets (25uM)	1μl
		GC- reinforcing agents	1μl
UQ-P1(25uM)	1μl
		Cumulative volume	50μl

PCR programs are as follows：

E) 95 DEG C 10 minutes；

F) 20 cyclic programs are as follows：

95 DEG C 30 seconds

62 DEG C 30 seconds

72 DEG C 1 minute

G) 72 DEG C 7 minutes

H) 4 DEG C of preservations.

Purified using 60 μ l Ampure XP beads, 20 μ l elution buffers wash-out.

13. whole amplified libraries

Reaction system such as table 9 below：

Table 9

PCR specific enrichment products	20μl
		HIFI Ready Mix(KAPA BIOSYSTEMS)	25μl
UQ-P1+UQ-P2 (each 5uM)	5μl
		Cumulative volume	50μl

PCR programs are as follows：

A) 98 DEG C 45 seconds；

B) 10 cyclic programs are as follows：

98 DEG C 15 seconds

60 DEG C 30 seconds

72 DEG C 30 seconds

C) 72 DEG C 1 minute

D) 4 DEG C of preservations.

Taking wherein 5 μ l products carries out 2% agarose gel electrophoresis detection, as a result as shown in Figure 4.

Purified using 60 μ l Ampure XP beads, 30 μ l elution buffers wash-out.

14. final libraries using Illumina companies NextSeq500 by after quantitative fluorescent PCR Quality Control, carrying out 75bp double End sequencing.

15. by machine data under high-flux sequence by after Quality Control filtering, carrying out BWA comparisons, calculate the mutation of target site Frequency, as a result see the table below 10：

Table 10

The results show, accurately trace can be detected using experimental technique of the invention from Various Complex hybrid dna Amount target dna mutation.

Above content is to combine specific embodiment further description made for the present invention, it is impossible to assert this hair Bright specific implementation is confined to these explanations.For general technical staff of the technical field of the invention, do not taking off On the premise of present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to protection of the invention Scope.

SEQUENCE LISTING

<110>People and future biological science and technology（Changsha）Co., Ltd

<120>A kind of method and reagent of the super sensitivity detection trace amount DNA from various hybrid dnas

<130> 16I23775

<160> 34

<170> PatentIn version 3.3

<210> 1

<211> 73

<212> DNA

<213>Joint sequence

<220>

<221> misc_feature

<222> (25)..(32)

<223> n is a, c, g, or t

<400> 1

caagcagaag acggcatacg agatnnnnnn nnggaattag tgactggagt tcagacgtgt 60

gctcttccga tct 73

<210> 2

<211> 73

<212> DNA

<213>Joint sequence

<220>

<221> misc_feature

<222> (25)..(32)

<223> n is a, c, g, or t

<400> 2

caagcagaag acggcatacg agatnnnnnn nnatccggcg tgactggagt tcagacgtgt 60

gctcttccga tct 73

<210> 3

<211> 73

<212> DNA

<213>Joint sequence

<220>

<221> misc_feature

<222> (25)..(32)

<223> n is a, c, g, or t

<400> 3

caagcagaag acggcatacg agatnnnnnn nncaggccgg tgactggagt tcagacgtgt 60

gctcttccga tct 73

<210> 4

<211> 12

<212> DNA

<213>Joint sequence

<400> 4

gatcggaaga gc 12

<210> 5

<211> 20

<212> DNA

<213>Primer sequence

<400> 5

ccttaccctc cacaccccca 20

<210> 6

<211> 16

<212> DNA

<213>Primer sequence

<400> 6

gttaatatcc gcaaat 16

<210> 7

<211> 21

<212> DNA

<213>Primer sequence

<400> 7

gtgacatgtt ctaatatagt c 21

<210> 8

<211> 21

<212> DNA

<213>Primer sequence

<400> 8

taatatgcat attaaaacaa g 21

<210> 9

<211> 20

<212> DNA

<213>Primer sequence

<400> 9

tattttacag agtaacagac 20

<210> 10

<211> 20

<212> DNA

<213>Primer sequence

<400> 10

agatcagcca aattcagtta 20

<210> 11

<211> 20

<212> DNA

<213>Primer sequence

<400> 11

acaattgcca gttaacgtct 20

<210> 12

<211> 20

<212> DNA

<213>Primer sequence

<400> 12

cagacatgag aaaaggtggg 20

<210> 13

<211> 20

<212> DNA

<213>Primer sequence

<400> 13

agcttttcct ccatgagtac 20

<210> 14

<211> 18

<212> DNA

<213>Primer sequence

<400> 14

gatgcccagc aggcggca 18

<210> 15

<211> 18

<212> DNA

<213>Primer sequence

<400> 15

tccaccgtgc agctcatc 18

<210> 16

<211> 20

<212> DNA

<213>Primer sequence

<400> 16

atctcccctc cccgtatctc 20

<210> 17

<211> 20

<212> DNA

<213>Primer sequence

<400> 17

acttggagga ccgtcgcttg 20

<210> 18

<211> 19

<212> DNA

<213>Primer sequence

<400> 18

agtgggaagg cagcctggt 19

<210> 19

<211> 50

<212> DNA

<213>Primer sequence

<400> 19

cgtcggcagc gtcagatgtg tataagagac agtctcatgg cactgtactc 50

<210> 20

<211> 51

<212> DNA

<213>Primer sequence

<400> 20

cgtcggcagc gtcagatgtg tataagagac agggacatac tggatacagc t 51

<210> 21

<211> 54

<212> DNA

<213>Primer sequence

<400> 21

cgtcggcagc gtcagatgtg tataagagac aggtatcgtc aaggcactct tgcc 54

<210> 22

<211> 50

<212> DNA

<213>Primer sequence

<400> 22

cgtcggcagc gtcagatgtg tataagagac agaacttgtg gtagttggag 50

<210> 23

<211> 52

<212> DNA

<213>Primer sequence

<400> 23

cgtcggcagc gtcagatgtg tataagagac agtgtgactc catagaaaat ct 52

<210> 24

<211> 50

<212> DNA

<213>Primer sequence

<400> 24

cgtcggcagc gtcagatgtg tataagagac agatttctac acgagatcct 50

<210> 25

<211> 52

<212> DNA

<213>Primer sequence

<400> 25

cgtcggcagc gtcagatgtg tataagagac agttccttgt tggctttcgg ag 52

<210> 26

<211> 51

<212> DNA

<213>Primer sequence

<400> 26

cgtcggcagc gtcagatgtg tataagagac agaagttaaa attcccgtcg c 51

<210> 27

<211> 50

<212> DNA

<213>Primer sequence

<400> 27

cgtcggcagc gtcagatgtg tataagagac agctccagga agcctacgtg 50

<210> 28

<211> 51

<212> DNA

<213>Primer sequence

<400> 28

cgtcggcagc gtcagatgtg tataagagac agagatgccc agcaggcggc a 51

<210> 29

<211> 52

<212> DNA

<213>Primer sequence

<400> 29

cgtcggcagc gtcagatgtg tataagagac agggaggcag ccgaagggca tg 52

<210> 30

<211> 51

<212> DNA

<213>Primer sequence

<400> 30

cgtcggcagc gtcagatgtg tataagagac agaggaagcc tacgtgatgg c 51

<210> 31

<211> 52

<212> DNA

<213>Primer sequence

<400> 31

cgtcggcagc gtcagatgtg tataagagac aggtattctt tctcttccgc ac 52

<210> 32

<211> 52

<212> DNA

<213>Primer sequence

<400> 32

cgtcggcagc gtcagatgtg tataagagac aggtcaagat cacagatttt gg 52

<210> 33

<211> 21

<212> DNA

<213>Primer sequence

<400> 33

caagcagaag acggcatacg a 21

<210> 34

<211> 62

<212> DNA

<213>Primer sequence

<400> 34

aatgatacgg cgaccaccga gatctacact cgtcggcagc gtcagatgtg tataagagac 60

ag 62

Claims

1. a kind of method of the super sensitivity detection trace amount DNA from various hybrid dnas, it is characterised in that methods described includes：

A () carries out probe using probe to the joint connection product of the target dna containing site to be detected or region to be detected pre- Enrichment, wherein the probe is double chain oligonucleotide, the joint at the two ends of the joint connection product includes universal primer Binding site；

B () uses the upstream specific primer and downstream specific primer for the site to be detected or region to be detected, with And the universal primer at two ends carries out specific enrichment to probe preenrichment product, then universal primer is carried out to specific enrichment product Amplification.

2. the method for the super sensitivity detection trace amount DNA from various hybrid dnas according to claim 1, it is characterised in that institute It is the double chain oligonucleotide that the one or more pairs of coverings site to be detected or region to be detected for obtaining is expanded by PCR to state probe.

3. the method for the super sensitivity detection trace amount DNA from various hybrid dnas according to claim 1, it is characterised in that institute Probe is stated with affinity labeling；Preferably, the affinity labeling is the biotin labeling at the 5 ' ends for being located at two chains of the probe.

4. the method for the super sensitivity detection trace amount DNA from various hybrid dnas according to claim 1, it is characterised in that institute State what step (b) was constituted using the primer sets and a plurality of downstream specific primer of a plurality of upstream specific primer composition Primer sets.

5. the method for the super sensitivity detection trace amount DNA from various hybrid dnas according to claim 1, it is characterised in that institute Probe length is stated for 150-200bp.

6. the method for the super sensitivity detection trace amount DNA from various hybrid dnas according to claim 1, it is characterised in that every The distance of one upstream specific primer and downstream specific primer apart from the site to be detected or region to be detected is 1-20 base, preferably 1-5 base.

7. the method for the super sensitivity detection trace amount DNA from various hybrid dnas according to claim 1, it is characterised in that institute State at least one that site to be detected or region to be detected are included in point mutation, insertion, missing and Gene Fusion.

8. the method for the super sensitivity detection trace amount DNA from various hybrid dnas according to claim 1, it is characterised in that institute Stating method also includes：

C () is sequenced to the amplified production of the step (b), to detect the site to be detected or region to be detected.

9. a kind of reagent of the super sensitivity detection trace amount DNA from various hybrid dnas, it is characterised in that the reagent includes：

A () probe, the probe is double chain oligonucleotide, for the target dna containing site to be detected or region to be detected Joint connection product carry out probe preenrichment, wherein, the joint at the two ends of the joint connection product includes universal primer Binding site；

(b) upstream specific primer and downstream specific primer, and universal primer, for carrying out spy to probe preenrichment product Specific enrichment, and universal primer amplification is carried out to specific enrichment product.

10. the reagent of the super sensitivity detection trace amount DNA from various hybrid dnas according to claim 9, it is characterised in that The probe is the Double stranded oligonucleotide that the one or more pairs of coverings site to be detected or region to be detected for obtaining is expanded by PCR Acid.