CN106191045A - Index and primer for multiple nucleic acid order-checking - Google Patents
Index and primer for multiple nucleic acid order-checking Download PDFInfo
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Abstract
The present invention relates to technical field of molecular biology, be particularly used for the Index of multiple nucleic acid order-checking and build the PCR primer of 16SrRNA multiple nucleic acid sequencing library.The Index that the present invention provides fully takes into account the sequence difference degree between index tab, base discrimination and the sequencing reaction data output Preference to different Index sequences, after being contained in order-checking or building storehouse primer, the sequencing data balance that mixing sequencing reaction obtains is preferable.The PCR primer that the present invention provides is highly suitable for pedotheque, high specificity, and purpose amplified band is clear, without diffusing phenomenon so that in pedotheque Phylogenetic diversity of bacteria study the most efficient convenient accurately.The Index provided containing the present invention in the PCR primer that the present invention provides, it is thus achieved that amplified production in containing two Index labels, this Index is used for biased sample order-checking, it is possible to realize the differentiation to different sequencing libraries.
Description
Technical field
The present invention relates to technical field of molecular biology, be particularly used for Index and the structure of multiple nucleic acid order-checking
The PCR primer of 16SrRNA multiple nucleic acid sequencing library.
Background technology
The numerous areas such as microbial diversity, especially Phylogenetic diversity of bacteria are micro-and agriculture, medical science, food and commercial production
There are close ties.Along with the development of Protocols in Molecular Biology, microbial diversity research method the most gradually improves and ripe.
During microbial evolution, ribosomal RNA (the especially 16SrRNA or the 18S rRNA of fungus of antibacterial), have certain
Evolutionary conservatism, conserved region sequence is that all same organism are common, exists and cause owing to evolving between conserved sequence
The Variable Area of sequence difference between species, is therefore considered as one of gene being best suitable for system classification.By to these sequences
The mensuration of row Variable Area and comparison, it is possible to probe into and disclose the multiformity of Soil Microorganism species and structure of community.Along with
The development of gene sequencing technology, high throughput sequencing technologies is also introduced in the investigative technique of microbial diversity.
In high throughput sequencing technologies, in order to save order-checking cost or research needs, it will usually difference sample mix is surveyed
Sequence, the most multiple order-checking (Multiplexedsequencing) technology, i.e. when building sequencing library, by PCR primer, use
After different index tab (Index) labelling difference samples, the upper machine that difference sample mix got up checks order, and order-checking is passed through after terminating
Identify that Index distinguishes the different sample datas in same swimming lane (Lane).Reducing of multiple sequencing technologies high degree
Order-checking cost, saves order-checking resource, and the convenient means provided to biological study.Such as, the microorganisms such as antibacterial are being carried out
During Study on Diversity, it usually needs the multiformity of hundreds of samples is analyzed, if there is no multiple sequencing technologies, scientific research cost
To be greatly increased with time cost.
On the one hand, actual sequencing reaction has certain data output Preference to different Index sequences, causes detection
The fluctuation of light intensity parameter during Insert Fragment, the quality of data of impact output and balance, i.e. Index sequence to a certain degree affects mixed
Closing the accuracy of the sequencing data of sample, the most any nucleotide sequence is suitable for as Index.Therefore it provides more may be used
Extremely important with Index sequence pair multiple sequencing technologies field.
On the other hand, inventor finds when carrying out Microbial diversity Journal of Sex Research: the most conventional is many for bacteria samples
The 16S rRNA amplimer of sample Journal of Sex Research (the 16S rRNA amplimer provided such as Illunima company) although can be to intestinal
Road sample obtains relatively good expanding effect；But when for complexity compared with strong, impurity is more pedotheque time, amplified production
Specificity is the highest, and electrophoresis showed amplified band disperse is serious, and this will have a strong impact on follow-up order-checking and precision of analysis.
In sum, it is provided that one group of sequencing data balance relatively preferably Index sequence and one group are applicable to build
The PCR primer of the multiple sequencing library of bacterial 16 S rRNA in pedotheque, it is possible to soil bacteria Study on Diversity is worked more
The most convenient.
Summary of the invention
Unless otherwise defined, all technology used herein and scientific terminology have and the technical field of the invention
The same meaning that those of ordinary skill is generally understood that.
Term " Index " in the present invention refers to index tab, refers specifically to one section for the nucleoside distinguishing different order-checking sample
Acid sequence；Term " PCR " refers to polymerase chain reaction；Term " connexon " refer to any one section and nucleotide sequence to be checked order it
Between there is not the nucleotide sequence of the specific pairs exceeding more than continuous 5 bases.
The present invention considers that index tab, on the amplification efficiency of PCR primer and the impact of the Preference of data output, carries simultaneously
One group of index tab for labelling nucleic acid samples to be checked order, a described group index label has been supplied to comprise following 34 Index
In 2 or multiple；The nucleotide sequence of 34 described Index is as shown in the table:
Present invention also offers above-mentioned Index for the structure of sequencing library and/or for the purposes checked order: by described
Index is included in and treats in the PCR primer of sequencing sequence for amplification, thus constitutes corresponding Index-PCR primer, passes through
Above-mentioned Index is introduced and treats in sequencing sequence by PCR method；Described Index-PCR primer 3 ' the end primers that can serve as PCR
Or 5 ' end primer, or be used simultaneously as 3 ' end primers and 5 ' end primers.
Wherein, in described Index-PCR primer, described Index passes through or does not pass through connexon and be used for expanding
5 ' the ends or the 3 ' ends that increase the PCR primer treating sequencing sequence are connected.
Wherein, described treat that sequencing sequence is preferably bacterial 16 S rRNA sequence.
Due in soil in addition to bacteria, possibly together with the hereditary material of a large amount of funguses, animal, plant etc., utilizing
When 16SrRNA order-checking carries out Phylogenetic diversity of bacteria research, pedotheque often has more compared to other kinds of samples such as intestinals
High complexity.The 16SrRNA amplimer of existing structure sequencing library is when intestinal samples, it is possible to ensure certain spy
The opposite sex；But for pedotheque time, due to its complexity, often cannot obtain preferable amplified production, it is thus achieved that amplification produce
Thing poor specificity, electrophoretic band disperse is serious.
Another object of the present invention is to provide one group for the PCR primer building the multiple sequencing library of 16SrRNA.Institute
One group stated includes forward primer group and reverse primer group for the PCR primer building the multiple sequencing library of 16SrRNA；Described
Any one primer in forward primer group and any one primer in reverse primer group are composed of primer pair two-by-two, pass through
PCR reaction is for expanding the 16S rRNA in sample；The upstream and downstream of the amplified production obtained respectively contains an Index, this
Two Index, as the specific marker of this sample, are used for and other samples to be checked order make a distinction.The amplified production obtained is even
After connecing sequence measuring joints, machine order-checking can be mixed；Can not be identical containing comprising in described forward primer group and reverse primer group
The primer of Index.
Described forward primer group is made up of one or more in following primer:
Primers F 1: comprise Index1 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:1；
Primers F 2: comprise Index2 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:2；
Primers F 3: comprise Index3 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:3；
Primers F 4: comprise Index4 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:4；
Primers F 5: comprise Index5 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:5；
Primers F 6: comprise Index6 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:6；
Primers F 7: comprise Index7 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:7；
Primers F 8: comprise Index8 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:8；
Primers F 9: comprise Index9 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:9；
Primers F 10: comprise Index10 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:10；
Primers F 11: comprise Index11 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:11；
Primers F 12: comprise Index12 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:12；
Primers F 13: comprise Index13 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:13；
Primers F 14: comprise Index14 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:14；
Primers F 15: comprise Index15 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:15；
Primers F 16: comprise Index16 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:16；
Primers F 17: comprise Index17 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:17；
Primers F 18: comprise Index18 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:18；
Primers F 19: comprise Index19 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:19；
Primers F 20: comprise Index20 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:20；
Primers F 21: comprise Index21 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:21；
Primers F 22: comprise Index22 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:22；
Primers F 23: comprise Index23 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:23；
Primers F 24: comprise Index24 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:24；
Primers F 25: comprise Index25 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:25；
Primers F 26: comprise Index26 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:26；
Primers F 27: comprise Index27 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:27；
Primers F 28: comprise Index28 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:28；
Primers F 29: comprise Index29 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:29；
Primers F 30: comprise Index30 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:30；
Primers F 31: comprise Index31 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:31；
Primers F 32: comprise Index32 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:32；
Primers F 33: comprise Index33 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:33；
Primers F 34: comprise Index34 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:34.
Described reverse primer group is made up of one or more in following primer:
Primer R1: comprise Index1 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:35；
Primer R2: comprise Index2 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:36；
Primer R3: comprise Index3 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:37；
Primer R4: comprise Index4 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:38；
Primer R5: comprise Index5 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:39；
Primer R6: comprise Index6 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:40；
Primer R7: comprise Index7 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:41；
Primer R8: comprise Index8 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:42；
Primer R9: comprise Index9 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:43；
Primer R10: comprise Index10 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:44；
Primer R11: comprise Index11 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:45；
Primer R12: comprise Index12 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:46；
Primer R13: comprise Index13 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:47；
Primer R14: comprise Index14 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:48；
Primer R15: comprise Index15 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:49；
Primer R16: comprise Index16 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:50；
Primer R17: comprise Index17 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:51；
Primer R18: comprise Index18 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:52；
Primer R19: comprise Index19 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:53；
Primer R20: comprise Index20 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:54；
Primer R21: comprise Index21 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:55；
Primer R22: comprise Index22 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:56；
Primer R23: comprise Index23 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:57；
Primer R24: comprise Index24 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:58；
Primer R25: comprise Index25 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:59；
Primer R26: comprise Index26 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:60；
Primer R27: comprise Index27 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:61；
Primer R28: comprise Index28 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:62；
Primer R29: comprise Index29 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:63；
Primer R30: comprise Index30 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:64；
Primer R31: comprise Index31 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:65；
Primer R32: comprise Index32 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:66；
Primer R33: comprise Index33 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:67；
Primer R34: comprise Index34 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:68.
A kind of multiple sequencing library of bacterial 16 S rRNA of what the present invention also provided for builds test kit, it is characterised in that: described
Test kit comprise of the present invention one group for building the PCR primer of the multiple sequencing library of 16SrRNA.
Above-mentioned a kind of multiple sequencing library of bacterial 16 S rRNA build test kit can also comprise extracting genome DNA reagent,
Archaeal dna polymerase, 16SrRNA sequence measuring joints add reagent.
Present invention also offers above-mentioned a kind of multiple sequencing library of bacterial 16 S rRNA and build test kit in Phylogenetic diversity of bacteria inspection
Application in survey.
Compared with prior art, the invention have the benefit that
(1), the Index that the present invention provides fully takes into account the sequence difference degree between index tab, base discrimination
And the data output Preference that sequencing reaction is to different Index sequences.The sequence of the Index that the present invention provides avoids sequence
Between occur more than 3 or 3 the identical bases of continuous print appearance, reducing in its sequent synthesis or sequencing procedure of high degree
The error rate being likely to occur；After label comprises and builds in storehouse or sequencing primer, be avoided as much as occurring hairpin structure or with
Sequencing primer and the identical phenomenon of reverse complementary sequence thereof；After being contained in order-checking or building storehouse primer, mixing sequencing reaction obtains
The sequencing data balance arrived is preferable.
(2), the PCR primer for building the multiple sequencing library of 16SrRNA that the present invention provides is in conventional 16SrRNA amplification
The Index that the present invention provides, and the joint sequence of a length of 28-36 base, and unexpected is with the addition of on the basis of primer
The PCR primer the most thus constituted is in addition to can obtaining, to samples such as intestinals, the 16SrRNA amplified production that specificity is higher, also
Being highly suitable for pedotheque, when expanding the 16SrRNA in pedotheque, high specificity, purpose amplified band is clear, without more
Dissipate phenomenon, ensure that the accuracy of follow-up sequencing reaction and result of study dramatically so that in pedotheque, antibacterial is various
Journal of Sex Research the most efficient convenient accurately.
(3), the present invention provide for building the PCR primer of 16S rRNA sequencing library, anti-by forward primer combination
Primer in primer sets introduces the Index that the present invention provides, it is thus achieved that amplified production in containing two Index labels, should
Index checks order for biased sample, it is achieved the differentiation to different sequencing libraries.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of the amplified production in experimental example 1 step (2).
Fig. 2 is the agarose gel electrophoresis figure of the amplified production in comparative example 1.
Detailed description of the invention
The explanation of following example is only intended to help to understand method and the core concept thereof of the present invention.It is right to it should be pointed out that,
For those skilled in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out
Some improvement and modification, these improve and modify in the protection domain also falling into the claims in the present invention.To disclosed enforcement
The description below of example, makes professional and technical personnel in the field be capable of or uses the present invention.Multiple amendment to these embodiments
Will be apparent from for those skilled in the art, generic principles defined herein can be without departing from this
In the case of the spirit or scope of invention, realize in other embodiments.Therefore, the present invention is not intended to be limited to illustrated herein
These embodiments in, but can apply to meet the broader model consistent with principles disclosed herein and features of novelty
Enclose.Although implementing or can use and heretofore described any method similar or of equal value and material in test in the present invention
Material, place enumerates preferred method and material herein.
Unless otherwise defined, all technology used herein and scientific terminology have and the technical field of the invention
The same meaning that those of ordinary skill is generally understood that.
Term " ddH in the present invention2O " refer to distilled water.
Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition
(such as with reference to works such as J. Pehanorm Brookers, " the Molecular Cloning: A Laboratory guide " that yellow training hall etc. is translated, the third edition, Science Press) or
Person is carried out according to product description.
Soil DNA Kit is purchased from Omega company；KOD-Plus-Neo is purchased from TOYOBO company；Primer closes
Student on commission work biological engineering limited company is become to carry out；The pedotheque of 100 separate sources is by Chinese Academy of Sciences's Beijing genome
Institute provides.
Embodiment 1 one groups is for building the PCR primer of the multiple sequencing library of 16SrRNA
It is made up of 20 primers,
Wherein forward primer group is made up of following 10 primers:
Primers F 1 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:1；
Primers F 2 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:2；
Primers F 4 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:4；
Primers F 5 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:5；
Primers F 7 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:7；
Primers F 8 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:8；
Primers F 9 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:9；
Primers F 10 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:10；
Primers F 12 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:12；
Primers F 13 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:13；
Wherein reverse primer group is made up of following 10 primers:
Primer R14 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:14；
Primer R15 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:15；
Primer R16 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:16；
Primer R18 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:18；
Primer R19 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:19；
Primer R20 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:20；
Primer R22 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:22；
Primer R23 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:23；
Primer R25 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:25；
Primer R26 of the present invention, its nucleotide sequence is as shown in SEQ ID NO:26.
The detection application of experimental example 1 Phylogenetic diversity of bacteria
Build the multiple sequencing library of bacterial 16 S rRNA of the pedotheque of 100 separate sources
(1), the extraction of sample gene group DNA:
WithSoil DNA Kit extracts the genomic DNA in pedotheque, and extraction step sees product and says
Bright book.
(2), PCR amplification 16S rRNA:
The genomic DNA extracted with step (1), as template, utilizes the PCR primer amplification sample provided in the embodiment of the present invention 1
16S rRNA in product, wherein the primer in forward primer group and reverse primer group matches two-by-two, it is thus achieved that 100 different primers
Right, expand for the PCR of 100 different samples.
PCR reaction system is as shown in the table:
| Composition | Content |
| Genomic DNA (10ng/ μ L) | 2μL |
| Primer (10 μMs) | Each 0.75 μ L |
| MgSO4(25mM) | 3μL |
| dNTPs(2mM) | 5μL |
| 10×KOD-Plus-Neo Buffer | 5μL |
| KOD-Plus-Neo | 1μL |
| ddH2O | 32.5μL |
| Amount to | 50μL |
PCR program is as shown in the table:
100 amplified productions obtained carry out agarose gel electrophoresis, gum concentration 0.8%, and electrophoresis result display uses
The 16SrRNA amplified production band that the primer that the present invention provides obtains is clear, without diffusing phenomenon.Owing to sample size is more,
This shows the electrophoresis result of 1-24 sample, as it is shown in figure 1, purpose band is clear, without diffusing phenomenon；25-100 sample
Electrophoresis result and No. 1-24 same or similar.
(3), sequence measuring joints is added:
100 amplified productions obtained using step (2) respectively as template, utilize following primer to carrying out PCR amplification, with
Lower primer contains the sequence measuring joints of Illumina MiSeq order-checking platform.
5’-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTG-3’；
5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3’。
PCR reaction system is as shown in the table:
PCR program is as shown in the table:
Use 100 amplified productions that Agencourt AMPure XP magnetic beads for purifying step (3) obtains, 100 after purification
The multiple sequencing library of bacterial 16 S rRNA of the pedotheque of 100 separate sources it is after the mixing of individual amplified production.
Comparative example 1
2 the most conventional 16SrRNA amplimers and Illunima company are provided by following primer by 1 and primer
16SrRNA sequencing library builds primer.
Primer is to 1:
5’-CCTACGGGNGGCWGCAG-3’
5’-GGACTACHVGGGTWTCTAAT-3’
Primer is to 2:
5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’；
5’-GTCTCGTGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’。
Utilize above-mentioned two primer in experimental example 1 of the present invention use 100 separate sources pedotheque in
16SrRNA in 50 pedotheques of machine expands.
The extracting method of sample gene group DNA is with experimental example 1 step (1) of the present invention, and PCR system and PCR program are with this
Bright experimental example 1 step (2).
2 parts of 50 the different amplified productions obtained are carried out agarose gel electrophoresis, gum concentration and deposition condition with experiment
Example 1 step (2).Electrophoresis result shows: use primer unclear to the 2 16SrRNA amplified production purpose bands obtained to 1 and primer
Clear, disperse is serious.Owing to sample size is more, show the primer amplified production electricity to 1 pair of 1-24 sample in comparative example 1 at this
Swimming result, as in figure 2 it is shown, amplified production purpose band is unintelligible, disperse is serious；The electrophoresis result of remaining sample in this comparative example
Same or similar with Fig. 2.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Within god and principle, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (9)
1. one group of index tab for labelling nucleic acid samples to be checked order, it is characterised in that: a described group index label comprises
In 34 Index 2 or multiple below, the nucleotides sequence of 34 described Index is classified as:
2. a group described in claim 1 is used for the index tab structure for sequencing library of labelling nucleic acid samples to be checked order
And/or the purposes for order-checking, it is characterised in that: described Index is included in the PCR primer treating sequencing sequence for amplification
In, thus constitute corresponding Index-PCR primer, by PCR method, above-mentioned Index introducing is treated in sequencing sequence；Described
Index-PCR primer with can serve as PCR 3 ' end primers or 5 ' end primers, or be used simultaneously as 3 ' end primers and 5 ' end draw
Thing.
3. purposes as claimed in claim 2, it is characterised in that: in described Index-PCR primer, described Index leads to
Cross or be not connected with 5 ' ends or the 3 ' ends of the PCR primer treating sequencing sequence for amplification by connexon.
4. one group of PCR primer being used for building the multiple sequencing library of 16SrRNA, it is characterised in that: one group of described PCR primer bag
Include forward primer group and reverse primer group；
Described forward primer group is made up of one or more in following primer:
Primers F 1: containing the Index1 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:1
Sequence；
Primers F 2: containing the Index2 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:2
Sequence；
Primers F 3: containing the Index3 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:3
Sequence；
Primers F 4: containing the Index4 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:4
Sequence；
Primers F 5: containing the Index5 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:5
Sequence；
Primers F 6: containing the Index6 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:6
Sequence；
Primers F 7: containing the Index7 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:7
Sequence；
Primers F 8: containing the Index8 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:8
Sequence；
Primers F 9: containing the Index9 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:9
Sequence；
Primers F 10: containing the Index10 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:10
Sequence；
Primers F 11: containing the Index11 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:11
Sequence；
Primers F 12: containing the Index12 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:12
Sequence；
Primers F 13: containing the Index13 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:13
Sequence；
Primers F 14: containing the Index14 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:14
Sequence；
Primers F 15: containing the Index15 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:15
Sequence；
Primers F 16: containing the Index16 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:16
Sequence；
Primers F 17: containing the Index17 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:17
Sequence；
Primers F 18: containing the Index18 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:18
Sequence；
Primers F 19: containing the Index19 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:19
Sequence；
Primers F 20: containing the Index20 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:20
Sequence；
Primers F 21: containing the Index21 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:21
Sequence；
Primers F 22: containing the Index22 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:22
Sequence；
Primers F 23: containing the Index23 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:23
Sequence；
Primers F 24: containing the Index24 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:24
Sequence；
Primers F 25: containing the Index25 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:25
Sequence；
Primers F 26: containing the Index26 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:26
Sequence；
Primers F 27: containing the Index27 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:27
Sequence；
Primers F 28: containing the Index28 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:28
Sequence；
Primers F 29: containing the Index29 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:29
Sequence；
Primers F 30: containing the Index30 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:30
Sequence；
Primers F 31: containing the Index31 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:31
Sequence；
Primers F 32: containing the Index32 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:32
Sequence；
Primers F 33: containing the Index33 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:33
Sequence；
Primers F 34: containing the Index34 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:34
Sequence；
Described reverse primer group is made up of one or more in following primer:
Primer R1: containing the Index1 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:35
Sequence；
Primer R2: containing the Index2 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:36
Sequence；
Primer R3: containing the Index3 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:37
Sequence；
Primer R4: containing the Index4 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:38
Sequence；
Primer R5: containing the Index5 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:39
Sequence；
Primer R6: containing the Index6 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:40
Sequence；
Primer R7: containing the Index7 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:41
Sequence；
Primer R8: containing the Index8 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:42
Sequence；
Primer R9: containing the Index9 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:43
Sequence；
Primer R10: containing the Index10 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:44
Sequence；
Primer R11: containing the Index11 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:45
Sequence；
Primer R12: containing the Index12 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:46
Sequence；
Primer R13: containing the Index13 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:47
Sequence；
Primer R14: containing the Index14 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:48
Sequence；
Primer R15: containing the Index15 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:49
Sequence；
Primer R16: containing the Index16 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:50
Sequence；
Primer R17: containing the Index17 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:51
Sequence；
Primer R18: containing the Index18 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:52
Sequence；
Primer R19: containing the Index19 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:53
Sequence；
Primer R20: containing the Index20 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:54
Sequence；
Primer R21: containing the Index21 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:55
Sequence；
Primer R22: containing the Index22 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:56
Sequence；
Primer R23: containing the Index23 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:57
Sequence；
Primer R24: containing the Index24 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:58
Sequence；
Primer R25: containing the Index25 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:59
Sequence；
Primer R26: containing the Index26 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:60
Sequence；
Primer R27: containing the Index27 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:61
Sequence；
Primer R28: containing the Index28 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:62
Sequence；
Primer R29: containing the Index29 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:63
Sequence；
Primer R30: containing the Index30 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:64
Sequence；
Primer R31: containing the Index31 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:65
Sequence；
Primer R32: containing the Index32 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:66
Sequence；
Primer R33: containing the Index33 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:67
Sequence；
Primer R34: containing the Index34 described in claim 1 in its sequence, its nucleotides sequence is classified as shown in SEQ ID NO:68
Sequence；
Described forward primer group can not have the primer containing identical Index with in reverse primer group.
One group of PCR primer the most as claimed in claim 4, it is characterised in that: described PCR primer in use: described just
Any one primer in primer sets and any one primer in reverse primer group are composed of primer pair two-by-two, pass through
PCR reaction is for expanding the 16S rRNA in sample；The upstream and downstream of the amplified production obtained respectively contains an Index, this
Two Index, as the specific marker of this sample, are used for and other samples to be checked order make a distinction.
6. a group described in claim 4 and 5 any one is for building the PCR primer of the multiple sequencing library of 16SrRNA carefully
Application in the detection of bacterium multiformity.
7. a group described in claim 4 and 5 any one is for building the PCR primer of the multiple sequencing library of 16SrRNA in system
Application in standby Phylogenetic diversity of bacteria detection kit.
8. the multiple sequencing library of bacterial 16 S rRNA builds test kit, it is characterised in that: described test kit comprises claim
Described in 4 and 5 any one one group is for building the PCR primer of the multiple sequencing library of 16SrRNA.
9. a kind of multiple sequencing library of bacterial 16 S rRNA described in claim 8 builds test kit in Phylogenetic diversity of bacteria detects
Application.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110869519A (en) * | 2017-05-26 | 2020-03-06 | Md保健株式会社 | Method for diagnosing autism by analyzing bacterial metagenome |
| CN112735530A (en) * | 2021-01-22 | 2021-04-30 | 中国科学院北京基因组研究所(国家生物信息中心) | Method for tracing sample based on flora structure |
| CN113293238A (en) * | 2021-07-16 | 2021-08-24 | 烟台海关技术中心 | Soil virus detection technology based on macroviromics |
| CN117004700A (en) * | 2023-10-07 | 2023-11-07 | 北京爱博生生物技术有限公司 | Method for high-throughput sequencing of monoclonal antibody variable region genes, composition and kit used by method |
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| US20150337364A1 (en) * | 2014-01-27 | 2015-11-26 | ArcherDX, Inc. | Isothermal Methods and Related Compositions for Preparing Nucleic Acids |
| CN105316320A (en) * | 2014-07-30 | 2016-02-10 | 天津华大基因科技有限公司 | DNA tags, PCR primer and application thereof |
| CN105671644A (en) * | 2016-02-26 | 2016-06-15 | 武汉冰港生物科技有限公司 | Preparation method of genome mixing sequencing library |
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| US20150337364A1 (en) * | 2014-01-27 | 2015-11-26 | ArcherDX, Inc. | Isothermal Methods and Related Compositions for Preparing Nucleic Acids |
| CN105316320A (en) * | 2014-07-30 | 2016-02-10 | 天津华大基因科技有限公司 | DNA tags, PCR primer and application thereof |
| CN105671644A (en) * | 2016-02-26 | 2016-06-15 | 武汉冰港生物科技有限公司 | Preparation method of genome mixing sequencing library |
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| HAMADY M等: "Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex", 《NATURE METHODS》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110869519A (en) * | 2017-05-26 | 2020-03-06 | Md保健株式会社 | Method for diagnosing autism by analyzing bacterial metagenome |
| CN112735530A (en) * | 2021-01-22 | 2021-04-30 | 中国科学院北京基因组研究所(国家生物信息中心) | Method for tracing sample based on flora structure |
| CN113293238A (en) * | 2021-07-16 | 2021-08-24 | 烟台海关技术中心 | Soil virus detection technology based on macroviromics |
| CN117004700A (en) * | 2023-10-07 | 2023-11-07 | 北京爱博生生物技术有限公司 | Method for high-throughput sequencing of monoclonal antibody variable region genes, composition and kit used by method |
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| CN106191045B (en) | 2019-10-11 |
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