CN108866220B - Method and detection kit for detecting nasal flora - Google Patents
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Abstract
The invention discloses a method and a detection kit for detecting nasal flora. The invention provides a set of primers for detecting sample flora, which comprises a primer for amplifying a V4-V5 region of bacteria 16S rDNA and a primer for amplifying a V1-V9 region of the bacteria 16S rDNA; the invention firstly adopts the V1-V9 primer with high amplification efficiency, strong specificity and low human homology to enrich the 16S rDNA segment, and then utilizes nested PCR to amplify the target segment V4V5 double segment, thus overcoming the problems that the sequence homology of the V4V5 segment amplification primer and the human genome is higher, and serious non-specific amplification phenomenon can occur when the nasal swab sample with higher human gDNA content is amplified.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method and a detection kit for detecting nasal flora.
Background
Hundreds of millions of microorganisms in the nasal cavity play an important role in human health and diseases, and researches show that diseases such as nasosinusitis, allergic rhinitis, hyposmia, even Alzheimer's disease and the like are related to nasal cavity flora imbalance. The nasal cavity flora mainly influences human health from two aspects, on one hand, a few pathogenic bacteria can directly cause acute inflammation to harm human health, and on the other hand, the whole nasal cavity symbiotic bacteria form a relatively stable ecological niche, so that the human body is helped to resist the invasion of pathogens, and meanwhile, the immune tolerance is enhanced by regulating mucosal immunity, and the occurrence of allergic diseases is avoided. The 16S rDNA is the most commonly used "molecular clock" in bacterial taxonomic studies, and its sequence contains 9 variable regions and 10 conserved regions. 16S rDNA sequencing typically involves amplification of one or more variable regions followed by genus classification of the flora using high throughput sequencing and sequence analysis. The method can break through the defect that the traditional microorganism can not be cultured, and can quickly obtain a large amount of flora information.
The 16S rDNA amplicon sequencing does not analyze the whole genome sequence of bacteria, only the technical characteristics of conservative region sequence analysis endows the advantages of economic cost, convenient analysis and rapid horizontal classification, and is widely applied to the research fields of environmental microorganisms, human microorganisms and the like in recent years.
Due to the limitation of the current high-throughput sequencing read length, the strategy of completely sequencing 9 variable regions of 16S rDNA is rarely adopted, and 1-3 variable regions are generally selected as amplification fragments. Due to the complex structure of the flora, the areas of different variable regions have different resolving power for the flora, and when the different variable regions are used for analysis, the coverage of the flora and the accuracy and stability of structural analysis can be different. For example, when the coverage of flora by different V regions of 16S rDNA is studied, the V4 region has the best coverage at five levels in the Meneodaceae, and the adjacent double-fragment region with the highest coverage is the V4-V5 segment. When the difference of the 16S rDNA gene is studied to overestimate the diversity of prokaryotes, the V4-V5 region is found to show the lowest overestimation degree (about 3.0%), and the V6 region is found to have the highest overestimation degree (about 13%). Therefore, in the study of the diversity of flora, the V4-V5 region of the 16S rDNA gene is more suitable for the target fragment of sequencing. However, the current scheme for sequencing nasal flora 16S rDNA generally selects a single V region which is easier to amplify for detection, but not a V4V5 region with the best effect, mainly because the common V4V5 region amplification primer has higher sequence homology with human 18S rDNA, and when a nasal swab sample with low flora content and relatively high human gDNA content is amplified, a serious non-specific amplification phenomenon occurs. Technical optimization is required in order to improve the amplification specificity and enrichment efficiency of nasal microorganisms.
In addition, the current 16S rDNA sequencing technology usually adopts an amplification primer with index to carry out 16S rDNA amplification. Although the method is simple, the length of the primer is generally over 60nt, and the synthesis cost is high. Meanwhile, the primer length is longer, and the amplification efficiency is low. In addition, the amplification primer has a single sequence, and the sequencing quality is poor due to the unbalance of the initial base easily during high-throughput parallel sequencing. First, 16S rDNA amplification was also used. Sequencing adapters are then added by ligation. The construction steps of the sequencing library are complicated, and the large-scale automatic sample library construction is not facilitated.
Disclosure of Invention
The invention aims to provide a set of primers for detecting the flora of a sample.
The set of primers provided by the invention comprises a primer for amplifying the V4-V5 region of the 16S rDNA of the bacterium and a primer for amplifying the V1-V9 region of the 16S rDNA of the bacterium; the primers for amplifying the V4-V5 region of the 16S rDNA of the bacteria consist of a primer shown in a sequence 4, a primer shown in a sequence 5, a primer shown in a sequence 6, a primer shown in a sequence 7, a primer shown in a sequence 8, a primer shown in a sequence 9, a primer shown in a sequence 10 and a primer shown in a sequence 11.
In the above set of primers, the primers for amplifying the V1-V9 region of the 16S rDNA of the bacterium consist of a primer (FP1) shown in sequence 1, a primer (RP1-1) shown in sequence 2 and a primer (RP1-2) shown in sequence 3.
In the above set of primers, the kit further comprises an upstream primer for sample discrimination and a downstream primer for sample discrimination,
the upstream primer for sample discrimination consists of a fragment shown as a sequence 12 (another part of one sequencing adaptor of the illumina, which forms one sequencing adaptor with a sequencing adaptor region in the upstream primer), a tag sequence (index) for sample discrimination and a fragment shown as a sequence 13 (a region complementary to the sequencing adaptor segment); embodied in the embodiment as FP3*；
The upstream primer for sample discrimination consists of a fragment shown as sequence 14 (another part of the sequencing linker region in the downstream primer forms the sequencing linker region in the sequence of illumina), another tag sequence for sample discrimination and a fragment shown as sequence 15 (a region complementary to another sequencing linker segment); embodied in the embodiment as RP3*；
Each sample corresponds to a combination of one tag sequence for sample discrimination and another tag sequence for sample discrimination. In the examples the different tag sequences consist of 8 different bases.
Another object of the present invention is to provide a PCR reagent or a kit comprising the primer set as described above.
The PCR reagent set containing the primers comprises a PCR reagent containing the primers for amplifying the V1-V9 region of the 16S rDNA of the bacteria, a PCR reagent containing the primers for amplifying the V4-V5 region of the 16S rDNA of the bacteria and a PCR reagent containing the primers for distinguishing samples;
in the PCR reagent containing the primers for amplifying the V1-V9 region of the 16S rDNA of the bacteria, the molar ratio of the primers shown in the sequence 1, the primers shown in the sequence 2 and the primers shown in the sequence 3 is 2:1: 1;
in the PCR reagent containing the primers for amplifying the V4-V5 region of the 16S rDNA of the bacterium, the molar ratio of the primer shown in the sequence 4, the primer shown in the sequence 5, the primer shown in the sequence 6, the primer shown in the sequence 7, the primer shown in the sequence 8, the primer shown in the sequence 9, the primer shown in the sequence 10 and the primer shown in the sequence 11 is 5:5:3: 1:1: 1;
in the PCR reagent containing the primers for sample discrimination, the molar ratio of the upstream primer for sample discrimination to the downstream primer for sample discrimination is 1: 1.
The application of the primer set or the PCR reagent or the kit in the preparation of the product for detecting the flora in the sample is also within the protection scope of the invention;
or, the application of the primer set or the PCR reagent or the kit in constructing a sequencing library of sample flora is also within the protection scope of the invention;
or the application of the primer set or the PCR reagent or the kit in the preparation of a product for constructing a sample flora sequencing library is also within the protection scope of the invention;
or the application of the primer for amplifying the V4-V5 region of the bacteria 16S rDNA and the primer for amplifying the V1-V9 region of the bacteria 16S rDNA in preparing a product for detecting the flora of the sample is also within the protection range of the invention;
or the application of the primer for amplifying the V4-V5 region of the bacteria 16S rDNA and the primer for amplifying the V1-V9 region of the bacteria 16S rDNA in constructing a sequencing library of sample flora is also within the protection scope of the invention;
or the application of the primer for amplifying the V4-V5 region of the bacteria 16S rDNA and the primer for amplifying the V1-V9 region of the bacteria 16S rDNA in preparing a product for constructing a sample flora sequencing library is also within the protection scope of the invention.
The invention also aims to provide a method for constructing the sequencing library of the sample flora.
The method provided by the invention comprises the following steps:
1) extracting DNA of an in vitro sample,
2) carrying out PCR amplification by using the DNA as a template and a primer for amplifying the V1-V9 region of the bacteria 16S rDNA in the kit to obtain a V1-V9 fragment of the 16S rDNA;
3) carrying out PCR amplification by using the primers for amplifying the V4-V5 region of the bacteria 16S rDNA in the kit by using the V1-V9 fragment of the 16S rDNA as a template to obtain a V4-V5 fragment of the 16S rDNA;
4) the 16S rDNA V4-V5 fragment was amplified (for Index addition) with the upstream primer for sample discrimination and the downstream primer for sample discrimination in the above-described primer set to obtain a nasal flora sequencing library.
The invention also provides a method for extracting DNA of the in vitro sample, which comprises the following steps:
1) cleaving the in vitro sample by proteinase K to obtain a cleavage product;
2) incubating the cracked product at 95 ℃ for 5min, discarding the swab, and obtaining a heat-treated cracked product;
3) mechanically cracking the thermally cracked product by using zirconia beads to obtain a mechanically cracked product;
4) and purifying the product after mechanical cracking to realize DNA extraction of the in vitro sample.
The sample is derived from nasal cavity, oral cavity or feces, and can be nasal swab.
The primer for amplifying the V4-V5 region of the 16S rDNA has the following characteristics:
(1) good conservation and high coverage. The specific sequence of the primer is to increase the coverage of the flora, and degenerate basic groups are introduced into different positions of the primer to increase the amplification sensitivity of the flora.
(2) Linker sequences of varying lengths (joining the illumina sequencing primers and the N between the specific primers) follow the specific sequence, while sequences comprise the illumina sequencing universal sequence, and these linker sequences can consist of 2-6 random bases (i.e., N). In the subsequent library preparation process, the target product amplification is carried out by mixing the linker sequence with different lengths and the primer without the linker sequence. Therefore, on one hand, the introduction of random base at the beginning of sequencing can improve the base balance of the initial position, thereby improving the quality of sequencing initial signals, and on the other hand, the linker sequences with different lengths and primers without the linker sequences are mixed for use, so that the distances between the target sequence and the amplification initial site are diversified, thereby increasing the overall base balance.
In summary, the present invention has the following advantages:
1) aiming at the problem of too low content of flora genome DNA in a nasal swab sample, a 16S rDNA primer with high amplification efficiency, strong specificity and low human homology is designed, a 16S V1-V9 region is enriched, a target fragment is further amplified through nested PCR, and the interference of human gDNA is reduced;
2) the sequencing quality is influenced by poor base balance due to high sequence similarity of the 16S rDNA library, and aiming at the problem, the sequencing quality of the 16S rDNA flora is improved by introducing random bases into the primers. By combining primers containing and not containing the linker sequence, the diversity of initial sequencing signals is improved, and the sequencing quality is improved.
3) Aiming at the problem of low detection coverage of a single hypervariable region in 16S rDNA sequencing, the invention detects a V4V5 segment which is a double-fragment region with the highest coverage reported in the literature.
4) Aiming at the problem of complicated library construction caused by adding a sequencing joint by a ligation method, the invention combines an amplification primer with an illumina sequencing original, completes library construction through PCR amplification, simplifies the construction steps of a 16S rDNA flora sequencing library, establishes a simple and efficient library construction reaction system and method, and is suitable for large-scale 16S rDNA flora sequencing.
5) Simplifies the steps of constructing the 16S rDNA flora sequencing library, establishes a simple and efficient library construction reaction system and method, and is suitable for large-scale 16S rDNA flora sequencing.
In a word, the invention firstly adopts the V1-V9 primer with high amplification efficiency, strong specificity and low human homology to enrich the 16S rDNA segment, and then utilizes nested PCR to amplify the target segment V4V5 double segment, thus overcoming the problems that the sequence homology of the V4V5 segment amplification primer and the human genome is higher, and serious non-specific amplification phenomenon can occur when a nasal swab sample with high human gDNA content is amplified.
Drawings
FIG. 1 is a flow chart of the preparation of the nasal flora 16S rDNA sequencing library of the present invention.
FIG. 2 is the electrophoresis chart of the V4-V5 library prepared in example 1.
Fig. 3 is a heat map of the percentage of nasal flora in example 1.
Fig. 4 is a histogram of nasal flora distribution in example 1.
FIG. 5 is a comparison of library preparation schemes of the method of the present invention and the control method in example 2.
FIG. 6 shows 2100 assays of the V4-V5 library prepared by the control method of example 2.
FIG. 7 shows the results of 2100 assays of the V4-V5 library prepared by the method of the invention in example 2.
FIG. 8 is the data percentage of the library Q20 prepared from the experimental group and the control group in example 3.
FIG. 9 shows the percentage of effective data of the library prepared in example 3.
Detailed Description
The technical solution and advantages of the present invention will be further described in detail with reference to specific embodiments. It is obvious that the present invention is not limited to the following embodiments.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The samples used in the following examples were obtained from healthy persons, and the subjects previously known the purpose of the sample and agreed to the collection.
The primer solutions used in the following examples were each at a concentration of 10. mu.M, unless otherwise specified.
In the quantitative tests in the following examples, three replicates were set up and the results averaged.
Example 1 preparation of kit for nasal flora detection and application thereof
Design of primer and preparation of detection kit
1. Primers for amplifying V1-V9 region of bacterial 16S rDNA
The following 3 primers were designed based on the V1-V9 region of bacterial 16S rDNA:
upstream primer FP 1: sequence 1 in the sequence table: 5 '-AGRGTTYGATYMTGGCTCAG-3' (SEQ ID NO: 1)
The downstream primer RP 1-1: sequence 2:5 '-TACAGYTACCWTGTTACGACTTNN-3' (sequence 2) in the sequence table
The downstream primer RP 2-1: sequence 3:5 '-GGYTACCTTGTDACGACTTNN-3' (sequence 3) in the sequence table
2. Primers for amplifying V4-V5 region of bacterial 16S rDNA
Due to single sequencing product, when the illumina platform sequencing is carried out, the 5 ends of the PCR products are the same sequence, so that the sequencing starting signal is single, and the sequencing quality is seriously reduced. According to the invention, a linker sequence is introduced between a sequencing linker sequence and a V4V5 specific amplification sequence, and the primer combination using the linker sequence and the primer not containing the linker sequence is adopted, so that the diversity of initial sequencing signals is improved, and the sequencing quality is improved.
The V4V5 specific amplification sequence can be provided with degenerate bases, the linker sequence can be 2-6 random bases (2-6N), the embodiment is described by taking 2N as an example, and N is any one of ATCGs.
In the present invention,
the upstream primer for amplifying the V4-V5 region consists of 2 primers, namely an upstream primer FP2-1 and an upstream primer FP 2-2:
the upstream primer FP 2-1: a sequence 4 in a sequence table;
5 '-CTTTCCCTACACGACGCTCTTCCGATCTCCAGCAGCCGCGGTAAH-3' (wherein, italics is the sequencing linker region and bold is the upstream specific primer segment);
the upstream primer FP 2-2: sequence 5 in the sequence table:
5'-CTTTCCCTACACGACGCTCTTCCGATCTNNCCAGCAGCTGCGGTAAD-3' (wherein the italic is the sequencing linker region; the linker sequence, consisting of 2-6 random bases N, is underlined; the upstream specific primer segment is bolded);
the downstream primer for amplifying the V4-V5 region consists of 2 primers, namely a downstream primer A and a downstream primer B:
the downstream primer RP 2-1: sequence 6 in the sequence table:
5 '-GGAGTTCAGACGTGTGCTCTTCCGATCTCCGTCAATTCVTTTAAGT-3' (where italics is another sequencing linker region and bold is the downstream specific primer segment);
the downstream primer RP 2-2: sequence 7 in the sequence table:
5'-GGAGTTCAGACGTGTGCTCTTCCGATCTNNCCGTCAATTCHTTTGAGT-3' (where the italics is the region of the other sequencing linker; the linker sequence is underlined, the sequence consisting of 2-6 random bases N; the bold is the downstream specific primer segment);
the downstream primer RP 2-3: sequence 8 in the sequence table:
5'-GGAGTTCAGACGTGTGCTCTTCCGATCTCCGTCAATTTCTTTGAGTM-3’
(wherein, italics is another sequencing linker region, bold is the downstream specific primer segment)
The downstream primer RP 2-4: sequence 9 in the sequence table:
5'-GGAGTTCAGACGTGTGCTCTTCCGATCTNNCCGTCAATTTCTTTGAGTK-3' (where the italics is the region of the other sequencing linker; the linker sequence is underlined, the sequence consisting of 2-6 random bases N; the bold is the downstream specific primer segment;);
the downstream primer RP 2-5: sequence 10 in the sequence table:
5 '-GGAGTTCAGACGTGTGCTTCCGATCTTCTTTGATCGBT-3' (wherein the italics is another sequencing linker region and the bold is the downstream specific primer segment);
the downstream primer RP 2-6: sequence 11 in the sequence table:
5'-GGAGTTCAGACGTGTGCTCTTCCGATCTNNCCGTCTATTCCTTTGAVT-3' (where the italic is the region of the other sequencing adapter; the linker sequence, consisting of 2-6 random bases N, is underlined; the downstream specific primer segment is bolded).
3. Detection kit
The detection kit comprises the primer for amplifying the V1-V9 region of the bacteria 16S rDNA and the primer for amplifying the V4-V5 region of the bacteria 16S rDNA;
the detection kit also comprises a primer for sample discrimination, which consists of an upstream primer for sample discrimination and a downstream primer for sample discrimination;
in which the upstream primer (FP 3 shown in Table 1 below) was used for sample discrimination*) Sequentially sequencing another portion of an illumina sequencing adaptor (forming an illumina sequencing adaptor with the sequencing adaptor region in the upstream primer), an index sequence for sample discrimination, and a region complementary to the sequencing adaptor segment;
in which the downstream primer (RP 3 shown in Table 1 below) was used for sample discrimination*) Sequentially from another part of the illumina further sequencing linker (forming an illumina further sequencing linker with the sequencing linker region in the downstream primer) for sample applicationThe index sequence of the instant discrimination and the region complementary to another sequencing adapter segment.
Second, application of kit
The used materials are flocked swab samples collected from the left and right nasal cavities of a healthy male and a healthy female, and the sample numbers are (CBNC180015 (left nasal cavity of healthy male), CBNC180016 (right nasal cavity of healthy male), CBNC180020 (left nasal cavity of healthy female) and CBNC180021 (right nasal cavity of healthy female).
1. Nasal swab sample collection
(1) Taking out a sterile flocked swab, and inserting the swab into the nasal middle passage from bottom to top;
(2) gently rotate the swab counter-clockwise for 30 sec.
(3) The swab was slowly withdrawn from the nasal cavity and placed in a 1.5mL centrifuge tube.
(4) Breaking the swab, fastening the tube cover and indicating the sampling information.
2. Nasal cavity sample gDNA extraction
In this embodiment, an improved version of the genomic DNA extraction kit for oral swabs by the paramagnetic particle method (DP 703-01, bio-technologies ltd.) is used to extract gDNA from nasal swabs, and conditions are optimized for nasal flora, where the optimization conditions include: firstly, a 95 ℃ thermal cracking process is added to ensure that the cell walls of most bacteria are destroyed; secondly, one step of mechanical cracking of zirconia beads is added, so that the cell wall of gram-positive bacteria with hard cell wall to be damaged can be also fully damaged. The specific experimental steps are as follows:
(1) to a 1.5mL centrifuge tube containing the swab sample, 500. mu.L of buffer GHA was added.
(2) Add 10. mu.L of protease K to the centrifuge tube, vortex for 10sec, and incubate at 65 ℃ for 30 min.
(3) Incubate at 95 ℃ for 5min, discard the swab.
(4) Add 250. mu.L of 0.2mm zirconia beads (Yasuwang, YTZ-0.2) and shake with hand for 10 min.
(5) Centrifuge briefly, aspirate the supernatant and transfer to a new 2mL centrifuge tube.
(6) Add 600. mu.L buffer GHC, shake and mix well. The swab is discarded.
(7) Adding 15 μ L of magnetic bead suspension G, shaking and mixing for 12min (standing on a magnetic frame for 30sec, and discarding the supernatant.
(8) Adding 900 μ L deproteinized solution PD, shaking and mixing for 3min, standing on magnetic frame for 30sec, and removing liquid.
(9) Repeating the steps once.
(10) Adding 900 μ L of rinsing liquid PWD, shaking and mixing for 3min, standing on magnetic frame for 30sec, and removing liquid. And (3) placing the centrifugal tube on a magnetic frame, and airing for 10-15 min at room temperature.
(11) The tube was removed from the magnetic frame, 50. mu.L EB Buffer was added, mixed well with shaking, and incubated at 56 ℃ for 10 min.
(12) The centrifuge tube was placed on a magnetic stand and allowed to stand for 2min, after which the solution was transferred to a collection tube.
(13) RnaseA (RT 405-02, Tiangen Biochemical technology Co., Ltd.) was added to the above solution in an amount of 2. mu.L, mixed well and incubated in a metal bath at a constant temperature of 37 ℃ for 30 min.
(14) The DNA concentration was determined using the Qubit 2.0 and the extracted DNA concentrations were all >20 ng/. mu.L.
3. Sequencing library construction
16S sequencing library construction is carried out on the extracted nasal gDNA sample, the process comprises three steps of PCR reaction as shown in figure 1, namely, 16S rDNA V1-V9 region is enriched, and the product fragment is about 1470 bp; amplifying the 16S rDNA V4-V5 fragment in a nested manner, wherein the product fragment is about 480 bp; and (3) connecting sequencing adaptors of the illumina to obtain a product fragment of about 560 bp.
Primers designed according to one of the above-mentioned methods are shown in Table 1
TABLE 1
Note: index1 index2 is a tag sequence used for sample differentiation (4 samples, i.e. 4 tag sequence combinations), and the tag sequences used for 4 samples in this example are shown in table 2 below:
TABLE 2
| index1(5'-3') | index2(5'-3') | |
| CBNC180015 | GCGATCTA | GCTCAGGA |
| CBNC180016 | GCGATCTA | AGGAGTCC |
| CBNC180020 | GCGATCTA | GTAGAGAG |
| CBNC180021 | GCGATCTA | CCTCTCTG |
The primer FP3 and the primer RP3 can also be included in the kit.
The PCR reaction in the 3 steps is as follows:
step 1: enrichment of 16S rDNA V1-V9 region
(1) The DNA template was diluted to 4 ng/. mu.L with Nuclear-free water (Thermo Fisher, AM9930) and the first round of PCR amplification was prepared as follows:
TABLE 3
Note: the forward and reverse primers mix is a mixture of FP1, RP1-1 and RP1-2, the molar mixing ratio is 2:1:1, and the final concentration of the mixed primers is 10 mu M.
(2) The above amplification system was subjected to PCR amplification under the following reaction conditions of Table 4:
TABLE 4
(3) mu.L of PCR product was taken, and 15. mu.L of Agencour AMPure XP Beads (Beckman, A63881) were added to purify and recover the target fragment according to the manufacturer's standard operation procedure.
Step 2: enrichment of 16S rDNA V4-V5 region
(4) Taking 2.5 μ L of the PCR purified product in the step (3), preparing a second round PCR reaction system according to the following table 5:
TABLE 5
Note: the forward and reverse primers mix is a mixture of FP2-1, FP2-2, RP2-1, RP2-2, RP2-3, RP2-4, RP2-5 and RP2-6, the mixing molar mixing ratio is 5:5:3:3:1:1:1, and the final concentration of the mixed primers is 10 mu M.
(5) The above amplification system was subjected to PCR amplification under the following reaction conditions of Table 6:
TABLE 6
(6) mu.L of PCR product was taken, and 15. mu.L of Agencour AMPure XP Beads (Beckman, A63881) were added to purify and recover the target fragment according to the manufacturer's standard operation procedure.
And 3, step 3: illumina sequencing adaptor ligation
(7) Taking 2.5 μ L of the PCR product obtained in the step (6), preparing a 3 rd round PCR reaction system according to the following table 7:
TABLE 7
Note: the forward and reverse primers mix was a mixture of FP3 and RP3, both at a 1:1 ratio, with a final concentration of 10. mu.M.
(8) Performing PCR amplification on the amplification system according to the following reaction conditions of the table 8 to obtain a sequencing library:
TABLE 8
(9) Taking 20 mu L of PCR product, adding 15 mu L of Agencour AMPure XP Beads (Beckman, A63881), and purifying and recovering the target fragment according to the standard operation flow of manufacturers; obtaining a purified sequencing library.
(10) The DNA library concentration was determined using a Qubit 2.0, with four sample concentrations each >40 ng/. mu.L. Using 1% agarose gel electrophoresis, the library band was single, about 500bp in size (FIG. 2).
Sequencing:
(11) and (3) mixing 20ng of each purified library, and performing PE 300bp sequencing by adopting an Illumina miseq sequencing platform.
For the results obtained by sequencing the 16S rDNA in this example, the representative sequences of the operation classification units (OTUs) classified by greenene database were obtained by classifying the operation units using QIIME software, and the distribution of the kingdom, phylum, class, order, family and genus of the microorganism in the sample was obtained.
From the results obtained, a percentage rhinophyma chart of fig. 3 and a histogram of rhinophyma abundance of fig. 4 were prepared. From FIG. 3, the difference between the species of the nasal cavity of different individuals and the species of the nasal cavity of the same individual can be clearly observed. The proportion of the first 20 dominant genera in each individual nasal cavity can be clearly seen from FIG. 4, including the 6 common genera in the nasal cavity mentioned in published reviews (Diana M.Proctor and David A.Relma, Cell Host & Microbe, 2017; IF ═ 17.9): staphylococcus (g __ Staphylococcus), Corynebacterium (g __ Corynebacterium), Moraxella (g __ Moraxella), Difference (g __ Alloococcus), Haemophilus (g __ Haemophilus) Streptococcus (g __ Streptococcus). In 376 kinds of bacteria detected in this example, the average abundance of the above 6 genera was ranked at the 3 rd, 1 st, 4 th, 2 nd, 16 th and 9 th positions, and the sum of the abundances exceeded 50%.
Example 2 comparison of nested amplification with conventional amplification library
In order to verify the influence of the nested amplification method on the amplification efficiency of microorganisms in nasal gDNA samples, 9 parts of nasal gDNA samples with the quantitive concentration of the Qubit of 10-100 ng/. mu.L are selected, 10ng of each gDNA sample is uniformly mixed, and the mixture is divided into two parts for subsequent library construction.
Experimental groups: 2 parts of the uniformly mixed nasal gDNA sample are subjected to library construction according to the sequencing library construction method of 3 in the second embodiment 1, and 3 steps of PCR amplification are included;
control group: different from the experimental group, the V1-V9 region enrichment PCR amplification of the step 1 is not carried out, and the V4-V5 region enrichment PCR amplification of the step 2 is directly carried out, which comprises the following steps: the 2 homo-nasal gDNA samples were pooled according to the 2 nd and 3 rd PCR in the three-step PCR in the sequencing library construction method of 3 in example 1, without performing the 1 st PCR amplification (V1-V9 amplification) and purification (see FIG. 5 for a simplified flow chart).
The size distribution of the library fragments was examined using the Agilent High Sensitivity DNA Kit (Agilent, 5067-4626).
The results of the control and experimental groups are shown in fig. 6 and fig. 7, respectively, and it can be seen that the experimental group (the method of the present invention) can significantly reduce the interference of the human genome to the V4V5 library.
Example 3 comparison of sequencing quality of the colony-specific amplification primer set used in this protocol with the conventional 16S rDNA amplification primer set
When the illumina platform sequencing is carried out, the 5' ends of PCR products are the same sequence, and the sequencing products are single, so that the sequencing starting signal is single, and the sequencing quality is low. According to the invention, by adding 2-6 random N at the 5' end of the specific primer, the diversity of the sequencing initiation site is increased, and then primers with linker sequences of different lengths are mixed to stagger the sequencing initiation site, so that the diversity of the whole amplification library is further increased, and the overall sequencing quality is improved.
In this embodiment, a sample of the intestinal flora is taken as an example, and the test results of 2 primer sets are shown.
Firstly, amplifying 40 cases of intestinal flora samples by adopting a primer V4V5_ orign group (a control group) without a random linker sequence, and sequencing; thereafter, a total of 140 intestinal samples were sequenced using a primer set V4V5_ Mix (experimental set) containing a random linker sequence.
The specific primer sequences used in this experiment are shown in table 9 below:
TABLE 9
The specific experimental steps are as follows:
1. extraction of nucleic acid sample of intestinal flora
200mg of each fecal sample is taken for nucleic acid extraction, the nucleic acid extraction is carried out by using a fecal genome extraction kit (DP328) of Tiangen Biochemical technology (Beijing) Co., Ltd, the extraction is carried out according to a reference instruction, and the nucleic acid quantification is carried out by using the Qubit.
2. Sequencing library preparation procedure
(1) Stool DNA was diluted to 4 ng/. mu.L for use according to the Qubit quantification. The reaction Mix was formulated as in table 10 below.
Watch 10
Note: for the experimental group, the forward and reverse primers mix are a mixture of FP2-1, FP2-2, RP2-1, RP2-2, RP2-3, RP2-4, RP2-5 and RP2-6, the mixing molar mixing ratio is 5:5:3:3:1:1:1, and the final concentration of the mixed primers is 10 mu M. For the control group, the forward and reverse primers mix were a mixture of FP2-1, RP2-1, RP2-3 and RP2-5, the mixing molar mixing ratio was 5:3:1:1, and the final concentration of the mixed primers was 10. mu.M.
(2) The PCR reaction plate was placed in a PCR amplification apparatus, and PCR amplification reaction was performed according to the thermal cycling program in Table 11.
TABLE 11
Note: in parentheses, the rise/fall rates are indicated
(3) mu.L of PCR product was taken, and 15. mu.L of Agencour AMPure XP Beads (Beckman, A63881) were added to purify and recover the target fragment according to the manufacturer's standard operation procedure.
(4) And (3) taking 1 mu L of PCR product obtained in the step (3), and diluting the product by 10 times with sterile water. A second PCR amplification Mix was prepared as in Table 12.
TABLE 12
Note: the forward and reverse primers mix was a mixture of FP3 and RP3 at a 1:1 ratio and a final concentration of 10. mu.M.
(5) The PCR tube was placed in a PCR amplification apparatus and PCR amplification was performed according to the thermal cycling protocol in Table 13.
Watch 13
(6) mu.L of PCR product was taken, and 15. mu.L of Agencour AMPure XP Beads (Beckman, A63881) were added to purify and recover the target fragment according to the manufacturer's standard operation procedure.
(7) The DNA library concentration was determined using a Qubit 2.0, with 180 samples each at a concentration >10 ng/. mu.L.
(8) According to the quantitative of the library Qubit, 20ng of each library is mixed, and the sequencing is carried out by PE 300bp by using the illumina Miseq.
(9) Results of the experiment
The V4V5_ Mix primer set showed a greater improvement in sequencing quality Q20 (FIG. 8) and in effective clean _ reads rate than the V4V5_ orign (FIG. 9). The average Q20 ratio of the V4V5_ Mix primer set group was 98%, and the clean _ reads ratio was 90.26%. And the average Q20 of the V4V5_ orign primer group is 60.99%, and the clean _ reads rate is 54.30%. The patented method is improved by about 30% and 40% in the ratio of Q20 and the ratio of clean _ reads, respectively, over the conventional method.
In example 3 of the present invention, the quality control results obtained by 16S rDNA sequencing in this example were collated and plotted as the data rate and clean read rate distribution graph of fig. 8Q 20, and it can be seen that the method of the present invention can significantly improve the sequencing data quality of the V4V5 library.
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Claims (7)
1. A set of primers for detecting the flora of a nasal cavity sample comprises a primer for amplifying a V4-V5 region of a bacterium 16S rDNA and a primer for amplifying a V1-V9 region of the bacterium 16S rDNA;
the primers for amplifying the V4-V5 region of the 16S rDNA of the bacteria consist of a primer shown in a sequence 4, a primer shown in a sequence 5, a primer shown in a sequence 6, a primer shown in a sequence 7, a primer shown in a sequence 8, a primer shown in a sequence 9, a primer shown in a sequence 10 and a primer shown in a sequence 11;
the primer for amplifying the V1-V9 region of the 16S rDNA of the bacteria consists of a primer shown in a sequence 1, a primer shown in a sequence 2 and a primer shown in a sequence 3.
2. PCR reagents comprising the set of primers of claim 1, which comprises PCR reagents comprising the primers for amplifying the V1-V9 region of the bacterial 16S rDNA, PCR reagents comprising the primers for amplifying the V4-V5 region of the bacterial 16S rDNA, and PCR reagents comprising the primers for nasal sample differentiation.
3. A kit comprising the primer set of claim 1 or the PCR reagent of claim 2.
4. Use of the primer set of claim 1 or the PCR reagent of claim 2 or the kit of claim 3 for the preparation of a product for detecting the flora of a nasal sample;
or, the use of the primer set of claim 1 or the PCR reagent of claim 2 or the kit of claim 3 for constructing a nasal cavity sample flora sequencing library;
or, the use of the primer set of claim 1 or the PCR reagent of claim 2 or the kit of claim 3 for preparing a product for constructing a nasal cavity sample flora sequencing library.
5. Use of the primer for amplifying the V4-V5 region of the bacterium 16S rDNA in the primer set according to claim 1 and the primer for amplifying the V1-V9 region of the bacterium 16S rDNA in the primer set according to claim 1 in the preparation of a product for detecting the flora in a nasal sample.
6. Use of a primer for amplifying the V4-V5 region of the bacterium 16S rDNA in the set of primers of claim 1 and a primer for amplifying the V1-V9 region of the bacterium 16S rDNA in the set of primers of claim 1 for constructing a nasal sample flora sequencing library;
or, the use of the primer for amplifying the V4-V5 region of the bacterium 16S rDNA in the primer set of claim 1 and the primer for amplifying the V1-V9 region of the bacterium 16S rDNA in the primer set of claim 1 for preparing a product for constructing a nasal cavity sample flora sequencing library.
7. A method for constructing a nasal cavity sample flora sequencing library comprises the following steps:
1) extracting DNA of an in vitro sample,
2) performing PCR amplification using the DNA as a template and the primers for amplifying the V1-V9 region of the 16S rDNA of the bacterium of claim 1 to obtain a V1-V9 fragment of the 16S rDNA;
3) performing PCR amplification by using the primers for amplifying the V4-V5 region of the bacterium 16S rDNA in claim 1 by using the V1-V9 fragment of the 16S rDNA as a template to obtain a V4-V5 fragment of the 16S rDNA;
4) performing PCR amplification on the 16S rDNA V4-V5 fragment by using an upstream primer for sample discrimination and a downstream primer for sample discrimination in the kit of claim 3 to obtain a nasal flora sequencing library.
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