CN109486922B - Method for detecting microbial target sequence based on single primer probe capture - Google Patents
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Abstract
A method for detecting a microbial target sequence based on single primer probe capture, comprising: fragmenting a DNA sample containing a microbial target sequence, and then performing end repair; connecting the DNA fragments with the repaired ends with a joint; annealing and extending a probe binding site of a target sequence for target sequence capture using a single primer probe, wherein the probe binding site is located upstream or downstream of the target sequence, the single primer probe comprising a probe sequence portion and a common sequence portion located 5' of the probe sequence portion; amplifying the captured target sequence using a pair of universal PCR primers; and sequencing and analyzing the amplified products. The method of the invention saves elution for separating the target fragment, simplifies the operation steps, reduces unnecessary loss of the target fragment, has relatively short hybridization time, and is suitable for the requirement of rapid detection; the single primer probe can be captured, so that the complexity of the design of the early-stage probe is reduced; the heterogeneity caused by enrichment of different primers is removed by using a common primer during amplification.
Description
Technical Field
The invention relates to the technical field of microbial detection, in particular to a method for detecting a microbial target sequence based on single-primer probe capture.
Background
With the development of sequencing technology, the advantages of the Next Generation Sequencing (NGS) detection method in various fields are gradually highlighted, compared with the traditional research method, the method is rapid, time-saving and accurate in result, and the advantage of accurately and sensitively detecting species DNA sequences is widely applied to various research fields. However, the relatively high sequencing cost still limits the application range of the Whole Genome Sequencing (WGS) because of the relatively long cycle of the WGS. The application of the target sequence capturing technology just solves the problems of long time and high cost of WGS sequencing, and the capturing and enriching of the specific target sequence reduces the proportion of unnecessary data in a sequencing result, improves the detection proportion of the target sequence, greatly reduces the data amount required by sequencing and further reduces the detection cost. Especially for the research of microorganisms, the detection results of different sample types contain different degrees of background sequences, which results in higher requirements on sequencing data amount and also causes interference on the analysis of the detection result of the target microorganism.
The specific detection technology of the target sequence by the capture method comprises the following steps: the Roche NimbleGen sequence capturing technology captures target fragments through a solid phase chip or a liquid phase chip, and the length of a probe is 50-105 bp. The main process comprises genome fragmentation and end repair, adaptor connection, hybridization, chip elution, target fragment elution, amplification and sequencing. An Agilent SureSelect DNA capture array technology is similar to the NimbleGen technology, and also captures a target fragment through a solid phase or liquid phase chip, wherein the length of a probe is about 120 bp. The main process comprises genome fragmentation, end repair and addition of A, adaptor connection, amplification, hybridization, elution, amplification and sequencing. The AmpliSeq On-Demand coils technology for capture sequencing enriches target fragments through multiplex PCR, and then performs library building and sequencing On the enriched fragments.
The probe length of the NimbleGen sequence capture technology and the Agilent SureSelect DNA capture array technology is long, and the synthesis cost is high compared with that of a short probe. Two elution steps are required after hybridization in the NimbleGen library construction process, so that the operation time, complexity and unnecessary loss of samples are increased, and the library construction cost is increased by the elution reagent. The longer hybridization and elution time of the Agilent technology is not suitable for the requirement of quick detection, and simultaneously, one-step amplification reaction is added, and the library construction operation time is also increased. The AmpliSeq technology is not suitable for quantitative analysis because the enrichment results of different target fragments are not uniform due to the difference of the amplification efficiency of different primers and the mutual influence between the primers when the multiplex PCR is used for enrichment. Most PCR amplification cycles are easy to introduce site mutation, which affects result judgment.
Disclosure of Invention
The invention provides a method for detecting a microbial target sequence based on single-primer probe capture, wherein a joint is connected to the upstream of a probe binding site in a library building process, so that only captured fragments can be enriched during subsequent amplification, the step of separating the target fragments by elution in the conventional method is omitted, the operation steps are simplified, unnecessary loss of the target fragments is reduced, the hybridization time is relatively short, and the method is suitable for the rapid detection requirement; the single primer probe can be captured, the complexity of early probe design is reduced, the length of the probe is shorter, and the synthesis cost is lower; the heterogeneity caused by enrichment of different primers is removed by using a common primer during amplification.
The invention is realized by the following technical scheme:
a method for detecting a microbial target sequence based on single primer probe capture, comprising:
fragmenting a DNA sample containing a microbial target sequence, and then performing end repair;
connecting the DNA fragments with the repaired ends with a joint;
annealing and extending a probe binding site of a target sequence for target sequence capture using a single primer probe, wherein said probe binding site is located upstream or downstream of said target sequence, said single primer probe comprising a probe sequence portion for annealing to said probe binding site and a common sequence portion located 5' of said probe sequence portion for serving as a binding site for a universal PCR primer;
amplifying the captured target sequence by using a pair of universal PCR primers to obtain an amplification product; and sequencing and analyzing the amplification product to obtain the sequence information of the target sequence.
The universal PCR primers in the above scheme can be paired with the adaptor sequence and the public sequence for PCR amplification.
Further, the DNA samples are fragmented to small fragments of 150bp to 300 bp.
Further, the above linker comprises a tag sequence; preferably, said linker further comprises a random sequence, said random sequence being adjacent to said tag sequence. The random sequence is a sequence of random combination of nucleotides, and can be used for distinguishing each amplified target sequence, and the length of the random sequence is generally 4-12nt, preferably 6 nt.
Further, the length of the probe sequence portion is 35 to 40 bp.
Further, the single primer probe is gradient annealed to bind to the probe binding site.
Further, the gradient annealing is to perform gradient cooling from 80 ℃ to 60 ℃.
Further, the sequence of the above linker is SEQ ID NO: 5 and SEQ ID NO: 6.
further, the sequence of the common linker sequence part of the sequencing platform is SEQ ID NO: 7.
further, the sequence of the universal PCR primer of the sequencing platform is SEQ ID NO: 8 and SEQ ID NO: 9.
the method reduces the detection cost of applying a second-generation sequencing technology, reduces the detection of non-target sequences by enriching the target sequences through the single-primer probe, improves the detection rate of the target sequences, further reduces the data volume used by sequencing, greatly reduces the detection cost, and simultaneously makes the analysis of sequencing result data easier; compared with the PCR enrichment technology, the invention ensures that the common primers are used for amplification in the subsequent PCR link because the same joints are connected at the two ends of the fragments after the capture, thereby eliminating the inaccuracy of the detection result caused by the amplification bias of the primers generated when the conventional different PCR primers are used for amplification; the single primer probe only needs one probe for capturing, thereby reducing the complexity of designing a primer pair, and simultaneously eliminating the condition of non-specific amplification caused by primer cross reaction in multiple PCR, so that the detection result is more accurate and reliable. In addition, sequencing by using a BGISEQ-100 platform can effectively shorten sequencing time and realize rapid detection of a target sequence.
Drawings
FIG. 1 is a schematic diagram showing the principle of construction of a microorganism detection library in the embodiment of the present invention;
FIG. 2 is a graph of Agilent 2100 results for a library that is qualified by library quality testing in an embodiment of the invention;
fig. 3 is a capturing result of 4 probes for staphylococcus aureus in the embodiment of the present invention, the abscissa represents the 4 probes for staphylococcus aureus, the ordinate represents the read length (reads) number of the staphylococcus aureus genome in the upper database, the black column represents the positive sample, the gray column represents the negative control (lower value), and the result indicates that the detection of the positive sample can be definitely determined by the embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present invention have not been shown or described in the specification in order to avoid obscuring the present invention from the excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they can be fully understood from the description in the specification and the general knowledge in the art.
FIG. 1 shows the construction principle of the microorganism detection library in the embodiment of the present invention, and the single primer probe capture is to design an oligonucleotide probe upstream or downstream of the target sequence and link a linker public sequence to the 5' end of the oligonucleotide probe. Extracting a sample DNA to be detected, fragmenting the sample DNA to a small fragment of 150-300bp, connecting the broken DNA to a tag sequence (Barcode) joint which can distinguish different samples after the tail end is repaired to a flat tail end, then combining an oligonucleotide probe with a target region through gradient annealing, covering the whole target sequence region through constant temperature extension, then enriching the captured sequence through common primer PCR amplification, sequencing through a sequencing platform, and further analyzing the base characteristics of the target sequence.
A common high-throughput sequencing technology platform is a Hiseq series of Illumina company, Hiseq _2500 can start up at most 600G base data at one time, but the cycle needs about 8 days, while the BGISEQ-100 platform can start up at one time and only needs about 6 hours. Therefore, the BGISEQ-100 platform is a more efficient choice. Thus, as a preferred embodiment, FIG. 1 shows a schematic single primer probe capture scheme based on the BGISEQ-100 platform, however, it should be understood that the method of the present invention can be implemented on other sequencing platforms.
As shown in fig. 1, the method of the embodiment of the present invention includes:
A. the extracted sample total-DNA was fragmented and end-repaired (open white boxes in fig. 1 represent target sequences, broken black filled boxes represent probe binding sites for target sequences).
B. The fragments after end repair are connected with the linker, and only the linker containing the tag sequence can be added, so that the two ends of the fragments are connected with the same linker sequence (black filled box part in FIG. 1), so that the fragments which are not captured subsequently cannot be enriched by PCR amplification; in other embodiments, the linker further comprises a random base, located before the tag sequence, to distinguish whether the products are from the same amplicon.
C. The single primer probe includes a probe sequence portion (grey arrow in fig. 1) and a common sequence portion (e.g., sequencing platform common linker sequence portion, open box to the left of the grey arrow in fig. 1) located 5' to the probe sequence portion, and is annealed to the probe binding site of the target sequence and extended for target sequence capture using the single primer probe. In FIG. 1, the probe binding site is located upstream or downstream of the target sequence, the probe sequence portion is used to anneal to the probe binding site, and the common sequence portion is used as a binding site for the universal PCR primers. In this step, the single-primer probe is subjected to gradient annealing to bind to the probe binding site, for example, by gradient cooling from 80 ℃ to 60 ℃. In a preferred embodiment, the length of the probe sequence part is 36bp, the length of the common joint sequence part of the sequencing platform (BGISEQ-100 platform) is 31bp, the total length is short, and the synthesis cost is low.
D. And amplifying the extended product by using BGISEQ-100 platform universal PCR primers (black arrow FWD and blank arrow REV in figure 1), wherein only the segment which is grafted into the universal joint primers at two ends after hybridization extension can be amplified, and the amplified product is the constructed DNA capture library. Then, the sequence of the DNA capture library was obtained by second-generation sequencing, and the base sequence information of the target sequence was obtained by analysis.
The technical solutions of the present invention are described in detail below by way of examples, and it should be understood that the examples are only illustrative and should not be construed as limiting the scope of the present invention. The reagent information used in the following examples is shown in table 1 below.
TABLE 1
Example 1
1. 4 probes were designed and captured for 2 specific regions of 250bp in the genome of Staphylococcus aureus (Sa), and library construction was performed according to the library construction procedure shown in FIG. 1. The portions of the probe sequences used (grey arrows in FIG. 1) are shown in Table 2.
TABLE 2
2. Bacterial liquid of staphylococcus aureus is used for extracting bacterial genome by a Tiangen DP-302 Kit, extracted nucleic acid is quantified by a Qubit dsDNA HS Assay Kit, and the genome copy number is calculated according to the genome size. The extraction and quantification of the blood DNA were carried out using Tiangen microsample DNA extraction kit DP-316. A nucleic acid of Staphylococcus aureus corresponding to 10000 copies was added to 10ng of the human nucleic acid.
3. Sample interruption: the sample breaking method comprises
Pico disruption, which breaks the sample into fragments ranging from about 200 bp to about 250 bp. In strict accordance with
Pico's instructions were performed using a 0.5mL centrifuge tube with a sample volume of 40. mu.L, sonication for 30s, pause for 30s, run 4 cyclesFor one cycle, samples were taken for transient centrifugation.
4. Library construction and sequencing analysis of fragmented DNA
(1) DNA fragment end repair
Fill in the gap at the 5 'end and connect with phosphate group, remove the sticky end at the 3' end. The reaction system is shown in table 3 below:
TABLE 3
(2) End repair product purification
Purifying by adopting 1 XAgencour AMPure XP magnetic Beads, and carrying out the operation flow according to the AMPure XP Beads purification instruction.
(3) Ligation linker tag sequences
Connecting the blunt-ended DNA fragments by T4 ligase; the linker is a blunt end linker containing a tag sequence, the sequence is: 5' -CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNNCGAT-3 ' (SEQ ID NO: 5), 5 ' -ATCGNNNNNNNNNCTGAGTCGGAGACACGCAGGGATGAGATGG/Thiol-C6S-S/T/Thiol-C6S-S/T-3 ' (SEQ ID NO: 6), whereinNNNNNNNNNIs a tag sequence.
The reaction system is shown in table 4 below:
TABLE 4
(4) Ligation product purification
Purifying by using 1 XAgencour AMPure XP magnetic beads, and carrying out the operation flow according to the instruction. After purification 8 samples were combined in one tube and concentrated to 41.5 μ L using a vacuum centrifuge concentrator.
(5) Capturing and extending probe
Probe Capture probes specifically designed to capture specific microbial sequences (sequences shown in Table 2) were hybridized to the sample and extended with Phusion DNA polymerase. The common joint sequence of the sequencing platform connected with the 5' end of the probe is as follows: 5'-TCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3' (SEQ ID NO: 7).
The reaction system is shown in table 5 below:
TABLE 5
(6) Purification of the captured product
And (3) purifying by using 0.8X Agencour AMPure XP magnetic beads twice.
(7) PCR amplification, purification, concentration detection
The PCR amplification adopts a platinum TMPfx DNA Polymerase kit to amplify for 10 cycles, and the primer sequence is as follows:
T_PCR_A:5'-CCATCTCATCCCTGCGTGTC-3’(SEQ ID NO：8)；
Plamp:5'-CCACTACGCCTCCGCTTTCCTCTCTATG-3'(SEQ ID NO：9)。
the reaction system is shown in table 6 below:
TABLE 6
The detection concentration adopts a Qubit dsDNA HS Assay kit 2.0Fluorometer kit.
(8) Detection of library quality
The Agilent 2100Bioanalyzer detector was used, the procedure was as described in the Agilent 2100Bioanalyzer instructions, and the Agilent 2100 results are shown in FIG. 2.
(9) BGISEQ-100 sequencing
Operating according to BGISEQ-100 instructions.
Comparing the off-line data with a target area to be detected of staphylococcus aureus, counting the read lengths (reads) which can only be compared with the target area of staphylococcus aureus, counting the reads captured by different probes of staphylococcus aureus in a negative sample and a positive sample (as shown in figure 3), and displaying the result that the detection of the positive sample can be definitely judged by the sequencing result of single-primer probe capture library building.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
SEQUENCE LISTING
<110> Shenzhen Hua Dagen shares GmbH
<120> method for detecting microbial target sequence based on single primer probe capture
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Claims (9)
1. A method for detecting a microbial target sequence based on single primer probe capture, the method comprising:
fragmenting a DNA sample containing a microbial target sequence, and then performing end repair;
connecting the DNA fragments with the repaired ends with a joint;
annealing and extending a probe binding site of a target sequence for target sequence capture using a single primer probe, wherein the probe binding site is located upstream or downstream of the target sequence, the single primer probe comprising a probe sequence portion for annealing to the probe binding site and a common sequence portion located 5' of the probe sequence portion for serving as a binding site for a universal PCR primer, the probe sequence portion being 35-40bp in length;
amplifying the captured target sequence by using a pair of universal PCR primers to obtain an amplification product;
and sequencing and analyzing the amplification product to obtain the sequence information of the target sequence.
2. The method of claim 1, wherein the DNA sample is fragmented to small fragments of 150bp-300 bp.
3. The method of claim 1, wherein the linker comprises a tag sequence.
4. The method of claim 3, wherein the linker further comprises a random sequence, wherein the random sequence is adjacent to the tag sequence.
5. The method of claim 1, wherein the single primer probe binds to the probe binding site via gradient annealing.
6. The method of claim 5, wherein the gradient anneal is a gradient ramp down from 80 ℃ to 60 ℃.
7. The method of claim 1, wherein the sequence of the linker is SEQ ID NO: 5 and SEQ ID NO: 6.
8. the method of claim 1, wherein the sequence of the sequencing platform common linker sequence portion is SEQ ID NO: 7.
9. the method of claim 1, wherein the sequence of the sequencing platform universal PCR primer is SEQ ID NO: 8 and SEQ ID NO: 9.
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| CN106987905A (en) * | 2017-04-06 | 2017-07-28 | 深圳华大基因股份有限公司 | A kind of construction method and kit in BRCA1/2 genetic tests library |
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