CN111020061B - Multiplex PCR primer group, kit and method for detecting HPV based on high-throughput sequencing - Google Patents
Multiplex PCR primer group, kit and method for detecting HPV based on high-throughput sequencing Download PDFInfo
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Abstract
The invention discloses a multiplex PCR primer group and a kit for detecting HPV based on high-throughput sequencing. The current methods are difficult to realize HPV high-throughput sample detection. When the requirement of detecting the sample size is large, a large amount of manpower is needed for operation. The method established by the invention can be used for amplifying and purifying by a one-step method, a library is established by the one-step method, and the method does not need to rotate the tube in the midway and open the cover, thereby effectively avoiding the confusion or cross contamination among samples; the library construction of 96 samples can be completed in 5 hours, the operation is simple and convenient, and the detection of a large number of samples can be easily realized. By increasing the number of sample tags in the adapter primers, the number of sample detections that can be performed in one sequencing run can be increased, further reducing the average manual time for a single sample.
Description
Technical Field
The invention belongs to the field of next-generation sequencing, and particularly relates to a multiple PCR primer set, a kit and a method for detecting HPV based on high-throughput sequencing.
Background
According to the statistical data of the world health organization in 2018, the cervical cancer is the fourth female malignant tumor worldwide, and about 57 ten thousand new cases are generated in 2018, and more than 31 ten thousand people die due to the cervical cancer. Worldwide, patients with cervical cancer are on the rise year by year and in a trend towards younger age. More than 10 ten thousand new cases in China are generated each year; the number of the diseases in other developing countries is more than 85 percent of the total number.
The cervical cancer is the only malignant tumor with definite etiology and capable of being prevented and cured in an early stage at present, 99.9 percent of cervical cancer is related to Human Papilloma Virus (HPV) infection, high-risk HPV persistent infection is an essential factor for the generation of the cervical cancer, and the risks caused by different types of HPV infection are different. Research shows that more than seven women have at least one HPV infection in one life, but most of HPV infections can be eliminated by the body's own immune system, and only 1% -4% of HPV infected people gradually develop lesions, which finally cause canceration. The world health organization recommends that cytological screening be performed every 3-5 years for women between the ages of 30 and 49 years, or HPV screening be performed every 5 years. Therefore, the key to the implementation of a normative and effective HPV screening strategy for cervical cancer prevention and treatment is realized.
High throughput sequencing, also known as massively parallel sequencing, or second generation sequencing. High-throughput sequencing, which can be performed by passing one sample through multiple target regions or multiple samples at a time, has also gained increasing attention in clinical applications, including pharmacogenomics, genetic disease research and screening, tumor mutation gene detection, and clinical microbial detection. The whole high-throughput sequencing process comprises the steps of nucleic acid extraction, library construction, sequencing, data analysis and the like of a sample. Firstly, before high-throughput sequencing, constructing a library for an extracted nucleic acid sample, namely, carrying a specific fragment which can be identified by a sequencer on the sample so as to facilitate template preparation of the sample, identification by the sequencer and sequencing; and secondly, a sample specificity label is carried on the sample to distinguish different samples, so that a plurality of samples can be sequenced at the same time, and the sequencing cost of the sample is reduced.
The main approaches to library construction today are hybrid capture and multiplex PCR.
Hybrid capture refers to the process of specific binding to a target region and then capture by designing a pool of probes that specifically recognize the target region. The hybrid capture process comprises: nucleic acid fragmentation-end repair-ligation and linker-amplification-pre-library purification-target region capture-library amplification-purification and the like. The library building method has long process, and the whole process needs more than 24 hours; the operation is complex, and the requirement on the capability of operators is high due to a plurality of fine steps. Therefore, hybrid capture is relatively difficult for library construction on large scale samples.
The multiplex PCR library building method is a scalable application of PCR technology. The operation flow of the currently used multiplex PCR library building method comprises the following steps: PCR, partial primer digestion, ligation and adapter addition, sample purification and the like. The operation is relatively simple, and the experimenter can master the method easily. However, in the operation process, one or more steps of uncovering exist after the PCR is finished so as to carry out subsequent operation, and a sample is easily polluted by environmental aerosol; meanwhile, aerosol generated after sample PCR may also pollute subsequent detection samples; in addition, the development of multiplex PCR is difficult, and in addition to the specificity of primers, the problems of primer mismatching and primer dimer formation need to be considered, and the more primers, the higher the possibility of forming mismatching or dimer.
In the operation process of hybridization or multiplex PCR, uncapping operation after tube rotation or PCR exists, and the possibility of cross contamination among samples exists; moreover, each sample requires a multi-step operation, which is complicated and difficult to handle with a large number of samples.
In light of the above problems, the present invention is directed to the study of a high throughput sequencing-based HPV detection method.
Disclosure of Invention
The invention aims to provide an HPV detection method based on high-throughput sequencing and application thereof, and solves the problems of easy sample pollution, inaccurate detection result, large sample amount and the like in the sample detection process by using a multiple PCR method.
The technical scheme adopted by the invention is as follows:
in the first aspect of the invention, a multiple PCR primer group for detecting HPV based on high-throughput sequencing is provided, wherein each multiple primer consists of a bridging sequence and specific sequences targeting different types of HPV; the bridging sequence matches with the adaptor primer; the nucleotides of the multiplex PCR primer set are as follows:
the nucleic acid sequence of the multiplex PCR primer group is numbered as follows: F001-F011 are numbered as SEQ ID NO. 1-SEQ ID NO. 11; the numbers of R001-R012 are SEQ ID NO. 12-SEQ ID NO. 23.
In a second aspect of the invention, a kit for detecting HPV based on high-throughput sequencing is provided, which comprises the multiplex PCR primer set and the adapter primer.
According to the embodiment of the invention, the adaptor primer comprises the following components from 5 'end to 3' end in sequence: a sequencer related universal sequence, an 8bp sample tag sequence and a bridging sequence matched with the 5' end of the multiplex PCR primer.
According to the embodiment of the invention, the adaptor primer and the multiplex PCR primer are connected by matching bridging sequences, and the nucleotide sequences are as follows:
according to the embodiment of the invention, the 8bp tag sequence can be replaced by a different 8bp tag sequence or a sample tag of 6-12bp in the example, so as to increase the number of tags and further expand the detection amount of the sample.
According to an embodiment of the invention, the nucleic acid sequence is numbered: P5-01-P5-08 is numbered as SEQ ID NO. 24-SEQ ID NO. 31; P7-01-P7-12 are numbered as SEQ ID NO. 32-SEQ ID NO. 43.
In a fourth aspect of the present invention, a method for detecting HPV based on high throughput sequencing is provided, comprising the following steps: extracting sample genome DNA, carrying out PCR amplification reaction by using the primer group and the primers, purifying an amplification product to construct a library, and sequencing to detect whether HPV nucleic acid exists in the sample.
According to an embodiment of the present invention, the nucleic acid sequence of the multiplex PCR primer sequence of the reference gene GADPH:
F-control CTGGAGTTCAGACGTGTGCTCTTCCGATCGCTTGCCCTGTCCAGTTA（SEQ ID NO.44）；
R-control TCTTTCCCTACACGACGCTCTTCCGATCTGCCAGGCTGGCCTGGCT（SEQ ID NO.45）。
according to the embodiment of the invention, the PCR amplification system is as follows:
according to an embodiment of the present invention, the PCR amplification procedure is:
in accordance with an embodiment of the present invention,
the preparation method of the primer library comprises the following steps: diluting the multiple PCR primer group to 50-200 mu M and mixing to obtain the primer group;
the sequence of the P5 joint is shown as SEQ ID NO. 24-SEQ ID NO. 31;
the sequence of the P7 joint is shown in SEQ ID NO. 32-SEQ ID NO. 43.
According to an embodiment of the present invention, the purification and construction of the library are performed as follows:
1) after the PCR reaction is finished, combining PCR extension products obtained by the PCR reaction, and purifying and concentrating to 30-100 mu L by using a nucleic acid purification kit;
2) adding 0.6-0.8 times volume of AMpure XP magnetic beads (capable of purifying nucleic acid or screening fragments), uniformly mixing, standing at room temperature for 2-5 min, putting on a magnetic frame, and washing with 150-200 mu L of fresh 70-80% ethanol for 2-3 times after the magnetic beads are completely adsorbed; after the magnetic beads are completely adsorbed, completely adsorbing residual ethanol by using a pipettor, drying at room temperature, adding 30-50 mu L of TE for elution, and standing at room temperature for 2-5 min;
3) transferring the supernatant to a new EP tube after adsorption by an upper magnetic frame, adding AMpureXP magnetic beads with the same volume, uniformly mixing, standing at room temperature for 2-5 min, loading the supernatant on the magnetic frame, and after the magnetic beads are completely adsorbed, absorbing and discarding the supernatant; washing with 150-200 mu L of freshly prepared 70-80% ethanol for 2-3 times, centrifuging for a short time, then loading on a magnetic frame, completely adsorbing magnetic beads, completely adsorbing residual ethanol by using a pipettor, drying at room temperature, adding 20-50 mu L of TE for elution, and standing at room temperature for 2-5 min;
4) and (4) loading the magnetic rack, transferring the supernatant to a new EP tube after the magnetic beads are completely adsorbed, and obtaining the supernatant which is the required library.
The invention has the beneficial effects that:
the method adopts a tubular one-step method to complete library construction, and the library construction can be completed through one round of PCR; meanwhile, double-end sample labels are added to the corresponding libraries of each sample, so that different samples can be distinguished, and the samples are prevented from being interfered with each other. The coverage is wide, and up to 40 HPV types can be simultaneously detected. The specificity is strong, in the existing methodology, if the wind intensity of a certain HPV type is extremely high, the signals of the types with low abundance are covered, and the result interpretation is not accurate enough. According to the invention, the result is interpreted through second-generation sequencing, and each type of signal can be found, so that the result is more accurate. HPV is a very large item to be detected. The detection method has the advantages of multiple HPV types, high sensitivity, complete and accurate detection of all types of HPV covered and high cost of manpower and material resources.
The method established by the invention can complete library construction of 96 samples in 5 hours by one-step amplification and purification, is simple and convenient to operate, and can easily realize detection of a large number of samples. By increasing the number of sample tags in the adapter primer, the number of sample detections that can be performed in one sequencing run can be increased. For example, 96 samples can be detected at a time with 20 sample tags (8P 5 adapter primers + 12P 7 adapter primers); 384 samples can be detected if 40 sample tags (16P 5 adapter primers + 24P 7 adapter primers) are used at a time; if it is 60 sample tags (24P 5 adapter primers + 36P 7 adapter primers), 864 samples can be detected at a time, so the present invention can further reduce the average manual handling time of a single sample.
Drawings
FIG. 1 is a library quality control map in a clinical specimen identity test.
Detailed Description
In order to better illustrate the technical solution of the present invention, the following description is made with reference to the embodiments and the accompanying drawings, but not limited thereto.
Test materials:
QIAamp DNA Blood Mini Kit was purchased from Qiagen corporation management (Shanghai) Inc.; the nucleic acid purification kit is purchased from bio-engineering (Shanghai) GmbH; PCR MixPratinum ™ Multiplex PCR Master Mix was purchased from Saimer Feishale science (China) Co., Ltd., and AMpureXP magnetic beads were purchased from Beckmann Kulter trade (China) Co., Ltd.
Example 1 primer design
Primer design comprises multiplex PCR primers and adapter primers.
The multiplex PCR primers contain specific sequences and bridging sequences. The specific sequences are designed aiming at 40 types of HPV, and are degenerated primers, and the annealing temperature is controlled to be 58-62 ℃; the bridge sequence is a portion which is the same for all the forward primers or the reverse primers, and is mainly a portion which is combined with the bridge sequence of the adaptor primer, and the length of the bridge sequence is adjusted according to actual conditions.
The joint primer comprises the following components from the 5 'end to the 3' end in sequence: a sequencer-related universal sequence, an 8bp sample tag sequence, and a bridging sequence matched with the 5' end of the multiplex PCR primer. The sample tags of each adapter primer band are different, and the annealing temperature of the bridging sequence part is 68-72 ℃. The 8bp sample label in the adapter primer can be replaced to meet the requirement of large sample amount; the specific part of the internal reference primer is designed aiming at the GADPH gene of the human genome. The adaptor primer is connected with the multiplex PCR primer through the matching of the bridging sequence.
In the example, there are 23 PCR primers obtained by the above design method, the sequences of the primers are shown as SEQ ID NO. 1-SEQ ID NO.23, and the PCR primers need to be diluted to 100. mu.M before the test and mixed into a primer library at equal volume ratio.
20 adaptor primers, comprising 8P 5 sample tags and 12P 7 sample tags, as follows:
nucleotide sequence of internal reference gene:
F-control CTGGAGTTCAGACGTGTGCTCTTCCGATCGCTTGCCCTGTCCAGTTA（SEQ ID NO.44）；
R-control TCTTTCCCTACACGACGCTCTTCCGATCTGCCAGGCTGGCCTGGCT（SEQ ID NO.45）。
EXAMPLE 2 Artificial Synthesis of plasmid specificity and sensitivity test
Artificially synthesizing 7 plasmids: HPV16, HPV18, HPV31, HPV33, HPV35, HPV58, HPV 68. 3 test concentrations are set, 10 respectively5Copy/test, 103Copy/test, 102Copy/test. Three positive controls (all mixtures of plasmids such as HPV16, HPV18, HPV31, HPV33, HPV58, HPV35, HPV68, and the like, and human genomic DNA, PC-1 (200 copies/test), PC-2 (1000 copies/test), PC-3 (5000 copies/test), and one Negative Control (NC) were set for a total of 25 samples, with the adapter primer set as shown in Table 1:
TABLE 1 linker primer set-up
Preparing a PCR amplification reaction system:
setting a PCR amplification reaction program:
constructing a library:
1) after the PCR reaction is finished, merging samples, and purifying and concentrating the samples to 50 mu L by using a nucleic acid purification kit;
2) adding 35 μ L LAMpure XP magnetic beads, mixing, standing at room temperature for 3min, loading on a magnetic rack, and washing with 150 μ L of freshly prepared 80% ethanol for 2 times after the magnetic beads are completely adsorbed; after the magnetic beads are completely adsorbed, completely adsorbing residual ethanol by using a pipettor, drying at room temperature, adding 30 mu L of TE for elution, and standing at room temperature for 2-5 min;
3) transferring the supernatant to a new EP tube after the supernatant is adsorbed by the magnetic frame, adding 30 mu LAMpureXP magnetic beads, uniformly mixing, standing at room temperature for 3min, loading the supernatant on the magnetic frame, and after the magnetic beads are completely adsorbed, absorbing and discarding the supernatant; washing with 150 μ L of freshly prepared 80% ethanol for 2 times, centrifuging for a short time, loading onto magnetic frame, completely adsorbing magnetic beads, removing residual ethanol with pipette, drying at room temperature, eluting with 30 μ L of TE, and standing at room temperature for 3 min;
4) and (4) loading the magnetic rack, transferring the supernatant to a new EP tube after the magnetic beads are completely adsorbed, and obtaining the supernatant which is the required library.
Sequencing by using an Illumina MiniSeq medium flux sequencing kit, wherein the sequencing data cluster density is 148K/mm2The cluster passage rate was 82.7% and Q30 was 90.8%, actually yielding 0.6M reads.
And (4) analyzing results: the results are shown in Table 2, all types of the three positive samples have reads, and the number of the reads of the negative samples is 0; of the 21 samples, there was good specificity and exhibited good gradient effect; can be detected for 100 copies of the plasmid.
Table 2 results of each sample after data analysis
Note: + indicates positive; negative is indicated.
Example 3 clinical sample conformance testing
Taking 92 clinical cervical swab samples, detecting the samples to be positive HPV by a fluorescence PCR method, extracting genomic DNA by using a QIAamp DNA blood mini kit, performing 1 test on each sample, performing 92 tests in total, and sequentially marking the samples as 1-92 samples; three positive controls, PC-1 (200 copies/test), PC-2 (1000 copies/test), PC-3 (5000 copies/test), and one Negative Control (NC), were set up for 96 samples. The assay was completed in a 96-well plate with the adapter primer set as shown in table 3:
TABLE 3 linker primer set-up
Preparing a PCR amplification reaction system:
setting a PCR amplification reaction program:
constructing a library:
1) after the PCR reaction is finished, merging samples, and purifying and concentrating the samples to 50 mu L by using a nucleic acid purification kit;
2) adding 35 μ L LAMpure XP magnetic beads, mixing, standing at room temperature for 3min, loading on a magnetic rack, and washing with 150 μ L of freshly prepared 80% ethanol for 2 times after the magnetic beads are completely adsorbed; after the magnetic beads are completely adsorbed, completely adsorbing residual ethanol by using a pipettor, drying at room temperature, adding 30 mu L of TE for elution, and standing at room temperature for 2-5 min;
3) transferring the supernatant to a new EP tube after the supernatant is adsorbed by the magnetic frame, adding 30 mu L AMpureXP magnetic beads, uniformly mixing, standing at room temperature for 3min, loading the supernatant on the magnetic frame, and after the magnetic beads are completely adsorbed, absorbing and discarding the supernatant; washing with 150 μ L of freshly prepared 80% ethanol for 2 times, centrifuging for a short time, loading onto magnetic frame, completely adsorbing magnetic beads, removing residual ethanol with pipette, drying at room temperature, eluting with 30 μ L of TE, and standing at room temperature for 3 min;
4) and (4) loading the magnetic rack, transferring the supernatant to a new EP tube after the magnetic beads are completely adsorbed, and obtaining the supernatant which is the required library.
The purified library was subjected to quality inspection using Perkin Elmer LabChip GX Touch HT, and the results are shown in FIG. 1.
Sequencing by using an Illumina MiniSeq medium flux sequencing kit, wherein the sequencing data cluster density is 110K/mm2The cluster passage was 95.2% and Q30 was 96.7%, yielding 1.9M reads.
And (4) analyzing results: as shown in Table 4, the results of this experiment showed good agreement with the fluorescent PCR method. In 1 of the samples (sample 43), the NGS method of this patent showed less type 81 positivity. In 7 cases (samples 46,48,49,51,52,57, 81), the NGS method showed more significant positive detection of HPV types on the basis of the detection of the same results as the PCR method.
TABLE 4PCR assay and NGS assay results
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Guangzhou Mijing Gene medicine science and technology Co., Ltd
<120> HPV detection multiplex PCR primer set, kit and method based on high-throughput sequencing
<130>
<160> 45
<170> PatentIn version 3.5
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<211> 69
<212> DNA
<213> Artificial sequence
<400> 34
aatgatacgg cgaccaccga gatctacaca gtcactaaca ctctttccct acacgacgct 60
cttccgatc 69
<210> 35
<211> 69
<212> DNA
<213> Artificial sequence
<400> 35
aatgatacgg cgaccaccga gatctacaca tcctgtaaca ctctttccct acacgacgct 60
cttccgatc 69
<210> 36
<211> 69
<212> DNA
<213> Artificial sequence
<400> 36
aatgatacgg cgaccaccga gatctacaca ttgaggaaca ctctttccct acacgacgct 60
cttccgatc 69
<210> 37
<211> 69
<212> DNA
<213> Artificial sequence
<400> 37
aatgatacgg cgaccaccga gatctacacc aaccacaaca ctctttccct acacgacgct 60
cttccgatc 69
<210> 38
<211> 69
<212> DNA
<213> Artificial sequence
<400> 38
aatgatacgg cgaccaccga gatctacacg actagtaaca ctctttccct acacgacgct 60
cttccgatc 69
<210> 39
<211> 69
<212> DNA
<213> Artificial sequence
<400> 39
aatgatacgg cgaccaccga gatctacacc aatggaaaca ctctttccct acacgacgct 60
cttccgatc 69
<210> 40
<211> 69
<212> DNA
<213> Artificial sequence
<400> 40
aatgatacgg cgaccaccga gatctacacc acttcgaaca ctctttccct acacgacgct 60
cttccgatc 69
<210> 41
<211> 69
<212> DNA
<213> Artificial sequence
<400> 41
aatgatacgg cgaccaccga gatctacacc agcgttaaca ctctttccct acacgacgct 60
cttccgatc 69
<210> 42
<211> 69
<212> DNA
<213> Artificial sequence
<400> 42
aatgatacgg cgaccaccga gatctacacc ataccaaaca ctctttccct acacgacgct 60
cttccgatc 69
<210> 43
<211> 69
<212> DNA
<213> Artificial sequence
<400> 43
aatgatacgg cgaccaccga gatctacacc cagttcaaca ctctttccct acacgacgct 60
cttccgatc 69
<210> 44
<211> 47
<212> DNA
<213> Artificial sequence
<400> 44
ctggagttca gacgtgtgct cttccgatcg cttgccctgt ccagtta 47
<210> 45
<211> 46
<212> DNA
<213> Artificial sequence
<400> 45
tctttcccta cacgacgctc ttccgatctg ccaggctggc ctggct 46
Claims (6)
1. The multiplex PCR primer group for detecting HPV based on high-throughput sequencing is characterized in that each multiplex primer consists of a bridging sequence and a specific sequence targeting different types of HPV; the nucleotide sequences of the multiplex PCR primer set are as follows:
。
2. A multiplex PCR kit for detecting HPV based on high-throughput sequencing, comprising the multiplex PCR primer set of claim 1 and a linker primer;
wherein the joint primer comprises the following components from 5 'end to 3' end in sequence: a universal sequence, a sample tag sequence, and a bridging sequence; the sequence of the joint primer is shown as SEQ ID NO. 24-SEQ ID NO. 43.
3. The method for using the multiplex PCR kit according to claim 2, comprising the steps of: extracting sample genome DNA, carrying out PCR amplification reaction by using the multiplex PCR primer group of claim 1 and the adaptor primer of claim 2, purifying an amplification product to obtain a constructed library, and determining the HPV type in the sample through sequencing and data analysis;
wherein, the method of use is not suitable for the diagnosis and treatment of diseases.
4. The use method according to claim 3, wherein the PCR amplification reaction system is:
the preparation method of the primer library comprises the following steps: diluting the multiplex PCR primer group of claim 1 to 50-200 μ M and mixing;
the sequence of the P5 joint is shown as SEQ ID NO. 24-SEQ ID NO. 31;
the sequence of the P7 joint is shown in SEQ ID NO. 32-SEQ ID NO. 43.
5. The use of claim 3, wherein the PCR amplification procedure is:
6. the use of claim 3, wherein the purification and library construction steps are as follows:
1) after PCR reaction, combining PCR amplification products obtained by the PCR reaction, and concentrating to 30-100 mu L;
2) adding 0.6-0.8 times of AMpure XP magnetic beads, mixing uniformly, adsorbing the magnetic beads until the solution is clarified, discarding the supernatant, retaining the magnetic beads, adding ethanol, adsorbing the magnetic beads until the solution is clarified, volatilizing the ethanol, retaining the magnetic beads, and eluting with TE;
3) transferring the supernatant to a new EP tube, adding an AMpure XP magnetic bead with the same volume,
4) mixing, adsorbing magnetic beads until the solution is clear, discarding the supernatant, retaining the magnetic beads, adding ethanol, adsorbing the magnetic beads until the solution is clear, volatilizing the ethanol, adding TE for elution, adsorbing the magnetic beads until the solution is clear, and obtaining the supernatant as a library.
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| CN112029842B (en) * | 2020-08-31 | 2021-04-27 | 深圳市血液中心(深圳市输血医学研究所) | Kit and method for ABO blood type genotyping based on high-throughput sequencing |
| CN112251501B (en) * | 2020-10-28 | 2023-07-25 | 深圳人体密码基因科技有限公司 | Internal reference gene set, screening method thereof, universal primer group, kit, reaction system and application |
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