HK1209797A1 - Primer set, kit and their use in whole genome detection of hpv - Google Patents

Abstract

The present invention discloses a set of primers comprising at least 2 pairs selected from the 14 pairs of primers of SEQ ID NOs: 1-28. This set of primers can specifically bind to different types of HPV and can be used to amplify the complete genome sequences of various types of HPV. The present invention also discloses the use of the set of primers, a kit, the use of the kit, and a method for obtaining the complete HPV gene sequence.

Description

Primer group, kit and application of primer group and kit in HPV whole genome detection

Technical Field

The invention relates to the field of virus detection, in particular to a group of primers, application of the group of primers in amplification of HPV genome and/or detection of HPV, a kit, application of the kit in amplification of HPV genome, HPV typing and/or detection of HPV, and a method for obtaining HPV whole genome sequence.

Background

Cervical cancer is the third most common female tumor in the world, and 86% of cases occur in developing countries [ Arbyn M, Castellsague X, de Sanjose S, Bruni L, Saraiya M, Bray F, FerlayJ: Worldwide garden of cervicalcancer in 2008.Ann Oncol2011,22: 2675-. According to the statistics in the world, about 46 million new cases occur every year, more than 20 million women die of cervical cancer, 75-80% of the worldwide annual onset is in developing countries, more than 13 million new cases occur every year in China due to numerous population, the number accounts for 25% of the worldwide onset, and the new cases are in a growing trend in recent years, the trend of increasing in vast rural areas is more obvious, and the onset age is advanced [ Kailibaru Eima, Ma Cai Ling.

ZurHause in 1977 first proposed the hypothesis that Human Papillomavirus (HPV) is the causative agent of cervical cancer; in 1992, the World Health Organization (WHO) announced that human papillomavirus was the leading cause of cervical carcinogenesis; in 1995, the international agency for research on cancer (IARC) topic discussion identified HPV infection as the major cause of cervical cancer, which was identified as an infectious disease. HPV infection is a major factor in the development of cervical cancer.

Human papillomaviruses are a group of DNA viruses within the A subgroup of the Papovaviridae (Papovaviridae) family, non-enveloped, and having a relative molecular mass of 5X 10⁶. The genome is double-stranded and closed-loop, and contains 7900 pairs of bases. Human papillomaviruses are a worldwide, widespread DNA virus that primarily infects human mucous membranes and skin. The study showed that HPV virus could be detected in 99.7% of cervical cancer tissues.

HPV double-stranded circular genomic DNA consists essentially of four parts, an early region, a late region, an upstream regulatory region and a very short, high mutation rate noncoding region between E5 and L2. Papilloma virus evolves slowly at 2. + -. 0.5X 10 per year^-8The frequency of individual base mutations was determined. At the same time, the evolution process also exhibits tissue and host specificity,HPV genomic polymorphisms were caused to occur only randomly during evolution within a few virus subtypes of each type [ Bernard H U, Burk R D, Chen Z, et al.Classification of Papillomavir (PVs) base on 189PV types and propofol soft inorganic references (J.) Virology,2010,401(1):70-79.]。

The existing research shows that the HPV pathogenic molecular mechanism is the replication and expression of virus genome DNA, wherein the early gene regions E1, E2, E4, E5, E6 and E7 transcription are the key steps of HPV proliferation in host cells [ IARC Working group to human. IARC monoclonal on the Evaluation of clinical trials to human beings [ M ] World Health Organization,2007 ], the research on HPV and cervical carcinoma occurrence is limited to high-risk HPV types, and few subtypes are discussed to cause carcinogenicity.

HPV detection is widely used for the study of the natural history and etiology of HPV-associated malignant lesions. The traditional method mainly detects the protein through morphology and immunology. The former includes pap smear cytology examination, colposcopy, cervical biopsy histopathology examination, electron microscopy (direct observation of virus particles), cervical photography examination, cervical fluoroscopy and the like. The latter includes detection of HPV by reaction of anti-L1 protein with coat protein using immunohistochemical method, determination of HPV16 antibody levels in the serum of cervical epithelial neoplasia (CIN) and cervical cancer patients using radioimmunoprecipitation method, detection of HPV E6, E7 specific antibody proteins in the serum using serum immunoadsorption assay (ELISA) [ Sehr P, Zumbach K, Pawlita M. Ageneric capture ELISAfor recombinant proteins fused to glutathione S-transferase: validation of HPV for use in the technology [ J ] Journal of immunological methods,2001,253(1):153-162 ]. The specificity and sensitivity of the traditional method are not ideal enough, high false negative rate and false positive rate exist, and HPV is not convenient to be typed, so that the main experiment or diagnosis technology of the HPV at present is to apply a molecular biological method to detect HPV-DNA.

The HPV-DNA detection methods currently used are mainly based on the currently common types of detection using the polymerase chain reaction including: universal primer PCR, Type-Specific PCR (Type-Specific PCR), and real-time fluorescent quantitative PCR. However, these methods are based on typing detection only on a certain part of genome region (mostly about 100-500 bp) of different subtypes.

Disclosure of Invention

The present invention is directed to at least one of the technical problems of the prior art or at least to a business method.

According to a first aspect of the present invention, there is provided a set of primers capable of specifically binding to different types of HPV, the set of primers comprising a sequence selected from the group consisting of SEQ ID NO: 1-28, and the sequences of said 14 pairs of primers are as shown in SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, SEQ ID NO: 21 and 22, SEQ ID NO: 23 and 24, SEQ ID NO: 25 and 26 and SEQ ID NO: 27 and 28. In some embodiments of the invention, the primer set comprises a primer selected from the group consisting of SEQ ID NOs: 1-28, at least 3, at least 4, at least 5, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all 14 of the 14 pairs of primers. In one embodiment of the present invention, the set of primers further comprises a primer having a sequence as set forth in SEQ ID NO: 29 and 30. The primer group is determined by the inventor through a large number of sequence design and a large number of test screening combinations, and can specifically identify the HPV of different types respectively, and the primer group can be specifically combined to respective target templates without mutual interference in the same reaction system, so that the HPV whole genome sequences of a plurality of types can be specifically amplified in the same reaction system.

According to a second aspect of the invention, there is provided the use of a set of primers of any one of the preceding embodiments in the amplification of the HPV genome and/or detection of HPV. Using the set of primers in one aspect or any embodiment of the invention, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and/or HPV66 can be amplified and genotyped. The method can not only quickly detect the HPV virus, but also analyze various characteristics of the genome of the HPV virus.

According to a third aspect of the invention, there is provided a kit comprising a set of primers as in any one of the preceding embodiments. The primers contained in the kit are primers which are determined by the inventor through a large number of sequence design and a large number of test screening combinations and can respectively and specifically identify different types of HPV, and the primers can be specifically combined to respective target templates without mutual interference in the same reaction system, so that the whole genome sequences of multiple types of HPV are amplified in the same reaction system.

According to a fourth aspect of the invention, the invention provides the use of the aforementioned kit in HPV genomic amplification, HPV typing and/or HPV detection. The kit of the invention can be used for carrying out whole-gene amplification and whole-gene typing on HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and/or HPV66 by using the primers contained in the kit. The method can not only quickly detect the HPV virus, but also analyze various characteristics of the genome of the HPV virus.

The detection method based on the complete genome information of the HPV virus is more favorable for identification and typing detection of the HPV virus, detection of the inter-type variants, prediction of the development of the cervical cancer patient and better auxiliary guidance of clinical strategies. Moreover, the HPV viral genome technology can also provide more effective opinions on development of HPV vaccines and prevention and treatment of HPV infection, and with the deep knowledge of the HPV viral genome, the HPV whole genome technology can possibly help to provide personalized medical opinions for patients. The invention provides a method for detecting HPV whole genome, aiming at the lack of a method for detecting HPV whole genome at present.

According to a fifth aspect of the present invention, there is provided a method for obtaining a complete HPV genome sequence, the method comprising the steps of: obtaining nucleic acid from a test sample; amplifying the nucleic acid using a set of primers according to one aspect or any embodiment of the invention described above to obtain an amplification product; performing sequence determination on the amplification product to obtain sequencing data, wherein the sequencing data comprises a plurality of reads; based on the sequencing data, HPV whole genome sequences were obtained. The sequencing data may be obtained by performing sequencing library preparation and machine sequencing on the nucleic acid sequence, and in one embodiment of the present invention, the obtaining of the sequencing data includes: preparing a sequencing library of the nucleic acid, and sequencing the sequencing library. The preparation method of the sequencing library is carried out according to the requirements of the selected sequencing method, the sequencing method can be selected according to different sequencing platforms, but is not limited to Hisq2000/2500 sequencing platform of Illumina, Ion Torrent platform of Life technologies and single-molecule sequencing platform, the sequencing mode can be single-ended sequencing or double-ended sequencing, and the obtained off-line data is a sequencing read fragment called reads. In one embodiment of the invention, after obtaining the amplification product, comprises: and (3) carrying out electrophoretic detection on the amplification product, and carrying out HPV typing according to the obtained electrophoretic detection result, specifically, a universal primer such as SEQ ID NO: 29 and 30, performing a first amplification of the nucleic acid obtained, electrophoretically detecting whether the first amplification has a product, i.e. whether the electrophoretogram has a band of the amplification product, wherein the band indicates that the nucleic acid of the sample contains HPV virus, and then performing a subsequent amplification using the nucleic acid sequence of SEQ ID NO: 1-28, and determining the HPV type contained in the sample nucleic acid based on the results of electrophoretic detection of the amplification product of the second amplification of the nucleic acid obtained by the second amplification of any of the 14 primer pairs. In one embodiment of the present invention, the obtaining of HPV whole genome sequence based on sequencing data comprises: and comparing the reading in the sequencing data with the HPV reference sequence to obtain a comparison result, and assembling the HPV whole genome sequence according to the comparison result. The reference sequence refers to a predetermined sequence, and may be any reference template of a biological category to which a nucleic acid to be detected belongs, which is obtained in advance, for example, HG19 provided by the NCBI database may be selected as the reference sequence if the nucleic acid to be detected is a human nucleic acid, or a sequence of a virus existing in the public database may be selected as the reference sequence if the target nucleic acid to be detected is a viral nucleic acid, or a resource library including more reference sequences may be configured in advance, for example, a more similar sequence may be selected or determined to be assembled as the reference sequence according to factors such as the source and region of the nucleic acid to be detected. The alignment can be performed using known alignment software, such as SOAP, BWA, and/or TeraMap, among others. In some embodiments of the invention, the HPV reference sequence comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all fourteen of an HPV16 reference sequence, an HPV18 reference sequence, an HPV31 reference sequence, an HPV33 reference sequence, an HPV35 reference sequence, an HPV39 reference sequence, an HPV45 reference sequence, an HPV51 reference sequence, an HPV52 reference sequence, an HPV56 reference sequence, an HPV58 reference sequence, an HPV59 reference sequence, an HPV66 reference sequence, and an HPV68 reference sequence. Assembly can be performed using the position of the aligned HPV reference sequence reads on the reference sequence and the overlap information between the reads.

In one embodiment of the invention, based on the comparison result, the mutation sites on the HPV nucleic acid sequence can be identified and detected, and the information such as the mutation frequency of the mutation sites is added to the assembled HPV whole genome sequence, so that the assembled HPV whole genome sequence has the mutation site information. Therefore, the HPV genome information is enriched, the identification and typing detection of HPV virus which is more complete and accurate later are facilitated, the more accurate detection of the intertype virus variant is facilitated, and the auxiliary prediction of the disease development of cervical cancer patients and the auxiliary guidance of clinical adopted treatment strategies are facilitated.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a diagram showing the result of electrophoresis for detecting an amplification product in one embodiment of the present invention.

FIG. 2 is a diagram showing the result of electrophoresis in detecting an amplification product in one embodiment of the present invention.

FIG. 3 is a diagram illustrating an alignment situation according to an embodiment of the present invention.

Detailed Description

According to one embodiment of the present invention, a set of primers is provided, the set of primers comprising a sequence selected from the group consisting of SEQ ID NOs: 1-28, and the sequences of said 14 pairs of primers are as shown in SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, SEQ ID NO: 21 and 22, SEQ ID NO: 23 and 24, SEQ ID NO: 25 and 26 and SEQ ID NO: 27 and 28. The group of primers are primers which are determined by the inventor through a large number of sequence design and a large number of test screening combinations and can respectively and specifically identify different types of HPV, and the group of primers can be specifically combined to respective target templates without mutual interference in the same reaction system, so that the whole genome sequences of multiple types of HPV are amplified in the same reaction system. Furthermore, only one pair of primers listed is required to obtain the complete genomic sequence of one type of HPV.

In some embodiments of the invention, the primer set comprises a primer selected from the group consisting of SEQ ID NOs: 1-28, at least 3, at least 4, at least 5, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all 14 of the 14 pairs of primers. The group of primers are primers which are determined by the inventor through a large number of sequence design and a large number of test screening combinations and can respectively and specifically identify different types of HPV, and the group of primers can be specifically combined to respective target templates without mutual interference in the same reaction system, so that the whole genome sequences of multiple types of HPV are amplified in the same reaction system. Furthermore, only one pair of primers designed can be used to obtain the complete genomic sequence of one type of HPV.

In one embodiment of the present invention, the set of primers further comprises a primer having a sequence as set forth in SEQ ID NO: 29 and 30. The group of primers are primers which are determined by the inventor through a large number of sequence design and a large number of test screening combinations and can respectively and specifically identify different types of HPV, and the group of primers can be specifically combined to respective target templates without mutual interference in the same reaction system, so that the whole genome sequences of multiple types of HPV are amplified in the same reaction system. Furthermore, only one pair of primers designed can be used to obtain the complete genomic sequence of one type of HPV. SEQ ID NO: 1-30 are shown in table 1.

TABLE 1

According to one embodiment of the invention, there is provided a use of a set of primers of any of the preceding embodiments in amplification of an HPV genome and/or detection of HPV. Using the set of primers in any of the preceding embodiments of the invention, it is possible to perform whole gene amplification and whole gene typing on HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and/or HPV 66. The method can not only quickly detect the HPV virus, but also analyze various characteristics of the genome of the HPV virus.

According to an embodiment of the present invention, there is provided a kit comprising a set of primers of any one of the preceding embodiments. The primers contained in the kit are primers which are determined by the inventor through a large number of sequence design and a large number of test screening combinations and can respectively and specifically identify different types of HPV, and the primers can be specifically combined to respective target templates without mutual interference in the same reaction system, so that the whole genome sequences of multiple types of HPV are amplified in the same reaction system.

According to one embodiment of the invention there is provided the use of a kit as in any preceding embodiment in HPV genomic amplification, HPV typing and/or HPV detection. The kit in any one of the preceding embodiments can be used for carrying out whole-gene amplification and whole-gene typing on HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 and/or HPV66 by using the primers contained in the kit. The method can not only quickly detect the HPV virus, but also analyze various characteristics of the genome of the HPV virus.

The detection method based on the complete genome information of the HPV virus is more favorable for identification and typing detection of the HPV virus, detection of the inter-type variants, prediction of the development of the cervical cancer patient and better auxiliary guidance of clinical strategies. Moreover, the HPV viral genome technology can also provide more effective opinions on development of HPV vaccines and prevention and treatment of HPV infection, and with the deep knowledge of the HPV viral genome, the HPV whole genome technology can possibly help to provide personalized medical opinions for patients. The invention provides a detection method of HPV whole genome, aiming at the lack of a detection method of HPV whole genome.

Detecting the HPV whole genome, including obtaining the HPV whole genome sequence, according to one embodiment of the invention, a method for obtaining the HPV whole genome sequence is provided, which comprises the following steps: obtaining nucleic acid from a test sample; amplifying the nucleic acid using a set of primers of any of the embodiments of the invention described above to obtain an amplification product; performing sequence determination on the amplification product to obtain sequencing data, wherein the sequencing data comprises a plurality of reads; based on the sequencing data, HPV whole genome sequences were obtained. The sequencing data may be obtained by performing sequencing library preparation and machine sequencing on the nucleic acid sequence, and in one embodiment of the present invention, the obtaining of the sequencing data includes: preparing a sequencing library of the nucleic acid, and sequencing the sequencing library. The preparation method of the sequencing library is carried out according to the requirements of the selected sequencing method, the sequencing method can be selected according to different sequencing platforms, but is not limited to Hisq2000/2500 sequencing platform of Illumina, Ion Torrent platform of Life Technologies and single-molecule sequencing platform, the sequencing mode can be single-ended sequencing or double-ended sequencing, and the obtained off-line data is a sequencing and reading fragment, which is called reads (reads).

In one embodiment of the invention, after obtaining the amplification product, comprises: and (3) carrying out electrophoretic detection on the amplification product, and carrying out HPV typing according to the obtained electrophoretic detection result, specifically, a universal primer such as SEQ ID NO: 29 and 30, performing a first amplification of the obtained nucleic acid, electrophoretically detecting whether the first amplification has a product, i.e. whether the electrophoretogram has a band of the amplification product, wherein the band indicates that the nucleic acid of the sample contains HPV virus, and then performing a subsequent amplification using the nucleic acid sequence of SEQ ID NO: 1-28, and determining the HPV type contained in the sample nucleic acid based on the results of electrophoretic detection of the amplification product of the second amplification of the nucleic acid obtained by the second amplification of any of the 14 primer pairs.

In one embodiment of the present invention, the obtaining of HPV whole genome sequence based on sequencing data comprises: and comparing the reading in the sequencing data with the HPV reference sequence to obtain a comparison result, and assembling the HPV whole genome sequence according to the comparison result. The reference sequence refers to a predetermined sequence, and may be any reference template of a biological category to which a nucleic acid to be detected belongs, which is obtained in advance, for example, HG19 provided by the NCBI database may be selected as the reference sequence if the nucleic acid to be detected is a human nucleic acid, or a sequence of a virus existing in the public database may be selected as the reference sequence if the target nucleic acid to be detected is a viral nucleic acid, or a resource library including more reference sequences may be configured in advance, for example, a more similar sequence may be selected or determined to be assembled as the reference sequence according to factors such as the source and region of the nucleic acid to be detected.

In some embodiments of the invention, the HPV reference sequence comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all fourteen of an HPV16 reference sequence, an HPV18 reference sequence, an HPV31 reference sequence, an HPV33 reference sequence, an HPV35 reference sequence, an HPV39 reference sequence, an HPV45 reference sequence, an HPV51 reference sequence, an HPV52 reference sequence, an HPV56 reference sequence, an HPV58 reference sequence, an HPV59 reference sequence, an HPV66 reference sequence, and an HPV68 reference sequence. Assembly can be performed using the position of the aligned HPV reference sequence reads on the reference sequence and the overlap information between the reads.

The alignment can be performed using known alignment software, such as SOAP, BWA, and/or TeraMap, among others. In one embodiment of the present invention, there is provided an HPV whole-gene detection method, comprising: based on the alignment, the mutation sites on the HPV nucleic acid sequence are identified. Software for detection of the recognition of the mutation site can utilize, but is not limited to, SOAPsnp, SOAPcnv, Varscan and GATK. In one embodiment of the present invention, based on the alignment result, information such as mutation frequency of these mutation sites is added to the assembled HPV whole genome sequence, so that the assembled HPV whole genome sequence carries the mutation site information. Therefore, the HPV genome information is enriched, the identification and typing detection of HPV virus which is more complete and accurate later are facilitated, the more accurate detection of the intertype virus variant is facilitated, and the auxiliary prediction of the disease development of cervical cancer patients and the auxiliary guidance of clinical adopted treatment strategies are facilitated.

In order to make the technical scheme and advantages of the present invention more clearly understood, a set of primers of the present invention and the use thereof in amplifying HPV whole genes, obtaining HPV whole genome sequences, and detecting HPV whole genomes are described in detail below with reference to specific embodiments. It is to be understood that the following examples are intended to illustrate, but not limit, the present invention. It should be noted that the terms "first," "second," and the like, as used herein, are used merely for convenience in description and are not to be construed as indicating or implying relative importance, nor order relationships therebetween. In the description of the present invention, "a plurality" means two or more unless otherwise specified.

Reagents, sequences (linkers, tags, and sequencing primers), software, and instruments referred to in the following examples are all conventional commercial products or open sources, such as the sequencing library construction kit purchased from Illumina, unless otherwise submitted.

Generally comprising:

A) HPV viral nucleic acid was extracted from biological samples using the commercial Kit QIAamp DNA Mini Kit (Qiagen, German).

B) Amplifying HPV whole genome fragments from the nucleic acid obtained in the step A) by using specific primers in the table 1.

C) Purifying the product obtained in the step B), mixing, and fragmenting into fragments of about 200 bp.

D) Using a library construction kit, for example, comprising filling 5 'cohesive ends with T4DNA polymerase, T4Phosphonucleotidekinase and Klenow fragment of Escherichia coli DNA polymerase and removing 3' cohesive ends; the complement was end-capped with A using d-ATP and Klenow3 '-5' Exo-enzyme; using specific tags containing custom coding sequences (8bp) amplified by high fidelity enzymatic PCR to the end of the product as products, each specific tag as a high throughput sequencing library, the tags, tag primers can be disclosed using CN 102409049A; sequencing was then performed using the standard Miseq sequencing protocol, which yielded sequences of corresponding length and 8bp index sequence per cycle.

E) And filtering the high-throughput detection original result to obtain a useless sequence and a sequence with poor quality, comparing the qualified sequence data with an HPV reference sequence, judging an HPV typing result, and obtaining various types of infected HPV genome sequences in the sample.

The method utilizes high-throughput detection and Long PCR (Long-PCR) technology to detect, sequence and/or type HPV in a biological sample. Can be used for the occurrence and development of various diseases caused by high-risk HPV infection in various biological samples and the prognosis guide or auxiliary detection of the diseases. Meanwhile, the invention can also accumulate more data for various scientific researches and provide a technical means for further determining the pathogenic mechanism of the HPV virus. The invention has high flux, short detection time, low cost and high detection specificity and sensitivity, and is a reliable method for HPV detection and research.

Example one

Sample nucleic acids were extracted using the commercial Kit QIAamp DNAMini Kit (Qiagen, German).

1. A cervical exfoliated cell sample (liquid-based, cervical sampling brush sampling) from a hospital is added into a 2ml centrifuge tube,preserving the preservation solution, centrifuging for 5min at 14000rmp, discarding the supernatant, and washing once by adding 400 μ L PBS buffer;

2. add 400. mu.L buffer, add 20. mu.L proteinase K, incubate at 56 ℃ on a constant temperature heater until complete dissolution;

3. adding 400 mu L of absolute ethyl alcohol into a centrifuge tube, whirling for 5 seconds, and centrifuging for a short time;

4. carefully transferring the liquid in the centrifugal tube to a QIAamp Mini column, centrifuging at 8000rpm for 1min, placing the QIAamp Mini column in a collection tube, and discarding the original collection tube;

5. adding 500 mu Lb Buffer AW1Buffer, covering a tube cover, centrifuging at 8000rpm for 1min, placing a QIAamp Mini column in a collection tube, and discarding the original collection tube;

6. adding 500 μ L Buffer AW2Buffer, covering the tube cover, centrifuging at 14000rpm for 3min, placing QIAamp Mini column in a collection tube, and discarding the original collection tube;

7.14000rpm for 3min, placing QIAamp Mini column in a new 1.5ml centrifuge tube, removing the collecting tube, adding 50 μ L AE buffer, incubating at room temperature for 1min, and centrifuging at 8000rpm for 1 min;

8. the QIAamp Mini column was discarded and the tube was labeled after elution to preserve the sample.

Example two

Amplification was performed using the L1 universal primer sequence.

1) L1 general amplification experiment reaction system:

0.125. mu.L of LA Taq (5U/. mu.L) (TaKaRa, RR02MB), 2.5. mu.L of 10xPCR Buffer (Mg) were used²⁺) (TaKaRa, RR02MB), 2. mu.L of dNTP mix (2.5 mM each) (TaKaRa, RR02MB), 1. mu.L of DNA (the concentration of DNA obtained in example one is required to be 10 ng/. mu.L or more), 10. mu.L of upstream and downstream primers (10. mu.M), and home-made double distilled water were mixed to prepare a 25. mu.L reaction system.

2) Reaction conditions are as follows:

using a conventional PCR instrument (Applied Biosystems,96-Well Thermal Cycler), the reaction conditions were as follows: 94 ℃ for 5minutes, (94 ℃ for 30seconds,55 ℃ for 30seconds,72 ℃ for 30 seconds) 30 cycles, 12 ℃ incubation

3) And (3) amplification results:

after electrophoresis for 30min using 1% Agarose gel (Agarose) at a voltage of 140V and a current of 75mA, the gel was stained with ethidium bromide for 10 minutes and then observed on a Tanon1600 full-automatic digital gel image analysis system. The electrophoresis result is shown in fig. 1, which comprises a marker, the PCR amplification product and a negative control from left to right, wherein the lane of the PCR amplification product shows the HPV L1 gene band, indicating that the sample is infected with HPV.

EXAMPLE III

Amplification of other HPV genomic sequences.

1) Amplification experiment reaction system:

using 0.25. mu.LLA Taq (5U/. mu.L) (TaKaRa, RR02MB), 2.5. mu.L 10x PCRbuffer (Mg)²⁺Plus) (TaKaRa, RR02MB), 4. mu.L dNTP mix (2.5 mM each) (TaKaRa, RR02MB), 1. mu.L DNA (the concentration of DNA obtained in example one is required to be 10 ng/. mu.L or more), each L of upstream and downstream primers (10. mu.M), and home-made double distilled water were mixed to prepare a 25. mu.L reaction system.

2) Reaction conditions are as follows:

using a conventional PCR instrument (Applied Biosystems,96-Well Thermal Cycler), the reaction conditions were as follows: 94 ℃ for 7minutes, (94 ℃ for 30seconds,54 ℃ for 30seconds,72 ℃ for 6 minutes) 30 cycles, 12 ℃ incubation.

3) And (3) amplification results:

after electrophoresis for 30min using 2% Agarose gel (Agarose) at a voltage of 140V and a current of 75mA, the gel was stained with ethidium bromide for 10 minutes and then observed on a Tanon1600 full-automatic digital gel image analysis system.

HPV virus typing was carried out based on the results of the gel diagram shown in FIG. 2, in which 16 indicates HPV16, 18 indicates HPV18, and so on, it can be seen that bands of about 8Kb appear in HPV16 type and HPV31 type, and therefore the sample infection type was considered to be HPV 16/31.

Example four

The amplified product of the viral genome was purified, and the amplified sample was purified using AMPure DNA Purification kit (SPRI beads).

1. The AMPure seeds stored at 4 ℃ are taken out and are kept at room temperature for 30min for balancing.

2. After shaking uniformly, the sample was added to a 1.5mL tube in a volume of 1.5 times the volume of the sample.

3. Taking out the PCR product, placing the PCR product on a magnetic frame, and sucking the supernatant; adding the supernatant, mixing, standing for 10min, and centrifuging for 3 s.

4. Transferring a 1.5mL centrifuge tube, placing on a magnetic frame, and standing for 5-10min until the centrifuge tube is clear.

5. After the supernatant was aspirated, 500. mu.L of 70% ethanol was added, the beads were blown up and mixed well, and then adsorbed, and the supernatant was discarded and repeated once.

6. Drying at 37 ℃ until the magnetic beads are dried.

7. Add 20. mu.L of deionized water to a 1.5mL centrifuge tube, mix well, stand for 5min, and then place on a magnetic rack for about 5min to clarify.

EXAMPLE five

Obtaining viral genomes

1) Viral sequence mixing

The purified product of example four was subjected to Qubit (Life Technologies, Grand Island, NY) quantification, and based on the quantification, copy number (copies), (6.02X 10, NY) was calculated according to the following formula²³) X (g/ml)/(DNAlength × 660) ═ copies/ml, and the samples were mixed in equal amounts according to the copy number.

2) Fragmentation of genomic DNA

The obtained genomic DNA was fragmented into fragments of about 200bp in size using sonication (Covaris, S2).

3) End repair and 3' ligation of "A" bases in DNA fragments

The fragmented DNA obtained in step 1 was subjected to end repair using T4DNA Polymerase (enzymes, P708L), KlaneowFragment (enzymes, P706L) and T4Polynucleotide Kinase (enzymes, Y904L) according to the manufacturer's instructions, to form blunt-ended DNA fragments. The "A" bases were then ligated to the 3' end of the blunt-ended DNA fragment using Klaneow (3, -5, exo-) enzyme (enzymics, P701-LC-L).

4) Attachment of Label adapters (Index Adapter)

The DNA fragments obtained in step 3) and having "A" bases ligated to their 3' ends were ligated to different tag linkers (Index adapters) using T4DNA Ligase (enzymes, L603-HC-L) (the sequence was designed with reference to CN102409045A and synthesized by Invitrogen). The eluted samples were amplified using Phusion Flash High-Fidelity PCR Master Mix (NEB, F-531L) and purified using the MinElute PCR Purification Kit (Qiagen,28006) to obtain sequencing libraries for each sample.

5) Sequencing

Sequencing the sequencing library of each sample using the Illumina Miseq sequencing platform (Illumina, Miseq) according to the manufacturer's instructions, wherein the read length of sequencing is 100bp and the average sequencing depth of each sample is at least 100x, to obtain genomic sequencing data for each sample.

EXAMPLE six

Analysis of viral gene sequencing data, sequencing data of individual samples were processed and analyzed as follows to obtain HPV viral genome sequences.

1. Obtaining sequencing data for each sample as described in example five;

2. removing sequencing fragments (reads) containing the adaptor sequences (adapter connectors) in step 3 of example five for each sample; the off-line data from each single lane (lane) was processed by sequencing fragment deduplication in order to remove some of the data redundancy caused by sequencing.

The method comprises the following specific steps:

firstly, obtaining a specific adapter sequence according to the report of the off-line data of each single lane, and removing sequencing fragments containing the adapter sequence by traversing each sequencing fragment of the off-line data (generally, there are two methods, one is to remove only the adapter sequence and keep the sequencing fragments after intercepting the adapter sequence, and the other is to directly throw away the sequencing fragments containing the adapter sequence, and since the quantity of the sequencing fragments containing the adapter sequence is less and not enough to affect the subsequent analysis, we adopt a method of directly throwing away the sequencing fragments containing the adapter sequence for the sake of sequence orderliness and the subsequent analysis).

Secondly, the completely identical sequencing fragments generated by the amplification in example five in the off-line data of each single lane are repetitive sequences, the larger the data amount is, the higher the repetition (duplication) ratio is, the completely repetitive sequencing fragments are redundant, and the data redundancy is removed according to the individuality of the sequencing fragments.

Third, in the off-line data, the partial sequence quality is low, and in the case of sufficient data quantity, the data can be removed, for example, the data quality fraction is removed to be less than 10 points, and the single sequencing sequence contains reads with uncertain base N total number greater than 7%, so as to improve the accuracy of the final assembly sequence.

3. The sequencing data obtained from the processing of step 2 were aligned with the standard sequences of human papillomaviruses from database NCBI, HPV16 (GeneBank-accepted K02718), HPV18(X05015), HPV31(J04353), HPV33(M12732), HPV35(X74477), HPV39(M62849), HPV45(X74479), HPV51(M62877), HPV52(X74481), HPV56(X74483), HPV58(D90400), HPV59(X77858), HPV66(U31794), HPV68(DQ080079) using sequence alignment software bwa (http:// bio-bw.sourcefor. net/software: Burrows-Wheelerligner) to obtain alignment results.

Alignment is shown in FIG. 3, in which the HPV16 reference sequence is located below the two sequences, and the sequencing fragment (read) is aligned above the top.

4. The quality, sequencing depth, and area coverage of the sequencing data of each sample were statistically obtained by applying the following processing to the alignment results of each sample.

The specific execution step:

firstly, reading an alignment result to obtain the total sequencing fragment number, further obtaining the sequencing fragment number aligned to the human papilloma virus, and the sequencing fragment number and the region site number of the non-redundantly aligned human papilloma virus region. Table 2 illustrates alignment statistics.

TABLE 2

5. The sequencing data for The single nucleotide polymorphisms, i.e.the sites at which mutations occur, in The sequencing data for each sample were identified using The alignment software Varscan (http:// Varscan. sourceforce. net) and GATK (http:// www.broadinstitute.org/gsa/wiki/index. ph/The Genome Analysis Toolkit), again using HPV16 (GenBank-accepted K02718), HPV18(X05015), HPV31(J04353), HPV33(M12732), HPV35(X74477), HPV39(M62849), HPV45(X74479), HPV51(M62877), HPV52(X74481), HPV56(X74483), HPV58(D90400), HPV59(X77858), HPV66(U31794), HPV68(DQ 0809) as reference sequences, i.e.g..

As shown in FIG. 3, taking the sequence of the ZHPVS0021 sample at 209 of a cervical cancer patient from a hospital as an example, the base is G, and the base C at the same site on the reference sequence HPV16(K02718) is considered to be aligned: 209C > G, where the mutation occurs.

6. The reads aligned to the reference sequence were assembled into the full-length sequence of HPV genome according to the reference sequences HPV16 (GeneBank-accepted K02718), HPV18(X05015), HPV31(J04353), HPV33(M12732), HPV35(X74477), HPV39(M62849), HPV45(X74479), HPV51(M62877), HPV52(X74481), HPV56(X74483), HPV58(D90400), HPV59(X77858), HPV66(U31794), and HPV68(DQ080079), while mutation information was added to the full-length sequence of HPV genome according to mutation frequency.

Claims (10)

1. A set of primers comprising a sequence selected from the group consisting of SEQ ID NOs: 1-28, and the sequences of the 14 pairs of primers are SEQ ID NOs: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, seq id NO: 21 and 22, SEQ ID NO: 23 and 24, SEQ ID NO: 25 and 26 and SEQ ID NO: 27 and 28;

optionally, comprising a sequence selected from SEQ ID NO: 1-28, at least 4 of the 14 primer pairs;

optionally, comprising a sequence selected from SEQ ID NO: 1-28 of at least 6 primers of the 14 primer pairs;

optionally, comprising a sequence selected from SEQ ID NO: 1-28 of at least 8 primers of the 14 pairs of primers;

optionally, comprising a sequence selected from SEQ ID NO: 1-28, at least 10 of the 14 primer pairs;

optionally, comprising a sequence selected from SEQ ID NO: 1-28 of at least 12 primers of the 14 primer pairs;

optionally, comprising SEQ ID NO: 1-28.

2. The set of primers of claim 1, further comprising a primer having the sequence of SEQ ID NO: 29 and 30.

3. Use of a set of primers according to claim 1 or 2 for amplification of the HPV genome and/or detection of HPV.

4. A kit comprising a set of primers of claim 1 or 2.

5. Use of the kit of claim 4 for HPV genomic amplification, HPV typing and/or HPV detection.

6. A method for obtaining HPV whole genome sequence, which is characterized by comprising the following steps:

obtaining nucleic acid from a test sample;

amplifying the nucleic acid using a set of primers of claim 1 or 2 to obtain an amplification product;

performing sequence determination on the amplification product to obtain sequencing data, wherein the sequencing data comprises a plurality of reads;

based on the sequencing data, obtaining HPV whole genome sequences.

7. The method of claim 6, after obtaining the amplification product, comprising:

and (4) carrying out electrophoresis detection on the amplification product, and carrying out HPV typing according to the obtained electrophoresis detection result.

8. The method of claim 6, wherein obtaining HPV whole genome sequences based on sequencing data comprises:

comparing the reads in the sequencing data with an HPV reference sequence to obtain a comparison result,

and assembling the HPV whole genome sequence according to the comparison result.

9. The method of claim 8, wherein the HPV reference sequences comprise an HPV16 reference sequence, an HPV18 reference sequence, an HPV31 reference sequence, an HPV33 reference sequence, an HPV35 reference sequence, an HPV39 reference sequence, an HPV45 reference sequence, an HPV51 reference sequence, an HPV52 reference sequence, an HPV56 reference sequence, an HPV58 reference sequence, an HPV59 reference sequence, an HPV66 reference sequence, and an HPV68 reference sequence.

10. The method of claim 8, characterized in that the HPV whole genome sequence carries mutation site information.