WO2008074182A1

WO2008074182A1 - Method for the detection and typing of human papillomavirus, and the reagent kit thereof

Info

Publication number: WO2008074182A1
Application number: PCT/CN2006/003447
Authority: WO
Inventors: Yanjun Bai; Jianer Yao; Jiutong Li
Original assignee: Shanghai Tellgen Life Science Co., Ltd.
Priority date: 2006-12-18
Filing date: 2006-12-18
Publication date: 2008-06-26

Abstract

A method for the detection and typing of human papillomavirus (HPV) and a kit for use in the method are provided. The method and the kit use primers and nucleic acid probes, especially the primers and nucleic acid probes for the detection and typing of HPV. Oligonucleotide probes specific for different HPV subtypes are immobilized on microspheres to form microsphere probes for the detection of HPV subtypes and prepare fluidic chips for the typing of HPV.

Description

Human papillomavirus detection typing method and kit

The present invention relates to nucleic acid probes, and more particularly to nucleic acid probes for detecting human papillomavirus (HPV) typing and kits thereof. Background technique

Human papillomavirus (HPV) is an epithelial virus that is widely distributed in humans and animals and has a high degree of specificity. It has long been known that HPV can cause benign tumors and tendons in humans, such as growing in Human common warts, genital warts, and papillomas that grow on the mucous membranes on the skin and mucous membranes near the reproductive organs. There are about 80 types of HPV types that have been identified. According to the location of the infected epithelium, it is divided into skin type HPV and genital tract epithelial HPV. About 35 types can infect women's reproductive tract, and about 20 types are related to tumors. According to different types of HPV and the risk of tumorigenesis, it is divided into low-risk type and high-risk type HPV. Low-risk type HPV includes HPV6, 11, 40, 42, 43, 44, 53 and 54. It often causes benign lesions such as external genital warts, including low-grade cervical intraepithelial lesions (CIN 1), and high-risk HPV including HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 Types 61, 66, 68, 73, 82, and 83 are associated with the development of cervical cancer and cervical intraepithelial lesions (CIN II/III), especially HPV 16 and 18.

Traditional cervical cancer screening methods are as follows: visual observation, Pap cytology smear, and thin-layer liquid-based cytology. Since the above methods can produce some cytological diagnosis results, but how to deal with the next step, the correct judgment is often difficult, and HPV testing will help guide further processing. At the same time, the combined cytology and HPV tests have higher negative predictive values (>99<3⁄4), provide objective indicators for assessing women's risk status, and reduce concerns about negative test results. HPV typing detection, simple method, high efficiency, high sensitivity, strong specificity, good reproducibility, suitable for large sample screening, is the most effective and accurate means of early detection of cervical cancer. The following common methods for detecting typing and their advantages and disadvantages are summarized in Table 1. They are mentioned in some related patent documents.

Table 1 common HPV detection typing methods and their advantages and disadvantages

Method Advantages Disadvantages

In situ hybridization, good specificity, cumbersome, time-consuming and labor-intensive, requires a lot of pure

DNA, which requires a large number of type-specific probes, is not suitable for screening large-volume clinical samples.

DNA direct capture method (HC[], there is a large amount of data to support the application of this technology, sensitivity is lower than PCR amplification method, can not be determined with DigeneCorp., USA) HPV sub-segment capable of separating low-risk and high-risk HPV samples Type, and there is a cross-reaction, affecting the accuracy of its detection.

Subtype-specific PCR is more specific and labor intensive

Universal primers have high sensitivity and can amplify multiple subtypes at a time.

Direct sequencing of PCR products is not very sensitive in determining the currently unknown HPV subtypes, especially in clinical samples that are particularly useful for HPV mixed infection infections and mutation studies. Break method.

PCR-enzyme-linked immunosorbent assay with high sensitivity and high sample consumption

PCR-solid reverse hybridization method is highly efficient, and multiple HPV chambers can be separated in one experiment.

Subtype. In the clinical testing of some genotyping, it is not practical to determine the rare genotypes in the population, and it is also a waste of resources. As reported in the current report, there are nearly 100 HPV subtypes, and the proportion of HPV subtypes in the population is different. Different literature reports have certain differences, but almost all reported data show HPV6, HPV11, HPV16. And HPV18 is the main type, accounting for the vast majority of HPV infections, accounting for more than 98%. According to clinical needs, these major types should be distinguished, so the corresponding probes must be strictly distinguished; for other rare types, especially low-risk types, the clinical value of each type is not significant.

Therefore, there is an urgent need in the art for a high sensitivity, high throughput, stable result, and good reproducibility; an HPV detection typing method that satisfies clinical needs without complicating the detection work and increases the detection cost. Summary of the invention

SUMMARY OF THE INVENTION The object of the present invention is to provide a nucleic acid probe for HPV detection and a kit thereof which are highly sensitive, high-throughput, stable in results, reproducible, and simple in operation.

In a first aspect of the invention, there is provided a nucleic acid probe microsphere set for detecting human papillomavirus HPV, characterized in that it comprises from 2 to 60 different fluorescently encoded microspheres, and each of said fluorescent codes 1 to 20 human papillomavirus-specific nucleic acid probes immobilized on the microspheres, and at least one fluorescent-encoded microsphere having 2-20 human papillomavirus-specific nucleic acid probes;

Wherein, the 1 to 20 human papillomavirus-specific nucleic acid probes are selected from the group consisting of:

(a) Human papillomavirus-specific nucleic acid probes for different subtypes of low-risk type HPV;

(b) Human papillomavirus-specific nucleic acid probes for different subtypes of high-risk type HPV.

In another preferred embodiment, the low-risk type HPV is selected from the group consisting of: HPV 6, 11, 40, 42, 43, 44, 53 or 54;

The high-risk type HPV is selected from the group consisting of: HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 61, 66, 68, 73, 82 or 83 .

In another preferred embodiment, the human papillomavirus-specific nucleic acid probe immobilized on the microspheres is selected from the group consisting of:

(i) HPV6, HPV1K HPV53, HPV54;

(ii) HPV40, HPV42, HPV43 and HPV44;

(iii) HPV16, HPV18, HPV31, HPV51

(iv) HPV33, HPV35, HPV39, HPV56;

(v) HPV45, HPV52, HPV58HPV59, HPV66; and

The probe is coated on a fluorescently encoded microsphere.

In another preferred embodiment, the surface of the fluorescently encoded microspheres is covalently linked to a nucleic acid probe. In another preferred embodiment, the sequence of the human papillomavirus-specific nucleic acid probe is as follows:

HPV06 5'AGACGTTCGATTTCCACTAC3 ' SEQ ED NO: l

HPV11 5'GGGGGTAATAACAGATCATC3' SEQ ID NO: 2

HPV53 5 TTCCTCACCAATAACGC3' SEQ ID NO: 3

HPV54 5OGAGTTGCAGCATAAATAGA3 ' SEQ ID NO: 4

HPV40 5'CAATAGGAGTCCGACCAG3' SEQ ID NO: 5

HPV42 5'GGCAGACATAATTTAGGTAGTA3' SEQ ID NO:6

HPV43 5 CTAATACCAGGTCCAAAAT3' SEQ ID NO:7

HPV44 5'GCACTTTTAATAACCAGATCCT3' SEQ ID NO: 8

HPV16 5'GAAAATAATTTGAACTGGCT3' SEQ ID NO:9

HPV18 5'CAGGTATGCCTGCTTCAC3' SEQ ID NO: 10

HPV31 5'CAGTAGGGACCGATTCAC3' SEQ ID NO: 11

HPV33 5'ACTACTGCCTCTATTCAAAGC3' SEQ ID NO: 12

HPV35 5'GCTGGAACTGTAGGTGAAA3' SEQ ID NO: 13

HPV39 5'CCACCATACCACCACGAT3' SEQ ID NO : 14

HPV45 5'ACGCATATTAGCGCTAGTG3 ' SEQ ID NO: 15

HPV52 5'ATACAAGGGTCTAACTCTGGC3' SEQ ID NO: 16

HPV58 5OGGTCCGGTAATACTGC3 ' SEQ ID NO: 17

HPV26 5'CCCCTACATCTTCTATTTATTC3' SEQ ID NO : 18

HPV51 5TTCCCCAACACCTACAAG3 ' SEQ ID NO: 19

HPV55 5'GGCATTAGGAACTGTACTTTT3' SEQ ID NO :20

HPV56 5'CCCCTCCGAGTTCTGTAT3' SEQ ID NO: 21

HPV59 5AACCCAGGCAGTTATTTAT3 ' SEQ ID NO : 22

HPV61 5 TAAGGGTGCGAATGACA3 ' SEQ ID NO: 23

HPV66 5'GCCACCCTTCCAATACAA3' ' SEQ ID NO :24

HPV68 5 AAGGGCACTGACATTCG3' SEQ ID NO: 25

HPV73 5'CTGGGATTTTATCACCGG3 ' SEQ ID NO:26

HPV83 5'GGTGCTGCCTACCTCTTA3' SEQ ID NO-.27

HPV82 5 TAAGGGTACTGGTGCTGG3 'SEQ ID NO: 28o In a second aspect of the invention, there is provided a kit comprising: a container and a nucleic acid probe microsphere set for detecting human papillomavirus according to the invention described above in the container.

In another preferred embodiment, the kit further comprises a universal primer, a hybridization reagent, and a signal labeling reagent. More preferably, the primer is a set of primer systems (BT/12F-BT/12R) designed for a region within the L1 region of the HPV genome.

More preferably, the primers also have a pair of primers that amplify a fragment of the Human Golbin gene for use as an internal pair of photographs.

In another preferred embodiment, the nucleic acid probe is an HPV-type flow chip.

In another preferred embodiment, the human papillomavirus-specific nucleic acid probe immobilized on said microspheres is selected from the group consisting of:

(i) HPV6, HPV11, HPV53, HPV54

(ii) HPV40, HPV42, HPV43 and HPV44

(iii) HPV16, HPV18, HPV31, HPV51;

(iv) HPV33, HPV35, HPV39, HPV56;

(v) HPV45, HPV52, HPV58, HPV59, HPV66;

And the probe is coated on a fluorescently encoded microsphere. In a third aspect of the invention, there is provided the use of the above-described nucleic acid probe microsphere for detecting human papillomavirus (HPV) of the invention, said use being selected from the group consisting of: (a) detecting the presence of HPV in vitro and its sub- Application in the form; (b) Preparation of a kit for detecting human papillomavirus.

In a fourth aspect of the invention, there is provided a method of detecting the presence or absence of HPV in a sample in vitro, the method comprising -

(a) extracting the DNA of the sample and amplifying it with HPV-specific primers to obtain an amplification product;

(b) mixing the amplification product of step (a) with the above-described nucleic acid probe microspheres for detecting human papillomavirus (HPV);

(c) detecting whether the nucleic acid probe on the microsphere is bound to the amplification product, wherein binding of the nucleic acid probe on the microsphere to the amplification product indicates the presence of human papillomavirus in the sample.

In another preferred embodiment, the detection method in step (c) is Luminex xMAP. detailed description

The inventors have conducted extensive and in-depth research and found that subtypes of low-risk HPV different subtypes or different subtypes of high-risk HPV or low- or high-risk populations with very low infection rates can be fixed in the same medium position or in On the same medium, one medium position or medium type can be detected at the same time to detect the presence of several different genotypes; in addition, the inventors used the Luminex xMAP technology platform to immobilize HPV-specific oligonucleoside probes on the microspheres. A microsphere probe for detecting HPV subtypes is formed and mixed together in a certain amount ratio to form an HPV fractionation flow chip, and a corresponding kit is prepared together with a universal primer, a hybridization reagent and a signal labeling reagent. The HPV flow chip referred to in the present invention refers to a mixture of different fluorescent coded microspheres, wherein the surface of different fluorescent coded microspheres is covalently linked to specific oligonucleotides for different HPV subtype nucleotide sequences. needle.

The HPV flow chip is specifically prepared by chemically modifying the 5' end of the oligonucleotide probe of a specific sequence to have an NH2 group, and forming a acyl group with a carboxyl group on the surface of the fluorescent coded polystyrene microsphere. Ammonia bond, thereby linking the two together; different specific oligonucleoside probes are immobilized on different microspheres to form microsphere probes for detecting different HPV subtypes; The microspheres of the acid probe are mixed together in a certain amount ratio to form an HPV fractionation flow chip; the above method of immobilizing the oligonucleotide probe to the microsphere is separately coated with each oligonucleotide probe On a different fluorescent numbered microsphere, a one-to-one relationship is formed.

The sequences of specific oligonucleotide probes for different HPV subtypes are as follows:

Name Sequence SEQ ID NO

HPV06 5'AGACGTTCGATTTCCACTAC3' 01

HPV11 5OGGGGTAATAACAGATCATC3 ' 02

HPV53 5TTTCCTCACCAATAACGC3' 03

HPV54 5OGAGTTGCAGCATAAATAGA3' 04

HPV40 5'CAATAGGAGTCCGACCAG3 ' 05

HPV42 5OGCAGACATAATTTAGGTAGTA3 ' 06

HPV43 5 CTAATACCAGGTCCAAAAT3' 07

HPV44 5OCACTTTTAATAACCAGATCCT3 ' 08 HPV16 5'GAAAATAATTTGAACTGGCT3 ' 09

HPV18 5'CAGGTATGCCTGCTTCAC3' 10

HPV31 5'CAGTAGGGACCGATTCAC3 ' 11

HPV33 5'ACTACTGCCTCTATTCAAAGC3' 12

HPV35 5'GCTGGAACTGTAGGTGAAA3' 13

HPV39 5'CCACCATACCACCACGAT3' 14

HPV45 5'ACGCATATTAGCGCTAGTG3' 15

HPV52 5'ATACAAGGGTCTAACTCTGGC3' 16

HPV58 5OGGTCCGGTAATACTGC3 ' 17

HPV26 5'CCCCTACATCTTCTATTTATTC3' 18

HPV51 5 TCCCCAACACCTACAAG3' 19

HPV55 5'GGCATTAGGAACTGTACTTTT3' 20

HPV56 5'CCCCTCCGAGTTCTGTAT3' 21

HPV59 5'AACCCAGGCAGTTATTTAT3 ' 22

HPV61 5 TAAGGGTGCGAATGACA3 ' 23

HPV66 5'GCCACCCTTCCAATACAA3' 24

HPV68 5TAAGGGCACTGACATTCG3 ' 25

HPV73 5 'CTGGGATTTTATCACCGG3 ' 26

HPV83 5'GGTGCTGCCTACCTCTTA3' 27

HPV82 5TAAGGGTACTGGTGCTGG3' 28 To meet the clinical needs, in order to save costs and simplify the testing work, the present invention also provides a method for hybrid coating of oligonucleotide probes, which will be directed to different subtypes of low-risk HPV or Different subtypes of high-risk HPV or low- or high-risk populations of sub-types with very low infection rates are coated on the same microsphere, and several subtypes are simultaneously detected without specific distinction. The following example illustrates this probe fixing idea. The genotypes to be detected include Al, A2, A3, A4, A5, A6, A7, A8, and A9 (the number can be arbitrarily changed), and the specific probes designed to detect the above types are Bl, B2, B3, and B4. , B5, B6, B7, B8 and B9, the probes Bl, B2, B3, B4, B5, B6, B7, B8 and B9 are respectively fixed at different positions of the medium or different types of media Cl, C2, C3, C4 On C5, C6, C7, C8 and C9, a media position or medium type is formed corresponding to a probe to detect a genotype.

When the detected genotypes have the following conditions:

1. Some genotypes such as Al, A2, A5 and A9 have the same clinical significance. Al, A2, A5 and A9 alone or simultaneously have no cumulative, additive dose effects, which can provide the same for clinical treatment. The information, while distinguishing the type between Al, A2, A5 and A9 has no practical meaning.

2. Some genotypes such as Al, A2, A5, and A9 account for a small proportion of the total genotypes in the tested subjects.

It is not necessary to fix the probes Bl, B2, B5 and B9 corresponding to the genotypes Al, A2, A5 and A9 to Cl, C2, C5 and C9, respectively, according to the conventional method, and the probes Bl and B2 can be used. , B5 and B9 are fixed at one position of C1. This allows one media location or media type to detect the presence of four different genotypes, although it is not possible to distinguish which one or which of the four.

The universal primer referred to in the present invention is specifically designed for a region in the L1 region of the HPV genome. The set of primer systems (BT/12F-BT/12R), including 12 forward and 12 reverse primers, and a pair of primers that amplify the Human Golbin gene fragment were used as amplification of the internal control fragment.

The present invention provides for HPV06, 11, 53, 54, 40, 42, 43, 44, 16, 18, 31, 33, 35, 39, 45, 52, 58, 26, 51, 55, 56, 59, Primer system BT/12F-BT/12R for 61, 66, 68, 73, 83, 82 subtype DNA amplification, all primers were labeled Biotin at the 5' end.

The sequence of the BT/12F-BT/12R primer system is as follows -

The hybridization referred to in the present invention is a process in which the PCR product is denatured and sufficiently mixed with the HPV chip to carry out nucleic acid hybridization under certain conditions.

The signal labeling referred to in the present invention is the addition of Strepavidin labeled with phycoerythrin (PE) after completion of hybridization, and finally forms a microsphere-probe-PCR product-Biotin-StrepAvidin-PE complex.

The present invention uses Luminex xMAP to detect the results of hybridization and to determine according to the criteria provided by the present invention. The main advantages of the invention are:

1. High sensitivity, high throughput, stable results and good repeatability; 2. While meeting clinical needs, it does not complicate the testing work and save costs. The invention is described in detail below with reference to specific embodiments, but is not to be construed as limiting. The examples described are merely illustrative of nucleic acid probes, kits, and methods of making and using them without limitation. Various modifications are conceivable within the scope of the invention and the appended claims. Example 1:

One-to-one probe method

1. Preparation of chips for detecting HPV6, HPV11, HPV16 and HPV18:

1. Synthesis of oligonucleotide probes according to the following sequence

2. Select No. 31, 32, 33, 34, 35, and 36. 6 kinds of microspheres [Luminex] The probe is coated as follows:

(I) 6 kinds of microspheres 31, 32, 33, 34, 35, and 36 and one EDC powder stored at -20 ° C were equilibrated at room temperature for 30 minutes;

(2) The HPV6, HPV11, HPV16, HPV18, Globin and NC probes were dissolved in double distilled water at a concentration of 0.01 niM (10 pmole L);

(3) mixing the microspheres uniformly with an oscillator;

(4) Take 50ul (6.0 X 10 ⁵ ) microspheres each, and put them into a clean Ep tube with a pre-marked number;

(5) Centrifuge the microspheres under centrifugation for 5 minutes under 8000 g of centrifugal force, and carefully discard the supernatant;

(6) adding lOOul double distilled water, fully oscillating the suspended microspheres, and centrifuging for 5 minutes under 8000 g centrifugal force to precipitate the microspheres;

(7) Discard the supernatant, resuspend the microspheres with 50ul 0.1 M MES (pH 4.5), and mix with a shaker;

(8) 2 ul (10 pmol _e ^ L) of each probe is added to the corresponding microsphere suspension in the above step, and mixed with a shaker;

(9) Freshly prepare EDC solution at 10 mg/mL;

(10) Take 2.5 ul of the EDC solution from the previous step into each microsphere suspension (25 g or ^ [0.5 pg/L] final) and mix with a shaker;

(I I) protected from light, allowed to stand at 37 ° C for 30 minutes;

(12) Repeat steps (10)-(11);

(13) centrifugation for 5 minutes under 8000 g centrifugal force to precipitate microspheres; (14) Discard the supernatant, resuspend the microspheres with 1.0 mL of 0.1% SDS, and mix with a shaker;

(15) Centrifugation for 5 minutes under 8000 g centrifugal force;

(16) Discard the supernatant, resuspend the microspheres with 100 ul TE (pH 8.0), and mix with a shaker;

(17) Microsphere counting - a. The connected microsphere suspension is diluted 50 times with water;

b. Mix the oscillator;

c. Take lOul to the counter board;

d. counting the microspheres in the counting chamber on the four corners;

e. Calculating the concentration of the microsphere suspension;

The count results of the five kinds of microspheres are all about: 6X 10 ³ /ul

(18) Store the connected microspheres in the dark at 2-10 °C.

3. Chip preparation:

The same volume of the above-mentioned microspheres coated with the probe is mixed, and the final concentration of each microsphere is 1.2×10 ³ /ul, which is stored in the dark at 2-10 ° C, that is, HPV6, HPV1U HPV16 is detected. And the streaming chip of HPV18.

Second, the preparation of the sample

Samples of clinical samples of cervical epidermal exfoliated cells from 1 to 5 were extracted according to the following procedure:

1. Vortex the commercially available cell preservation solution containing the sample for 5 seconds to suspend the exfoliated cells;

2. Take 1 ml of the preservation solution in a 1.5 ml centrifuge tube and centrifuge at 8000 rpm for 5 minutes.

3. Carefully dump and discard the supernatant;

4. Add 500μ1ρΗ7.4 phosphate buffer, vortex for 10 seconds;

Centrifuge at 5.8000 rpm for 5 minutes, carefully pour the discarded supernatant;

6. Add 90μ1ρΗ7.4 phosphate buffer, vortex for 20 seconds, add 10 μM commercial digest, vortex for 5 seconds;

7. The centrifuge tube is digested in a 55 ° C water bath for 1 hour;

Denaturation at 8.100 ° C water bath for 15 minutes.

The extracted product is directly used for PCR, and can be stored at - 20 ° C if it is not used temporarily. Sample

Third, PCR amplification

1. Synthesize primers for HPV PCR amplification according to the following sequence:

BT/12F

5' biotin -AAATATCCAGATTATTTAAAAATG 3'

5' biotin -AAATATCCTGACTATTTAAAAATG 3'

5' biotin - AAATACCCTGATTACTTAAAAATG 3'

5' biotin -AAATATCCTGATTATCTAAAAATG 3'

5' biotin - AAATATCCTGATTACCTTAAAATG 3'

5' biotin-AAATATCCAGATTATATTAAAATG 3' 5' biotin-AAATATCCTGATTATTTGCAAATG 3'

5' biotin - AAATATCCTGACTATTTGCAAATG 3'

5' biotin - AAATATCCTGATTATTTACAAATG 3 '

5' biotin -AAATATCCTGACTATTTACAAATG 3'

5' Biotin - AAATATCCAGATTATTTGGGAATG 3'

5' biotin -AAATACCCAGATTATTTAGGCATG 3'

5' biotin -GGCCAATCTACTCCCAGGG-3' GF

BT/12R

5' biotin -CCAATATGGTTTATTAAATAATTG 3'

5' Biotin - CCAATATGGCTTATTAAACAATTG 3'

5' biotin -CCATAAGGGTTTGTTAAACAATTG 3'

5' biotin -CCAATATGGTTTATTAAATATTTG 3'

5' Biotin - CCAATAAGGTTTATTGAATATTTG 3'

5' Biotin - CCAATATGGTTTATTAAAAATTTG 3'

5' biotin -CCAGTAGGGCTTATTAAATAGTTG 3'

5' Biotin - CCAATAAGGCTTATTAAATAACTG 3'

5' biotin -CCAATAGGGCTTGTTAAATAACTG 3'

5' Biotin - CCAATATGGTTTATTAAACAACTG 3'

5' Biotin - CCAATAAGGCTTATTAAAAATCTG 3'

5' biotin -CCACAATGGCTTGTTAAATATCTG 3'

5' Biotin -AGGTTGTCCAGGTGAGCCA-3' GR

2. Preparation of mixed primer working solution:

(1) Each primer synthesized is formulated into a stock solution of lOOpmol/L;

(2) Take 4ul of each primer stock solution into the same container, add ddH ₂ 0 288ul to make up the volume to 400ul, and mix evenly to mix the primer working solution;

3. HPV PCR amplification:

reaction system:

V ₀ = 20ul

Premixed Buffer (2 X ) lO.Oul Mixed Primer Working Fluid 5.0ul

Template 5.0ul Amplification procedure: 95 ° C for 5 minutes - 95 ° C for 30 seconds, 52 ° C for 1 minute, 72 ° C for 1 minute (40 cycles total) - 72 ° C for 5 minutes - 4 ° C insulation.

Fourth, hybrid

1. Take 5 ul to 1-5 clean EP tubes from 5 clinical samples of the above 1-5, and add 12 ul of TE buffer to the hook and denature at 95 °C for 5 minutes. 5 minutes on ice;

2. Add 33ull.5 X hybrid solution (containing six kinds of microspheres, the number is 2000 / species), mix and mix at 48 °C for 15 minutes; 5. Signal Marking and Detection

1. Add 150 ul (containing 2 ng/ml PE) of TE buffer, mix and incubate at 48 °C for 5 minutes to form microsphere-probe-PCR product-Biotin-StrepAvidin-PE complex;

2. The results of the hybridization were tested using Luminex xMAP and the results were determined according to the criteria provided by the present invention.

Sample

The result is as follows -

Sixth, data analysis

According to the kit determination rule, the test results of the above samples No. 1-5 are as follows: Test reading / negative probe reading 2.0 is positive;

Test reading / negative probe reading <2.0 is negative

5 positive negative positive negative

Result description

Sample No. 1 was HPV06 infection;

Sample No. 2 was not infected with the above subtype of HPV;

Sample No. 3 was HPV06, HPV11 and HPV18 mixed infection;

Sample No. 4 is a mixed infection of HPV16 and HPV18;

Sample No. 5 was a mixed infection of HPV06 and HPV16. Example 2:

Probe mixing method

1. Preparation of chips for detection of HPV0, HPV11, HPV53, HPV54, HPV40, HPV42, HPV43, HPV44, HPV16, HPV18, HPV3K HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV59, HPV58 and HPV66:

1. Synthesis of oligonucleotide probes according to the following sequence

2. Select 7 kinds of microspheres [Luminex] No. 31, 32, 33, 34, 35, 36 and 42, and carry out the coating of the probe according to the following corresponding methods and methods: Corresponding way:

HPV6, HPV11, HPV53, HPV54 correspond to a microsphere LI

HPV40 HPV42 HPV43 and HPV44 correspond to a microsphere L2

HPV16, HPV18 HPV31, HPV51 correspond to a microsphere -HI

HPV33, HPV35, HPV39> HPV56 corresponds to a microsphere -H2

HPV45, HPV52, HPV58, HPV59, HPV66 correspond to a microsphere H3

Globin corresponds to a microsphere

NC corresponds to a microsphere

Method:

(1) Seven kinds of microspheres 31, 32, 33, 34, 35, 36 and 42 and one EDC powder stored at -20 ° C were equilibrated at room temperature for 30 minutes;

2) Dissolve HPV6, HPV11, HPV53, HPV54, HPV40, HPV42, HPV43, HPV44, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV52 and HPV58 probes in double distilled water at a concentration of

3) Mix the microspheres evenly with an oscillator;

:4) Take 50ul (6.0 X 10 ⁵ ) microspheres each, and put them into a clean Ep tube with a pre-marked number;

5) Centrifuge the microspheres under 8000g centrifugal force for 5 minutes, carefully discard the supernatant;

6) Add lOOul double distilled water, fully oscillate the suspended microspheres, and centrifuge for 5 minutes under 8000g centrifugal force to precipitate the microspheres;

7) Discard the supernatant and resuspend the microspheres with 5(^1 0.1 ]^^8 114.5) and mix with a shaker;

8) pre-form the probe at the appropriate concentration so that the final concentration of each combination probe remains at lOpmole / L;

9) Each probe is taken 2 ul (10 pmole ^ L) and added to the corresponding microsphere suspension in the above step, and quenched by shaking;

10) Freshly prepare EDC solution at 10 mg/mL;

11) Take 2.5 ul of the EDC solution from the previous step into each microsphere suspension (25 μ _β or ^ [0.5 μ _β /μ3⁄4 ίίηα1), and mix with a shaker;

12) Protect from light and let stand at 37 °C for 30 minutes;

13) Repeat steps (11)-(12);

14) Precipitating the microspheres by centrifugation for 5 minutes under 8000 g centrifugal force;

15) Discard the supernatant, resuspend the microspheres with 1.0 mL of 0.1% SDS, and mix with a shaker;

16) Precipitating the microspheres by centrifugation for 5 minutes under 8000 g centrifugal force;

17) Discard the supernatant, resuspend the microspheres with 100 ul TE (pH 8.0), and mix with a shaker;

18) Microsphere count:

a. The connected microsphere suspension is diluted 50 times with water; b. Mix the oscillator;

c. Take lOul to the counter board;

d. counting the microspheres in the counting chamber on the four corners;

e. Calculating the concentration of the microsphere suspension;

The count results of the five kinds of microspheres are about: 6 10 ³ ^1

(19) Store the connected microspheres in the dark at 2-10 °C.

3. Chip preparation:

The equal volume of the above-mentioned microspheres coated with the probe is mixed, and the final concentration of each microsphere is adjusted to be 1.2×10 ³ /ul by centrifugation, and stored under the condition of 2-10 ° C in the dark, which is to detect HPV6. Streaming chips for HPV11, HPV53, HPV54, HPV40, HPV42, HPV43, HPV44> HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV52 and HPV58.

Second, the preparation of the sample

Clinical samples of cervical epidermal exfoliated cells from 1 to 5 were extracted according to the following procedure _:

• 3. Carefully dump and discard the supernatant;

4. Add 500μ1ρΗ7.4 phosphate buffer, vortex for 10 seconds;

7. The centrifuge tube is digested in a 55 ° C water bath for 1 hour;

Denaturation at 8.100 ° C water bath for 15 minutes.

The extracted product is directly used for PCR, and can be stored at - 20 ° C if it is not used temporarily.

Sample sample

Third, PCR amplification

Amplify the above sample DNA No. 1-5 as follows:

BT/12F

5' biotin -AAATATCCAGATTATTTAAAAATG 3'

5' biotin -AAATATCCTGACTATTTAAAAATG 3'

5' biotin - AAATACCCTGATTACTTAAAAATG 3'

5' Biotin - AAATATCCTGATTATCTAAAAATG 3'

5' Biotin - AAATATCCTGATTACCTTAAAATG 3'

5' biotin -AAATATCCAGATTATATTAAAATG 3'

5' biotin-AAATATCCTGATTATTTGCAAATG 3' 5' Biotin - AAATATCCTGACTATTTGCAAATG 3 '

5' Biotin - AAATATCCTGATTATTTACAAATG 3'

5' biotin -AAATATCCTGACTATTTACAAATG 3'

5' biotin -AAATATCCAGATTATTTGGGAATG 3'

5' biotin -AAATACCCAGATTATTTAGGCATG 3'

5' biotin -GGCCAATCTACTCCCAGGG-3' GF

BT/12R

5' Biotin -CC AATATGGTTTATTAAATAATTG 3 '

5' Biotin - CCAATATGGCTTATTAAACAATTG 3'

5' biotin -CCATAAGGGTTTGTTAAACAATTG 3'

5' biotin -CCAATATGGTTTATTAAATATTTG 3'

5' biotin - CCAATAAGGTTTATTGAATATTTG 3'

5' Biotin - CCAATATGGTTTATTAAAAATTTG 3'

5' biotin -CCAGTAGGGCTTATTAAATAGTTG 3'

5' biotin - CCAATAAGGCTTATTAAATAACTG 3 '

5' Biotin - CCAATAGGGCTTGTTAAATAACTG 3'

5' Biotin - CCAATATGGTTTATTAAACAACTG 3'

5' Biotin - CCAATAAGGCTTATTAAAAATCTG 3'

5' biotin -CCACAATGGCTTGTTAAATATCTG 3'

5' biotin -AGGTTGTCCAGGTGAGCCA-3' GR

2. Preparation of mixed primer working solution:

(1) Each primer synthesized is formulated into a stock solution of lOOpmol/L;

3. HPV PCR amplification:

reaction system:

V ₀ = 20ul

Premixed Buffer (2X) lO.Oul Mixed Primer Working Fluid 5.0ul

Template 5.0ul amplification program: 95 ° C 5 minutes - 95 30 seconds, 52 Ό 1 minute, 72 ° C 1 minute (40 cycles total) - 72 ° C5 Minutes → 4 ° C insulation. Fourth, hybrid

1. Take 5 ul to 1-5 clean EP tubes from 5 clinical samples of the above 1-5, add 12 ul of TE buffer, mix and denature at 95 °C for 5 minutes, immediately place on ice. Last 5 minutes;

2. Add 33uI1.5 X hybrid solution (containing six kinds of microspheres, the number is 2000/species), mix and mix at 48 °C for 15 minutes; 5. Signal Marking and Detection

1. Add 150 ul of TE buffer containing 2 ng/ml PE, mix well, and incubate at 48 ° C for 5 minutes to form microsphere-probe-PCR product-Biotin-StrepAvidin-PE complex;

2. The results of the hybridization were tested using Luminex xMAP and the results were determined according to the criteria provided by the present invention. The results are as follows:

Sixth, data analysis

According to the kit determination rule, the test results of the above samples No. 1-5 are determined as follows:

Test reading / negative probe reading 2.0 is positive;

Test reading / negative probe reading <2.0 is negative

Result description Sample No. 1 is a mixed infection of one or more of HPV6, HPV11, HPV53, HPV54 and one or more of HPV40, HPV42, HPV43, HPV44;

Sample No. 2 is not infected with any of HPV6, HPV11, HPV53, HPV54, HPV40, HPV42, HPV43, HPV44, HPV16, HPV18, HPV3 HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, and HPV66 ;

Sample No. 3 is a mixed infection of one or more of HPV6, HPV1 HPV53 HPV54 and one or more of HPV16, HPV18, HPV31, HPV51 and one or more of HPV33, HPV35, HPV39, HPV56;

Sample No. 4 is a mixed infection of one or more of HPV40, HPV42, HPV43, HPV44 and one or more of HPV16, HPV18, HPV31, HPV51;

Sample No. 5 is one or several of HPV6, HPV1K HPV53, HPV54 and HPV16, HPV18, HPV31,

One or more of HPV51 and a mixed infection of one or several of HPV33, HPV35, HPV39 HPV56. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entirety as if they are individually incorporated by reference. In addition, it should be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the present invention.

Claims

Rights request

A nucleic acid probe microsphere set for detecting human papillomavirus HPV, characterized in that it comprises 2-60 different fluorescently encoded microspheres, and 1~ fixed on each of said fluorescent coded microspheres 20 human papillomavirus-specific nucleic acid probes, and at least one fluorescent-encoding microsphere has 2-20 human papillomavirus-specific nucleic acid probes; wherein, 1 to 20 human papillomavirus specific The nucleic acid probe is selected from the group consisting of:

The nucleic acid probe microsphere set according to claim 1, wherein the low risk type HPV is selected from the group consisting of: HPV 6, 11, 40, 42, 43, 44, 53 or 54 type ;

The nucleic acid probe microsphere set according to claim 1, wherein the human papillomavirus-specific nucleic acid probe immobilized on the microsphere is selected from the group consisting of:

(i) HPV6, HPV11, HPV53, HPV54;

(ii) HPV40, HPV42, HPV43 and HPV44;

(iii) HPV16, HPV18, HPV31, HPV51

(iv) HPV33, HPV35, HPV39, HPV56;

(v) HPV45, HPV52, HPV58HPV59, HPV66; and

The probe is coated on a fluorescently encoded microsphere.

The nucleic acid probe microsphere set according to claim 1, wherein the surface of the fluorescent coded microsphere is covalently linked to the nucleic acid probe.

The nucleic acid probe microsphere set according to any one of claims 1 to 3, wherein the sequence of the human papillomavirus-specific nucleic acid probe is as follows:

HPV06 5'AGACGTTCGATTTCCACTAC3' SEQ ID NO: l

HPV11 5'GGGGGTAATAAC AGATC ATC3 ' SEQ ID NO: 2

HPV53 5 TTCCTCACCAATAACGC3' SEQ ID NO: 3

HPV54 5'GGAGTTGC AGC ATAAATAGA3 ' SEQ ID NO: 4

HPV40 5'CAATAGGAGTCCGACCAG3' SEQ ID NO: 5

HPV42 5*GGCAGACATAATTTAGGTAGTA3' SEQ ID NO:6

HPV43 5 CTAATACCAGGTCCAAAAT3' SEQ ID NO:7

HPV44 5'GCACTTTTAATAACCAGATCCT3' SEQ ID NO:8

HPV16 5'GAAAATAATTTGAACTGGCT3' SEQ ID NO:9

HPV18 5'CAGGTATGCCTGCTTCAC3' SEQ ID NO: 10

HPV31 5'CAGTAGGGACCGATTCAC3' SEQ ID NO: 11

HPV33 5'ACTACTGCCTCTATTCAAAGC3* SEQ ID NO: 12 HPV35 5OCTGGAACTGTAGGTGAAA3' SEQ ID NO: 13

HPV39 5'CCACCATACCACCACGAT3' SEQ ID NO: 14

HPV45 5'ACGC ATATTAGCGCTAGTG3 ' SEQ ID NO: 15

HPV52 5'ATACAAGGGTCTAACTCTGGC3' SEQ ID NO-.16

HPV58 5'GGGTCCGGTAATACTGC3' SEQ ID NO: 17

HPV26 5'CCCCTACATCTTCTATTTATTC3' SEQ BD NO:18

HPV51 5 TCCCCAACACCTACAAG3' SEQ ID NO: 19

HPV55 5'GGCATTAGGAACTGTACTTTT3' SEQ ID NO:20

HPV56 5'CCCCTCCGAGTTCTGTAT3 ' SEQ ID NO: 21

HPV59 5'AACCCAGGCAGTTATTTAT3 ' SEQ ID NO: 22

HPV61 5 TAAGGGTGCGAATGACA3' SEQ ID NO:23

HPV66 5'GCCACCCTTCCAATACAA3' SEQ ID NO:24

HPV68 5TAAGGGCACTGAC ATTCG3 ' SEQ ID NO: 25

HPV73 5'CTGGGATTTTATCACCGG3' SEQ ID NO:26

HPV83 5'GGTGCTGCCTACCTCTTA3 ' SEQ ID NO:27

HPV82 5 AAGGGTACTGGTGCTGG3 ' SEQ ID NO: 28o

A kit according to the invention, comprising: a container and a nucleic acid probe microsphere set for detecting human papillomavirus according to claim 1 in the container.

The kit according to claim 6, wherein the kit further comprises a universal primer, a hybridization reagent, and a signal labeling reagent.

The kit according to claim 6, wherein the nucleic acid probe is an HPV fractionated flow chip.

9. The use of a nucleic acid probe microsphere for detecting human papillomavirus (HPV) according to claim 1, wherein said use is selected from the group consisting of: (a) detecting the presence of HPV in vitro and its sub- Application in the form; (b) Preparation of a kit for detecting human papillomavirus.

10. A method of detecting the presence or absence of HPV in a sample in vitro, the method comprising:

(b) mixing the amplification product of step (a) with the nucleic acid probe microsphere of the human papillomavirus (HPV) according to claim 1;