CN104020291B - A kind of HPV nucleic acid detection kit and its application based on ELISA - Google Patents

Abstract

The invention belongs to molecular biology, immunology and nucleic acid chemistry field, be specifically related to a kind of HPV nucleic acid detection kit based on enzyme-linked immuno assay and application thereof.This kit detects the immunologic detection method of high-risk HPV16 type E6, E7RNA based on a kind of Sdr of utilization monoclonal antibody, testing process does not need the technology such as reverse transcription and PCR to increase to target sequence, but utilization does not need the DNA probe of mark and HPV16 type E6, E7RNA to hybridize, form DNA:RNA heterozygote, then utilize Sdr monoclonal antibody to carry out identifying and the amplification of follow-up signal to heterozygote.Detection method has fast, low cost, highly sensitive, do not need complicated amplification and detecting instrument, and the advantage of original viral carrying capacity can be detected.

Description

A kind of HPV nucleic acid detection kit and its application based on ELISA

technical field

The invention belongs to the technical fields of molecular biology, immunology and nucleic acid chemistry. In particular, it relates to an enzyme-linked immunoassay-based HPV nucleic acid detection kit and an application thereof.

Background technique

Human papillomavirus (hereinafter referred to as HPV) is a circular double-stranded DNA virus belonging to the family Papillomaviridae and the genus Alphapapillomavirus. Since it was first discovered in the 1970s, more than 100 genotypes of HPV have been published. Some of these genotypes have been shown to be associated with various epithelial or mucosal malignancies, especially cervical cancer. Cervical cancer is the second most common malignancy after breast cancer among women worldwide. In the United States, 12,000 women are diagnosed with cervical cancer each year. It is now well established that HPV is the cause of almost all cervical cancers worldwide. HPV causes 99% of cervical cancers, and high-risk HPV genotypes 16 and 18 account for 70%. More importantly, the E6 and E7 genes in the virus and their transcription and translation products are key factors that determine its ability to invade and treat diseases.

For decades, women have relied on cytology as a tool to detect the presence or absence of cervical cancer. Women need access to better screening tools, including HPV primary screening, to reduce their risk of cervical cancer. Now there are many methods for diagnosing HPV infection, such as: histology, serology and molecular biology. However, based on the characteristics of the HPV virus itself, it cannot be cultured in vitro so far, so the culture method is not realistic; in addition, due to the lack of sufficient sensitivity and specificity for HPV immunoassays, serum or protein detection has not been applied clinically . Therefore, the methods currently used for HPV detection also rely on its nucleic acid (DNA or RNA). Signal amplification method of high-risk HPV detection method, target amplification analysis based on Real-Time PCR and integrated HPV sequence PCR (DIPS-PCR); for RNA detection, there are reverse transcription PCR, nucleic acid sequence based amplification (NASBA) method and transcription Mediated amplification (TMA) method, etc. Among them, it is worth noting that Qiagen's HybridCapture2 method approved by FDA in 2003 and Roche's cobasHPVTest method approved by FDA in 2014.

However, the above detection methods for HPV nucleic acid also have their shortcomings. For example, some methods are relatively complicated and difficult to operate, resulting in high costs, and some methods cannot truly reflect the initial viral load except for corresponding amplification and detection instruments. quantity. The viral load is an important factor related to the course of the patient's disease and the degree of harm of virus transmission. Current studies have shown that with high-risk HPV genomic DNA>1pg/ml (100,000 HPV copies) as the threshold, the positive rate of cervical epithelial sarcoidosis (CIN) II-III is 97.5%, the positive rate of CIN III is 100%, and the positive rate of cervical cancer is positive. The rate is 100%. Therefore, an ideal HPV detection method should be based on nucleic acid hybridization analysis without amplification of HPV target nucleic acid.

Monoclonal antibody Sdr is a mouse-derived antibody with high specificity and affinity against DNA:RNA hybrids. Our patented technology is to establish an immunological detection method using Sdr monoclonal antibody to detect high-risk HPV16 E6 and E7 RNA. This method does not need reverse transcription and PCR technology to amplify the target sequence to be detected, but uses The labeled DNA probe is hybridized with HPV16 type E6 and E7 RNA to form a DNA: RNA hybrid, and then the Sdr monoclonal antibody is used for recognition and subsequent signal amplification. The detection method has the advantages of rapidity, low cost, high sensitivity, no need for complex amplification and detection instruments, and the ability to detect the initial viral load.

Contents of the invention

The problem to be solved by the present invention: the disadvantages of existing HPV nucleic acid detection methods, such as relatively complicated operation and high cost due to difficulties, corresponding amplification and detection instruments are required, and the initial viral load cannot be truly reflected. quantity. Provide a method and reagent for rapid detection of HPV, and assemble it into a detection kit, and determine the method of use of the kit, which can detect high-risk HPV16 type E6 and E7 RNA quickly, at low cost, and with high sensitivity, and the detection process does not require complicated procedures Amplification and detection instrument that detects initial viral load.

Overall technical route of the present invention: sample collection, processing, extract HPV16 type DNA or RNA from disease sample (blood, tissue, cervical secretion, cell exfoliation etc.); Analysis; according to the standard sequence published by GenBank, the oligonucleotide fragment (probe) that hybridizes with the target nucleic acid is designed; under specific hybridization conditions, the oligonucleotide probe is hybridized with the nucleic acid to be tested; the hybridization product is bound to the PLL On a pre-coated microtiter plate; add monoclonal antibodies against DNA-RNA hybrids; add anti-monoclonal antibody enzyme-labeled secondary antibodies; add enzyme substrates for color development, and measure absorbance after the reaction is terminated. The key reaction conditions, reagent components and concentrations in the experimental process were optimized, and the probe mixture, annealing buffer, primary antibody, secondary antibody dilution, substrate chromogenic solution, and reaction termination solution were developed to detect HPV16 types E6, Kit for E7RNA.

Use this kit to detect in vitro transcribed HPV16 type E6 and E7 RNA, and conduct methodological investigation. The detection method can detect HPV16E6 and E7 RNA with detection limits of 0.923pg/mL and 0.424pg/mL, respectively, and the concentration linear ranges are from 92.3pg/mL to 0.923pg/mL and from 42.4pg/mL to 0.424pg/mL, respectively.

Technical scheme of the present invention: 1. extract the genomic DNA in the disease sample; 2. design in vitro transcription primers according to the standard sequence published by GenBank, clone the target sequence, and sequence analysis; 3. design and target nucleic acid according to the standard sequence published by GenBank Oligonucleotide fragments (probes) for hybridization; 4. Establish an immunological detection method for HPV16 E6 and E7 RNA; 5. Optimize key reaction conditions, reagent components and concentrations, and determine their use; 6. HPV16 E6 and E7 RNA Sensitivity experiments of the immunoassay method. The details are as follows:

1. Extract genomic DNA or RNA from disease samples

Phenol/chloroform extraction or genomic DNA or RNA extraction kit directly extract HPV16 disease sample DNA or RNA from disease samples (blood, tissue, cervical secretions, cell exfoliation, etc.). Measure the nucleic acid concentration and adjust the nucleic acid concentration to 10ng/uL.

2. Design in vitro transcription primers according to the standard sequence published by GenBank, clone the target sequence, and sequence analysis

According to the complete genome sequence of HPV16 type published by NCBI (reference sequence: NC_001526.2), primers for in vitro transcription of the entire gene fragments of E6 and E7 were designed. Primer sequence (5'-3'): HPV16E6 gene upstream primer TAATACGACTCACTATAGGG ATGCACCAAAAGAGAACTGCAATGTTTCAG, HPV16E6 gene downstream Primer TTACAGCTGGGTTTCTCTACGTGTTCTTG; HPV16E7 gene upstream primer TAATACGACTCACTATAGGG ATGCATGGAGATACACCTACATTGCAT, HPV16E7 gene downstream primer TTATGGTTTCTGAGAACAGATGGGGCACAC. PCR amplification of the nucleic acid template extracted above; recovery of positive PCR products by gel; transformation into competent cells after connection to the carrier, expansion of culture after picking positive clones, and sequencing analysis after the extracted plasmid was identified as positive. It was determined that the templates used were HPV16 type E6 and E7 gene fragments.

3. Design the oligonucleotide fragment (probe) that hybridizes with the target nucleic acid according to the standard sequence published by GenBank

According to the complete genome sequence of HPV16 type published by NCBI (reference sequence: NC_001526.2), the oligonucleotide probes for E6 and E7 gene fragments are designed, and the sequences are as follows:

4. Establish the immunological detection method of HPV16 E6 and E7 RNA

In step 2, the PCR product amplified by primers with an in vitro transcription promoter is transcribed in vitro; the combination of the transcription product and one or more probes in step 3 in a certain annealing buffer, a certain annealing Hybridization is carried out under conditions such as temperature; after incubating the hybridization product with the PLL pre-treated microtiter plate, add the mouse monoclonal antibody against the DNA-RNA hybrid, then add the enzyme-labeled secondary antibody against the mouse monoclonal antibody, and finally The substrate was added to develop the color, and the absorbance value was detected after the reaction was terminated.

5. Optimize key reaction conditions, reagent components and concentrations, and determine their usage methods

After verifying that the system can be used to detect RNA in samples, optimize the indicators such as probe concentration, quantity, annealing buffer, annealing time, annealing temperature, blocking solution, primary antibody, secondary antibody concentration and its dilution. Under optimized conditions, the components in the detection system are assembled into a kit, and the components of the kit are:

Pre-coated ELISA plate: PLL-treated polystyrene microwell plate, sealed with BSA, stored at 4°C, with a shelf life of half a year.

Probe mixture 1: a combination of probes targeting HPV16 E6 RNA fragments, E6DNA1, E6DNA2, and E6DNA3, all at a concentration of 10uM.

Probe mixture 2: probe combinations targeting HPV16E7 RNA fragments, E7DNA1 and E7DNA2, both at a concentration of 10uM.

10×Annealing buffer: Tris-HCl 100mM, pH 7.5, EDTA 10mM, NaCl 1M.

Mouse monoclonal antibody Sdr solution against DNA-RNA.

Enzyme-labeled secondary antibody solution against Sdr: HRP-Goatanti-mouseIgG (H+L) solution.

Substrate chromogenic solution: TMB solution.

Washing buffer: 10×PBS solution.

Reaction termination solution: 2MH ₂ SO ₄ .

The optimized hybridization reaction conditions and immunoassay process are used as the instructions of the kit, which are described in detail as follows:

(1) Hybridize the in vitro transcribed RNA template or the RNA directly extracted from the disease sample with probe mixture 1 or 2, hybridization system: 10uL 10× annealing buffer, 3uL or 2uL probe mixture, 10uL RNA template solution, Supplement DEPCH ₂ O to 100uL; hybridization conditions: 95°C for 5min, 65°C for 2h, cooling on ice for 5min.

(2) Add 100uL hybridization product to each well of the pre-coated ELISA plate, incubate at room temperature for 1h, wash 5 times with 300uLPBS (pH7.4, containing 1‰Tween-20); add 100uLSdr monoclonal antibody solution to each well, and Incubate for 1h, wash as above; add 100uL enzyme-labeled secondary antibody solution to each well, incubate at room temperature for 1h, wash as above; add 100uL TMB substrate color development solution, incubate at 37°C for 20min; add 50uL reaction stop solution to each well to terminate the reaction, measure the Abs value, The measurement wavelength is 450nm, and the reference wavelength is 570nm.

6. Sensitivity experiment of immunodetection method for HPV16 E6 and E7 RNA

The HPV16 type E6 and E7 RNA templates transcribed in vitro were diluted 10 times and then detected.

Beneficial effects of the present invention: aiming at the deficiencies of existing HPV nucleic acid detection methods, the patented technology of the present invention is to establish a kind of immunodetection method utilizing Sdr monoclonal antibody to detect high-risk HPV16 type E6, E7 RNA. Reverse transcription and PCR technology amplify the target sequence to be tested, but use unlabeled DNA probes to hybridize with HPV16 type E6 and E7 RNA to form DNA: RNA hybrids, and then use Sdr monoclonal antibodies to identify and follow-up signal amplification. The detection method can detect HPV16E6 and E7 RNA with detection limits of 0.923pg/mL and 0.424pg/mL, respectively, and the concentration linear ranges are from 92.3pg/mL to 0.923pg/mL and from 42.4pg/mL to 0.424pg/mL, respectively. The detection method has the advantages of rapidity, low cost, high sensitivity, no need for complex amplification and detection instruments, and the ability to detect the initial viral load.

Description of drawings

Figure 1 Schematic diagram of the principle of the invention

1. Poly-L-lysine 2. DNA: RNA hybrid 3. Sdr monoclonal antibody 4. Goat anti-mouse antibody 5. Horseradish peroxidase 6. TMB substrate 7. Chromogenic product

Figure 2 PCR product (A) of HPV16E6, E7 ORF fragment and its in vitro transcription product electrophoresis (B)

A1.HPV16E62.HPV16E7M.DL2000DNAMarker

B1.HPV16E62.HPV16E6RNA3.HPV16E74.HPV16E7RNAM.DL2000DNAMarker

The invention method of Fig. 3 detects HPV16 type E6, E7 RNA sensitivity

A. Hybridization product of E6 DNA probe 1/2/3 mixture and serially diluted E6 RNA fragment

B. Hybridization product of E7 DNA probe 1/2 mixture and serially diluted E7 RNA

detailed description

Specific examples are provided below to further illustrate the technical solution of the present invention, but the application of the technology of the present invention is not limited to the examples.

Cloning, sequence analysis and in vitro transcription of 1HPV16 E6 and E7 gene fragments

Genomic DNA in the disease sample was extracted, HPV16 type E6, E7 gene (296bp-477bp length range) was amplified by PCR, HPV16 type E6, E7 ORF primer sequence (5'-3') with T7RNAPolyase promoter (underlined part): HPV16E6 gene upstream primer TAATACGACTCACTATAGGG ATGCACCAAAAGAGAACTGCAATGTTTCAG, HPV16E6 gene downstream primer TTACAGCTGGGTTTCTCTACGTGTTCTTG; HPV16E7 gene upstream primer TAATACGACTCACTATAGGG ATGCATGGAGATACACCTACATTGCAT, HPV16E7 gene downstream primer TTATGGTTTCTGAGCACAGATGG.

PCR reaction system: a single reaction contains 2.5uL10×PCRBuffer, 1uL2.5mMeachdNTPMix, _2uL25mMMgCl2 , 0.4uLTaq polymerase (5U/μL), 1uLSenseprimer(10uM), 1uLAnti _- senseprimer(10uM), 5uL template DNA, add ddH2O to make up 25uL; amplification conditions: pre-denaturation at 95°C for 5min, 1 cycle; denaturation at 95°C for 40s, HPV16E6 annealing temperature at 62.5°C, HPV16E7 annealing temperature at 71.0°C for 1min, extension at 72°C for 1min, the above three steps were performed 40 times Cycling; supplementary extension at 72°C for 8 min, 1 cycle. Run 1% agarose gel to recover HPV16E6, E7 positive fragments. Connect the gel-recovered fragments to pMD-19TVector; transform pMD-19T-HPV16E6 and E7 into DH5α competent cells, spread them on LB agar plates containing ampicillin (Amp), and culture them upside down at 37°C for 12-16 hours until single Colony formation. Pick a single colony and inoculate it in a test tube of LB medium containing Amp, and culture at 37°C with shaking at 200rpm for 12-16h. Extract pMD-19T-HPV16E6, E7 plasmids. Sequencing analysis was carried out after the plasmid was identified as positive by PCR (system conditions as before). After the sequence was compared with the standard sequence, the gene fragments consistent with the standard sequence were selected for in vitro transcription experiments.

Perform in vitro transcription of the above PCR products. The concentration of the PCR products HPV16E6 and HPV16E7 are both 0.2ug/uL. In vitro transcription system, each reaction contains 2uL10×TranscriptionBuffer, 2uL each of ATP, GTP, CTP, UTPSolution, 0.5uL RNase Inhibitor, 2uLT7RNA Polymerase, 5uL HPV16E6 or E7 DNA template, supplemented with DEPCH ₂ O to 20uL. The reaction conditions are: 42°C, 2 hours of reaction. The product was stored in a -80°C refrigerator for later use.

2Establishment of immunoassay method for E6 and E7 RNA of HPV16

2.1 Annealing hybridization

The probe sequences designed according to the HPV16 type E6 and E7 gene sequences published by NCBI are shown in Table 1.

Table 1 Probe sequence

Hybridize the above probe system with in vitro transcription template or directly extract RNA from disease samples, hybridization system: 10uL 10× annealing buffer, 3uL or 2uL probe mixture, 10uL RNA template solution, supplement DEPCH ₂ O to 100uL; hybridize Conditions: 95°C for 5min, 65°C for 2h, cooling on ice for 5min.

2.2 ELISA

Add 100uL hybridization product to each well of the pre-coated ELISA plate, incubate at room temperature for 1h, wash 5 times with 300uLPBS (pH7.4, containing 1‰Tween-20); add 100uLSdr monoclonal antibody solution to each well, incubate at room temperature for 1h, Wash as above; add 100uL enzyme-labeled secondary antibody solution to each well, incubate at room temperature for 1h, wash as above; add 100uL TMB substrate chromogenic solution, incubate at 37°C for 20min; add 50uL reaction stop solution to each well to terminate the reaction, measure the Abs value, and measure at a wavelength of 450nm , reference wavelength 570nm.

Each experiment was repeated more than twice, which was regarded as good repeatability and stability.

<110> Nanjing University

<120> A detection kit for HPV nucleic acid based on enzyme-linked immunoassay and its application

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taatacgactcactatagggatgcaccaaaagagaactgcaatgtttcag

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ttacagctgggtttctctacgtgttcttg

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taatacgactcactatagggatgcatggagatacacctacattgcat

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ttatggtttctgagaacagatggggcacac

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Claims (2)

1., based on HPV16 type E6, the E7RNA detection kit of enzyme-linked immuno assay, it is characterized in that the ingredient of kit is:

(1) pre-coated ELISA Plate: the polystyrene micropore plate of PLL process, BSA closes, and storage condition is 4 DEG C, half a year shelf-life;

(2) probe mixed liquor 1: for the probe combinations of HPV16E6RNA fragment, E6DNA1, E6DNA2, E6DNA3, concentration is 10 μMs;

Probe mixed liquor 1 probe E6DNA1:SEQIDNO5, and sequence (5 '-3 '): GCTCTGTGCATAACTGTGGTAACTTTCTGGGTCG;

Probe mixed liquor 1 probe E6DNA2:SEQIDNO6, and sequence (5 '-3 '): TCCCGAAAAGCAAAGTCATATACCTCACGTCGCAGTAACT;

Probe mixed liquor 1 probe E6DNA3:SEQIDNO7, and sequence (5 '-3 '): CAGCTGGGTTTCTCTACGTGTTCTTGATGATCTGCAACAA;

(3) probe mixed liquor 2: for the probe combinations of HPV16E7RNA fragment, E7DNA1, E7DNA2, concentration is 10 μMs;

Probe mixed liquor 2E7DNA1:SEQIDNO8, and sequence (5 '-3 '): TACGCACAACCGAAGCGTAGAGTCACACTTGC;

Probe mixed liquor 2E7DNA2:SEQIDNO9, and sequence (5 '-3 '): AGTGTGCCCATTAACAGGTCTTCCAAAGTACGAATGTCTACGTGTGTGC;

(4) 10 × annealing buffers: Tris-HCl100mM, pH7.5, EDTA10mM, NaCl1M;

(5) for the mouse resource monoclonal antibody Sdr solution of DNA-RNA;

(6) for the ELIAS secondary antibody solution of Sdr;

(7) substrate chromophoric solution: TMB solution;

(8) lavation buffer solution: 10 × PBS solution;

(9) reaction terminating liquid: 2MH ₂sO ₄.

2. kit is used for the detection method of in-vitro transcription RNA template according to claim 1, it is characterized in that being made up of following steps:

(1) in-vitro transcription RNA template and probe mixed liquor 1 or 2 are hybridized, hybridization system: 10 μ L10 × annealing buffers, 3 μ L or 2 μ L probe mixed liquors, RNA template solution 10 μ L, supplements DEPCH ₂o to 100 μ L; Hybridization conditions: 95 DEG C of 5min, 65 DEG C of 2h, cooled on ice 5min;

(2), in pre-coated ELISA Plate, every hole adds 100 μ L hybrid product, incubated at room 1h, and 300 μ LPBST wash 5 times; Every hole adds 100 μ LSdr monoclonal antibody solutions, and incubated at room 1h, as above washs; Every hole adds 100 μ L ELIAS secondary antibody solution, and incubated at room 1h, as above washs; Add 100 μ LTMB substrate chromophoric solutions, hatch 20min for 37 DEG C; Every hole adds 50 μ L reaction terminating liquid cessation reactions, surveys Abs value, measures wavelength 450nm, reference wavelength 570nm.