CN111607667A - Human papilloma virus genotyping nucleic acid labeling kit and use method thereof - Google Patents
Human papilloma virus genotyping nucleic acid labeling kit and use method thereof Download PDFInfo
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Abstract
The application relates to a human papilloma virus genotyping nucleic acid marker kit and a use method thereof; the nucleic acid labeling kit comprises 33 primer pairs and probes labeled by human papilloma virus subtype nucleic acids. The nucleic acid labeling kit comprises a PCR expansion reaction system: clinical specimen DNA or negative quality control template 5.0. mu. L, PCR amplification reagent mixture 12.5. mu. L, PCR amplification primer mixture 7.5. mu. L, PCR enzyme mixture 1. mu.L, total volume 25.0. mu.L. The PCR extension reaction parameters of the nucleic acid labeling kit are as follows: 5min × 1cycle at 95 ℃; (94 ℃ 30s, 42 ℃ 50s, 65 ℃ 45s) x 35 cycles; 10min × 1cycle at 72 deg.C; storing at 4 deg.C; and so on. The application adopts a fluorescent labeling mode to label 33 human papilloma virus subtype nucleic acids, and the displayed fluorescent labeling map can provide help for later analysis of medical experts.
Description
Technical Field
The application belongs to the technical field of nucleic acid marker kit contents, in particular to the technical field of nucleic acid marker kit contents capable of genotyping human papilloma virus genes, and the fluorescence marked result can provide help for later-stage expert analysis.
Background
Human Papilloma Virus (HPV), a small molecule, enveloped-free circular double-stranded DNA virus, has a genome length of about 8000 base pairs and is divided into 3 functional regions, namely an early transcribed region (E region), a late transcribed region (L region) and a non-transcribed region (long control region, LCR). HPV infects humans by direct or indirect contact with contaminated articles or by sexual transmission. The virus not only has host specificity, but also has tissue specificity, and can only infect skin and mucosal epithelial cells of human, causing various papilloma or wart of human skin and reproductive tract epithelial hyperplastic injury. The types of HPV which are determined at present are about more than 100, and can be divided into two types of high-risk types and low-risk types according to the pathogenicity or carcinogenic risk of different types. The existing kit product has the defects of less marked subtypes, complex and complicated operation, low recognizability, easy occurrence of misjudgment and the like, and a novel gene chip is provided for marking the nucleic acid of the virus rapidly and accurately at one time by considering that the virus has a plurality of subtypes, so that the technical problem to be solved is solved.
Disclosure of Invention
The present application provides a human papillomavirus genotyping nucleic acid labeling kit and a use method thereof, aiming at solving the above problems and defects.
The technical scheme is adopted for realizing the method.
The nucleic acid marking kit for human papilloma virus genotyping, which is described in the application, comprises a general primer pair (I): GP5+ (1), Cy3-GP6+ (1) or a universal primer pair (c): GP5+ (2), Cy3-GP6+ (2) and probes for labeling.
Further, the probe for labeling in the nucleic acid labeling kit described herein includes a probe for labeling HPV 6: NH2-Oligo (15T) -CGTAACTACATCTTCCACATACACCAA;
or probes for labeling HPV 11: NH2-Oligo (15T) -TGTGTCTAAATCTGCTACATACAC;
or probes for labeling HPV 16: NH2-Oligo (15T) -TTATGTGCTGCCATATCTACTT;
or probes for labeling HPV 18: NH2-Oligo (15T) -TCTACACAGTCTCCTGTACCTGGGCA;
or probes for labeling HPV 26: NH2-Oligo (15T) -CATTATCTGCAGCATCTGCATCCACT;
or probes for labeling HPV 31: NH2-Oligo (15T) -TGTGCTGCAATTGCAAACAGTGATAC;
or probes for labeling HPV 33: NH2-Oligo (15T) -TTTATGCACACAAGTAACTAGTGAC;
or probes for labeling HPV 34: NH2-Oligo (15T) -CAATCCACAAGTACAAATGCACCA;
or probes for labeling HPV 35: NH2-Oligo (15T) -GTGTGTTCTGCTGTGTCTTCTAGT;
or probes for labeling HPV 39: NH2-Oligo (15T) -TCTACCTCTATAGAGTCTTCCATA;
or probes for labeling HPV 40: NH2-Oligo (15T) -CACACAGTCCCCCACACCAACCC;
or probes for labeling HPV 42: NH2-Oligo (15T) -CTGCAACATCTGGTGATACATA;
or probes for labeling HPV 43: NH2-Oligo (15T) -CTGACCCTACTGTGCCCAGTACATA;
or probes for labeling HPV 44: NH2-Oligo (15T) -ACTACACAGTCCCCTCCGTCTAC;
or probes for labeling HPV 45: NH2-Oligo (15T) -AAAATCCTGTGCCAAGTACAT;
or probes for labeling HPV 51: NH2-Oligo (15T) -CTGCCACTGCTGCGGTTTCCCCA;
or probes for labeling HPV 52: NH2-Oligo (15T) -GAGGTTAAAAAGGAAAGCACATA;
or probes for labeling HPV 53: NH2-Oligo (15T) -TCTACATATAATTCAAAGCAAAT;
or probes for labeling HPV 54: NH2-Oligo (15T) -GCATCCACGCAGGATAGCTTTAAT;
or probes for labeling HPV 55: NH2-Oligo (15T) -CTGCTACAACTCAGTCTCCATCTACA;
or probes for labeling HPV 56: NH2-Oligo (15T) -CTGCTACAGAACAGTTAAGTAAATATG;
or probes for labeling HPV 58: NH2-Oligo (15T) -ATGCACTGAAGTAACTAAGGAAGGTAC;
or probes for labeling HPV 59: NH2-Oligo (15T) -TACTGCTTCTATTCCTAATGTATAC;
or probes for labeling HPV 61: NH2-Oligo (15T) -GCTACATCCCCCCCTGTATCTGAATA;
or probes for labeling HPV 62: NH2-Oligo (15T) -CTGCAGCAGAATACACGGCTACCAACT;
or probes for labeling HPV 66: NH2-Oligo (15T) -TAATGCAGCTAAAAGCACATTAACTAA;
or probes for labeling HPV 68: NH2-Oligo (15T) -TACTACTGAATCAGCTGTACCAAT;
or probes for labeling HPV 70: NH2-Oligo (15T) -GCCTGCACCGAAACGGCCATACCTGCTG;
or probes for labeling HPV 72: NH2-Oligo (15T) -CACAGCGTCCTCTGTATCAGAATATAC;
or probes for labeling HPV 73: NH2-Oligo (15T) -AGGTACACAGGCTAGTAGCTCTACTA;
or probes for labeling HPV 83: NH2-Oligo (15T) -TGCTGCTACACAGGCTAATGAATACA;
or probes for labeling HPV 84: NH2-Oligo (15T) -CTGCTACCAACACCGAATCAGAATATA;
or probes for labeling HPV 81: NH2-Oligo (15T) -CTACATCTGCTGCTGCAGAATACA;
any one or more combinations thereof.
Further, the nucleic acid labeling kit described herein comprises an internal reference primer pair: IC-RP, Cy3-IC-FP with Probe: NH2-Oligo (15T) -CATTGTAGAAGGTGTGGTGCCAGA.
Further, the nucleic acid labeling kit described herein includes a hybridization control probe HC: NH2-Oligo (15T) -TCAGGGTGAGGATGCC.
Further, the nucleic acid labeling kit comprises negative quality control probes NC: NH 2-TTTTTTTTTTTTTTTT.
Further, the nucleic acid labeling kit described in the present application includes a PCR extension reaction system: clinical specimen DNA or negative quality control template 5.0. mu. L, PCR amplification reagent mixture 12.5. mu. L, PCR amplification primer mixture 7.5. mu. L, PCR enzyme mixture 1. mu.L, total volume 25.0. mu.L.
Further, the PCR extension reaction parameters of the nucleic acid labeling kit described in the present application: 5min × 1cycle at 95 ℃; (94 ℃ 30s, 42 ℃ 50s, 65 ℃ 45s) x 35 cycles; 10min × 1cycle at 72 deg.C; storing at 4 ℃.
Further, the nucleic acid labeling kit comprises a gene chip, wherein the gene chip is arranged into a rectangle, and is divided into an upper part and a lower part by N parts, each part is provided with M repeats, the 1 st column of the 1 st part is provided with a hybridization control site, and the last 1 st column of the 1 st part is provided with a negative quality control site; the 1 st column of the last 1 part is set as an internal reference control site, and the 1 st column of the last 1 part is set as a hybridization control site; and setting the rest sites as pre-marked sample sites to be detected.
Further, the nucleic acid labeling kit comprises an eluent I and an eluent II; eluent I: preparing in a chip elution container according to the proportion of distilled water or purified water to 20 XSSC to 10 percent SDS to 100 to 5 to 1; eluent II: preparing in a chip elution container according to the proportion of distilled water or purified water to 20 XSSC to 10 percent SDS to 400 to 1 to 4; the eluent is used after hybridization of the gene chip, specifically, eluent I is used firstly, and shaking elution is carried out for 1 min; then using eluent II, shaking and eluting for 1 min; and after the elution is finished, naturally drying the chip or spin-drying the chip at a low speed.
The application of the method for using the nucleic acid labeling kit comprises the following steps:
(1) preparing instruments and consumables: preparing a sterile operating platform, a biological safety cabinet, a gene amplification instrument, a sampler, a table centrifuge, a constant-temperature heating table, a water bath constant-temperature oscillator, a slide glass rack, a slide glass elution container and a chip scanner;
(2) preparing an eluent I and an eluent II;
(3) obtaining a nucleic acid sample or a suspected nucleic acid sample;
(4) PCR amplification and marking;
(5) and (3) hybridization: 5.1 heating the constant temperature heating table to 42 ℃, and fixing the chip by using a hybridization module preheated at 42 ℃;
5.2 placing the PCR product in a PCR instrument, heating to 95 ℃ for denaturation for 5min, and immediately carrying out ice bath for 3 min;
5.3 adding 75 mu L of hybridization buffer solution into a clinical sample amplification reaction tube, fully and uniformly mixing, and dripping the mixture into a corresponding microarray reaction chamber from a sample adding hole for hybridization for 60 min; in the operation process, the microarray area is prevented from being touched, so that mixed liquid drops are prevented from falling on the surface of the hybridization module;
(6) and (3) elution: preheating the eluent at 37 ℃ before elution, carefully taking the chip out of the hybridization module, and immediately putting the chip into an elution container to finish the elution process; eluting the eluent I for 1min by shaking; eluting the eluent II for 1min by shaking; after the elution is finished, the chip is naturally dried or spin-dried at low speed, and then scanning can be carried out;
(7) scanning: placing the dried chip in a laser scanner (crystal core LuxScan 10K) and scanning by using laser with the wavelength of 532nm to obtain scanning data and a map; completing the nucleic acid labeling.
The primer pair and the probe of the technical method have extremely high specificity, and can well mark pathogens, so that the time spent on data analysis is saved, and data information marked by various virus nucleic acids is obtained in a shorter time. The method adopts a fluorescent labeling mode, can label 33 HPV pathogen nucleic acids simultaneously, and the displayed fluorescent labeling map can provide help for later analysis of medical experts.
The present application is further explained below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a diagram showing the arrangement of one of the structures of the gene chip of the present application.
FIG. 2 is a HPV6 subtype marker map of the present application.
FIG. 3 is a HPV11 subtype marker map of the present application.
FIG. 4 is a HPV16 subtype marker map of the present application.
FIG. 5 is a HPV18 subtype marker map of the present application.
FIG. 6 is a HPV26 subtype marker map of the present application.
FIG. 7 is a HPV31 subtype marker map of the present application.
FIG. 8 is a HPV33 subtype marker map of the present application.
FIG. 9 is a HPV34 subtype marker map of the present application.
FIG. 10 is a HPV35 subtype marker map of the present application.
FIG. 11 is a HPV39 subtype marker map of the present application.
FIG. 12 is a HPV40 subtype marker map of the present application.
FIG. 13 is a HPV42 subtype marker map of the present application.
FIG. 14 is a HPV43 subtype marker map of the present application.
FIG. 15 is a HPV44 subtype marker map of the present application.
FIG. 16 is a HPV45 subtype marker map of the present application.
FIG. 17 is a HPV51 subtype marker map of the present application.
FIG. 18 is a HPV52 subtype marker map of the present application.
FIG. 19 is a HPV53 subtype marker map of the present application.
FIG. 20 is a HPV54 subtype marker map of the present application.
FIG. 21 is a HPV55 subtype marker map of the present application.
FIG. 22 is a HPV56 subtype marker map of the present application.
FIG. 23 is a HPV58 subtype signature of the present application.
FIG. 24 is a HPV59 subtype marker map of the present application.
FIG. 25 is a HPV61 subtype marker map of the present application.
FIG. 26 is a HPV62 subtype marker map of the present application.
FIG. 27 is a HPV66 subtype marker map of the present application.
FIG. 28 is a HPV68 subtype marker map of the present application.
FIG. 29 is a HPV70 subtype marker map of the present application.
FIG. 30 is a HPV72 subtype marker map of the present application.
FIG. 31 is a HPV73 subtype marker map of the present application.
FIG. 32 is a HPV81 subtype marker map of the present application.
FIG. 33 is a HPV83 subtype marker map of the present application.
FIG. 34 is a labeling chart of application negative control.
Detailed Description
Design of PCR primers
The performance of the PCR primers plays a decisive role in the PCR reaction. In a multiplex PCR amplification system, in order to improve the amplification homology, while keeping with the basic principle of primer design, similar annealing temperature and GC content need to be designed for each pair of primers, and the formation of secondary structures of the primers and dimers among the primers are reduced as much as possible, and the design general guiding principle is as follows:
A. the Tm of the primers should be 50-60 ℃ and the difference between Tm of the upstream primer and Tm of the downstream primer is not more than 2 ℃.
B. The GC content of the primer was around 50%.
C. The length of the primer is preferably 18-24nt, and the specificity and efficiency of amplification can be affected by the primer which is too long or too short.
D. The primer sequence avoids the 3' end ending with a.
E. Primer mismatching: the ambiguity of the 3 'end, i.e.the fact that the 3' end is not linked to three consecutive bases, should be avoided as much as possible, since even a single base mismatch at this position prevents primer extension and thus reduces the number of desired products.
Gc clamp: the inclusion of a G or C at the 3 'end of the primer increases the amplification efficiency of the primer, which promotes the proper binding of the 3' end of the primer to the template, since the GC clamp can form a more stable hydrogen bond.
G. The hairpin structure of the primer cannot be paired with 3 consecutive bases, which would otherwise limit the ability of the primer to bind to the site of interest.
H. In primer dimers, the pairing region should be avoided at the 3' end.
And (3) determining species specific sequences of different pathogens to be detected by consulting literature and comparing the sequences, and designing primers in corresponding regions according to the principle. The positive control site in the product is mainly used for marking the collection condition of human cells, and the design can be carried out by adopting human housekeeping gene beta-actin. Design and screening of primers were performed using gene chip primer-related design software. The design flow is as follows:
A. looking up related documents, referring to and determining the gene or region where the beta-actin primer is located;
B. searching and downloading corresponding nucleic acid sequences, and performing primer design and screening by using a primer design auxiliary tool, wherein the primer design parameter is that the length of a primer is 17-24 nt; tm 57 ± 3 ℃ with product lengths specified between 80 and 300 bp;
C. and (3) screening out optimal condition primers through joint verification of multiple primer designs and chip design auxiliary software.
TABLE 1 primer sequence Listing
By referring to relevant documents of molecular biology at home and abroad, the design of a probe for marking needs to meet the following conditions:
A. the length of the pathogen-labeled probe should be 27-35nt, and too long or too short will affect the specificity and hybridization efficiency.
B. The GC content of the probe is 30-60%, otherwise, the occurrence probability of nonspecific hybridization is increased.
C. The hybridization of the probe and the target molecule is influenced by the steric hindrance of the probe, and a 15nt Oligo dT linker can be connected to the 5' end of the probe to change the steric conformation. Alternatively, the probe may be modified by introducing a chemical group (e.g., -NH) at the end of the probe2) The high-freedom covalent combination of the probe and the substrate is realized, so that the hybridization kinetics are further improved, and the probe hybridization efficiency is obviously improved.
D. No bases exceeding 4nt should form reverse complementary base pairs inside the probe base, otherwise, a hairpin structure is easily formed.
E. The same base is prevented from appearing repeatedly in the probe sequence, and is generally less than or equal to 4 nt. Since the probes are under the same hybridization conditions, different probes should have similar annealing temperatures.
F. Since the chip probes are reacted under the same hybridization conditions, different probes have similar annealing temperatures as much as possible.
And designing and screening the probes in the corresponding primer amplification segment areas according to design requirements by using gene chip probe related design software.
NC in the product is a negative control probe, the sequence of the negative control probe is 15 continuous thymines (OligoT15), and the negative control probe is used for labeling non-specific hybridization reaction possibly occurring with a probe linker sequence and monitoring the labeling specificity of the kit.
HC is hybridization control probe, whose sequence is the reverse complementary sequence of the positive control (IC) labeled amplification primer, and is used to mark whether the chip hybridization system is normal.
IC is a sample extraction control and positive control probe, the sequence of which is a housekeeping gene beta-actin fragment in a human genome, and the probe is used for monitoring the quality of the sample and the effectiveness of a PCR amplification system.
Screening of probes
After the probes designed according to the principle are entrusted and synthesized, the determined clinical specimen is used as a template, the chip with the probes spotted is verified, and the optimal probes of all the marker sites are analyzed and screened by analyzing the hybridization signal intensity and specificity of all the probes of the hybridization chip.
The pathogen-labeled probes and control probes ultimately determined by the product from the above study are shown in table 2 below.
TABLE 2 Probe information Table
The nucleic acid marking kit for human papilloma virus genotyping, which is described in the application, comprises a general primer pair (I): GP5+ (1), Cy3-GP6+ (1) or a universal primer pair (c): GP5+ (2), Cy3-GP6+ (2) and probes for labeling.
Further, the probe for labeling in the nucleic acid labeling kit described herein includes a probe for labeling HPV 6: NH2-Oligo (15T) -CGTAACTACATCTTCCACATACACCAA;
or probes for labeling HPV 11: NH2-Oligo (15T) -TGTGTCTAAATCTGCTACATACAC;
or probes for labeling HPV 16: NH2-Oligo (15T) -TTATGTGCTGCCATATCTACTT;
or probes for labeling HPV 18: NH2-Oligo (15T) -TCTACACAGTCTCCTGTACCTGGGCA;
or probes for labeling HPV 26: NH2-Oligo (15T) -CATTATCTGCAGCATCTGCATCCACT;
or probes for labeling HPV 31: NH2-Oligo (15T) -TGTGCTGCAATTGCAAACAGTGATAC;
or probes for labeling HPV 33: NH2-Oligo (15T) -TTTATGCACACAAGTAACTAGTGAC;
or probes for labeling HPV 34: NH2-Oligo (15T) -CAATCCACAAGTACAAATGCACCA;
or probes for labeling HPV 35: NH2-Oligo (15T) -GTGTGTTCTGCTGTGTCTTCTAGT;
or probes for labeling HPV 39: NH2-Oligo (15T) -TCTACCTCTATAGAGTCTTCCATA;
or probes for labeling HPV 40: NH2-Oligo (15T) -CACACAGTCCCCCACACCAACCC;
or probes for labeling HPV 42: NH2-Oligo (15T) -CTGCAACATCTGGTGATACATA;
or probes for labeling HPV 43: NH2-Oligo (15T) -CTGACCCTACTGTGCCCAGTACATA;
or probes for labeling HPV 44: NH2-Oligo (15T) -ACTACACAGTCCCCTCCGTCTAC;
or probes for labeling HPV 45: NH2-Oligo (15T) -AAAATCCTGTGCCAAGTACAT;
or probes for labeling HPV 51: NH2-Oligo (15T) -CTGCCACTGCTGCGGTTTCCCCA;
or probes for labeling HPV 52: NH2-Oligo (15T) -GAGGTTAAAAAGGAAAGCACATA;
or probes for labeling HPV 53: NH2-Oligo (15T) -TCTACATATAATTCAAAGCAAAT;
or probes for labeling HPV 54: NH2-Oligo (15T) -GCATCCACGCAGGATAGCTTTAAT;
or probes for labeling HPV 55: NH2-Oligo (15T) -CTGCTACAACTCAGTCTCCATCTACA;
or probes for labeling HPV 56: NH2-Oligo (15T) -CTGCTACAGAACAGTTAAGTAAATATG;
or probes for labeling HPV 58: NH2-Oligo (15T) -ATGCACTGAAGTAACTAAGGAAGGTAC;
or probes for labeling HPV 59: NH2-Oligo (15T) -TACTGCTTCTATTCCTAATGTATAC;
or probes for labeling HPV 61: NH2-Oligo (15T) -GCTACATCCCCCCCTGTATCTGAATA;
or probes for labeling HPV 62: NH2-Oligo (15T) -CTGCAGCAGAATACACGGCTACCAACT;
or probes for labeling HPV 66: NH2-Oligo (15T) -TAATGCAGCTAAAAGCACATTAACTAA;
or probes for labeling HPV 68: NH2-Oligo (15T) -TACTACTGAATCAGCTGTACCAAT;
or probes for labeling HPV 70: NH2-Oligo (15T) -GCCTGCACCGAAACGGCCATACCTGCTG;
or probes for labeling HPV 72: NH2-Oligo (15T) -CACAGCGTCCTCTGTATCAGAATATAC;
or probes for labeling HPV 73: NH2-Oligo (15T) -AGGTACACAGGCTAGTAGCTCTACTA;
or probes for labeling HPV 83: NH2-Oligo (15T) -TGCTGCTACACAGGCTAATGAATACA;
or probes for labeling HPV 84: NH2-Oligo (15T) -CTGCTACCAACACCGAATCAGAATATA;
or probes for labeling HPV 81: NH2-Oligo (15T) -CTACATCTGCTGCTGCAGAATACA;
any one or more combinations thereof.
Further, the nucleic acid labeling kit described herein comprises an internal reference primer pair: IC-RP, Cy3-IC-FP with Probe: NH2-Oligo (15T) -CATTGTAGAAGGTGTGGTGCCAGA.
Further, the nucleic acid labeling kit described herein includes a hybridization control probe HC: NH2-Oligo (15T) -TCAGGGTGAGGATGCC.
Further, the nucleic acid labeling kit comprises negative quality control probes NC: NH 2-TTTTTTTTTTTTTTTT.
Further, the nucleic acid labeling kit described in the present application includes a PCR extension reaction system: clinical specimen DNA or negative quality control template 5.0. mu. L, PCR amplification reagent mixture 12.5. mu. L, PCR amplification primer mixture 7.5. mu. L, PCR enzyme mixture 1. mu.L, total volume 25.0. mu.L.
Further, the PCR extension reaction parameters of the nucleic acid labeling kit described in the present application: 5min × 1cycle at 95 ℃; (94 ℃ 30s, 42 ℃ 50s, 65 ℃ 45s) x 35 cycles; 10min × 1cycle at 72 deg.C; storing at 4 ℃.
Further, the nucleic acid labeling kit comprises a gene chip, wherein the gene chip is arranged into a rectangle, and is divided into an upper part and a lower part by N parts, each part is provided with M repeats, the 1 st column of the 1 st part is provided with a hybridization control site, and the last 1 st column of the 1 st part is provided with a negative quality control site; the 1 st column of the last 1 part is set as an internal reference control site, and the 1 st column of the last 1 part is set as a hybridization control site; and setting the rest sites as pre-marked sample sites to be detected. NC is a negative quality control probe; IC is an extracted control and internal control probe; HC is hybridization control probe; BC is a blank site. The accompanying drawings of the partial success test of this application are shown in fig. 1-34.
Further, the nucleic acid labeling kit comprises an eluent I and an eluent II; eluent I: preparing in a chip elution container according to the proportion of distilled water or purified water to 20 XSSC to 10 percent SDS to 100 to 5 to 1; eluent II: preparing in a chip elution container according to the proportion of distilled water or purified water to 20 XSSC to 10 percent SDS to 400 to 1 to 4; the eluent is used after hybridization of the gene chip, specifically, eluent I is used firstly, and shaking elution is carried out for 1 min; then using eluent II, shaking and eluting for 1 min; and after the elution is finished, naturally drying the chip or spin-drying the chip at a low speed.
The application of the method for using the nucleic acid labeling kit comprises the following steps:
(1) preparing instruments and consumables: preparing a sterile operating platform, a biological safety cabinet, a gene amplification instrument, a sampler, a table centrifuge, a constant-temperature heating table, a water bath constant-temperature oscillator, a slide glass rack, a slide glass elution container and a chip scanner;
(2) preparing an eluent I and an eluent II;
(3) obtaining a nucleic acid sample or a suspected nucleic acid sample;
(4) PCR amplification and marking;
(5) and (3) hybridization: 5.1 heating the constant temperature heating table to 42 ℃, and fixing the chip by using a hybridization module preheated at 42 ℃;
5.2 placing the PCR product in a PCR instrument, heating to 95 ℃ for denaturation for 5min, and immediately carrying out ice bath for 3 min;
5.3 adding 75 mu L of hybridization buffer solution into a clinical sample amplification reaction tube, fully and uniformly mixing, and dripping the mixture into a corresponding microarray reaction chamber from a sample adding hole for hybridization for 60 min; in the operation process, the microarray area is prevented from being touched, so that mixed liquid drops are prevented from falling on the surface of the hybridization module;
(6) and (3) elution: preheating the eluent at 37 ℃ before elution, carefully taking the chip out of the hybridization module, and immediately putting the chip into an elution container to finish the elution process; eluting the eluent I for 1min by shaking; eluting the eluent II for 1min by shaking; after the elution is finished, the chip is naturally dried or spin-dried at low speed, and then scanning can be carried out;
(7) scanning: placing the dried chip in a laser scanner (crystal core LuxScan 10K) and scanning by using laser with the wavelength of 532nm to obtain scanning data and a map; completing the nucleic acid labeling.
The above description is only for the specific embodiment of the present application, and the common general knowledge of the known specific structure and characteristics in the scheme is not described herein too much. It should be noted that the above-mentioned embodiments do not limit the present application in any way, and all technical solutions obtained by means of equivalent substitution or equivalent transformation for those skilled in the art are within the protection scope of the present application. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
<110> Kunming atlas-based biochip industries, Ltd
<120> human papilloma virus genotyping nucleic acid labeling kit and using method thereof
<160>42
<210>1
<211>27
<212>DNA
<213> Artificial sequence
<400>1
CGTAACTACATCTTCCACATACACCAA
<210>2
<211>24
<212>DNA
<213> Artificial sequence
<400>2
TGTGTCTAAATCTGCTACATACAC
<210>3
<211>22
<212>DNA
<213> Artificial sequence
<400>3
TTATGTGCTGCCATATCTACTT
<210>4
<211>26
<212>DNA
<213> Artificial sequence
<400>4
TCTACACAGTCTCCTGTACCTGGGCA
<210>5
<211>26
<212>DNA
<213> Artificial sequence
<400>5
CATTATCTGCAGCATCTGCATCCACT
<210>6
<211>26
<212>DNA
<213> Artificial sequence
<400>6
TGTGCTGCAATTGCAAACAGTGATAC
<210>7
<211>25
<212>DNA
<213> Artificial sequence
<400>7
TTTATGCACACAAGTAACTAGTGAC
<210>8
<211>24
<212>DNA
<213> Artificial sequence
<400>8
CAATCCACAAGTACAAATGCACCA
<210>9
<211>24
<212>DNA
<213> Artificial sequence
<400>9
GTGTGTTCTGCTGTGTCTTCTAGT
<210>10
<211>24
<212>DNA
<213> Artificial sequence
<400>10
TCTACCTCTATAGAGTCTTCCATA
<210>11
<211>23
<212>DNA
<213> Artificial sequence
<400>11
CACACAGTCCCCCACACCAACCC
<210>12
<211>22
<212>DNA
<213> Artificial sequence
<400>12
CTGCAACATCTGGTGATACATA
<210>13
<211>25
<212>DNA
<213> Artificial sequence
<400>13
CTGACCCTACTGTGCCCAGTACATA
<210>14
<211>23
<212>DNA
<213> Artificial sequence
<400>14
ACTACACAGTCCCCTCCGTCTAC
<210>15
<211>21
<212>DNA
<213> Artificial sequence
<400>15
AAAATCCTGTGCCAAGTACAT
<210>16
<211>23
<212>DNA
<213> Artificial sequence
<400>16
CTGCCACTGCTGCGGTTTCCCCA
<210>17
<211>23
<212>DNA
<213> Artificial sequence
<400>17
GAGGTTAAAAAGGAAAGCACATA
<210>18
<211>23
<212>DNA
<213> Artificial sequence
<400>18
TCTACATATAATTCAAAGCAAAT
<210>19
<211>24
<212>DNA
<213> Artificial sequence
<400>19
GCATCCACGCAGGATAGCTTTAAT
<210>20
<211>26
<212>DNA
<213> Artificial sequence
<400>20
CTGCTACAACTCAGTCTCCATCTACA
<210>21
<211>27
<212>DNA
<213> Artificial sequence
<400>21
CTGCTACAGAACAGTTAAGTAAATATG
<210>22
<211>27
<212>DNA
<213> Artificial sequence
<400>22
ATGCACTGAAGTAACTAAGGAAGGTAC
<210>23
<211>25
<212>DNA
<213> Artificial sequence
<400>23
TACTGCTTCTATTCCTAATGTATAC
<210>24
<211>26
<212>DNA
<213> Artificial sequence
<400>24
GCTACATCCCCCCCTGTATCTGAATA
<210>25
<211>27
<212>DNA
<213> Artificial sequence
<400>25
CTGCAGCAGAATACACGGCTACCAACT
<210>26
<211>27
<212>DNA
<213> Artificial sequence
<400>26
TAATGCAGCTAAAAGCACATTAACTAA
<210>27
<211>24
<212>DNA
<213> Artificial sequence
<400>27
TACTACTGAATCAGCTGTACCAAT
<210>28
<211>28
<212>DNA
<213> Artificial sequence
<400>28
GCCTGCACCGAAACGGCCATACCTGCTG
<210>29
<211>27
<212>DNA
<213> Artificial sequence
<400>29
CACAGCGTCCTCTGTATCAGAATATAC
<210>30
<211>26
<212>DNA
<213> Artificial sequence
<400>30
AGGTACACAGGCTAGTAGCTCTACTA
<210>31
<211>26
<212>DNA
<213> Artificial sequence
<400>31
TGCTGCTACACAGGCTAATGAATACA
<210>32
<211>27
<212>DNA
<213> Artificial sequence
<400>32
CTGCTACCAACACCGAATCAGAATATA
<210>33
<211>24
<212>DNA
<213> Artificial sequence
<400>33
CTACATCTGCTGCTGCAGAATACA
<210>34
<211>24
<212>DNA
<213> Artificial sequence
<400>34
CATTGTAGAAGGTGTGGTGCCAGA
<210>35
<211>16
<212>DNA
<213> Artificial sequence
<400>35
TCAGGGTGAGGATGCC
<210>36
<211>16
<212>DNA
<213> Artificial sequence
<400>36
TTTTTTTTTTTTTTTT
<210>37
<211>18
<212>DNA
<213> Artificial sequence
<400>37
TGTTACTGTTGTAGATAC
<210>38
<211>19
<212>DNA
<213> Artificial sequence
<400>38
AAAATAAACTGTAAATCAA
<210>39
<211>19
<212>DNA
<213> Artificial sequence
<400>39
TGTTACTGTTGTAGATACC
<210>40
<211>21
<212>DNA
<213> Artificial sequence
<400>40
AAAATAAACTGTAAATCAAAC
<210>41
<211>19
<212>DNA
<213> Artificial sequence
<400>41
AAGGTGACAGCAGTCGGTT
<210>42
<211>17
<212>DNA
<213> Artificial sequence
<400>42
TGGGGTGGCTTTTAGGA
Claims (10)
1. The human papilloma virus genotyping nucleic acid labeling kit is characterized by comprising a universal primer pair (I): GP5+ (1), Cy3-GP6+ (1) or a universal primer pair (c): GP5+ (2), Cy3-GP6+ (2) and probes for labeling.
2. The human papillomavirus genotyping nucleic acid labeling kit of claim 1, wherein the probes for labeling in the nucleic acid labeling kit comprise probes for labeling HPV 6:
NH2-Oligo(15T)-CGTAACTACATCTTCCACATACACCAA；
or probes for labeling HPV 11: NH2-Oligo (15T) -TGTGTCTAAATCTGCTACATACAC;
or probes for labeling HPV 16: NH2-Oligo (15T) -TTATGTGCTGCCATATCTACTT;
or probes for labeling HPV 18: NH2-Oligo (15T) -TCTACACAGTCTCCTGTACCTGGGCA;
or probes for labeling HPV 26: NH2-Oligo (15T) -CATTATCTGCAGCATCTGCATCCACT;
or probes for labeling HPV 31: NH2-Oligo (15T) -TGTGCTGCAATTGCAAACAGTGATAC;
or probes for labeling HPV 33: NH2-Oligo (15T) -TTTATGCACACAAGTAACTAGTGAC;
or probes for labeling HPV 34: NH2-Oligo (15T) -CAATCCACAAGTACAAATGCACCA;
or probes for labeling HPV 35: NH2-Oligo (15T) -GTGTGTTCTGCTGTGTCTTCTAGT;
or probes for labeling HPV 39: NH2-Oligo (15T) -TCTACCTCTATAGAGTCTTCCATA;
or probes for labeling HPV 40: NH2-Oligo (15T) -CACACAGTCCCCCACACCAACCC;
or probes for labeling HPV 42: NH2-Oligo (15T) -CTGCAACATCTGGTGATACATA;
or probes for labeling HPV 43: NH2-Oligo (15T) -CTGACCCTACTGTGCCCAGTACATA;
or probes for labeling HPV 44: NH2-Oligo (15T) -ACTACACAGTCCCCTCCGTCTAC;
or probes for labeling HPV 45: NH2-Oligo (15T) -AAAATCCTGTGCCAAGTACAT;
or probes for labeling HPV 51: NH2-Oligo (15T) -CTGCCACTGCTGCGGTTTCCCCA;
or probes for labeling HPV 52: NH2-Oligo (15T) -GAGGTTAAAAAGGAAAGCACATA;
or probes for labeling HPV 53: NH2-Oligo (15T) -TCTACATATAATTCAAAGCAAAT;
or probes for labeling HPV 54: NH2-Oligo (15T) -GCATCCACGCAGGATAGCTTTAAT;
or probes for labeling HPV 55: NH2-Oligo (15T) -CTGCTACAACTCAGTCTCCATCTACA;
or probes for labeling HPV 56: NH2-Oligo (15T) -CTGCTACAGAACAGTTAAGTAAATATG;
or probes for labeling HPV 58: NH2-Oligo (15T) -ATGCACTGAAGTAACTAAGGAAGGTAC;
or probes for labeling HPV 59: NH2-Oligo (15T) -TACTGCTTCTATTCCTAATGTATAC;
or probes for labeling HPV 61: NH2-Oligo (15T) -GCTACATCCCCCCCTGTATCTGAATA;
or probes for labeling HPV 62: NH2-Oligo (15T) -CTGCAGCAGAATACACGGCTACCAACT;
or probes for labeling HPV 66: NH2-Oligo (15T) -TAATGCAGCTAAAAGCACATTAACTAA;
or probes for labeling HPV 68: NH2-Oligo (15T) -TACTACTGAATCAGCTGTACCAAT;
or probes for labeling HPV 70: NH2-Oligo (15T) -GCCTGCACCGAAACGGCCATACCTGCTG;
or probes for labeling HPV 72: NH2-Oligo (15T) -CACAGCGTCCTCTGTATCAGAATATAC;
or probes for labeling HPV 73: NH2-Oligo (15T) -AGGTACACAGGCTAGTAGCTCTACTA;
or probes for labeling HPV 83: NH2-Oligo (15T) -TGCTGCTACACAGGCTAATGAATACA;
or probes for labeling HPV 84: NH2-Oligo (15T) -CTGCTACCAACACCGAATCAGAATATA;
or probes for labeling HPV 81: NH2-Oligo (15T) -CTACATCTGCTGCTGCAGAATACA;
any one or more combinations thereof.
3. The human papillomavirus genotyping nucleic acid labeling kit of claim 2, wherein the nucleic acid labeling kit comprises internal reference control primer pairs: IC-RP, Cy3-IC-FP with Probe:
NH2-Oligo(15T)-CATTGTAGAAGGTGTGGTGCCAGA。
4. the human papillomavirus genotyping nucleic acid labeling kit according to claim 2, characterized in that it comprises hybridization control probes HC: NH2-Oligo (15T) -TCAGGGTGAGGATGCC.
5. The human papillomavirus genotyping nucleic acid labeling kit according to claim 2, characterized in that the nucleic acid labeling kit comprises negative quality control probes NC: NH 2-TTTTTTTTTTTTTTTT.
6. The human papillomavirus genotyping nucleic acid labeling kit according to claim 1, wherein the nucleic acid labeling kit comprises a PCR extension reaction system: clinical specimen DNA or negative quality control template 5.0. mu. L, PCR amplification reagent mixture 12.5. mu. L, PCR amplification primer mixture 7.5. mu. L, PCR enzyme mixture 1. mu.L, total volume 25.0. mu.L.
7. The human papillomavirus genotyping nucleic acid labeling kit according to claim 1, wherein the PCR extension reaction parameters of the nucleic acid labeling kit are: 5min × 1cycle at 95 ℃; (94 ℃ 30s, 42 ℃ 50s, 65 ℃ 45s) x 35 cycles; 10min × 1cycle at 72 deg.C; storing at 4 ℃.
8. The human papillomavirus genotyping nucleic acid marker kit according to claim 1, wherein the nucleic acid marker kit comprises a gene chip, the gene chip is arranged in a rectangle, and is divided into upper and lower N parts, each part is provided with M repeats, the 1 st column of the 1 st part is provided with a hybridization control site, and the 1 st column of the 1 st part is provided with a negative quality control site; the 1 st column of the last 1 part is set as an internal reference control site, and the 1 st column of the last 1 part is set as a hybridization control site; and setting the rest sites as pre-marked sample sites to be detected.
9. The human papillomavirus genotyping nucleic acid labeling kit according to claim 1, wherein the nucleic acid labeling kit comprises eluent I and eluent II; eluent I: preparing in a chip elution container according to the proportion of distilled water or purified water to 20 XSSC to 10 percent SDS to 100 to 5 to 1; eluent II: preparing in a chip elution container according to the proportion of distilled water or purified water to 20 XSSC to 10 percent SDS to 400 to 1 to 4; the eluent is used after hybridization of the gene chip, specifically, eluent I is used firstly, and shaking elution is carried out for 1 min; then using eluent II, shaking and eluting for 1 min; and after the elution is finished, naturally drying the chip or spin-drying the chip at a low speed.
10. The method of using the nucleic acid labeling kit of any of claims 1-9, comprising the steps of:
(1) preparing instruments and consumables: preparing a sterile operating platform, a biological safety cabinet, a gene amplification instrument, a sampler, a table centrifuge, a constant-temperature heating table, a water bath constant-temperature oscillator, a slide glass rack, a slide glass elution container and a chip scanner;
(2) preparing an eluent I and an eluent II;
(3) obtaining a nucleic acid sample or a suspected nucleic acid sample;
(4) PCR amplification and marking;
(5) and (3) hybridization: 5.1 heating the constant temperature heating table to 42 ℃, and fixing the chip by using a hybridization module preheated at 42 ℃;
5.2 placing the PCR product in a PCR instrument, heating to 95 ℃ for denaturation for 5min, and immediately carrying out ice bath for 3 min;
5.3 adding 75 mu L of hybridization buffer solution into a clinical sample amplification reaction tube, fully and uniformly mixing, and dripping the mixture into a corresponding microarray reaction chamber from a sample adding hole for hybridization for 60 min; in the operation process, the microarray area is prevented from being touched, so that mixed liquid drops are prevented from falling on the surface of the hybridization module;
(6) and (3) elution: preheating the eluent at 37 ℃ before elution, carefully taking the chip out of the hybridization module, and immediately putting the chip into an elution container to finish the elution process; eluting the eluent I for 1min by shaking; eluting the eluent II for 1min by shaking; after the elution is finished, the chip is naturally dried or spin-dried at low speed, and then scanning can be carried out;
(7) scanning: placing the dried chip in a laser scanner to scan by using laser with the wavelength of 532nm to obtain scanning data and a map; completing the nucleic acid labeling.
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