Composition, kit, detection method, model construction method, and prediction method

Abstract

Provided are a composition, a kit, use for detecting a human papillomavirus in a sample, a method for detecting the presence or type of a human papillomavirus in a sample, a model construction method for predicting the efficiency of a recombinase polymerase amplification reaction of a virus template strand, and a prediction method for the efficiency of a recombinase polymerase amplification reaction. The composition comprises: a plurality of different decoy oligonucleotides, wherein the plurality of different decoy oligonucleotides are configured to hybridize to a plurality of DNA molecules. The plurality of DNA molecules are derived from at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one or more of the sequences of SEQ ID NOs: 91-100. The composition can be used for detecting a human papillomavirus of the described genotype.

Description

Compositions, kits, detection methods, model construction methods, and prediction methods TECHNICAL FIELD

[0001] Embodiments of the present disclosure relate to a composition, a kit, a use for detecting human papillomavirus in a sample, a method for detecting the presence or type of human papillomavirus in a sample, a model construction method for predicting the efficiency of a recombinase polymerase amplification reaction of a viral template strand, and a prediction method for the efficiency of a recombinase polymerase amplification reaction. BACKGROUND

[0002] In recent years, the state has been continuously strengthening the prevention and monitoring of major infectious diseases, and has been continuously regulating the rational use of antibacterial drugs. The infectious disease and infection rapid detection industry is also in a stage of rapid development. At present, the infectious disease and infection rapid detection technology is mainly based on colloidal gold, fluorescence immunization and other immunodiagnosis technology platforms, the market is seriously homogenized, and molecular diagnosis technology platforms begin to be applied to this field with constant temperature amplification technology, microfluidic chip technology, biosensor technology, and gradually enter the commercialization and application stage, and a high-end market dominated by foreign capital trend appears. For example, multinational enterprises such as Roche, Abbott, and BD occupy the Chinese infectious disease and infection rapid detection high-end product market and the terminal market of large-scale third-grade hospitals with high-end technology and brand influence, and domestic molecular diagnosis rapid detection products are in the early stage of development. At present, the methods for detecting HPV infection in China include liquid phase hybridization capture and real-time fluorescence PCR, and such methods are mainly applied to hospital laboratories or related detection institutions, which have the characteristics of complicated operation and long time-consuming, and cannot meet the application requirements of outpatient service, emergency or home self-detection for privacy protection. Therefore, higher requirements are put forward for the molecular rapid detection technology for HPV.

[0003] SUMMARY

[0004] At least one embodiment of the present disclosure provides a composition, a kit, a use for detecting human papillomavirus in a sample, a method for detecting the presence or type of human papillomavirus in a sample, a model construction method for predicting the efficiency of a recombinase polymerase amplification reaction of a viral template strand, and a prediction method for the efficiency of a recombinase polymerase amplification reaction. The kit for detecting human papillomavirus can detect human papillomavirus of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, and HPV53 genotypes. The primer pair and the combination of the primer pair and the probe provided by the embodiments of the present disclosure are designed on the specific sequences of each type of viral genome, which can effectively avoid missed detection and non-specificity, and the RPA-nfo probe lateral flow chromatography system is used, so that the operation process of detection becomes simpler and more convenient.

[0005] The composition comprises a plurality of different decoy oligonucleotides configured to hybridize to a plurality of DNA molecules derived from at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the sequences of any one or more of SEQ ID NOs: 91-100.

[0006] For example, in the composition provided by at least one embodiment of the present disclosure, the plurality of DNA molecules are derived from at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the sequences of any one or more of SEQ ID NOs: 91-100.

[0007] For example, in the composition provided by at least one embodiment of the present disclosure, the plurality of DNA molecules are derived from at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the sequences of any one or more of SEQ ID NOs: 91-100.

[0008] For example, in the composition provided by at least one embodiment of the present disclosure, the decoy oligonucleotides are one or more of primers and probes.

[0009] For example, in the composition provided by at least one embodiment of the present disclosure, the probes are nfo probes.

[0010] The kit further comprises a hybridization reaction system in which the plurality of decoy oligonucleotides hybridize to the plurality of DNA molecules.

[0011] For example, in the kit provided by at least one embodiment of the present disclosure, the hybridization reaction system is an RPA amplification system.

[0012] For example, in the kit provided by at least one embodiment of the present disclosure, the RPA amplification system comprises an enzyme mixture comprising T4 UvsY protein, T4 gene32 protein, T4 UvsX protein, Bsu DNA polymerase, and creatine kinase.

[0013] For example, in the kit provided by at least one embodiment of the present disclosure, the enzyme mixture comprises T4 UvsY protein at a concentration of 30-100 ng / μL, T4 gene32 protein at a concentration of 500-1100 ng / μL, T4 UvsX protein at a concentration of 100-200 ng / μL, Bsu DNA polymerase at a concentration of 10-50 ng / μL, and creatine kinase at a concentration of 30-150 ng / μL.

[0014] For example, in the kit provided in at least one embodiment of the present disclosure, the diluent is a NaHCO3 diluent.

[0015] For example, in the kit provided in at least one embodiment of the present disclosure, the diluent is a NaHCO3 diluent with a mass fraction of 0.1%-5%.

[0016] For example, in the kit provided in at least one embodiment of the present disclosure, the kit comprises a buffer comprising any one of Tris-Hcl, dithiothreitol, PEG2000 and PEG20000.

[0017] For example, in the kit provided in at least one embodiment of the present disclosure, the buffer comprises Tris-Hcl with a molar concentration of 50-100 mM / L, dithiothreitol with a molar concentration of 2-6 mM / L, and any one of PEG2000 with a mass concentration of 10%-20% W / V and PEG20000 with a mass concentration of 2%-8% W / V.

[0018] For example, in the kit provided in at least one embodiment of the present disclosure, the kit comprises a base solution comprising ATP, creatine phosphate, potassium acetate and heparin sodium.

[0019] For example, in the kit provided in at least one embodiment of the present disclosure, the base solution comprises ATP with a molar concentration of 10-60 mM / L, creatine phosphate with a molar concentration of 300-500 mM / L, potassium acetate with a molar concentration of 500-1000 mM / L, and heparin sodium with a concentration of 10-50 ng / μL.

[0020] For example, in the kit provided in at least one embodiment of the present disclosure, the kit further comprises magnesium acetate, dNTP and endonuclease.

[0021] For example, in the kit provided in at least one embodiment of the present disclosure, the kit further comprises a nucleic acid releasing agent.

[0022] For example, in the kit provided in at least one embodiment of the present disclosure, the nucleic acid releasing agent and the hybridization reaction system are both freeze-dried microspheres.

[0023] For example, in the kit provided in at least one embodiment of the present disclosure, the nucleic acid releasing agent and the hybridization reaction system act synchronously.

[0024] For example, in the kit provided in at least one embodiment of the present disclosure, the hybridization reaction system comprises a component that eliminates the influence of the nucleic acid releasing agent on the hybridization reaction system.

[0025] For example, in the kit provided in at least one embodiment of the present disclosure, the nucleic acid releasing agent comprises sodium hydroxide, EDTA and Tris-HCl buffer, or the nucleic acid releasing agent comprises sodium hydroxide, SDS, EDTA and Tris-HCl buffer.

[0026] For example, in the kit provided in at least one embodiment of the present disclosure, the nucleic acid releasing agent comprises sodium hydroxide with a molar concentration of 20 mM / L, guanidine hydrochloride with a molar concentration of 1 M / L, EDTA with a molar concentration of 1 mM / L and Tris-HCl buffer with a molar concentration of 10 mM / L, or the nucleic acid releasing agent comprises sodium hydroxide with a molar concentration of 10 mM / L, 3% SDS, EDTA with a molar concentration of 1 mM / L and Tris-HCl buffer with a molar concentration of 10 mM / L.

[0027] For example, in the kit provided in at least one embodiment of the present disclosure, the hybridization reaction system comprises acetaldehyde.

[0028] For example, in the kit provided in at least one embodiment of the present disclosure, the hybridization reaction system comprises acetaldehyde with a molar concentration of 1 M / L.

[0029] For example, in the kit provided in at least one embodiment of the present disclosure, the hybridization reaction system comprises a polyether copolymer.

[0030] For example, in the kit provided in at least one embodiment of the present disclosure, the hybridization reaction system comprises a polyether copolymer with a mass percentage of 3%.

[0031] The present disclosure also provides a use of the composition according to any one of the above for detecting human papillomavirus in a sample.

[0032] For example, in the use provided in at least one embodiment of the present disclosure, the human papillomavirus is one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52 and HPV53.

[0033] The present disclosure also provides a method for detecting the presence or type of human papillomavirus in a sample, comprising performing a recombinase polymerase amplification reaction using the composition according to any one of the above, wherein the human papillomavirus in the sample is one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52 and HPV53.

[0034] For example, in the method provided by at least one embodiment of the present disclosure, the reaction temperature of the recombinase polymerase amplification is 30-45°C, and the reaction time is 5-30 minutes.

[0035] For example, in the method provided by at least one embodiment of the present disclosure, the method further comprises qualitatively and quantitatively analyzing the components of the sample by at least one of spectroscopy, chromatography, mass spectrometry, energy spectrum method and thermal spectrum method.

[0036] For example, in the method provided by at least one embodiment of the present disclosure, the method further comprises releasing and detecting nucleic acids from the sample.

[0037] At least one embodiment of the present disclosure also provides a model construction method for predicting the efficiency of recombinase polymerase amplification reaction of a viral template strand, comprising: constructing a data set, wherein the data set comprises template strand sequences and decoy oligonucleotide sequence features and corresponding recombinase polymerase amplification reaction efficiencies, and the decoy oligonucleotide sequence is derived from at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% of the sequence of any one or more of SEQ ID NO: 91-100; dividing the data set into a training set and a test set according to a predetermined proportion; training a regression model using the training set and adjusting at least part of the parameters of the regression model to optimal parameters; testing the test set using the regression model with the optimal parameters to evaluate the trained regression model, wherein the trained regression model is configured to predict the efficiency of recombinase polymerase amplification reaction; and the template strand sequences and the decoy oligonucleotide sequence features comprise k-mer sequence-based bag-of-words features, separators and template strand lengths.

[0038] For example, in the method provided by at least one embodiment of the present disclosure, the k-mer sequence-based bag-of-words features are obtained by the following steps: converting the template strand sequences and the corresponding decoy oligonucleotide sequence features into k-mer sequences; and converting the k-mer sequences into vector representations using a bag-of-words model method to obtain the bag-of-words features.

[0039] For example, in the method provided by at least one embodiment of the present disclosure, the length of the template strand is obtained by the following steps: extracting the k-mer sequence length of the template strand sequence, and normalizing the k-mer sequence length to a value between 0 and 1.

[0040] For example, in the method provided by at least one embodiment of the present disclosure, the regression model is a random forest regression model.

[0041] For example, in the method provided in at least one embodiment of the present disclosure, during the process of training the regression model, the at least part of parameters of the regression model to be adjusted include n_estimators and random seed.

[0042] For example, in the method provided in at least one embodiment of the present disclosure, the optimal parameters include that the n_estimators is 150 and the random seed is 9120.

[0043] For example, in the method provided in at least one embodiment of the present disclosure, the evaluation index for evaluating the trained regression model includes mean squared error, root mean squared error and mean absolute error.

[0044] For example, in the method provided in at least one embodiment of the present disclosure, the viral template strand includes one or more of the corresponding template strands of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52 and HPV53.

[0045] For example, in the method provided in at least one embodiment of the present disclosure, the predetermined ratio is (8.5-9.5):(1.5-0.5).

[0046] For example, in the method provided in at least one embodiment of the present disclosure, the amplification reaction efficiency of the training set is distributed between 0-1, and the amplification reaction efficiency of the test set is distributed between 0-0.5.

[0047] For example, in the method provided in at least one embodiment of the present disclosure, the k-mer represents k consecutive bases, and the k is an integer greater than or equal to 3.

[0048] For example, in the method provided in at least one embodiment of the present disclosure, the separator is a marker connecting the bait oligonucleotide sequence and the template strand sequence.

[0049] At least one embodiment of the present disclosure also provides a method for predicting the efficiency of recombinase polymerase amplification reaction, comprising using the trained regression model in any one of the above model construction methods for predicting the efficiency of recombinase polymerase amplification reaction. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present disclosure, but not limit the present disclosure.

[0051] Figure 1 shows an example of HPV template strands and primer pairs and their amplification efficiency, Figure 1 verifies the detection results of nucleic acid release system + PCR, the positive control group is other positive standard, the experimental positive is the positive standard of HPV, and the experimental negative is the negative standard of HPV.

[0052] Figure 2 shows the distribution histogram of amplification efficiency in the training set and the test set.

[0053] Figure 3 shows the influence of different n_estimators values on the mean square error of the test set.

[0054] Figure 4 shows the influence of different random number values on the mean square error of the test set.

[0055] Figure 5 shows the distribution scatter plot of the predicted value and the true value of the amplification efficiency on the training set and the test set.

[0056] Figure 6A shows the amplification results of the screening results of the primer combination of HPV16 (left) and HPV18 (right).

[0057] Figure 6B shows the amplification results of the screening results of the primer combination of HPV31 (left) and HPV33 (right).

[0058] Figure 6C shows the amplification results of the screening results of the primer combination of HPV35 (left) and HPV39 (right).

[0059] Figure 6D shows the amplification results of the screening results of the primer combination of HPV45 (left) and HPV51 (right).

[0060] Figure 6E shows the amplification results of the screening results of the primer combination of HPV52 (left) and HPV53 (right).

[0061] Figure 7 shows the detection results of different reaction temperatures of RPA for detecting HPV type 16 (left) and HPV type 18 (right) papillomavirus.

[0062] Figure 8 shows the detection results of different reaction times of RPA for detecting HPV type 16 (left) and HPV type 18 (right) papillomavirus.

[0063] Figure 9 shows the detection results of RPA reaction 1, Figure 9 is the detection results of reaction system 1 nucleic acid release system + RPA of Example 5.1.

[0064] Figure 10 shows the detection results of RPA reaction 2, which shows the results of two subtypes HPV16 and HPV18 performed on a test strip, Figure 10 is the detection results of reaction system 1 nucleic acid release agent + RPA of Example 5.2.

[0065] Figure 11 shows a tube type reaction system.

[0066] Figure 12 shows the curve of amplification performance of the reagent system.

[0067] Figure 13 shows the amplification curve of the commercial kit.

[0068] Figure 14 shows the amplification curve system optimization of the reagent system.

[0069] Figure 15 shows the amplification results (creatine kinase solution prepared with enzyme-free water).

[0070] Figure 16 shows the amplification results (creatine kinase solution prepared with Tris-Hcl buffer).

[0071] Figure 17 shows the amplification results (creatine kinase solution prepared with NaHCO3 buffer).

[0072] Figure 18 shows the amplification results (enzyme mixture addition amount optimization).

[0073] Figure 19 shows the amplification results (creatine kinase and Bsu DNA polymerase addition amount optimization).

[0074] Figure 20 shows the amplification results (ATP and MgOAc addition amount optimization).

[0075] Figure 21 shows the color development results of the HPV18 type test strip.

[0076] Figure 22 shows the color development results of the HPV16 type test strip.

[0077] Figure 23 is a model construction method for predicting the efficiency of recombinase polymerase amplification reaction of viral template strands based on kits provided by the embodiments of the present disclosure.

[0078] Figure 24 shows the mass spectrum of RHPV16P1, the theoretical molecular weight is 10828.09, and the molecular weight error is ≤0.05%.

[0079] Figure 25 shows the mass spectrum of RHPV18P1, the theoretical molecular weight is 9403.16, and the molecular weight error is ≤0.05%.

[0080] Figure 26 shows the mass spectrum of RHPV31P3, the theoretical molecular weight is 10443.84, and the molecular weight error is ≤0.05%.

[0081] Figure 27 shows the mass spectrum of RHPV33P2, the theoretical molecular weight is 10087.63, and the molecular weight error is ≤0.05%. DETAILED DESCRIPTION

[0082] In order to make the purposes, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions of the embodiments of the present disclosure will be described clearly and completely below with reference to the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by those of ordinary skill in the art without any creative effort belong to the protection scope of the present disclosure.

[0083] Unless otherwise defined, technical terms or scientific terms used in the present disclosure shall have the common meaning understood by one of ordinary skill in the art to which the present disclosure belongs. The terms “first”, “second” and similar terms used in the present disclosure do not indicate any order, number or importance, but are only used to distinguish different components. The terms “comprise”, “include” and similar terms mean that the elements or objects before the terms encompass the elements or objects listed after the terms and their equivalents, and do not exclude other elements or objects.

[0084] Unless otherwise defined, “at least one” in the embodiments of the present disclosure means one or more, and “a plurality of” means at least two.

[0085] Generally, the rapid detection method includes isothermal amplification methods such as RPA and LAMP, and the detection result of amplification can be determined by using a fluorescence signal or a naked-eye coloration method. For example, LAMP isothermal amplification and CRISPR Cas12a lateral flow chromatography system can be used to detect human papilloma virus (HPV) types 16 and 18.

[0086] Compared with other isothermal amplification technologies, RPA isothermal amplification method can be carried out under the condition of 23-45℃, and the most suitable temperature condition is 37-42℃, and does not need heat denaturation, and can complete the amplification process at room temperature. The speed of nucleic acid amplification is faster, and the purpose amplification product can be obtained within 20 min, and does not need temperature control equipment, and can truly realize portable rapid nucleic acid detection. RPA-lateral flow chromatography system refers to adding a probe and a corresponding nuclease in the basic RPA system, the probe is labeled at the 5'-end (for example, with fluorescein), has a blocking agent at the 3'-end, and an internal abasic site (THF or dSpacer). Endonuclease IV cuts at this abasic site of the probe and generates an extendable 3'-OH group for polymerization. However, unlike E. coli exonuclease III, which degrades most of the amplicon during the RPA reaction, endonuclease IV produces a slow signal and incomplete cleavage to avoid amplicon degradation. At the same time, the amplicon is placed on a chromatographic test paper with relevant labels, and relevant color signals can be obtained if the amplicon has relevant labels. The RPA-Nfo lateral flow chromatography system can ensure its performance without adding the CRISPR Cas reaction step, can avoid pollution to some extent, and has an advantage in cost compared with the CRISPR Cas12 system.

[0087] RPA-nfo probe lateral flow chromatography system detection principle

[0088] Lateral flow chromatographic test strip: on the basis of RPA, add endonuclease IV (endonuclease IV, i.e. nfo), nfo probe and biotin-labeled reverse primer. When the nfo probe is complementary to the template strand, the nfo enzyme recognizes and cuts the THF site, and the amplicon with both probe and primer labels is obtained by amplification. The results are identified by lateral flow chromatography, such as antibody or antibody / streptavidin sandwich method. One C line and one T line capture.

[0089] Embodiments of the present disclosure utilize the RPA-Nfo lateral flow chromatography system to design specific primers and probes for HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52 and HPV53 type human papillomavirus, respectively, to achieve identification of the above types, and have the advantages of short detection time, simple detection process, reduced pollution and reduced cost.

[0090] For example, the RPA reagent system in the embodiments of the present disclosure has a faster peak time than the commercially available finished kit, can reach the plateau earlier, can obtain the detection result in a shorter time, shortens the detection time and improves the detection efficiency, thereby having higher market competitiveness.

[0091] For example, in order to solve the problems of complicated operation, easy pollution and high cost, the embodiments of the present disclosure provide a detection system for HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52 and HPV53 type human papillomavirus by using RPA-nfo amplification system and based on nucleic acid flow layer chromatography technology. The detection system is simpler to operate, can effectively reduce pollution and has good accuracy.

[0092] For example, the name, corresponding sequence information and detection type of the primer pair provided by the embodiments of the present disclosure are shown in Table 1.

[0093] Table 1: Name, corresponding sequence information and detection type of primer pair

[0094] For example, the design principles of the RPA primer pair include: (1) the amplicon region is selected after sequence alignment; (2) the length of the upstream primer and the downstream primer is 30-35 bases; (3) the length of the amplicon is 80-400 bases; (4) multiple Gs should be avoided at the 5' end of the primer, and Gs and Cs should be contained at the 3' end of the primer as much as possible; (5) a large number of palindromic structures and primer dimers should be avoided; (6) the mass percentage of GC is 40%-60%.

[0095] The embodiments of the present disclosure provide a composition, which comprises: a plurality of different decoy oligonucleotides, wherein the plurality of different decoy oligonucleotides are configured to hybridize to a plurality of DNA molecules, the plurality of DNA molecules are derived from at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% of the sequence of any one or more of SEQ ID NOs: 91-100, and the kit prepared by using the composition can detect human papillomavirus.

[0096] For example, in one example, the plurality of DNA molecules are derived from at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% of the sequence of any one or more of SEQ ID NOs: 91-100, i.e. the plurality of DNA molecules are derived from the sequence of multiple of SEQ ID NOs: 91-100, so as to enrich the types and quantities of DNA molecules.

[0097] For example, in one example, the plurality of DNA molecules are derived from at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% or 80% of the sequence of any one or more of SEQ ID NOs: 91-100, i.e. the plurality of DNA molecules are derived from the sequence of multiple of SEQ ID NOs: 91-100, so as to enrich the types and quantities of DNA molecules.

[0098] For example, in one example, the bait oligonucleotide is one or more of a primer and a probe.

[0099] For example, in one example, the probe is an nfo probe.

[0100] At least one embodiment of the present disclosure provides a kit for detecting human papillomavirus, comprising the above-mentioned composition, and further comprising a hybridization reaction system in which a plurality of bait oligonucleotides are hybridized to a plurality of DNA molecules. For example, the kit for detecting human papillomavirus provided by the embodiments of the present disclosure can detect HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, and HPV53 genotype human papillomavirus, respectively, and the corresponding primer probes mentioned later are designed on the specific sequences of each type of virus genome, which can effectively avoid missed detection and non-specificity, and the RPA-nfo probe lateral flow chromatography system is used, so that the detection operation is more simple and convenient.

[0101] The kit for detecting human papillomavirus provided by at least one embodiment of the present disclosure comprises one or more primer pairs as follows: (1) SEQ ID NO: 1-2; (2) SEQ ID NO: 4-5; (3) SEQ ID NO: 7-8; (4) SEQ ID NO: 10-11; (5) SEQ ID NO: 13-14; (6) SEQ ID NO: 16-17; (7) SEQ ID NO: 19-20; (8) SEQ ID NO: 22-23; (9) SEQ ID NO: 25-26; and (10) SEQ ID NO: 28-29, and the human papillomavirus is one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, and HPV53.

[0102] For example, the kit provided by at least one embodiment of the present disclosure further comprises at least one probe selected from the following: (1) a probe of SEQ ID NO: 3 matched with a primer pair of SEQ ID NO: 1-2; (2) a probe of SEQ ID NO: 6 matched with a primer pair of SEQ ID NO: 4-5; (3) a probe of SEQ ID NO: 9 matched with a primer pair of SEQ ID NO: 7-8; (4) a probe of SEQ ID NO: 12 matched with a primer pair of SEQ ID NO: 10-11; (5) a probe of SEQ ID NO: 15 matched with a primer pair of SEQ ID NO: 13-14; (6) a probe of SEQ ID NO: 18 matched with a primer pair of SEQ ID NO: 16-17; (7) a probe of SEQ ID NO: 21 matched with a primer pair of SEQ ID NO: 19-20; (8) a probe of SEQ ID NO: 24 matched with a primer pair of SEQ ID NO: 22-23; (9) a probe of SEQ ID NO: 27 matched with a primer pair of SEQ ID NO: 25-26; and (10) a probe of SEQ ID NO: 30 matched with a primer pair of SEQ ID NO: 28-29.

[0103] For example, in one example, the hybridization reaction system is an RPA amplification system.

[0104] For example, in one example, the RPA amplification system comprises an enzyme mixture, which comprises T4 UvsY protein, T4 gene32 protein, T4 UvsX protein, Bsu DNA polymerase and 30-150 ng / µL of creatine kinase.

[0105] For example, in one example, the enzyme mixture comprises T4 UvsY protein with a concentration of 30-100 ng / µL, T4 gene32 protein with a concentration of 500-1100 ng / µL, T4 UvsX protein with a concentration of 100-200 ng / µL, Bsu DNA polymerase with a concentration of 10-50 ng / µL and creatine kinase with a concentration of 30-150 ng / µL.

[0106] For example, in one example, the kit comprises a diluent, and the diluent is a NaHCO3 diluent.

[0107] For example, in one example, the diluent is a NaHCO3 diluent with a mass fraction of 0.1%-5%.

[0108] For example, in one example, the kit comprises a buffer, and the buffer comprises any one of Tris-Hcl, dithiothreitol, PEG2000 and PEG20000.

[0109] For example, in one example, the buffer solution comprises Tris-Hcl with a molar concentration of 50-100 mM / L, dithiothreitol with a molar concentration of 2-6 mM / L, and PEG2000 with a mass concentration of 10%-20% W / V and PEG20000 with a mass concentration of 2%-8% W / V.

[0110] For example, in one example, the kit comprises a base solution, and the base solution comprises ATP, creatine phosphate, potassium acetate, and heparin sodium.

[0111] For example, in one example, the base solution comprises ATP with a molar concentration of 10-60 mM / L, creatine phosphate with a molar concentration of 300-500 mM / L, potassium acetate with a molar concentration of 500-1000 mM / L, and heparin sodium with a concentration of 10-50 ng / μL.

[0112] For example, in one example, the kit further comprises magnesium acetate, dNTP, and endonuclease.

[0113] For example, in one example, the kit further comprises a nucleic acid releasing agent.

[0114] For example, in one example, the nucleic acid releasing agent and the hybridization reaction system are both freeze-dried microspheres.

[0115] For example, in one example, the nucleic acid releasing agent and the hybridization reaction system act synchronously.

[0116] For example, in one example, the hybridization reaction system comprises a component that eliminates the effect of the nucleic acid releasing agent on the hybridization reaction system.

[0117] For example, in one example, the nucleic acid releasing agent comprises sodium hydroxide, EDTA, and Tris-HCl buffer, or the nucleic acid releasing agent comprises sodium hydroxide, SDS, EDTA, and Tris-HCl buffer.

[0118] For example, in one example, the nucleic acid releasing agent comprises sodium hydroxide with a molar concentration of 20 mM / L, guanidine hydrochloride with a molar concentration of 1 M / L, EDTA with a molar concentration of 1 mM / L, and Tris-HCl buffer with a molar concentration of 10 mM / L, or the nucleic acid releasing agent comprises sodium hydroxide with a molar concentration of 10 mM / L, 3% SDS, EDTA with a molar concentration of 1 mM / L, and Tris-HCl buffer with a molar concentration of 10 mM / L.

[0119] For example, in one example, the hybridization reaction system comprises acetaldehyde.

[0120] For example, in one example, the hybridization reaction system comprises acetaldehyde with a molar concentration of 1 M / L.

[0121] For example, in one example, the hybridization reaction system comprises a polyether copolymer.

[0122] For example, in one example, the hybridization reaction system comprises a polyether copolymer with a mass percentage of 3%.

[0123] For example, in one example, the length of the nfo probe is 45-50 bp, wherein tetrahydrofuran (THF) is modified at least 30 bp away from the 5' end and at least 15 bp away from the 3' end, the THF-substituted bases are complementary to the template nucleotides, and the substituted bases can be A, G, C or T, and the upstream primer can have a partial overlap of 10-20 bp with the nfo probe. The 5' end of the nfo probe and the 3' end of the downstream primer are modified with a combination of FAM or FITC and block.

[0124] For example, a plurality of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, and HPV53 genome sequences are downloaded from NCBI, and the MegAlign software in the DNAStar software package is used to compare the homology of the above-mentioned high-risk human papillomavirus genome sequences. In the corresponding interval, specific and intra-type conserved segments are found, and RPA primers and nfo probes are designed in the corresponding conserved segments. Three sets of primer probe combinations are designed for each genotype.

[0125] For example, in one example, the kit further comprises at least one container, and the at least one container comprises a first container containing a 20X enzyme mixture, and the 20X enzyme mixture comprises T4 UvsY protein with a concentration of 30-100 ng / μL, T4 gene32 protein with a concentration of 500-1100 ng / μL, T4 UvsX protein with a concentration of 100-200 ng / μL, Bsu DNA polymerase with a concentration of 10-50 ng / μL, and creatine kinase with a concentration of 30-150 ng / μL.

[0126] For example, the at least one container further comprises a second container, a third container, a fourth container, a fifth container, a sixth container, a seventh container, an eighth container and a ninth container, the second container contains a dilution solution, the dilution solution is NaHCO3 with a mass fraction of 0.1%-5%; the third container contains a 2X buffer solution, the 2X buffer solution contains Tris-Hcl with a molar concentration of 50-100mM / L, dithiothreitol with a molar concentration of 2-6mM / L, and one of PEG2000 with a mass concentration of 10%-20% W / V and PEG20000 with a mass concentration of 2%-8% W / V. The fourth container contains a 10X base solution, the 10X base solution contains ATP with a molar concentration of 10-60mM / L, creatine phosphate with a molar concentration of 300-500mM / L, potassium acetate with a molar concentration of 500-1000mM / L, heparin sodium with a concentration of 10-50ng / μL; the fifth container contains magnesium acetate with a molar concentration of 100-300mM / L; the sixth container contains dNTP; the seventh container contains primers; the eighth container contains a template; and the ninth container contains sterile water.

[0127] For example, the steps of testing the RPA amplification system include taking out the required components of the kit 30min in advance, melting at room temperature, shaking and mixing, which specifically includes:

[0128] 1. Add 25μL 2X reaction buffer, 1μL dNTP, 2μL upstream primer, 2μL downstream primer, 5μL 10X enzyme mixture, 3μL template and 5μL 10X base solution to the reaction tube in turn, and finally supplement with ultrapure water to 47.5μL.

[0129] 2. Add 2.5μL of 100-300mM / L MgOAc or Mgcl2 to the inside of the test tube cover, and mix by inverting it 20 times.

[0130] 3. After mixing, centrifuge the reaction solution to the bottom of the test tube, incubate at 30-39℃ for 15-20min.

[0131] 4. Take 5μL of the above liquid and dilute it 10 times with 45μL of ultrapure water, then drop it on the test strip, and observe whether the test strip develops color after 2min.

[0132] For example, in order to verify the amplification performance of the reagent system, a certain amount of fluorescent probe needs to be added to the system, a constant temperature of 30-39℃ is set on the fluorescence PCR instrument to simulate the normal temperature reaction environment, and the system performance is directly observed by detecting the change of fluorescence value after the reaction starts. The relevant test situation is shown in Figure 12. As can be seen from Figure 12, the fluorescence value basically enters the plateau period at 15-20min, which proves that the amplification reaction can meet the result within 20min in the normal temperature environment, which shows that this technology has great market potential in the application scene of urgent and rapid detection results.

[0133] Meanwhile, compared with the same type of commercially available kit at present, the amplification system shows higher fluorescence value signal, earlier peak time and faster into the platform period under the same environment, which indicates that the system has better amplification effect. The related test conditions are shown in FIG. 13 and FIG. 14, FIG. 13 is an amplification curve of a commercially available kit, and FIG. 14 is an amplification curve of the reagent system in the embodiment of the present disclosure.

[0134] For example, during the preparation of the reagent system, it is found that the preparation of creatine kinase solution has a significant effect on the final amplification result. The creatine kinase buffer is prepared by diluting creatine kinase with enzyme-free water, Tris-Hcl (pH = 7.5-8.5) and NaHCO3 buffer (mass fraction 0.1%-5%) respectively, and the amplification results are shown in FIG. 15-FIG. 17. As can be seen from FIG. 15-FIG. 17, the amplification effect is obvious after dilution with NaHCO3 buffer, while no amplification phenomenon occurs with the other two buffers. For example, FIG. 15 is the amplification result of creatine kinase solution prepared with enzyme-free water, FIG. 16 is the amplification result of creatine kinase solution prepared with Tris-Hcl buffer, and FIG. 17 is the amplification result of creatine kinase solution prepared with NaHCO3 buffer.

[0135] Curves 1-4 in FIG. 18 are the optimization of the amount of enzyme mixture added, and curves 1-4 are the enzyme mixture added in an amount of 3 μL, 1.5 μL, 2 μL and 1 μL respectively. It can be found that except for curve 1, the other amplification curves are poor, but the time to reach the equilibrium period of curve 1 is about 30 min, the amplification time is long, and it cannot meet the requirements of the study on amplification, so the amount of enzyme mixture added is still selected as 2.5 μL.

[0136] Curves 1-4 in FIG. 19 are the optimization of the amount of creatine kinase and Bsu DNA polymerase added. In FIG. 19, curves 1 and 2 are the experimental results of increasing (curve 2) and decreasing (curve 1) the concentration of creatine kinase (CPK) in the enzyme mixture by 50%; curves 3 and 4 are the experimental results of increasing (curve 3) and decreasing (curve 4) the concentration of Bsu DNA polymerase in the enzyme mixture by 50%. The results prove that adjusting the concentration of the two enzymes in the enzyme mixture will make the amplification effect worse, and it is speculated that this may be because the change of the concentration of the two enzymes will affect the activity of other enzymes, thereby affecting the amplification efficiency.

[0137] Figure 20 is a graph of the results of optimization of ATP and MgOAc addition amounts, in which amplification curve 1 and 3 are the results of experiments in which the concentration of ATP in the 10X base solution was increased by 50% (amplification curve 1) and decreased by 50% (amplification curve 3); and amplification curves 2, 4, 5, 6 are the results of experiments in which the amount of MgOAc was optimized, in which the four amplification curves are the results of experiments in which 3 μL, 2 μL, 1.5 μL and 1 μL of MgOAc were added, respectively. The results prove that the original ATP concentration and the addition of 2.5 μL of MgOAc are the optimal conditions.

[0138] Figure 20 is a graph of the results of optimization of ATP and MgOAc addition amounts, in which amplification curve 1 and 3 are the results of experiments in which the concentration of ATP in the 10X base solution was increased by 50% (amplification curve 1) and decreased by 50% (amplification curve 3); and amplification curves 2, 4, 5, 6 are the results of experiments in which the amount of MgOAc was optimized, in which the four amplification curves are the results of experiments in which 3 μL, 2 μL, 1.5 μL and 1 μL of MgOAc were added, respectively. The results prove that the original ATP concentration and the addition of 2.5 μL of MgOAc are the optimal conditions.

[0139] For example, the kit further comprises an endonuclease.

[0140] For example, the kit further comprises a one-tube reaction tube, the one-tube reaction tube comprising a first compartment and a second compartment, the first compartment and the second compartment being separated by a water-soluble film, the first compartment having a sample inlet and comprising the nucleic acid release agent microspheres, and the second compartment comprising the nucleic acid detection agent microspheres.

[0141] For example, in one example, the nucleic acid release agent microspheres and the nucleic acid detection agent microspheres are both lyophilized microspheres.

[0142] For example, in one example, the nucleic acid release agent microspheres comprise: a strong base, a surfactant, a lysis salt, an organic solvent, an ion chelator and a buffer.

[0143] For example, the strong base has a molar concentration of 1-100 mM / L, and preferably the strong base is one or more of: sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide.

[0144] For example, the surfactant has a mass percentage content in the nucleic acid release agent microspheres of 1%-5%, and preferably the surfactant is one or more of: sodium dodecyl sulfonate, Triton X-100, Tween, ethyl phenyl polyethylene glycol, stearic acid, sulfonate, betaine, polysorbate and cysteine.

[0145] For example, in one example, the lysis salt has a molar concentration of 100-3000 M / L. For example, the lysis salt comprises one or more of: guanidine hydrochloride, guanidine isothiocyanate, non-guanidine salt lysis salt.

[0146] For example, in one example, the molar concentration of the ion chelator is 0.5-5 mM / L. For example, the ion chelator is one or more of EDTA, EGTA, and citric acid.

[0147] For example, in one example, the molar concentration of the buffer is 1-100 mM / L. For example, the buffer is one or more of PBS, Tris-HCl, HEPES, and MOPS.

[0148] For example, in one example, the nucleic acid detection reagent includes a combination of a reaction enzyme, a deoxyribonucleotide triphosphate, a primer probe, a metal ion, an enhancer, a buffer, and an auxiliary ingredient, and the nucleic acid detection reagent can form a nucleic acid detection reagent microsphere by freeze-drying.

[0149] For example, the molar concentration of the reaction enzyme is 1-1000 ng / μL. For example, in one example, the reaction enzyme is one or more of Taq enzyme, Pfu enzyme, Bsu enzyme, Sau enzyme, T4 UvsX protein, T4 UvsY protein, and T4 gp32 protein.

[0150] For example, the molar concentration of the dNTP is 10-500 μM / L. For example, in one example, the dNTP includes one or more of dATP, dTTP, dGTP, and dCTP.

[0151] For example, in one example, the molar concentration of the probe and primer is 100-800 nM / L.

[0152] For example, in one example, the molar concentration of the metal ion is 5-60 mM / L. For example, the metal ion is one or more of potassium ion, calcium ion, and magnesium ion.

[0153] For example, in one example, the molar concentration of the enhancer is 1-100 mM / L. For example, the enhancer is one or more of trehalose, bovine serum albumin, gelatin, and glycine.

[0154] For example, in one example, the molar concentration of the buffer is 1-100 mM / L. For example, the buffer is one or more of PBS, Tris-HCl, HEPES, and MOPS.

[0155] For example, in one example, the molar concentration of the auxiliary ingredient is 100-5000 mM / L, and the auxiliary ingredient is one or more of sulfuric acid, nitric acid, perchloric acid, hydrochloric acid, phosphoric acid, aldehyde, polysiloxane, polyether copolymer, fatty acid, fatty acid ester, phosphate ester, and dimethicone.

[0156] For example, in one example, the material of the water-soluble film is one or more of polyvinyl alcohol, starch, sodium carboxymethyl cellulose.

[0157] For example, in one example, the thickness of the water-soluble film is 0.1-100 microns.

[0158] For example, in one example, the kit further comprises a nucleic acid releasing agent and a nucleic acid detection agent, the nucleic acid releasing agent comprises sodium hydroxide with a molar concentration of 20mM / L, guanidine hydrochloride with a molar concentration of 1M / L, EDTA with a molar concentration of 1mM / L, and Tris-HCl buffer with a molar concentration of 10mM / L, and the nucleic acid detection agent comprises Taq enzyme with a mass concentration of 100ng / μL, dNTP with a molar concentration of 200μM / L, primer probe with a molar concentration of 400nM / L, magnesium ion with a molar concentration of 30mM / L, Tris-HCl buffer with a molar concentration of 10mM / L, hydrochloric acid with a molar concentration of 20mM / L, and acetaldehyde with a molar concentration of 1M / L; or wherein the nucleic acid releasing agent comprises sodium hydroxide with a molar concentration of 10mM / L, 3% SDS, EDTA with a molar concentration of 1mM / L, and Tris-HCl buffer with a molar concentration of 10mM / L, and the nucleic acid detection agent comprises Sau enzyme with a mass concentration of 15ng / μL, T4 UvsX protein with a mass concentration of 100ng / μL, T4 UvsY protein with a mass concentration of 50ng / μL, T4 gp32 protein with a mass concentration of 500ng / μL, dNTP with a molar concentration of 200μM / L, primer probe with a molar concentration of 400nM / L, magnesium ion with a molar concentration of 30mM / L, Tris-HCl buffer with a molar concentration of 10mM / L, PEG with a mass percentage of 5%, dithiothreitol with a molar concentration of 2mM / L, phosphocreatine with a molar concentration of 50mM / L, creatine kinase with a mass concentration of 100ng / μL, adenosine phosphate with a molar concentration of 3mM / L, and polyether copolymer with a mass percentage of 3%.

[0159] For example, in one example, the nucleic acid detection agent and the nucleic acid releasing agent are physically separated or mixed in equal mass proportions.

[0160] For example, in one example, the kit further comprises a recombinase polymerase amplification solution, wherein the recombinase polymerase amplification solution comprises 20X enzyme mix, 2X buffer, 10X base, magnesium acetate, dNTP, primer, template, and diluent, the 20X enzyme mix accounts for 5% of the volume of the recombinase polymerase amplification solution; the 2X buffer accounts for 50% of the volume of the recombinase polymerase amplification solution; the 10X base accounts for 10% of the volume of the recombinase polymerase amplification solution; and the magnesium acetate has a molar concentration of 5-15 mM / L.

[0161] The at least one embodiment of the present disclosure also provides a method for detecting the presence or type of human papillomavirus in a sample, comprising performing a recombinase polymerase amplification reaction using any of the kits described above, wherein the human papillomavirus in the sample is one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, and HPV53.

[0162] For example, in the method provided by the at least one embodiment of the present disclosure, the reaction temperature of the recombinase polymerase amplification is 30-45°C, and the reaction time is 5-30 minutes.

[0163] For example, in one example, the reaction temperature of the recombinase polymerase amplification is 30-45°C, and the reaction time is 8-25 minutes.

[0164] For example, in another example, the reaction temperature of the recombinase polymerase amplification is 30-45°C, and the reaction time is 10-20 minutes.

[0165] For example, the method provided by the at least one embodiment of the present disclosure further comprises qualitatively and quantitatively analyzing the components of the sample by at least one of spectroscopy, chromatography, mass spectrometry, energy spectrum, and thermal spectrum.

[0166] For example, the method provided by the at least one embodiment of the present disclosure further comprises releasing and detecting nucleic acid from the sample, wherein the releasing and detecting nucleic acid from the sample is performed by using a tube reaction tube, the method comprising adding the sample to a tube reaction tube via a sample inlet, contacting the sample with nucleic acid releasing agent microspheres to dissolve the nucleic acid releasing agent microspheres to obtain a first dissolution solution, and contacting the first dissolution solution with nucleic acid detection agent microspheres to dissolve the nucleic acid detection agent microspheres to obtain a second dissolution solution after the water-soluble film is dissolved.

[0167] For example, in the method provided by the at least one embodiment of the present disclosure, the second dissolution solution is detected by a PCR amplification method.

[0168] For example, the method provided by at least one embodiment of the present disclosure further comprises contacting the sample with the nucleic acid releasing agent or the freeze-dried microspheres thereof to obtain a third lysate, and contacting the third lysate with the nucleic acid detection agent or the freeze-dried microspheres thereof to obtain a fourth lysate.

[0169] For example, in one example, the fourth lysate is detected by a PCR amplification method.

[0170] For example, the method provided by at least one embodiment of the present disclosure further comprises contacting the sample with the nucleic acid releasing agent or the freeze-dried microspheres thereof and the nucleic acid detection agent or the freeze-dried microspheres thereof to obtain a fifth lysate.

[0171] For example, in the method provided by at least one embodiment of the present disclosure, the fifth lysate is detected by a PCR amplification method.

[0172] At least one embodiment of the present disclosure also provides a method for diagnosing a papillomavirus infection or a papillomavirus infection related disease in a subject, comprising detecting the presence or type of papillomavirus in a sample of the subject using the kit of any of the above embodiments.

[0173] For example, the method provided by at least one embodiment of the present disclosure comprises a recombinase polymerase amplification reaction, wherein the temperature of the recombinase polymerase amplification reaction is 30-45°C, and the reaction time is 5-30 minutes.

[0174] For example, in one example, the recombinase polymerase amplification is performed in the recombinase polymerase amplification solution described above.

[0175] For example, the sample is a sample of a subject or an environmental sample. For example, the subject is a mammal, preferably a human.

[0176] For example, in one example, the sample is one or more of a buccal swab, nasopharyngeal or nasal sample, cervical swab, cervical smear, blood, urine, lymphatic fluid, spinal fluid, synovial fluid, aqueous humor, tear fluid, saliva, sputum, fecal, environmental and food samples.

[0177] For example, the method further comprises qualitatively and quantitatively analyzing the components of the sample by one or more methods selected from the group consisting of spectroscopy, chromatography, mass spectrometry, energy spectrometry and thermal spectrometry.

[0178] For example, in one example, the spectroscopy is one or more of ultraviolet spectrometry, infrared spectrometry and nuclear magnetic spectrometry.

[0179] For example, in one example, the chromatography is one or more of gas chromatography, liquid chromatography and ion chromatography.

[0180] For example, in one example, the mass spectrometry is gas chromatography-mass spectrometry and / or liquid chromatography-mass spectrometry.

[0181] For example, in one example, the spectroscopy method is fluorescence spectroscopy and / or diffraction spectroscopy.

[0182] For example, in one example, the thermography method is thermogravimetric analysis and / or differential scanning calorimetry.

[0183] For example, the method provided by at least one embodiment of the present disclosure further comprises releasing and detecting the nucleic acid from the sample, wherein the releasing and detecting the nucleic acid from the sample is achieved by using a tube reaction tube, the method comprising adding the sample to a tube reaction tube through a sample inlet, contacting the sample with nucleic acid releasing agent microspheres to dissolve the nucleic acid releasing agent microspheres to obtain a first dissolution solution, and contacting the first dissolution solution with nucleic acid detection agent microspheres to dissolve the nucleic acid detection agent microspheres after the water-soluble film is dissolved to obtain a second dissolution solution.

[0184] For example, the releasing and detecting the nucleic acid by using a tube reaction tube can achieve the following: complete release of nucleic acid molecules in the sample, effective treatment of interferents in the sample, one-tube implementation of nucleic acid release detection, and stability of the reagent after reconstitution of the freeze-dried microspheres to ensure the effectiveness of the reagent.

[0185] For example, in the method provided by at least one embodiment of the present disclosure, the second dissolution solution is detected by a PCR amplification method.

[0186] For example, the method provided by at least one embodiment of the present disclosure further comprises contacting the sample with a nucleic acid releasing agent or freeze-dried microspheres thereof to obtain a third dissolution solution, and contacting the third dissolution solution with the nucleic acid detection agent or freeze-dried microspheres thereof to obtain a fourth dissolution solution.

[0187] For example, in the method provided by at least one embodiment of the present disclosure, the fourth dissolution solution is detected by a PCR amplification method.

[0188] For example, the method provided by at least one embodiment of the present disclosure further comprises contacting the sample with a nucleic acid releasing agent or freeze-dried microspheres thereof and a nucleic acid detection agent or freeze-dried microspheres thereof to obtain a fifth dissolution solution.

[0189] For example, in the method provided by at least one embodiment of the present disclosure, the fifth dissolution solution is detected by a PCR amplification method.

[0190] For example, the composition of the liquid can be detected by spectroscopy, chromatography, energy spectrum method and thermal spectrum method, etc. The spectroscopy includes ultraviolet spectrometer, infrared spectrometer or nuclear magnetic spectrometer. The chromatography includes gas chromatograph, liquid chromatograph or ion chromatograph. The mass spectrum method includes mass spectrometer, gas chromatograph-mass spectrometer or liquid chromatograph-mass spectrometer. The energy spectrum method includes fluorescence spectrometer or diffraction spectrometer. The thermal spectrum method includes thermal gravimetric analyzer or differential scanning calorimeter. Therefore, the RPA reagent system can be quantitatively analyzed by multiple analysis methods.

[0191] For example, the fluorescence spectrometer can detect the information of various substances, mainly including the chemical structure and concentration of the substance. When the fluorescence spectrometer is used to detect the composition of the liquid, the chemical structure of the substance can be determined by analyzing the fluorescence spectrum characteristics of different compounds in the excited state. At the same time, since the fluorescence intensity is positively correlated with the concentration of the substance, the fluorescence spectrometer can also be used to measure the concentration of the substance in the solution, the excitation spectrum, the emission spectrum, the quantum yield, the fluorescence lifetime and the three-dimensional fluorescence of the substance, which can help understand the fluorescence characteristics and energy level structure of the substance.

[0192] For example, the fluorescence spectrometer can also be widely used in various fields, such as biological field, chemical field, food field, pharmaceutical analysis field and environmental monitoring field, etc. For example, in the biological field, it is used to study the dynamics, rigidity and structural information of DNA, protein and virus; in the chemical field, it is used for environmental monitoring to detect organic and inorganic substances in air, water and soil; in the food field, it is used to analyze minerals, vitamins, pesticide residues, etc.; in the pharmaceutical analysis field, it is used for effective component identification and metabolic kinetics research of drugs; in the environmental monitoring field, it is used to detect pollutants in water and atmosphere.

[0193] The XRD test is mainly used for the analysis of the composition of the solid phase material, the analysis of the crystallinity, the determination of the lattice parameters and the analysis of the crystal defects.

[0194] For example, when analyzing the composition of the solid phase material, the XRD can determine the solid phase composition in the material, i.e. the type and content of the solid phase material with different crystal structures. By analyzing the diffraction peak of the material, each crystal phase in the material can be determined, and their relative content can be calculated.

[0195] For example, crystallinity refers to the degree of perfection and order of the crystal in the material. When analyzing the crystallinity of a solid-phase substance, XRD testing can evaluate the crystallinity of the material by analyzing the intensity and width of the diffraction peaks, thereby determining the crystal quality and purity of the material.

[0196] For example, lattice parameters refer to the size and shape of the unit cell in the crystal. When determining the lattice parameters of a solid-phase substance, XRD testing can calculate the lattice parameters of the unit cell, including cell constants, cell volume, and cell shape, by analyzing the position and intensity of the diffraction peaks.

[0197] For example, crystal defects refer to point defects, line defects, and surface defects in the crystal. When analyzing the crystal defects of a solid-phase substance through XRD testing, the type and number of crystal defects can be studied by analyzing the shape and position changes of the diffraction peaks, thereby evaluating the quality and performance of the material.

[0198] Mass spectrometers can be used to determine the molecular weight, molecular formula, chemical composition and composition, structure and conformation, content and purity of the sample.

[0199] For example, mass spectrometers can determine the molecular weight and molecular formula of the sample by ionization and ion mass analysis. For organic compounds and biological macromolecules, mass spectrometers can determine their molecular weight and molecular formula through molecular ion peaks or molecular ion fragment peaks. For inorganic compounds and metal ions, mass spectrometers can determine their molecular weight and molecular formula through ion peaks in the mass spectrum.

[0200] For example, when determining the chemical composition and composition of the sample using a mass spectrometer, for complex mixtures, the mass spectrometer can determine the relative content and molecular formula of each component through the ion peaks in the mass spectrum. For biological macromolecules, mass spectrometers can further determine their sequence and glycosyl composition.

[0201] For example, when determining the structure and conformation of the sample using a mass spectrometer, for organic compounds and biological macromolecules, mass spectrometers can determine their structure and conformation through fragment ion peaks in the mass spectrum.

[0202] For example, mass spectrometers can determine the content and purity of the sample by ion signal intensity.

[0203] For example, chromatographs can determine the number of sample components and perform qualitative and quantitative analysis of the sample.

[0204] For example, when testing the number of sample components, the minimum number of sample components can be initially determined according to the number of chromatographic peaks.

[0205] For example, when performing qualitative and quantitative analysis on a sample, qualitative analysis can be performed by the retention value of the chromatographic peak. According to the peak area and peak height, quantitative analysis can be performed on the sample to determine the content of each component in the sample.

[0206] For example, the energy spectrometer can determine the thermal stability, reaction kinetics, and phase transition behavior of the sample, and can identify the purity of the sample, identify the type of substance, analyze the components of the substance, and evaluate the compatibility and compatibilizer effect of the substance.

[0207] For example, the thermal stability of a substance can be analyzed by thermal analysis, i.e., determining the stability of a substance at different temperatures, including thermal decomposition temperature, melting point, glass transition temperature, etc. These information is crucial for evaluating the behavior of materials at high temperatures or during processing.

[0208] For example, thermal analysis can help study the kinetics of chemical reactions, including reaction rate, activation energy, and reaction mechanism. These information helps understand the nature of the reaction, optimize reaction conditions, and predict reaction products.

[0209] For example, thermal analysis can reveal the phase transition behavior of a substance during heating or cooling, such as solid-state phase transition, melting, crystallization, etc. By analyzing the heat change during phase transition, the crystal structure, molecular arrangement, and interaction of the substance can be understood.

[0210] For example, impurities or additives in a sample can be detected by thermal analysis for purity identification, as the presence of impurities or additives often leads to abnormal thermal behavior on the thermogram. By analyzing these abnormalities, the purity of the sample can be evaluated.

[0211] For example, different substances have unique thermal spectrum characteristics, so thermal analysis can be used for identification and classification of substances. By comparing the thermal spectrum of different samples, their similarities or differences can be determined.

[0212] For example, in a mixture, each component will exhibit different thermal behavior during heating. By thermal analysis, different components in the mixture can be distinguished and their content can be estimated.

[0213] For example, the compatibility and compatibilizer effect of a sample can be evaluated by thermal analysis, which can be used in material science to evaluate the compatibility between different materials and the effect of compatibilizers on material performance.

[0214] For example, the process of verifying the clinical sample includes: selecting 16 clinical samples for testing, the sampling site is the cervical epithelial cells, which have been extracted and stored at -20℃ for later use. Using the detection reagent, the steps of the RPA amplification system test are detected, and the same sample nucleic acid is detected using the fluorescence quantitative PCR reagent as a control. The results of the test strip and the fluorescence PCR are as follows:

[0215] For example, in one test process, the HPV18 type test strip and the fluorescence PCR detection results are:

[0216] Note: "+" indicates that there is a color signal, "-" indicates that there is no color signal, and "√" indicates that the results are completely consistent.

[0217] For example, in another test process, the HPV16 type test strip and the fluorescence PCR detection results are:

[0218] Note: "+" indicates that there is an amplification signal, "-" indicates that there is no amplification signal, and "√" indicates that the results are completely consistent.

[0219] For example, the characterization means of the primer pair and the probe combination is amplicon sequencing combined with fragment analysis and mass spectrometry.

[0220] For example, the process of configuring the primer pool Pool includes: preparing a mixture of all primer pairs and probe combinations according to the amplification instructions, shaking and mixing, and then storing for later use.

[0221] For example, the reaction system of PCR amplification is:

[0222] The amplification conditions of PCR are:

[0223] For example, the magnetic beads are purified 1.2 times, and 30-50 μL is eluted. The Qubit determines the concentration of the PCR product.

[0224] For example, the reaction program of the PCR product phosphorylation is: reacting at 20℃ for 30min; then reacting at 65℃ for 30min; and finally incubating at 4℃ for a period of time.

[0225] For example, the names of the components of the PCR product phosphorylation and the volumes of the individual components added are as follows:

[0226] For example, the reaction condition for forming the ligation junction is 20 min at 20 °C, and the magnetic beads are purified 0.88 times, and eluted with 22 μL.

[0227] For example, the names of the components for forming the phosphate linker and the volumes of the individual components added are as follows:

[0228] For example, when the library is amplified, the PCR reaction system is as follows:

[0229] The PCR amplification conditions are as follows:

[0230] For example, the magnetic beads are purified 0.5 times to remove large fragments, and the target fragments are purified 1.2 times, and eluted with 40 μL.

[0231] For example, sequencing the amplicon includes: sequencing the amplicon of the purified product obtained in the previous step, and analyzing the sequencing data to obtain the target fragment and the positions of the upstream and downstream primers.

[0232] For example, analyzing the primer sequence includes: using an Agilent 4200 automatic electrophoresis instrument or an ABI 3500 to analyze the nucleic acid fragments for the primer probe combination, to obtain the primer length information, and verifying the primer length ± 3 bp obtained by analyzing the upstream and downstream primers in combination with the sequencing results, to obtain the primer sequence information.

[0233] For example, analyzing the probe sequence includes: using the primer probe mixture to perform high-resolution agarose gel electrophoresis, and since the lengths of the primers and probes are different, they can be separated by electrophoresis, and the longer fragments obtained by separation are the probes. The probes are obtained by gel recovery and purified by magnetic beads, and the molecular weight of the probes is obtained by mass spectrometry analysis, and the probe fragment sequence is obtained in combination with the label and the position of the target fragment.

[0234] At least one embodiment of the present disclosure also provides a use of the composition of any one of the above for detecting human papillomavirus in a sample.

[0235] For example, in one example, the human papillomavirus is one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, and HPV53.

[0236] The present disclosure at least one embodiment further provides a use of a primer pair in preparing a kit for detecting the presence or type of human papillomavirus, or for diagnosing a human papillomavirus infection or a disease associated with a human papillomavirus infection, the human papillomavirus being one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, and HPV53; the primer pair being one or more of (1) SEQ ID NO: 1-2; (2) SEQ ID NO: 4-5; (3) SEQ ID NO: 7-8; (4) SEQ ID NO: 10-11; (5) SEQ ID NO: 13-14; (6) SEQ ID NO: 16-17; (7) SEQ ID NO: 19-20; (8) SEQ ID NO: 22-23; (9) SEQ ID NO: 25-26; and (10) SEQ ID NO: 28-29.

[0237] The present disclosure at least one embodiment further provides a method for detecting the presence or type of human papillomavirus in a sample, comprising performing a recombinase polymerase amplification reaction using the composition of any of the above embodiments, the human papillomavirus in the sample being one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, and HPV53.

[0238] For example, in the method provided by the present disclosure at least one embodiment, the reaction temperature of the recombinase polymerase amplification is 30-45℃, and the reaction time is 5-30 minutes.

[0239] For example, in the method provided by the present disclosure at least one embodiment, the method further comprises qualitatively and quantitatively analyzing the components of the sample by at least one of spectroscopy, chromatography, mass spectrometry, energy spectrum method, and thermal spectrum method.

[0240] For example, in the method provided by the present disclosure at least one embodiment, the method further comprises releasing and detecting the nucleic acid from the sample.

[0241] The present disclosure also provides use of a combination of primers and probes in the preparation of a kit for detecting the presence or type of human papillomavirus, or for diagnosing a papillomavirus infection or a papillomavirus infection-related disease, the human papillomavirus being one or more of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52 and HPV53; the combination of primers and probes being one or more of: (1) a primer pair of SEQ ID NOs: 1-2 and a probe of SEQ ID NO: 3; (2) a primer pair of SEQ ID NOs: 4-5 and a probe of SEQ ID NO: 6; (3) a primer pair of SEQ ID NOs: 7-8 and a probe of SEQ ID NO: 9; (4) a primer pair of SEQ ID NOs: 10-11 and a probe of SEQ ID NO: 12; (5) a primer pair of SEQ ID NOs: 13-14 and a probe of SEQ ID NO: 15; (6) a primer pair of SEQ ID NOs: 16-17 and a probe of SEQ ID NO: 18; (7) a primer pair of SEQ ID NOs: 19-20 and a probe of SEQ ID NO: 21; (8) a primer pair of SEQ ID NOs: 22-23 and a probe of SEQ ID NO: 24; (9) a primer pair of SEQ ID NOs: 25-26 and a probe of SEQ ID NO: 27; and (10) a primer pair of SEQ ID NOs: 28-29 and a probe of SEQ ID NO: 30.

[0242] For example, primer probe combination experiment screening / verification

[0243] PCR reaction reagents were prepared using different groups of primers and probes for different genotypes, and standard samples of the corresponding types were detected, and the color development effects of test strips of different primer probe combinations of the same type were observed to screen better primer probe combinations.

[0244] Sample preparation included: taking HPV16 and HPV18 as examples, using enzyme-free water to dilute HPV16 and HPV18 standard samples to 1x10 5 copies / mL, and storing at -20°C for standby.

[0245] Reagent preparation included: taking out each component reagent, placing it on the desktop, and gently shaking and mixing after equilibrating to room temperature, and then instantaneous centrifugation. Each set of primers and probes was used to prepare the corresponding amplification reagent, and the specific system preparation was as follows:

[0246] Prepare RPA mixed solution according to the following table

[0247] The freeze-dried microspheres were dissolved and mixed thoroughly, 2 μL of the sample and 2.5 μL of 280 mM MgOAc were added in sequence, and mixed (shaken) thoroughly to start the reaction. Centrifuge briefly.

[0248] The amplification detection includes: incubation at 39°C for 20 min, after the reaction, add diluent to 100 μL to the product, and add nucleic acid detection test strip, stand for 5 min, and observe the results. If only the C line appears on the test strip, the result is negative, if the T line and the C line of the test strip appear, the result is positive, if the C line does not appear, the detection result is invalid, and needs to be re-verified.

[0249] The test results were analyzed: after the reaction, the amplification results of each group of primers and probes were observed.

[0250] For example, the reaction system can include a nucleic acid release system and a corresponding nucleic acid detection reagent.

[0251] For example, in order to reduce the influence of the nucleic acid release reagent on the nucleic acid detection reagent system, the main components of the nucleic acid release agent according to different sample types include one or more of the following: strong alkali, surfactant, lysis salt, organic solvent, ion chelating agent, buffer, etc. Among them, the strong alkali (1-100 mM) includes one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, the surfactant (1%-5%) includes one or more of sodium dodecyl sulfate (SDS), Triton X-100, Tween 20, ethyl phenyl polyethylene glycol (NP40), stearic acid, sulfonate, betaine, polysorbate, cysteine, the lysis salt (100-3000 M) includes one or more of guanidine hydrochloride, guanidine isothiocyanate, non-guanidine salt lysis salt, the ion chelating agent (0.5-5 mM) contains one or more of EDTA, EGTA and citric acid, and the buffer (1-100 mM) includes one or more of PBS, Tris-HCl, HEPES, MOPS.

[0252] The main components of the corresponding nucleic acid detection reagent include one or more of the following: reaction enzyme, deoxyribonucleotide triphosphate (dNTP), primer probe, metal ion, enhancer, buffer, auxiliary component, etc. The reaction enzyme (1-1000 ng / μL) includes one or more of Taq enzyme, Pfu enzyme, Bsu enzyme, Sau enzyme, T4 UvsX protein, T4 UvsY protein, and T4 gp32 protein, the dNTP (10-500 μM) includes one or more of dATP, dTTP, dGTP, and dCTP, the probe primer (100-800 nM) is designed according to different detection targets, the metal ion (5-60 mM) includes one or more of potassium ion, calcium ion, and magnesium ion, the enhancer (1-100 mM) includes one or more of trehalose, bovine serum albumin, gelatin, and glycine, the buffer (1-100 mM) includes one or more of PBS, Tris-HCl, HEPES, and MOPS, and the auxiliary component (100-5000 mM) includes one or more of sulfuric acid, nitric acid, perchloric acid, hydrochloric acid, phosphoric acid, aldehyde, polysiloxane, polyether copolymer, fatty acid, fatty acid ester, phosphate ester, and dimethicone.

[0253] For example, in one example, the components of the nucleic acid release agent include 20 mM sodium hydroxide, 1 M guanidine hydrochloride, 1 mM EDTA, and 10 mM Tris-HCl buffer. The components of the corresponding nucleic acid detection reagent include 100 ng / μL Taq enzyme, 200 μM dNTP, 400 nM primer probe, 30 mM magnesium ion, 10 mM Tris-HCl buffer, 20 mM hydrochloric acid, and 1 M acetaldehyde. The nucleic acid release reagent and the detection reagent are freeze-dried to form microspheres by freeze-drying.

[0254] The experimental steps are as follows:

[0255] 1) Take 100 μL of the virus sample to dissolve the nucleic acid release reagent microspheres, and mix uniformly by vortexing or blowing.

[0256] 2) Incubate at room temperature or 38°C for 5 minutes;

[0257] 3) Dissolve the nucleic acid detection reagent microspheres in the solution after the lysis solution;

[0258] 4) Perform amplification detection in a PCR instrument according to the common method of polymerase chain reaction (PCR), including denaturation, annealing, and extension.

[0259] For example, in another example, the components of the nucleic acid release agent include 10 mM sodium hydroxide, 3% SDS, 1 mM EDTA, and 10 mM Tris-HCl buffer.

[0260] The components of the corresponding nucleic acid detection reagent include: 15 ng / μL Sau enzyme, 100 ng / μL T4 UvsX protein, 50 ng / μL T4 UvsY protein, 500 ng / μL T4 gp32 protein, 200 μM dNTP, 400 nM primer probe, 30 mM magnesium ion, 10 mM Tris-HCl buffer, 5% PEG, 2 mM dithiothreitol, 50 mM phosphocreatine, 100 ng / μL creatine kinase, 3 mM adenosine phosphate, 3% polyether copolymer. The nucleic acid release reagent and the detection reagent are freeze-dried into microspheres by freeze-drying method.

[0261] The experimental steps are as follows:

[0262] 1) Take 100 μL of the virus sample to dissolve the nucleic acid release reagent microspheres, and mix uniformly by vortexing or blowing;

[0263] 2) Incubate at room temperature or 38°C for 5 minutes;

[0264] 3) Dissolve the nucleic acid detection microspheres in the solution after the lysis solution;

[0265] 4) Incubate at 38°C for 20 minutes to achieve isothermal amplification, and use the test strip to detect the results.

[0266] In this embodiment, the main component of the nucleic acid release reagent is a surfactant that reduces surface tension to form a foam effect to release the nucleic acid in the sample. The polyether copolymer in the corresponding nucleic acid release reagent can produce a defoaming effect, thereby eliminating its influence on the nucleic acid detection system, achieving the purpose of one-pot reaction of nucleic acid release and detection.

[0267] For example, in order to realize the release and detection of nucleic acid in one reaction tube and facilitate storage and transportation, the nucleic acid release reagent and the nucleic acid detection reagent need to be freeze-dried into microspheres to ensure that they do not affect the detection performance after reconstitution. Then store the nucleic acid release reagent microspheres and the nucleic acid detection microspheres in the same test tube, and use a water-soluble film to divide them into two parts.

[0268] For example, in one example, the experimental process of the one-pot reaction tube includes:

[0269] 1) Add the above-mentioned nucleic acid release reagent or nucleic acid detection reagent to the centrifuge tube according to a certain proportion, and vortex mix uniformly to obtain a mixed solution;

[0270] 2) Take the mixed solution with a volume of 10 μL and drop it into liquid nitrogen to condense into a circular small ball;

[0271] 3) Put the condensed microspheres into a pre-cooled freeze dryer, the cold trap temperature is -69°C, and vacuum freeze-dry for more than 10 h to obtain freeze-dried microspheres of the nucleic acid release reagent and freeze-dried microspheres of the nucleic acid detection reagent.

[0272] For example, in another example, the experimental process of the one-tube reaction tube includes:

[0273] 1) Store the nucleic acid release reagent microspheres and nucleic acid detection microspheres in the same test tube, and divide them into two parts using a water-soluble film.

[0274] 2) Take 100 μL of the virus sample to dissolve the nucleic acid release reagent microspheres, and mix them uniformly by blowing.

[0275] 3) Incubate at room temperature or 38 degrees Celsius for 5 minutes, and the water-soluble film gradually dissolves during the incubation period.

[0276] 4) Then dissolve the nucleic acid detection microspheres as the solvent;

[0277] 5) Finally, after mixing uniformly, perform PCR or isothermal amplification detection.

[0278] The material of the water-soluble film can be selected from one of polyvinyl alcohol, starch, and sodium carboxymethyl cellulose. According to the time requirement of nucleic acid release, a water-soluble film with a corresponding thickness (0.1-100 microns) is designed, so that after the reaction system dissolves the water-soluble film after the release of the nucleic acid, the amplification reaction is performed in the nucleic acid detection system.

[0279] In this embodiment, the main component of the nucleic acid release reagent is guanidine hydrochloride, which releases the nucleic acid in the sample by destroying the hydrogen bond. The corresponding acetaldehyde in the nucleic acid release reagent can react with guanidine hydrochloride, thereby eliminating its influence on the nucleic acid detection system, achieving the purpose of one-tube reaction of nucleic acid release and detection.

[0280] The model construction method for predicting the recombinase polymerase amplification reaction efficiency of the viral template strand based on the kit according to any one of the above embodiments is also provided in at least one embodiment of the present disclosure. FIG. 23 is a model construction method for predicting the recombinase polymerase amplification reaction efficiency of the viral template strand based on the kit provided in an embodiment of the present disclosure, as shown in FIG. 23, the model construction method includes the following steps:

[0281] Step S101: Construct a data set, which includes template strand sequences and bait oligonucleotide sequence characteristics and corresponding recombinase polymerase amplification reaction efficiency, the bait oligonucleotide sequence is derived from at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the sequence of any one or several of SEQ ID NO: 91-100.

[0282] Step S102: Divide the data set into a training set and a test set according to a predetermined ratio.

[0283] Step S103: training the regression model using the training set, and adjusting at least part of the parameters of the regression model to optimal parameters.

[0284] Step S104: testing the regression model with optimal parameters on the test set to evaluate the trained regression model configured to make a prediction of the efficiency of the recombinase polymerase amplification reaction, the template strand sequence and primer sequence features including the bag-of-words features based on k-mer sequences, the separator, and the template strand length.

[0285] For example, the predetermined ratio is (8.5-9.5):(1.5-0.5).

[0286] For example, in some examples, the predetermined ratio is 8.5:1.5, 8.8:1.3, 9.0:0.9, 9.2:0.8, 9.4:0.7, or 9.5:1.2.

[0287] For example, in an embodiment of the present disclosure, the efficiency of the isothermal molecular amplification reaction (RPA) is predicted based on the k-mer sequence of the primer pair-template. To achieve the purpose, first, the DNA sequence is divided into a set of k-mer short sequences, the token frequency is counted using the bag-of-words technique, and the frequency feature is combined with the template strand length feature as input; then, the training set and the test set are randomly divided, the random forest model is trained according to the existing label data, and the RPA amplification reaction efficiency on the test set is predicted. Under the premise of combining sequence frequency features and sequence length features, the embodiment of the present disclosure learns a regression model, effectively guiding the design engineering of HPV virus-primer pair RPA reaction, that is, the model for predicting the recombinase polymerase amplification reaction efficiency of the kit on the viral template strand can be obtained by the model construction method shown in FIG. 23.

[0288] For example, the use of k-mer technology improves the utilization of information on DNA sequences, increases the length feature of the template strand, and greatly reduces the prediction error. Using the bag-of-words technique and the random forest regression model, the RPA reaction efficiency of the HPV virus template strand-primer pair is effectively predicted.

[0289] For example, the bag-of-words features based on k-mer sequences are obtained by the following steps: converting the template strand sequence and the corresponding primer sequence features into k-mer sequences; converting the k-mer sequences into vector representations using the bag-of-words model method to obtain the bag-of-words features.

[0290] For example, the K-mer method is one of the common methods for processing DNA sequences in bioinformatics analysis. A codon is composed of 3 bases, and a k-mer sequence contains more information than a single base. The value of k is generally {3, 4, 5, 6,...}, and K-mer represents k consecutive bases.

[0291] For example, in one example, the length of the template chain is obtained by extracting the k-mer sequence length of the template chain sequence and normalizing the k-mer sequence length to a value between 0 and 1 as an input feature of the model.

[0292] For example, in some examples, the k-mer sequence length can be normalized to 0.2, 0.5, 0.5, 0.5, 0.7, 0.8, or 0.9.

[0293] For example, the regression model is a random forest regression model.

[0294] For example, in the process of training the regression model, the at least part of the parameters of the regression model to be adjusted include n_estimators and random number seed.

[0295] For example, in some examples, the parameters of the random forest can be debugged using a grid search algorithm, the value of n_estimators can be 200, 170, 150, 130, or 100, and the value of the random number seed can be 1, 42, 100, or 9120.

[0296] For example, the optimal parameters include n_estimators of 150 and random seed of 9120.

[0297] For example, the evaluation indicators for evaluating the trained regression model include mean squared error, root mean squared error, and mean absolute error.

[0298] For example, the viral template chain includes one or more of the corresponding template chains of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, and HPV53 types.

[0299] For example, the amplification reaction efficiency of the training set is distributed between 0 and 1, and the amplification reaction efficiency of the test set is distributed between 0 and 0.5.

[0300] For example, the k-mer represents k consecutive bases, and k is an integer greater than or equal to 3.

[0301] For example, the delimiter is a marker for connecting the primer sequence and the template chain sequence.

[0302] The at least one embodiment of the present disclosure also provides a method for predicting the efficiency of a recombinase polymerase amplification reaction, comprising using the regression model trained by any one of the above model construction methods to predict the efficiency of the recombinase polymerase amplification reaction.

[0303] For example, using k-mer technology to improve the information utilization rate of DNA sequence can increase the length characteristics of template chain, greatly reducing the prediction error. Using bag-of-words technology and random forest regression model, the RPA reaction efficiency of HPV virus template chain-primer pair is effectively predicted.

[0304] For example, the prediction algorithm of RPA primer amplification efficiency includes: predicting the efficiency of upstream and downstream primers amplifying HPV template chain. The specific implementation is as follows:

[0305] Data preprocessing: based on multiple sets of upstream and downstream primers, amplifying 10 subtypes of HPV virus {HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52 and HPV53}, the amplification efficiency of different upstream and downstream primers is different, and the larger the amplification efficiency value, the more template sequences are amplified.

[0306] For example, the 6-mer method can be used to convert the template sequence and the primer sequence into token sequence, for example, the DNA sequence “ATGGCTA”, its 6-mer sequence is {ATGGCT, TGGCTA}, ATGGCT is one of the tokens, which has certain biological significance; A special token {‘SEP’} is added as a separator to connect the primer pair sequence and the template chain sequence. Then, feature extraction is performed, and the bag-of-words (BOW) method is used to convert the k-mer sequence into a vector representation. The BOW method mainly counts the number of occurrences of each token in the 6-mer sequence text, and the CountVectorizer of the scikit-learn library can be used to realize the feature extraction of the BOW method. In addition, the length of the template chain k-mer sequence can also be extracted as a feature, and the length is normalized to 0-1 by using the normalization method, and the calculation formula of the normalization is as follows:

[0307] x^'=(x-min) / (max-min)

[0308] Where min represents the shortest k-mer sequence length of the template chain, min=40, and max represents the longest k-mer sequence length of the template chain, max=487. The length of the template chain will affect the amplification efficiency, and generally the longer the sequence, the worse the amplification efficiency.

[0309] For example, the k-mer bag-of-words features of the primer pair-template chain and the normalized features of the template chain k-mer sequence length can be combined as input features of the model.

[0310] For example, the training set and the test set are constructed by randomly dividing all data into the training set and the test set in a ratio of 9:1, wherein the training set contains 27 groups of primer pair-template strands and the test set contains 4 groups of primer pair-template strands, the amplification efficiency of the training set is evenly distributed between 0 and 1, and the amplification efficiency of the test set is mainly concentrated between 0 and 0.5.

[0311] In this document, the target gene sequence can be one or more sequences as follows:

[0312] HPV16

[0313] HPV18

[0314] HPV31

[0315] HPV33

[0316] HPV35

[0317] HPV39

[0318] HPV45

[0319] HPV51

[0320] HPV52

[0321] HPV53

[0322] Embodiment

[0323] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples without specific conditions are preferably referred to the instructions given in the present application, and can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0324] The reaction system and the reaction conditions are shown in the part of "sensitivity verification and reaction condition determination" in Example 4

[0325] Example 1: Design of primer and probe

[0326] Download multiple HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 53 genome sequences from NCBI, use MegAlign software in DNAStar software package to compare the homology of the above high-risk HPV genome sequences, find out the specific and intra-type conservative segments in the corresponding interval, design RPA primers and nfo probes in the corresponding conservative segments, and design three sets of primer probe combinations for each genotype. The principle of primer design: 1. The region of the amplicon is selected after sequence alignment; 2. The length of the upstream primer and the downstream primer is 30-35 bases; 3. The length of the amplicon is 80-400 bases; 4. Avoid multiple G at the 5' end of the primer, and the 3' end should contain G and C as much as possible; 5. Avoid a large number of palindromic structures and primer dimers; 6. The GC content is 40%-60%. The length of the nfo probe is 45-50 bp, among which the tetrahydrofuran (THF) modification is at least 30 bp away from the 5' end and at least 15 bp away from the 3' end. The THF substituted base is the complementary template nucleotide, which can be A, G, C or T. The upstream primer can have a 10-20 bp partial overlap with the nfo probe. The 5' end of the nfo probe and the 3' end of the downstream primer are modified with FAM or FITC and block combination.

[0327] The specific implementation is as follows:

[0328] Example 2: Prediction of RPA primer amplification efficiency algorithm

[0329] For the primers involved in Example 2, a deep learning method is provided to predict the efficiency of the upstream and downstream primers in amplifying the HPV template strand. The specific implementation is as follows:

[0330] 1. Data preprocessing: based on 31 sets of upstream and downstream primers, 6 subtypes of HPV virus HPV45, HPV53, HPV51, HPV52, HPV16, HPV18 are amplified, the amplification efficiency of different upstream and downstream primers is different, the larger the amplification efficiency value, the more template sequences are amplified.

[0331] The k-mer method is one of the common methods for processing DNA sequences in bioinformatics analysis. It is known that a codon is composed of 3 bases, and a k-mer sequence contains more information than a single base. The value of k is generally {3, 4, 5, 6,...}, and K-mer represents k consecutive bases. Here, we use the 6-mer method to convert both the template sequence and the primer sequence into token sequences, for example, the DNA sequence "ATGGCTA", its 6-mer sequence is {ATGGCT, TGGCTA}, ATGGCT is one of the tokens, which has certain biological significance. We also add a special token: {‘SEP’} as a separator to connect the primer pair sequence and the template chain sequence. Then, feature extraction is performed, and the Bag-of-Words (BOW) method is used to convert the k-mer sequence into a vector representation. The BOW method mainly counts the number of occurrences of each token in the 6-mer sequence text, and the CountVectorizer library of scikit-learn can be used to implement the BOW method for feature extraction. In addition, we also extract the length of the template chain k-mer sequence as a feature, and use normalization to normalize the length to 0-1, the normalization formula is as follows:

[0332] where min represents the shortest k-mer sequence length of the template chain, min = 40, max represents the longest k-mer sequence length of the template chain, max = 487. The length of the template chain affects the amplification efficiency, generally speaking, the longer the sequence, the worse the amplification efficiency.

[0333] We combine the primer pair-template chain k-mer bag-of-words features and the template chain k-mer sequence length normalization features as the input features of the model.

[0334] 2. Construct training set and test set: all data are randomly divided into training set and test set in the ratio of 9:1, where the training set contains 27 primer pair-template chains, and the test set contains 4 primer pair-template chains. The amplification efficiency of the training set is evenly distributed between 0 and 1, and the amplification efficiency of the test set is mainly concentrated between 0 and 0.5. The distribution histogram of the RPA amplification efficiency of the primer pair-template chain in the two data sets is shown in Figure 2.

[0335] 3. Construct machine learning model: we choose the random forest regression model, where n_estimators = 150, random seed seed = 9120, and the rest of the parameters use the default value.

[0336] 4. Parameter tuning and optimization: We use grid search algorithm to tune the parameters of the random forest, the value range of n_estimators is {200, 170, 150, 130, 100}, and the value range of random seed is {1, 42, 100, 9120}. The experimental results are shown in Figure 3. Figure 3 shows the influence of different n_estimators values on the mean square error of the test set. In Figure 3, it can be seen that when n_estimators = 150, the mse value is the smallest. Figure 4 shows the influence of different random seeds on the mean square error, where it can be seen that when the seed value is 9120, the test set mean square error is the smallest.

[0337] 5. Model training and evaluation: The selected random forest model is trained using the training set, and then evaluated using the test set, with the evaluation indicators selected as mean square error, root mean square error and mean absolute error, and the results are shown in the following table:

[0338] The scatter plot of the predicted value and the true value of the amplification efficiency on the test set and the training set is shown in Figure 5:

[0339] As shown in Figure 5, the running time of model training and testing and evaluation is 0.77s, which is greatly improved in running efficiency compared with deep learning algorithm.

[0340] 6. Feature combination evaluation: We evaluate the influence of different feature combinations on the error on the test set, and the running time and results are shown in the following table:

[0341] It can be seen that the model of the three feature combinations has the smallest error on the test set.

[0342] 7. Predict the amplification efficiency of the template chain-primer pair on the other 4 HPV subtypes {HPV31, HPV33, HPV35, HPV39}, and the specific results are shown in the following table:

[0343] Based on the above results, further amplification experiments can be carried out to screen primers with high amplification efficiency.

[0344] Example 3: Screening of primer probe combination experiment

[0345] Different groups of primers and probes are used to prepare PCR reaction reagents for different genotypes, and standard samples of corresponding genotypes are detected, and the color development effect of test strips of different primer probe combinations of the same genotype is observed, and better primer probe combinations are screened out.

[0346] 1. Sample preparation

[0347] Take HPV16 and HPV18 as an example, dilute the HPV16 and HPV18 type standard to 1 x 10 5 copies / mL with enzyme-free water, and store at -20℃ for standby.

[0348] 2. Reagent preparation

[0349] Take out each component reagent, place it on the table, and gently shake it to mix after balancing to room temperature. Then centrifuge it. Use each set of primers and probes to prepare the corresponding amplification reagent, and the specific system preparation is as follows. Prepare the PCR mixture according to the following table

[0350] Dissolve the freeze-dried microspheres thoroughly, add 2 μL of sample and 2.5 μL of 280 mM MgOAc in turn, and mix (shake) thoroughly to start the reaction. Centrifuge briefly.

[0351] 3. Amplification detection

[0352] Incubate at 39℃ for 20 min, then add diluent to the product to 100 μL, add nucleic acid detection test strips, stand for 5 min, and observe the results.

[0353] 4. If only the C line appears on the test strip, the result is negative, if the T line and C line appear on the test strip, the result is positive, if the C line does not appear, the test result is invalid, and needs to be re-verified.

[0354] 5. Result analysis

[0355] After the reaction, observe the amplification results of each primer probe group (as shown in FIGS. 6A-6E). Determine whether the primer probe combination is available according to whether there is a colored band and whether the band is bright and complete, determine the optimal primer probe combination, and the final primer combination is as follows:

[0356] The experimental results show that the prediction RPA primer amplification efficiency algorithm has a correct rate of 75%.

[0357] Example 4: Sensitivity verification and reaction condition determination

[0358] Dilute the standard samples of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, and 53 genotypes with ultrapure water to obtain concentrations of 1 x 10 4 copies / mL (S1), 1 x 10 3The samples of 1 x 10 copies / mL (S1), 5 x 10 copies / mL (S2), and 500 copies / mL (S3) were detected according to the detection method, primer and probe combination of Example 3, and the detection results of the test strip were counted as follows.

[0359] "+" represents an amplification signal, and "-" represents no color development signal

[0360] Result analysis: the standard samples of different concentrations were detected by the HPV16, 18, 31, 33, 35, 39, 45, 51, 52, and 53 detection systems, respectively, and both of them could normally detect 1 x 10 4 copies / mL (S1) and 1 x 10 3 copies / mL (S2), and the samples of 500 copies / mL had no color development.

[0361] Reaction system optimization

[0362] Taking HPV16 and HPV18 as examples, the reaction system was further optimized in terms of RPA reaction temperature and time, and the specific time and temperature optimization combination was as follows:

[0363] 1. RPA reaction temperature condition

[0364] FIG. 7 shows the detection results of RPA different reaction temperatures for detecting HPV16 (left) and HPV18 (right) papillomavirus.

[0365] 2. RPA reaction time: time optimization based on the above confirmed reaction temperature condition

[0366] Result analysis: the optimization results show that the color development bands of HPV16 / 18 are brighter at a reaction temperature of 39°C and a reaction time of 20 min, and the optimal reaction program is determined to be 39°C for 20 min. FIG. 8 shows the detection results of RPA different reaction times for detecting HPV16 (left) and HPV18 (right) papillomavirus.

[0367] Example 5: reaction system optimization

[0368] The fluorescent probe sequence used in this example is as follows:

[0369] 5.1 Reaction system 1

[0370] The composition of the nucleic acid releasing agent includes: 20 mM sodium hydroxide, 1 M guanidine hydrochloride, 1 mM EDTA, and 10 mM Tris-HCl buffer.

[0371] The components of the corresponding nucleic acid detection reagent include: 100 ng / μL Taq enzyme, 200 μM dNTP, 400 nM primer probe, 30 mM magnesium ion, 10 mM Tris-HCl buffer, 20 mM hydrochloric acid, 1 M acetaldehyde. The nucleic acid release reagent and the detection reagent are freeze-dried into microspheres by freeze-drying method.

[0372] The experimental steps are as follows:

[0373] 1. Take 100 μL of the virus sample to dissolve the nucleic acid release reagent microspheres, and mix uniformly by vortexing or blowing;

[0374] 2. Incubate at room temperature or 38°C for 5 minutes;

[0375] 3. Dissolve the nucleic acid detection microspheres in the solution after lysing the solution as a solvent;

[0376] 4. Perform amplification detection in a PCR instrument according to the common method of polymerase chain reaction (PCR), including denaturation, annealing and extension. The detection results are shown in FIG. 1 and FIG. 9.

[0377] In this embodiment, the guanidine hydrochloride in the nucleic acid release reagent releases the nucleic acid in the sample by destroying the hydrogen bond. The acetaldehyde in the corresponding nucleic acid release reagent can react with guanidine hydrochloride, thereby eliminating its influence on the nucleic acid detection system, achieving the purpose of one-pot reaction of nucleic acid release and detection.

[0378] 5.2. Reaction system 2

[0379] The components of the nucleic acid release reagent include: 10 mM sodium hydroxide, 3% SDS, 1 mM EDTA, 10 mM Tris-HCl buffer.

[0380] The components of the corresponding nucleic acid detection reagent include: 15 ng / μL Sau enzyme, 100 ng / μL T4 UvsX protein, 50 ng / μL T4 UvsY protein, 500 ng / μL T4 gp32 protein, 200 μM dNTP, 400 nM primer probe, 30 mM magnesium ion, 10 mM Tris-HCl buffer, 5% PEG, 2 mM dithiothreitol, 50 mM phosphocreatine, 100 ng / μL creatine kinase, 3 mM adenosine phosphate, 3% polyether copolymer. The nucleic acid release reagent and the detection reagent are freeze-dried into microspheres by freeze-drying method.

[0381] The experimental steps are as follows:

[0382] 1. Take 100 μL of the virus sample to dissolve the nucleic acid release reagent microspheres, and mix uniformly by vortexing or blowing;

[0383] 2. Incubate for 5 minutes at room temperature or 38 degrees Celsius;

[0384] 3. Dissolve the nucleic acid detection microspheres with the solution after lysis solution as solvent;

[0385] 4. Incubate for 20 minutes at 38 degrees Celsius to achieve isothermal amplification, and use the test strip to detect the results as shown in Figure 10.

[0386] In this embodiment, the main component in the nucleic acid release agent is a surfactant that reduces surface tension to form a foam to release nucleic acids in the sample. The polyether copolymer in the corresponding nucleic acid release agent can produce a defoaming effect, thereby eliminating its impact on the nucleic acid detection system, achieving one-pot reaction for nucleic acid release and detection.

[0387] 5.3 Reaction system 3

[0388] In order to achieve the release and detection of nucleic acid in one reaction tube and facilitate storage and transportation, the nucleic acid release reagent and nucleic acid detection reagent need to be freeze-dried into microspheres to ensure that they do not affect the detection performance after reconstitution. Then store the nucleic acid release reagent microspheres and nucleic acid detection microspheres in the same test tube, and use a water-soluble film to divide them into two parts.

[0389] The specific implementation is as follows:

[0390] 1. Add each component to the centrifuge tube according to the ratio of the nucleic acid release agent or the nucleic acid detection reagent in 5.1 or 5.2, and vortex mix uniformly to obtain a mixed solution;

[0391] 2. Use a pipette to drop the mixed solution into liquid nitrogen at a volume of 10 μL, and condense it into a round ball;

[0392] 3. Place the condensed microspheres into a pre-cooled freeze dryer, set the cold trap temperature to -69°C, and vacuum freeze dry for more than 10 hours to obtain freeze-dried microspheres of the nucleic acid release reagent and freeze-dried microspheres of the nucleic acid detection reagent.

[0393] 5.4 One-pot reaction system

[0394] 1. Store the nucleic acid release reagent microspheres and nucleic acid detection microspheres in the same test tube, and use a water-soluble film to divide them into two parts, as shown in Figure 11.

[0395] 2. Take 100 μL of the virus sample to dissolve the nucleic acid release reagent microspheres, and mix uniformly by blowing;

[0396] 3. Incubate for 5 minutes at room temperature or 38 degrees Celsius, and the water-soluble film gradually dissolves during incubation;

[0397] 4. Then dissolve the nucleic acid detection microspheres with the solution as solvent;

[0398] 5. Finally, mix well and then perform PCR or isothermal amplification detection.

[0399] The material of the water-soluble film can be selected from one of polyvinyl alcohol, starch, and sodium carboxymethyl cellulose. According to the time requirement for releasing the nucleic acid, a water-soluble film with a corresponding thickness (0.1-100 microns) is designed, so that after the reaction system releases the nucleic acid, the water-soluble film is dissolved, and then the amplification reaction is performed in the nucleic acid detection system.

[0400] 5.5 RPA amplification system

[0401] This experiment is the verification of the RPA system. The probe is modified, and real-time fluorescence can be detected.

[0402] The allocation ratio of each component of the reaction system is as follows.

[0403] The 2X buffer component is: 50-100 mM Tris-Hcl (pH = 7.5-8.5), 2-6 mM dithiothreitol (DTT), 10%-20% (W / V) polyethylene glycol (PEG2000) or 2%-8% (W / V) polyethylene glycol (PEG20000)

[0404] The 10X base liquid component is: 10-60 mM ATP, 300-500 mM phosphocreatine, 500-1000 mM potassium acetate, 10-50 ng / μL of sodium heparin

[0405] 20X enzyme mixture: T4 UvsY protein 30-100 ng / μL, T4 gene32 protein 500-1100 ng / μL, T4 UvsX protein 100-200 ng / μL, Bsu DNA polymerase 10-50 ng / μL, creatine kinase (CPK) 30-150 ng / μL

[0406] The specific operation steps are as follows:

[0407] Take out the required components of the kit 30 min in advance, melt at room temperature, and shake to mix.

[0408] 1. Add 25 μL of 2X reaction buffer, 1 μL of d NTP, 2 μL of upstream primer, 2 μL of downstream primer, 5 μL of 10X enzyme mixture, 3 μL of template, and 5 μL of 10X base liquid to the reaction tube in turn, and finally supplement ultrapure water to 47.5 μL.

[0409] 2. Add 2.5 μL of 100-300 mM MgOAc or Mgcl2 to the inside of the test tube cover, and invert it up and down for 20 times to mix.

[0410] 3. After mixing, centrifuge the reaction solution to the bottom of the test tube, and incubate at 30-39℃ for 15-20 min.

[0411] 4. Take 5 μL and dilute 10 times in 45 μL ultrapure water, then drop on the test strip, and observe whether the test strip develops color after 2 min.

[0412] To verify the amplification performance of the reagent system, a certain amount of fluorescent probe was added to the system, a constant temperature of 30-39°C was set by using a fluorescence PCR instrument to simulate the normal temperature reaction environment, and the system performance was directly observed by detecting the change of fluorescence value after the reaction started. The relevant test conditions are shown in Figure 12. As can be seen from Figure 12, the fluorescence value basically enters the plateau period at 15-20 min, which proves that the amplification reaction can meet the result within 20 min in the normal temperature environment, which shows that the technology has great market potential in the application scene of urgently needing fast detection results. Compared with the same type of commercially available reagent kit, the amplification system shows higher fluorescence value signal, earlier peak time and faster plateau period in the same environment, which shows that the system has better amplification effect. The relevant test conditions are shown in Figures 13-14. Figure 13 shows the amplification curve of the commercially available reagent kit. Figure 14 shows the amplification curve of the reagent system.

[0413] 5.6 System optimization

[0414] 1. During the preparation of the reagent system, it was found that the preparation of creatine kinase solution had a significant effect on the final amplification result. Creatine kinase buffer was prepared by diluting creatine kinase with enzyme-free water, Tris-Hcl (pH = 7.5-8.5) and NaHCO3 buffer (mass fraction 0.1%-5%). The amplification results are shown in Figures 15-17. As can be seen from the figures, the amplification effect is obvious after dilution with NaHCO3 buffer, while the amplification basically does not occur with the other two buffers. Figure 15 shows the amplification result (preparation of creatine kinase solution with enzyme-free water). Figure 16 shows the amplification result (preparation of creatine kinase solution with Tris-Hcl buffer). Figure 17 shows the amplification result (preparation of creatine kinase solution with NaHCO3 buffer).

[0415] 2. Figure 18 shows the amplification result (enzyme mixture addition amount optimization). Figures 18 1-4 are the optimization of enzyme mixture addition amount, 1-4 are the addition amount of 3, 1.5, 2, 1 μL, respectively. It can be found that except for curve 1 which is better, the other amplification curves are poor, but the time to reach the equilibrium period of curve 1 is about 30 min, the amplification time is long, and it cannot meet our amplification requirements, so the addition amount of 2.5 μL is still selected.

[0416] 3. Figure 19 shows the amplification results (optimization of creatine kinase and Bsu DNA polymerase addition). The two amplification curves in Figure 19, 1-2, are the results of experiments in which the concentration of creatine kinase (CPK) in the enzyme mixture was increased by 50% (amplification curve 2) and decreased by 50% (amplification curve 1); the two amplification curves in Figure 19, 3-4, are the results of experiments in which the concentration of Bsu DNA polymerase in the enzyme mixture was increased by 50% (amplification curve 3) and decreased by 50% (amplification curve 4). The results show that adjusting the concentrations of the two enzymes in the enzyme mixture results in poorer amplification, presumably because changes in the concentrations of the two enzymes affect the activities of other enzymes, thereby affecting the amplification efficiency.

[0417] 4. Figure 20 shows the amplification results (optimization of ATP and MgOAc addition). The amplification curves in Figure 20, 1 and 3, are the results of experiments in which the concentration of ATP in the 10X base solution was increased by 50% (amplification curve 1) and decreased by 50% (amplification curve 3); 2, 4, 5, and 6 are the results of experiments in which the amount of MgOAc was optimized, and the four curves are the results of experiments in which 3, 2, 1.5, and 1 μL of MgOAc was added, respectively. The results show that the original ATP concentration and the addition of 2.5 μL of MgOAc are the optimal conditions.

[0418] Example 6, Verification of Clinical Samples

[0419] Sixteen clinical samples were selected, and the sampling site was the cervical epithelial cells. The nucleic acids had been extracted in advance and stored at -20°C for later use. The samples were detected using the test reagent according to the operation of the above examples, and the same samples were detected using a fluorescent quantitative PCR reagent as a control. The results of the test strips and the fluorescent PCR were compared. The results are as follows:

[0420] HPV18 Type Test Strip and Fluorescent PCR Detection Results

[0421] Note: "+" indicates a color development signal, "-" indicates no color development signal, and "√" indicates that the results are completely consistent.

[0422] HPV16 Type Test Strip and Fluorescent PCR Detection Results

[0423] Note: "+" indicates an amplification signal, "-" indicates no amplification signal, and "√" indicates that the results are completely consistent.

[0424] Figure 21 shows the color development results of the HPV18 type test strip. Figure 22 shows the color development results of the HPV16 type test strip. Result analysis: taking the fluorescence PCR detection results as the standard, the test strip detection results showed that a total of 16 HPV clinical sample nucleic acids were verified, of which 2 were positive for HPV18 type, 2 were positive for HPV16 type, and sample No. 15 was positive for HPV16 and 18, which was completely consistent with the fluorescence PCR results, indicating that the detection reagent was consistent with the existing conventional HPV molecular diagnosis method, and had good accuracy.

[0425] Example 7, primer probe characterization means - amplicon sequencing combined with fragment analysis and mass spectrometry

[0426] 1, primer pool Pool configuration

[0427] 1) All primer probe mixtures were prepared according to the amplification instructions, and shaken and mixed for standby;

[0428] 2, PCR amplification

[0429] PCR reaction system:

[0430] PCR amplification conditions:

[0431] Magnetic bead purification:

[0432] 1.2 times magnetic bead purification, 30-50 μL elution. Qubit determination of PCR product concentration.

[0433] 3, PCR product phosphorylation

[0434] Reaction program: 20℃, 30min; 65℃, 30min; 4℃, Hold.

[0435] 4, linker ligation

[0436] Reaction conditions: 20℃, 20min.

[0437] Magnetic bead purification:

[0438] 0.88 times Beads purification, 22 μL elution. Qubit determination of concentration.

[0439] 5, library amplification

[0440] PCR reaction system:

[0441] PCR amplification conditions:

[0442] Magnetic bead purification:

[0443] 0.5 times magnetic bead to remove large fragments, 1.2 times magnetic bead to purify target fragments, 40 μL elution.

[0444] 6. Amplicon sequencing

[0445] The purified product obtained in the previous step was subjected to amplicon sequencing, and the analysis according to the sequencing data obtained the target fragment and the position of the upstream and downstream primers.

[0446] 7. Primer sequence analysis

[0447] The Agilent 4200 automatic electrophoresis instrument or ABI 3500 was used to analyze the nucleic acid fragments against the primer probe combination, and the primer length information was obtained. The primer length ± 3 bp obtained by upstream and downstream analysis was verified combined with the sequencing result, and the primer sequence information was obtained.

[0448] 8. Probe sequence analysis

[0449] The primer probe mixture was used for high-resolution agarose gel electrophoresis. Due to the different lengths of the primer probes, they can be separated by electrophoresis. The longer fragments obtained by separation are the probes. The probes were obtained by gel recovery and purified by magnetic beads. The probe molecular weight was obtained by mass spectrometry analysis, and the probe fragment sequence was obtained combined with the marker and the position of the target fragment.

[0450] Figure 24 shows the mass spectrum of RHPV16P1, the theoretical molecular weight is 10828.09, and the molecular weight error is ≤0.05%. Figure 25 shows the mass spectrum of RHPV18P1, the theoretical molecular weight is 9403.16, and the molecular weight error is ≤0.05%. Figure 26 shows the mass spectrum of RHPV31P3, the theoretical molecular weight is 10443.84, and the molecular weight error is ≤0.05%. Figure 27 shows the mass spectrum of RHPV33P2, the theoretical molecular weight is 10087.63, and the molecular weight error is ≤0.05%.

[0451] The kit for detecting human papillomavirus, the method for detecting the presence or type of human papillomavirus in a sample, the method for diagnosing a papillomavirus infection or a papillomavirus infection related disease in a subject, the use of a primer pair in the preparation of a kit, the use of a combination of primers and probes in the preparation of a kit, the model construction method for predicting the recombination enzyme polymerase amplification reaction efficiency of a virus template strand based on the kit, and the prediction method of the recombination enzyme polymerase amplification reaction efficiency, which can detect human papillomavirus of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52 and HPV53 genotypes, the primer pair and the combination of the primer pair and the probe provided by the embodiments of the present disclosure are designed on the specific sequences of each type of virus genome, which can effectively avoid missed detection and non-specificity, and the RPA-nfo probe lateral flow chromatography system is used, so that the operation process of detection becomes simpler and more convenient.

[0452] The following points need to be explained:

[0453] (1) The drawings of the embodiments of the present disclosure only involve the structures involved in the embodiments of the present disclosure, and other structures can be referred to the general design.

[0454] (2) For the sake of clarity, the thickness of the layer or region is magnified or reduced in the drawings used to describe the embodiments of the present disclosure, that is, these drawings are not drawn according to the actual proportion.

[0455] (3) In the case of no conflict, the embodiments of the present disclosure and the features in the embodiments can be combined with each other to obtain new embodiments.

[0456] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto, and the protection scope of the present disclosure should be subject to the protection scope of the claims.

Claims

1. A composition comprising: Several different bait oligonucleotides, wherein the several different bait oligonucleotides are configured to hybridize to several DNA molecules, the several DNA molecules being at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of sequences derived from any one or more of SEQ ID NO: 91-100.

2. The composition according to claim 1, wherein, The plurality of DNA molecules are at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of sequences derived from several of the items in SEQ ID NO:91-100.

3. The composition according to claim 1 or 2, wherein, The plurality of DNA molecules are at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the entire sequence of SEQ ID NO:91-100.

4. The composition according to claim 1, wherein, The bait oligonucleotide is one or more of the primer and probe.

5. The composition according to claim 4, wherein, The probe is an nfo probe.

6. A kit comprising the composition according to any one of claims 1-5, and further comprising a hybridization reaction system in which the plurality of bait oligonucleotides hybridize to a plurality of DNA molecules.

7. The kit according to claim 6, wherein, The hybridization reaction system is an RPA amplification system.

8. The kit according to claim 7, wherein, The RPA amplification system includes an enzyme mixture containing T4 UvsY protein, T4 gene32 protein, T4 UvsX protein, Bsu DNA polymerase, and creatine kinase.

9. The kit according to claim 8, wherein, The enzyme mixture contains 30-100 ng / μL of T4 UvsY protein, 500-1100 ng / μL of T4 gene32 protein, 100-200 ng / μL of T4 UvsX protein, 10-50 ng / μL of Bsu DNA polymerase, and 30-150 ng / μL of creatine kinase.

10. The kit according to claim 6, wherein, The kit contains a diluent, which is a NaHCO3 diluent.

11. The kit according to claim 10, wherein, The diluent is a NaHCO3 diluent with a mass fraction of 0.1%-5%.

12. The kit according to claim 6, wherein, The kit contains a buffer solution containing any one of Tris-HCl, dithiothreitol, PEG2000, and PEG20000.

13. The kit according to claim 12, wherein, The buffer solution contains Tris-HCl at a molar concentration of 50-100 mM / L, dithiothreitol at a molar concentration of 2-6 mM / L, and any one of PEG2000 at a mass concentration of 10%-20% W / V and PEG20000 at a mass concentration of 2%-8% W / V.

14. The kit according to claim 6, wherein, The kit contains a base solution comprising ATP, creatine phosphate, potassium acetate, and sodium heparin.

15. The kit according to claim 14, wherein, The base solution contains ATP at a molar concentration of 10-60 mM / L, creatine phosphate at a molar concentration of 300-500 mM / L, potassium acetate at a molar concentration of 500-1000 mM / L, and sodium heparin at a molar concentration of 10-50 ng / μL.

16. The kit according to claim 6, wherein, The kit also contains magnesium acetate, dNTPs, and endonucleases.

17. The kit according to claim 6, wherein, The kit also contains a nucleic acid release agent.

18. The kit according to claim 17, wherein, Both the nucleic acid release agent and the hybridization reaction system are lyophilized microspheres.

19. The kit according to claim 17, wherein, The nucleic acid releasing agent and the hybridization reaction system act synchronously.

20. The kit according to claim 6, wherein, The hybridization reaction system includes components that eliminate the influence of nucleic acid releasing agents on the hybridization reaction system.

21. The kit according to claim 17, wherein, The nucleic acid release agent comprises sodium hydroxide, EDTA, and Tris-HCl buffer, or the nucleic acid release agent comprises sodium hydroxide, SDS, EDTA, and Tris-HCl buffer.

22. The kit according to claim 21, wherein, The nucleic acid release agent comprises sodium hydroxide at a molar concentration of 20 mM / L, guanidine hydrochloride at a molar concentration of 1 M / L, EDTA at a molar concentration of 1 mM / L, and Tris-HCl buffer at a molar concentration of 10 mM / L, or the nucleic acid release agent comprises sodium hydroxide at a molar concentration of 10 mM / L, 3% SDS, EDTA at a molar concentration of 1 mM / L, and Tris-HCl buffer at a molar concentration of 10 mM / L.

23. The kit according to claim 20, wherein, The hybridization reaction system includes acetaldehyde.

24. The kit according to claim 23, wherein, The hybridization reaction system includes acetaldehyde with a molar concentration of 1 M / L.

25. The kit according to claim 20, wherein, The hybridization reaction system includes polyether copolymers.

26. The kit according to claim 20, wherein, The hybridization reaction system includes a polyether copolymer with a mass percentage of 3%.

27. Use of the composition according to any one of claims 1-5 for detecting human papillomavirus in a sample.

28. The use according to claim 27, wherein, The human papillomavirus is one or more of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, and 53.

29. A method for detecting the presence or type of human papillomavirus in a sample, comprising performing a recombinase polymerase amplification reaction using the composition of any one of claims 1-5, wherein, The human papillomavirus in the sample is one or more of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, and 53.

30. The method according to claim 29, wherein, The reaction temperature for the recombinase polymerase amplification is 30-45℃, and the reaction time is 5-30 minutes.

31. The method according to claim 29, wherein, The method further includes qualitative and quantitative analysis of the components of the sample by at least one of spectroscopy, chromatography, mass spectrometry, energy dispersive spectroscopy, and thermal spectroscopy.

32. The method according to claim 29, wherein, The method also includes releasing and detecting nucleic acids from the sample.

33. A method for constructing a model to predict the efficiency of recombinase polymerase amplification reaction of a viral template strand, comprising: Construct a dataset comprising template strand sequence and decoy oligonucleotide sequence features and corresponding recombinase polymerase amplification reaction efficiency, wherein the decoy oligonucleotide sequence is derived from at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one or more sequences in SEQ ID NO: 91-100; The dataset is divided into a training set and a test set according to a predetermined ratio; The regression model is trained using the training set, and at least some parameters of the regression model are adjusted as follows: Optimal parameters; The regression model with the optimal parameters is tested on the test set to evaluate the trained regression model, wherein the trained regression model is configured to predict the efficiency of recombinase polymerase amplification reaction. The template chain sequence and the decoy oligonucleotide sequence features include bag-of-words features based on k-mer sequences, delimiters, and template chain length.

34. The method according to claim 33, wherein, The bag-of-words features based on k-mer sequences are obtained through the following steps: The template strand sequence and the corresponding decoy oligonucleotide sequence features are converted into k-mer sequences; The k-mer sequence is converted into a vector representation using the bag-of-words model method to obtain the bag-of-words features.

35. The method according to claim 33, wherein, The length of the template chain is obtained through the following steps: Extract the k-mer sequence length of the template chain sequence and normalize the k-mer sequence length to a value between 0 and 1.

36. The method according to any one of claims 33-35, wherein, The regression model is a random forest regression model.

37. The method of claim 36, wherein, During the training of the regression model, at least some of the parameters of the regression model being adjusted include n_estimators and a random number seed.

38. The method according to claim 37, wherein, The optimal parameters include n_estimators being 150 and the random seed being 9120.

39. The method according to claim 33, wherein, Evaluation metrics used to assess the trained regression model include mean squared error, root mean squared error, and mean absolute error.

40. The method according to claim 33, wherein, The viral template chain includes one or more corresponding template chains from HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52 and HPV53.

41. The method according to claim 33, wherein, The predetermined ratio is (8.5-9.5):(1.5-0.5).

42. The method according to claim 41, wherein, The amplification reaction efficiency of the training set is distributed between 0 and 1, and the amplification reaction efficiency of the test set is distributed between 0 and 0.

5.

43. The method according to claim 33, wherein, The k-mer represents k consecutive bases, where k is an integer greater than or equal to 3.

44. The method according to claim 33, wherein, The separator is a marker that connects the decoy oligonucleotide sequence and the template strand sequence.

45. A method for predicting the efficiency of recombinase polymerase amplification reaction, comprising predicting the efficiency of recombinase polymerase amplification reaction using the regression model trained by the model construction method according to any one of claims 33-44.

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