CN116855642A - Real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPVs - Google Patents

Real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPVs Download PDF

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CN116855642A
CN116855642A CN202311031974.1A CN202311031974A CN116855642A CN 116855642 A CN116855642 A CN 116855642A CN 202311031974 A CN202311031974 A CN 202311031974A CN 116855642 A CN116855642 A CN 116855642A
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hpv
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居金良
崔振玲
张帝
沈晓宁
钱卫国
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Shanghai Rendu Biotechnology Co ltd
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Abstract

The invention discloses a real-time fluorescent nucleic acid isothermal amplification detection kit for synchronously detecting 14 high-risk HPVs, and a special primer and a special probe thereof, belonging to the technical field of biomedical detection. The kit provided by the invention comprises nucleic acid extracting solutions, detection solutions and SAT enzyme solutions of two systems for 14 high-risk HPVs, primers and probes which are more suitable for detecting the high-risk HPVs are optimally designed, the detection systems are reasonably distributed, and each component is added step by step in the detection process to carry out step reaction, so that the real-time and rapid detection of the 14 high-risk HPVs can be synchronously realized, the sensitivity is high, the specificity is good, the RNA of an amplification product is easy to degrade, the environmental pollution is not caused, and the cross pollution of a sample is not caused in the detection process.

Description

Real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPVs
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a real-time fluorescent nucleic acid isothermal amplification detection kit capable of detecting 14 high-risk HPVs (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68), a special primer and a probe for detecting the 14 high-risk HPVs, and particularly relates to a primer, a probe and a related kit used in the real-time fluorescent nucleic acid isothermal amplification detection of the 14 high-risk HPVs by combining a specific target capturing technology and a real-time fluorescent nucleic acid isothermal amplification detection technology (Simultaneous Amplification and Test, SAT).
Background
Persistent infection with HPV is a major cause of cervical cancer in women, especially 14 of over 200 HPV subtypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) are considered to be high-risk HPV subtypes responsible for cervical cancer and precancerous lesions. Early diagnosis and early treatment are main means for preventing and treating cervical cancer.
The diagnosis of HPV at present mainly comprises colposcopy examination, cervical biopsy and PCR amplification detection. However, none of the colposology, colposcopy and cervical biopsy, among others, determine whether the infected HPV is a high risk HPV, which may be confusing for clinical administration and may create unnecessary mental stress on the patient. The PCR amplification detection method is a method commonly used for detecting high risk type HPV at present, for example, patent document CN107988432A (hereinafter referred to as document 1) discloses a kit for simultaneously detecting a plurality of high risk type human papillomaviruses in a single tube reaction, which at least comprises specific probes SEQ ID NOs 1-14 corresponding to 14 genotypes of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, respectively, and 6 forward primers and 6 reverse primers corresponding to the 14 genotypes are synthesized, and the minimum detection limit of the kit for 14 HPV genotypes can reach 100 copies/reaction (wherein the minimum detection limit for HPV16 and 18 can reach 10 copies/reaction). However, the method disclosed in the document 1 is based on the basic principle of PCR, which requires temperature rise and fall and circulation during the PCR reaction, and thus requires a long detection time (generally, 2 to 3 hours), and is inefficient; in addition, the reaction product of PCR is DNA, which is not easy to degrade and can easily cause sample cross contamination and experimental environment pollution. In order to overcome the defect of the PCR method in detecting HPV, patent document CN106834547A (hereinafter referred to as document 2) discloses a kit for detecting the E6/E7 gene of human papilloma virus based on real-time isothermal amplification, which comprises primers and probes designed for 14 high-risk HPVs, and can specifically detect and identify the mRNA of HPV E6/E7. However, the lowest limit of detection of RNA templates against 14 high-risk HPV viruses in this document 2 is only 100 copies/. Mu.l (i.e., 500 copies/reaction), and the detection sensitivity is low.
Disclosure of Invention
In view of one or more problems in the prior art, one aspect of the present invention provides a real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPV types, comprising:
(T1) nucleic acid extract 1: comprising a solid support comprising a first specific capture probe and a second specific capture probe for capturing detection sequences of 9 high-risk HPV types, and 9 primers for specifically binding to target sequences of 9 high-risk HPV types, respectively, wherein the 9 high-risk HPV types comprise HPV 16, 31, 33, 35, 51, 52, 56, 58, 66, the 9 primers comprising a fifth primer for specifically binding to target sequences of HPV 16, a sixth primer for specifically binding to target sequences of HPV 31, a seventh primer for specifically binding to target sequences of HPV 33, an eighth primer for specifically binding to target sequences of HPV 35, a ninth primer for specifically binding to target sequences of HPV 51, an eleventh primer for specifically binding to target sequences of HPV 52, an eleventh primer for specifically binding to target sequences of HPV 58, a twelfth primer for specifically binding to sequences of HPV 56 and HPV 66, and a universal primer;
(T2) nucleic acid extract 2: comprising a solid support comprising a third specific capture probe and a fourth specific capture probe for capturing detection sequences of 5 high-risk HPV types, and 4 primers for specifically binding to target sequences of 5 high-risk HPV types, respectively, wherein the 5 high-risk HPV types comprise HPV 18, 39, 45, 59, 68, the 4 primers comprising a seventeenth primer for specifically binding to target sequences of HPV 18, an eighteenth primer for specifically binding to target sequences of HPV 45, a nineteenth primer for specifically binding to target sequences of HPV 59, a twentieth primer for specifically binding to target sequences of HPV 39 and HPV 68, and one of the universal primers;
(T3) amplification assay 1.1: it comprises a first primer, a second primer, a third primer and a fourth primer, wherein the first primer is matched with the eleventh primer and the universal primer for amplifying the target sequence of HPV 58, the second primer is matched with the seventh primer, the tenth primer and the universal primer for amplifying the target sequence of HPV 33 and HPV 52, the third primer is matched with the twelfth primer and the universal primer for amplifying the target sequence of HPV 56 and HPV 66, and the fourth primer is matched with the fifth primer, the sixth primer, the eighth primer, the ninth primer and the universal primer for amplifying the target sequences of HPV 16, 31, 35 and 51 respectively;
(T4) amplification assay 1.2: comprising the universal primer, a first target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 51, a second target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 16, 31, 35, a third target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 33, 52, 58, and a fourth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 56, 66;
(T5) amplification assay solution 2.1: comprising a thirteenth primer, a fourteenth primer, a fifteenth primer and a sixteenth primer, wherein the thirteenth primer is complexed with the seventeenth primer and a universal primer for amplifying a target sequence of HPV 18, the fourteenth primer is complexed with the eighteenth primer and a universal primer for amplifying a target sequence of HPV 45, the fifteenth primer is complexed with the nineteenth primer and a universal primer for amplifying a target sequence of HPV 59, and the sixteenth primer is complexed with the twentieth primer and a universal primer for amplifying a target sequence of HPV 39 and HPV 68;
(T6) amplification assay solution 2.2: comprising the universal primer, a fifth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 18, a sixth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 45, a seventh target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 59, and an eighth target detection probe that specifically binds to amplified product RNA copies of target sequences of HPV 39 and HPV 68;
(T7) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase;
wherein:
the first specific capture probe to the fourth specific capture probe respectively comprise nucleotide sequences shown as SEQ ID NO. 42-SEQ ID NO. 45;
the first primer to the twentieth primer respectively comprise nucleotide sequences shown as SEQ ID NO. 1-SEQ ID NO. 20;
the universal primer comprises a nucleotide sequence shown as SEQ ID NO. 21; and the sequences of the 5' end of the fifth primer to the twelfth primer and the seventeenth primer to the twentieth primer are the sequences of the universal primers;
The first target detection probe to the eighth target detection probe respectively comprise nucleotide sequences shown as SEQ ID NO. 22-SEQ ID NO. 29.
In some embodiments, the first to eighth target detection probes each carry a fluorescent reporter group and a quencher group at both ends of the nucleotide sequence, and the fluorescent reporter groups carried by the eighth target detection probes are the same or different.
In some embodiments, the kit further comprises:
(M1) washing solution: it contains NaCl and SDS; preferably contains 5-50mM HEPES, 50-500mM NaCl, 0.5-1.5% SDS, 1-10mM EDTA; and/or
(M2) mineral oil; and/or
(M3) positive control: a system for E6/E7 in vitro transcription of RNA comprising HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68; and/or
(M4) negative control: a system that does not contain human papillomavirus target nucleic acid.
In some embodiments, the components of the nucleic acid extraction solution 1 include: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 μm of the first and second specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL of the fifth to twelfth primers, 25-150pmol/mL of the universal primers.
In some embodiments, the components of the nucleic acid extraction solution 2 include: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 μm of the third and fourth specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL of the seventeenth to twentieth primers, 25-150pmol/mL of the universal primers.
In some embodiments, the components of the amplification test solution 1.1 include: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of the first primer to the fourth primer.
In some embodiments, the components of the amplification test solution 1.2 include: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL of the universal primer, 143-857pmol/mL of the first target detection probe to the fourth target detection probe.
In some embodiments, the components of the amplification detection solution 2.1 include: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of the thirteenth to sixteenth primers.
In some embodiments, the components of the amplification detection solution 2.2 include: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL of the universal primer, 143-857pmol/mL of the fifth target detection probe to the eighth target detection probe.
In some embodiments, the components of the SAT enzyme solution include: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM of N-acetyl-L-cysteine, 0.04-0.4mM of zinc acetate, 10-100mM of trehalose, 40-200mM of Tris-HCl pH 8.0, 40-200mM of KCl, 0.01-0.5mM of EDTA, 0.1-1% (v/v) of Triton X-100 and 20-50% (v/v) of glycerol.
In another aspect, the invention provides an oligonucleotide combination for detecting 14 high risk HPV types and suitable for use in a real-time fluorescent nucleic acid isothermal amplification detection system, comprising:
(i) A specific capture probe combination comprising: first to fourth specific capture probes respectively comprising the nucleotide sequences shown as SEQ ID NO. 42-SEQ ID NO. 45;
(ii) A primer combination comprising: first to twentieth primers comprising the nucleotide sequences shown as SEQ ID NO. 1 to SEQ ID NO. 20, respectively;
(iii) A universal primer comprising the nucleotide sequence set forth in SEQ ID NO. 21;
(iv) A target detection probe combination comprising: first to eighth target detection probes respectively comprising nucleotide sequences as shown in SEQ ID NO. 22-SEQ ID NO. 29.
In some embodiments, the first to eighth target detection probes each carry a fluorescent reporter group and a quencher group at both ends of the nucleotide sequence, and the fluorescent reporter groups carried by the eighth target detection probes are the same or different.
In yet another aspect, the present invention provides a non-disease diagnostic method for detecting 14 high-risk HPV types, the 14 high-risk HPV types including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, the method comprising the steps of:
1) Sub-packaging a sample to be tested into a tube 1 and a tube 2, adding a nucleic acid extracting solution 1 into the tube 1 for nucleic acid extraction, and adding a nucleic acid extracting solution 2 into the tube 2 for nucleic acid extraction to obtain analysis detection samples 1 and 2 respectively;
2) Adding an amplification detection liquid 1.1 into the analysis detection sample 1 to perform a first-step reaction to obtain a first-step reaction liquid 1, and adding an amplification detection liquid 2.1 into the analysis detection sample 2 to perform a first-step reaction to obtain a first-step reaction liquid 2;
3) Adding SAT enzyme solution into the first-step reaction solution 1 to perform a second-step reaction to obtain a second-step reaction solution 1, and adding SAT enzyme solution into the first-step reaction solution 2 to perform a second-step reaction to obtain a second-step reaction solution 2;
4) Adding an amplification detection liquid 1.2 into the second-step reaction liquid 1 for a third-step reaction to obtain an amplification system 1, and simultaneously performing real-time fluorescence detection to obtain a dt value of the real-time fluorescence detection of the tube 1, and adding an amplification detection liquid 2.2 into the second-step reaction liquid 2 for a third-step reaction to obtain an amplification system 2, and simultaneously performing real-time fluorescence detection to obtain a dt value of the real-time fluorescence detection of the tube 2;
5) Performing result judgment according to the dt value of the real-time fluorescence detection obtained in the step 4);
if the channel corresponding to the amplification system 1 has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected of the tube 1 contains HPV 16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acids; if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be tested of the tube 1 does not contain HPV 16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acids;
if an S-shaped amplification curve appears in a channel corresponding to the amplification system 2 and dt is less than or equal to 35, the sample to be detected of the tube 2 contains HPV18, 35, 45, 59 and 68 genotype nucleic acids; if the "S" type amplification curve does not appear, or the "S" type amplification curve appears, but dt >35, then HPV18, 35, 45, 59, 68 genotype nucleic acids are not contained in the sample to be tested of tube 2.
In some embodiments, the conditions of the first step reaction in step 2) are 40 ℃ to 45 ℃ for 3 to 15 minutes.
In some embodiments, the SAT enzyme solution in step 3) is preheated prior to use at a temperature of 41-43 ℃.
In some embodiments, the conditions of the second reaction step in step 3) are 41 ℃ to 43 ℃ for 30 to 50 minutes.
In some embodiments, the sample to be tested comprises medical samples and non-medical samples of sources including food, blood products, dairy products.
The primers and probes for detecting the real-time fluorescent nucleic acid isothermal amplification of 14 high-risk HPVs (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) based on the technical scheme are obtained based on the optimized design of the 14 high-risk HPV E6/E7 RNA, a section of universal sequence is inserted into a downstream primer, and the universal primer (the sequence of which is the universal sequence) is added into the subsequent step-by-step reaction for detection, so that the inhibition effect in the reaction process can be greatly reduced, and the detection sensitivity is improved. The real-time fluorescent nucleic acid isothermal amplification detection kit provided based on the primer and the probe comprises a nucleic acid extracting solution 1, a nucleic acid extracting solution 2, an amplification detection solution 1.1, an amplification detection solution 1.2, an amplification detection solution 2.1, an amplification detection solution 2.2, a SAT enzyme solution and the like. The nucleic acid extract 1 may contain specific capture probes for capturing the detection sequences of HPV16, 31, 33, 35, 51, 52, 56, 58, 66, the nucleic acid extract 2 may contain specific capture probes for capturing the detection sequences of HPV18, 45, 39, 59, 68, the amplification detection solution 1.1 contains primers for specifically binding to the target sequences of HPV16, 31, 33, 35, 51, 52, 56, 58, 66, the amplification detection solution 1.2 contains target detection probes for respectively detecting HPV16, 31, 33, 35, 51, 52, 56, 58, 66, the amplification detection solution 2.1 contains primers for specifically binding to the target sequences of HPV18, 45, 39, 59, 68, the amplification detection solution 2.2 contains target detection probes for respectively detecting HPV18, 45, 39, 59, 68, and the SAT enzyme solution contains RNA polymerase, reverse transcriptase, and the like necessary for the reaction. Based on SAT isothermal amplification detection technology, through stepwise reaction of the reaction liquid in the use process, mutual interference between different primers and probes can be further avoided, the system reaction is simpler, and high-sensitivity detection of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 can be realized by using only two reaction systems. The invention can be used for detecting non-medical samples, such as food, blood products, dairy products and the like, besides HPV medical samples, and is suitable for being widely popularized and used.
Compared with the existing detection method, the method has the following advantages:
(1) The primer and the probe provided by the invention are obtained by optimizing design according to HPV E6/E7 RNA, a section of general sequence (namely the sequence of the general primer) is inserted into the downstream primer, 14 high-risk HPVs are divided into two tubes for detection, and higher sensitivity detection can be realized by adding different components step by step in the detection process. The results of the examples show that the detection limit of the kit and the method provided by the invention on 14 high-risk HPVs can reach 10 copies/reaction, which is obviously lower than the detection limit (100 copies/reaction) of the method disclosed in the above document 1 and is obviously lower than the detection limit (100 copies/μl, namely 500 copies/reaction) of the method disclosed in the above document 2.
(2) The invention adopts the real-time fluorescent nucleic acid isothermal amplification detection technology to detect 14 high-risk HPV RNAs, and the whole process has no temperature rise and fall and circulation, so compared with the above-mentioned document 1, the invention can avoid the operations of heating, centrifugation, in vitro reverse transcription and the like of extracting RNA in the traditional PCR, has simpler experimental steps and reaction systems, is easy to realize automation, reduces the error caused by personnel operation, reduces the risk of infection of operators, greatly shortens the required time, can improve the detection efficiency, and reduces the design and production cost of the PCR instrument used.
(3) The amplified product of the invention is RNA which is easy to degrade in nature, and compared with the PCR amplified DNA in the document 1, the amplified product has the advantages of easy control of pollution, small cross influence and no environmental pollution.
Drawings
FIG. 1 is a graph showing the detection of HPV16 and HPV18 genotype nucleic acids, respectively, using the 3-lot kit of example 3.
Detailed Description
The present invention will be described in detail with reference to specific embodiments and drawings.
Hereinafter, only certain exemplary embodiments are briefly described. As will be recognized by those of skill in the pertinent art, the described embodiments may be modified in various different ways without departing from the spirit or scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.
The methods used in the examples below are conventional methods unless otherwise specified, and specific steps can be found in: molecular cloning guidelines (Molecular Cloning: ALaboratory Manual) Sambrook, j., russell, david w., molecular Cloning: ALaboratory Manual,3rd edition,2001,NY,Cold Spring Harbor).
The various biomaterials described in the examples were obtained by merely providing a means of experimental acquisition for the purposes of specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.
All primers, specific capture probes, target detection probes and in vitro transcribed RNA products mentioned in the present invention are synthesized using the prior art.
Example 1: special primer and probe design for detecting 14 high-risk HPVs (human papilloma Virus) by real-time fluorescent nucleic acid isothermal amplification
The inventor starts from 14 high-risk type HPV E6/E7 RNA nucleic acid sequences (wherein HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) disclosed in Genbank databases, and designs primers and probe sequences which can simultaneously perform real-time fluorescent nucleic acid isothermal amplification detection on 9 high-risk type HPVs (HPV 16, 31, 33, 35, 51, 52, 56, 58, 66) in the same detection system (referred to herein as "system 1") according to primer and probe design principles, and can simultaneously perform amplification on the high-risk type HPVs in the same detection system (HPV 18, 39, 45, 59, 68) in the same detection system (referred to herein as "system 1"), and insert primers and probe sequences which can simultaneously perform high-sensitivity fluorescent amplification on the high-risk type HPVs in the same detection system (HPV 18, 39, 45, 59, 68) in the same system) in the same detection system (referred to herein as "system 2"), in the same high-risk type HPV 14 ") according to primer and probe design principles.
This example designed multiple sets of downstream primers for each of the 14 high-risk HPV types, two of which are shown in tables 1 and 2 below (the two sets of downstream primers respectively cooperate with the corresponding upstream primers to have high detection sensitivity for the corresponding HPV genotypes when each HPV genotype is detected individually, each up to 1 copy/reaction), and sequence information of specific capture probes, upstream primers, universal primers and target detection probes designed for the 14 high-risk HPV types.
Table 1: primer and probe sequence information in System 1
Table 2: primer and probe sequence information in System 2
In this example, based on the primer and probe sequence information listed in tables 1 and 2 above, various combinations of primers and probes were performed, 4 combinations (group 1 to group 4) were listed for system 1 and system 2, respectively, in table 3 below, and the 14 high-risk HPV positive controls (for in vitro transcription of RNA, described in detail below) and negative controls (without the 14 HPV high-risk target nucleic acid sequences, such as deionized water or sample preservation solution (commercially available)) were used in both systems to perform real-time fluorescent nucleic acid isothermal amplification detection (specific detection methods can be seen in example 4 below) to screen out primer and probe combinations that can be used to detect 14 high-risk HPV types with good sensitivity and specificity, respectively.
Table 3: primer and probe combination information in system 1 and system 2 for detecting 14 high-risk HPVs
The positive control in this example was prepared by the following steps:
(1) E6/E7 RNA fragments of HPVs 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 (corresponding to KP677556.1, KC662604.1, KC662597.1, KC662602.1, GQ465863.1, KT943747.1, KC662608.1, KF436866.1, KY077836.1, KX645763.1, JX896422.1, KU195240.1, JN661482.1, KU195243.1, respectively) were synthesized by chemical synthesis and constructed on a conventional plasmid vector containing a T7 promoter sequence;
(2) RNA fragments were transcribed using a commercial T7 promoter external transcription kit (sigma), and after purification, RNA copy numbers were calculated by UV and in vitro transcribed RNA was used as positive controls, respectively.
The positive control samples (in vitro transcribed RNA gradient concentrations were 10 each) containing each HPV RNA (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) were each subjected to the respective gradient concentration using the primers and probes of each set (set 1 to set 4) shown in Table 3 above 5 cobies/reaction, 10 4 cobies/reaction, 10 3 cobies/reaction, 10 2 cobies/reaction, 10 1 cobies/reaction, 10 0 cobies/reactions; each system contains RNA templates of HPV types) and negative controls. The results are shown in tables 4-7 below, wherein "+" indicates detected and "-" indicates undetected.
Table 4: detection results of primers and probes shown in group 1 respectively aiming at HPV nucleic acids with different concentrations and different types
Table 5: detection results of primers and probes shown in group 2 respectively aiming at HPV nucleic acids with different concentrations and different types
Table 6: detection results of primers and probes shown in group 3 respectively aiming at HPV nucleic acids with different concentrations and different types
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Table 7: the primer and the probe shown in the group 4 are respectively aimed at detection results of HPV nucleic acids with different concentrations and different types
As can be seen from the sensitivity and specificity results of each set of primer probes shown in tables 4-7 above for the detection of 14 high-risk HPV nucleic acids, respectively, different combinations of primers (downstream primers) may have an effect on the sensitivity and specificity results of the detection. Specifically, for 9 HPV detection in system 1, the use of the downstream primers of group 2 and group 3 will detect the target (HPV 39, HPV 68) in system 2, and the use of the downstream primer of group 4 will result in a minimum detection limit of HPV16, 31, 35, 52, 33, 58 of not reaching 10 copies/reaction. For 5 HPV detection in system 2, the use of the downstream primer of group 2 resulted in the lowest detection limit of HPV 45, 39, 68 not reaching 10 copies/reaction, the use of the downstream primer of group 3 detected the target in system 1 (HPV 51), and the use of the downstream primer of group 4 detected the target in system 1 (HPV 16, 31, 35). It can be seen that, of the four sets of primers and probes above, only the specific combination (set 1) has higher sensitivity and specificity when HPV is detected for system 1 and system 2, while the primers and/or probes in other combinations may interfere with each other to affect the sensitivity and specificity of detection. Therefore, the invention determines the primer and probe combination of the group 1 to be the optimal combination for detecting 14 high-risk HPVs and suitable for a real-time fluorescent nucleic acid isothermal amplification detection method.
In the embodiment, the primer probe combinations of the group 1 (the primer probe combinations of the system 1 and the system 2) are used for respectively detecting 5 low-risk type HPV nucleic acids (HPV 6, 11, 42, 43 and 44), and the results are negative, so that the primer probe combinations of the group 1 provided by the invention have better specificity when aiming at the low-risk type HPV detection, and can not obtain non-specific detection results.
In this example, the primer probe combination of group 1 was also used to perform multiplex real-time fluorescent nucleic acid isothermal amplification detection on mixed forms of HPV RNA templates, wherein HPV RNA templates for system 1 were mixed with equal concentrations of HPV16, 31, 33, 35, 51, 52, 56, 58, 66 in vitro transcribed RNA, the final RNA concentration was 10 copies/reaction, and HPV RNA templates for system 2 were mixed with equal concentrations of HPV18, 39, 45, 59, 68 in vitro transcribed RNA, the final RNA concentration was 10 copies/reaction. The results are shown in Table 8 below, where "+" indicates detected and "-" indicates undetected. As can be seen from the results shown in Table 8, even when the primer probe of the group 1 provided by the invention performs multiplex detection on HPV RNA templates in a mixed form, target RNA with a final RNA concentration of 10 copies/reaction can be detected, and the detection specificity is still good.
Table 8: detection results of primers and probes shown in group 1 against HPV RNA templates in mixed form
Example 2: real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPV RNAs
The real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk type HPV RNAs (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) provided in this embodiment is a kit based on the RNA nucleic acid isothermal synchronous amplification detection principle, and specifically may include the following components:
(T1) nucleic acid extract 1: comprising a solid support comprising a first specific capture probe (SEQ ID NO: 42) and a second specific capture probe (SEQ ID NO: 43) for capturing 9 high-risk HPVs, and 9 primers for specifically binding to target sequences of 9 high-risk HPVs, respectively, wherein the 9 high-risk HPVs comprise HPV 16, 31, 33, 35, 51, 52, 56, 58, 66, the 9 primers comprising a fifth primer (SEQ ID NO: 5) for specifically binding to target sequences of HPV 16, a sixth primer (SEQ ID NO: 6) for specifically binding to target sequences of HPV 31, a seventh primer (SEQ ID NO: 7) for specifically binding to target sequences of HPV 33, an eighth primer (SEQ ID NO: 8) for specifically binding to target sequences of HPV 51, a ninth primer (SEQ ID NO: 9) for specifically binding to target sequences of HPV 51, a tenth primer (SEQ ID NO: 10) for specifically binding to sequences of HPV 52, an eleventh primer (SEQ ID NO: 12) for specifically binding to target sequences of HPV 31, an eleventh primer (SEQ ID NO: 8) for binding to target sequences of HPV 35; specifically, the nucleic acid extraction solution 1 may contain: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 mu m of first and second specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL fifth to twelfth primers, 25-150pmol/mL universal primers;
(T2) nucleic acid extract 2: comprising a solid support comprising a third specific capture probe (SEQ ID NO: 44) and a fourth specific capture probe (SEQ ID NO: 45) for capturing 5 high-risk HPVs, and 4 primers for specifically binding to target sequences of 5 high-risk HPVs, respectively, wherein the 5 high-risk HPVs comprise HPV 18, 39, 45, 59, 68, the 4 primers comprising a seventeenth primer (SEQ ID NO: 17) for specifically binding to target sequences of HPV 18, an eighteenth primer (SEQ ID NO: 18) for specifically binding to target sequences of HPV 45, a nineteenth primer (SEQ ID NO: 19) for specifically binding to target sequences of HPV 59, a twentieth primer (SEQ ID NO: 20) for specifically binding to target sequences of HPV 39 and HPV 68, respectively, and a universal primer (SEQ ID NO: 21); specifically, the nucleic acid extraction solution 2 may contain: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 mu m of third and fourth specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL seventeenth to twentieth primers, 25-150pmol/mL universal primers;
(T3) amplification assay 1.1: comprising a first primer (SEQ ID NO: 1), a second primer (SEQ ID NO: 2), a third primer (SEQ ID NO: 3) and a fourth primer (SEQ ID NO: 4), wherein the first primer is complexed with the eleventh primer and the universal primer for amplifying the target sequence of HPV 58, the second primer is complexed with the seventh primer, the tenth primer and the universal primer for amplifying the target sequence of HPV 33 and HPV 52, the third primer is complexed with the twelfth primer and the universal primer for amplifying the target sequence of HPV 56 and HPV 66, and the fourth primer is complexed with the fifth primer, the sixth primer, the eighth primer, the ninth primer and the universal primer for amplifying the target sequences of HPV 16, 31, 35, 51; specifically, the amplification detection solution 1.1 may contain: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of first primer to fourth primer;
(T4) amplification assay 1.2: comprising the universal primer, a first target detection probe (SEQ ID NO: 22), a second target detection probe (SEQ ID NO: 23), and a third target detection probe (SEQ ID NO: 24)And a fourth target detection probe (SEQ ID NO: 25), wherein the first target detection probe specifically binds to an amplified product RNA copy of the target sequence of HPV 51, the second target detection probe specifically binds to an amplified product RNA copy of the target sequence of HPV 16, 31, 35, the third target detection probe specifically binds to an amplified product RNA copy of the target sequence of HPV 33, 52, 58, and the fourth target detection probe specifically binds to an amplified product RNA copy of the target sequence of HPV 56, 66; specifically, the amplification detection solution 1.2 may contain: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL universal primer, 143-857pmol/mL primary target detection probes to fourth target detection probes;
(T5) amplification assay solution 2.1: comprising a thirteenth primer (SEQ ID NO: 13), a fourteenth primer (SEQ ID NO: 14), a fifteenth primer (SEQ ID NO: 15) and a sixteenth primer (SEQ ID NO: 16), wherein the thirteenth primer is complexed with the seventeenth primer and a universal primer for amplifying a target sequence of HPV 18, the fourteenth primer is complexed with the eighteenth primer and the universal primer for amplifying a target sequence of HPV 45, the fifteenth primer is complexed with the nineteenth primer and the universal primer for amplifying a target sequence of HPV 59, and the sixteenth primer is complexed with the twentieth primer and the universal primer for amplifying a target sequence of HPV 39 and HPV 68; specifically, the amplification detection solution 2.1 may contain: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL thirteenth to sixteenth primers;
(T6) amplification assay solution 2.2: comprising the universal primer, a fifth target detection probe (SEQ ID NO: 26), a sixth target detection probe (SEQ ID NO: 27), a seventh target detection probe (SEQ ID NO: 28), and an eighth target detection probe (SEQ ID NO: 29), wherein the fifth target detection probe specifically binds to an amplified product RNA copy of a target sequence of HPV 18, the sixth target detection probe specifically binds to an amplified product RNA copy of a target sequence of HPV 45, and the seventh target detection probe specifically binds to a target sequence of HPV 59The eighth target detection probe specifically binds to amplified product RNA copies of the target sequences of HPV 39 and HPV 68; specifically, the amplification detection solution 2.2 may contain: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL universal primer, 143-857pmol/mL fifth target detection probes to eighth target detection probes;
(T7) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase; specifically, the SAT enzyme solution may comprise: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM of N-acetyl-L-cysteine, 0.04-0.4mM of zinc acetate, 10-100mM of trehalose, 40-200mM of Tris-HCl pH 8.0, 40-200mM of KCl, 0.01-0.5mM of EDTA, 0.1-1% (v/v) of Triton X-100 and 20-50% (v/v) of glycerol.
For ease of detection, this example provides a kit further comprising the following components:
(M1) washing solution: it contains NaCl and SDS; preferably contains 5-50mM HEPES, 50-500mM NaCl, 0.5-1.5% SDS, 1-10mM EDTA; and/or
(M2) mineral oil; and/or
(M3) positive control: E6/E7 in vitro RNA transcription system containing HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 (prepared in example 1); and/or
(M4) negative control: systems that do not contain human papillomavirus target nucleic acids, such as deionized water or sample preservation solutions (which contain high concentrations of detergent and physiological saline).
Example 3: stability of real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPV RNAs
To verify the stability of the kit of the present invention (provided in example 2), 3 sets of different batches of kits were prepared using 3 sets of different batches of raw material reagents in this example, and the resulting kits were used to simultaneously detect HPV positive controls at two concentrations (see example 4 below for specific detection methods). The results obtained showed that the detection results of the 3 batches of kits were very similar, as shown in Table 9 and FIG. 1 (HPV 16, 18 are examples).
Table 9: detection of two-concentration HPV positive control results by three different batches of kit
Example 4: method for detecting 14 high-risk HPV genotypes by real-time fluorescent nucleic acid isothermal amplification
The method of this embodiment is based on the principle of isothermal amplification detection of RNA, and comprises the steps of synchronously detecting HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 by two tubes, and detecting whether the sample contains the 14 high-risk HPV nucleic acids by using the kit provided in the above embodiment 2:
4.1, sample preparation
Taking 2mL of human cervical exfoliated cell sample stored in Hao Luo Jie company ThinPrep PreservCyt Solution, and uniformly mixing with 2mL of sample storage solution (Shanghai Reed degree biotechnology Co., ltd.) in a ratio of 1:1, and taking the mixture as a sample to be tested; 400. Mu.L of sample preservation solution was placed in a sample processing tube as a negative control.
4.2 nucleic acid extraction
(1) Respectively taking 400 mu L of a sample to be tested into a sample processing tube 1 and a sample processing tube 2, adding 100 mu L-800 mu L of a nucleic acid extracting solution 1 into the sample processing tube 1, adding 100 mu L-800 mu L of a nucleic acid extracting solution 2 into the sample processing tube 2, uniformly mixing, preserving heat at 60 ℃ for 10 minutes, and standing at room temperature for 10 minutes;
(2) And placing each sample treatment tube on a magnetic bead separation device, and standing for 3-5 minutes. After the magnetic beads are adsorbed on the tube wall, the sample treatment tube is kept on the magnetic bead separation device, waste liquid is sucked and discarded, and the magnetic beads are reserved. Adding 1mL of washing liquid, shaking uniformly, standing for 3-5 minutes, adding 800 mu L of washing liquid and 200 mu L of mineral oil, shaking uniformly, standing for 3-5 minutes, sucking waste liquid, and retaining magnetic beads;
(3) The sample processing tube is removed from the magnetic bead separation device and the tube is filled with a magnetic bead-nucleic acid complex for use (the beads should be clearly visible in this step).
4.3 SAT amplification assay
(1) Adding 40 mu L of amplification detection solution 1.1 into the sample treatment tube 1 (the magnetic bead-nucleic acid compound in the tube) treated in the step 4.2, adding 40 mu L of amplification detection solution 2.1 into the sample treatment tube 2 (the magnetic bead-nucleic acid compound in the tube), and oscillating to re-suspend the magnetic beads;
(2) Taking 40 mu L of detection liquid 1 (amplification detection liquid 1.1 in a sample treatment tube 1) and detection liquid 2 (amplification detection liquid 2.1 in a sample treatment tube 2) which are uniformly mixed by vibration, respectively adding 50 mu L of mineral oil into a micro-reaction tube, preheating the micro-reaction tube for 5-10min under the reaction condition of 42 ℃, adding 25 mu L of SAT enzyme liquid preheated to 42 ℃, and continuously preserving at the constant temperature of 42 ℃ for 5-10min;
(3) To a micro-reaction tube containing the amplification test solution 1.1 (test solution 1), 35. Mu.L of the amplification test solution 1.2 preheated to 42℃was added, to a micro-reaction tube containing the amplification test solution 2.1 (test solution 2), 35. Mu.L of the amplification test solution 2.2 preheated to 42℃was added, the micro-reaction tube was rapidly transferred to a constant temperature fluorescence detection apparatus, reacted at 42℃for 40 minutes, and the collection of a fluorescence signal every minute was set. Negative controls were processed and tested synchronously as above.
4.4, result determination
And automatically reading the dt value by software according to a curve obtained by the SAT amplification result, and judging the result.
The result judgment criteria were:
if the corresponding channel in the sample processing tube 1 has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected contains HPV16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acid (any one or more); if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be detected does not contain HPV16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acid;
if the corresponding channel in the sample processing tube 2 has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected contains HPV18, 35, 45, 59 and 68 genotype nucleic acid (any one or more); if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be tested does not contain HPV18, 35, 45, 59 and 68 genotype nucleic acids.
By using the method, 500 clinical samples are taken as detection objects, and the kit provided by the invention is compared with an HPV kit of Hao Luo Jie company. Similar to the kit of the invention, the Haoro kit also utilizes TMA isothermal detection technology to detect the 14 high-risk HPV genotypes. The detection of these samples was first completed by the state intellectual property test according to the protocol of the Hao Luo Jie panther kit. The remaining samples were tested by the kit of the invention. The comparison result shows that the positive consistency rate of the detection results of the two kits is 94.4%, the negative consistency rate is 96.5%, and the total consistency rate is 96.0% (table 10), so that the kit provided by the invention has higher accuracy in detecting clinical samples, and is basically equivalent to the detection results of the conventional HPV kit of Hao Luo Jie company.
Table 10: the kit and the Hao Luo Jie HPV kit of the invention take clinical samples as the objects to compare and detect results
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any equivalent substitutions, modifications and the like made within the spirit and principles of the present invention should fall within the present disclosure.

Claims (10)

1. A real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPV types, comprising:
(T1) nucleic acid extract 1: comprising a solid support comprising a first specific capture probe and a second specific capture probe for capturing detection sequences of 9 high-risk HPV types, and 9 primers for specifically binding to target sequences of 9 high-risk HPV types, respectively, wherein the 9 high-risk HPV types comprise HPV 16, 31, 33, 35, 51, 52, 56, 58, 66, the 9 primers comprising a fifth primer for specifically binding to target sequences of HPV 16, a sixth primer for specifically binding to target sequences of HPV 31, a seventh primer for specifically binding to target sequences of HPV 33, an eighth primer for specifically binding to target sequences of HPV 35, a ninth primer for specifically binding to target sequences of HPV 51, an eleventh primer for specifically binding to target sequences of HPV 52, an eleventh primer for specifically binding to target sequences of HPV 58, a twelfth primer for specifically binding to sequences of HPV 56 and HPV 66, and a universal primer;
(T2) nucleic acid extract 2: comprising a solid support comprising a third specific capture probe and a fourth specific capture probe for capturing detection sequences of 5 high-risk HPV types, and 4 primers for specifically binding to target sequences of 5 high-risk HPV types, respectively, wherein the 5 high-risk HPV types comprise HPV 18, 39, 45, 59, 68, the 4 primers comprising a seventeenth primer for specifically binding to target sequences of HPV 18, an eighteenth primer for specifically binding to target sequences of HPV 45, a nineteenth primer for specifically binding to target sequences of HPV 59, a twentieth primer for specifically binding to target sequences of HPV 39 and HPV 68, and one of the universal primers;
(T3) amplification assay 1.1: it comprises a first primer, a second primer, a third primer and a fourth primer, wherein the first primer is matched with the eleventh primer and the universal primer for amplifying the target sequence of HPV 58, the second primer is matched with the seventh primer, the tenth primer and the universal primer for amplifying the target sequence of HPV 33 and HPV 52, the third primer is matched with the twelfth primer and the universal primer for amplifying the target sequence of HPV 56 and HPV 66, and the fourth primer is matched with the fifth primer, the sixth primer, the eighth primer, the ninth primer and the universal primer for amplifying the target sequences of HPV 16, 31, 35 and 51 respectively;
(T4) amplification assay 1.2: comprising the universal primer, a first target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 51, a second target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 16, 31, 35, a third target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 33, 52, 58, and a fourth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 56, 66;
(T5) amplification assay solution 2.1: comprising a thirteenth primer, a fourteenth primer, a fifteenth primer and a sixteenth primer, wherein the thirteenth primer is complexed with the seventeenth primer and a universal primer for amplifying a target sequence of HPV 18, the fourteenth primer is complexed with the eighteenth primer and a universal primer for amplifying a target sequence of HPV 45, the fifteenth primer is complexed with the nineteenth primer and a universal primer for amplifying a target sequence of HPV 59, and the sixteenth primer is complexed with the twentieth primer and a universal primer for amplifying a target sequence of HPV 39 and HPV 68;
(T6) amplification assay solution 2.2: comprising the universal primer, a fifth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 18, a sixth target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 45, a seventh target detection probe that specifically binds to an amplified product RNA copy of a target sequence of HPV 59, and an eighth target detection probe that specifically binds to amplified product RNA copies of target sequences of HPV 39 and HPV 68;
(T7) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase;
wherein:
the first specific capture probe to the fourth specific capture probe respectively comprise nucleotide sequences shown as SEQ ID NO. 42-SEQ ID NO. 45;
the first primer to the twentieth primer respectively comprise nucleotide sequences shown as SEQ ID NO. 1-SEQ ID NO. 20;
the universal primer comprises a nucleotide sequence shown as SEQ ID NO. 21; and the sequences of the 5' end of the fifth primer to the twelfth primer and the seventeenth primer to the twentieth primer are the sequences of the universal primers;
The first target detection probe to the eighth target detection probe respectively comprise nucleotide sequences shown as SEQ ID NO. 22-SEQ ID NO. 29.
2. The kit for isothermal amplification detection of real-time fluorescent nucleic acids according to claim 1, wherein fluorescent reporter groups and quenching groups are respectively carried at both ends of the nucleotide sequences of the first target detection probe to the eighth target detection probe, and the fluorescent reporter groups carried by the eighth target detection probe are the same or different.
3. The real-time fluorescent nucleic acid isothermal amplification detection kit according to claim 1 or 2, further comprising:
(M1) washing solution: it contains NaCl and SDS; preferably contains 5-50mM HEPES, 50-500mM NaCl, 0.5-1.5% SDS, 1-10mM EDTA; and/or
(M2) mineral oil; and/or
(M3) positive control: a system for E6/E7 in vitro transcription of RNA comprising HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68; and/or
(M4) negative control: a system that does not contain human papillomavirus target nucleic acid.
4. The kit for real-time fluorescent nucleic acid isothermal amplification detection according to any one of claims 1-3,
the nucleic acid extraction solution 1 comprises the following components: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 μΜ of the first and second specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL of the fifth to twelfth primers, 25-150pmol/mL of the universal primers;
The nucleic acid extraction solution 2 comprises the following components: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 μΜ of the third and fourth specific capture probes, 50-500mg/L magnetic beads, 25-150pmol/mL of the seventeenth to twentieth primers, 25-150pmol/mL of the universal primers;
the components of the amplification detection liquid 1.1 comprise: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of the first primer to the fourth primer;
the components of the amplification detection liquid 1.2 comprise: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL of the universal primer, 143-857pmol/mL of the first target detection probe to fourth target detection probe;
the components of the amplification detection liquid 2.1 comprise: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of the thirteenth primer to sixteenth primer;
the components of the amplification detection liquid 2.2 comprise: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 143-857pmol/mL of the universal primer, 143-857pmol/mL of the fifth target detection probe through eighth target detection probe;
The SAT enzyme solution comprises the following components: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM of N-acetyl-L-cysteine, 0.04-0.4mM of zinc acetate, 10-100mM of trehalose, 40-200mM of Tris-HCl pH 8.0, 40-200mM of KCl, 0.01-0.5mM of EDTA, 0.1-1% (v/v) of Triton X-100 and 20-50% (v/v) of glycerol.
5. An oligonucleotide combination for detecting 14 high-risk HPV types and suitable for use in a real-time fluorescent nucleic acid isothermal amplification detection system, comprising:
(i) A specific capture probe combination comprising: first to fourth specific capture probes respectively comprising the nucleotide sequences shown as SEQ ID NO. 42-SEQ ID NO. 45;
(ii) A primer combination comprising: first to twentieth primers comprising the nucleotide sequences shown as SEQ ID NO. 1 to SEQ ID NO. 20, respectively;
(iii) A universal primer comprising the nucleotide sequence set forth in SEQ ID NO. 21;
(iv) A target detection probe combination comprising: first to eighth target detection probes respectively comprising nucleotide sequences as shown in SEQ ID NO. 22-SEQ ID NO. 29.
6. The oligonucleotide combination according to claim 5, wherein a fluorescent reporter group and a quencher group are carried at both ends of the nucleotide sequence of the first target detection probe to the eighth target detection probe, respectively, and the fluorescent reporter groups carried by the eighth target detection probe are the same or different.
7. A non-disease diagnostic method for detecting 14 high-risk HPV types, the 14 high-risk HPV types comprising HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, the method comprising the steps of:
1) Sub-packaging a sample to be tested into a tube 1 and a tube 2, adding a nucleic acid extracting solution 1 into the tube 1 for nucleic acid extraction, and adding a nucleic acid extracting solution 2 into the tube 2 for nucleic acid extraction to obtain analysis detection samples 1 and 2 respectively;
2) Adding an amplification detection liquid 1.1 into the analysis detection sample 1 to perform a first-step reaction to obtain a first-step reaction liquid 1, and adding an amplification detection liquid 2.1 into the analysis detection sample 2 to perform a first-step reaction to obtain a first-step reaction liquid 2;
3) Adding SAT enzyme solution into the first-step reaction solution 1 to perform a second-step reaction to obtain a second-step reaction solution 1, and adding SAT enzyme solution into the first-step reaction solution 2 to perform a second-step reaction to obtain a second-step reaction solution 2;
4) Adding an amplification detection liquid 1.2 into the second-step reaction liquid 1 for a third-step reaction to obtain an amplification system 1, and simultaneously performing real-time fluorescence detection to obtain a dt value of the real-time fluorescence detection of the tube 1, and adding an amplification detection liquid 2.2 into the second-step reaction liquid 2 for a third-step reaction to obtain an amplification system 2, and simultaneously performing real-time fluorescence detection to obtain a dt value of the real-time fluorescence detection of the tube 2;
5) Performing result judgment according to the dt value of the real-time fluorescence detection obtained in the step 4);
if the channel corresponding to the amplification system 1 has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected of the tube 1 contains HPV 16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acids; if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be tested of the tube 1 does not contain HPV 16, 31, 33, 35, 51, 52, 56, 58 and 66 genotype nucleic acids;
if an S-shaped amplification curve appears in a channel corresponding to the amplification system 2 and dt is less than or equal to 35, the sample to be detected of the tube 2 contains HPV18, 35, 45, 59 and 68 genotype nucleic acids; if the "S" type amplification curve does not appear, or the "S" type amplification curve appears, but dt >35, then HPV18, 35, 45, 59, 68 genotype nucleic acids are not contained in the sample to be tested of tube 2.
8. The method according to claim 7, wherein the conditions of the first reaction in step 2) are 40 ℃ to 45 ℃ for 3 to 15min.
9. The method according to claim 7 or 8, wherein the SAT enzyme solution in step 3) is preheated before use at a temperature of 41-43 ℃; and/or
The condition of the second step reaction in the step 3) is 41-43 ℃ for 30-50min.
10. The method according to any of claims 7-9, wherein the sample to be tested comprises medical samples and non-medical samples of sources including food, blood products, dairy products.
CN202311031974.1A 2023-08-16 2023-08-16 Real-time fluorescent nucleic acid isothermal amplification detection kit for detecting 14 high-risk HPVs Pending CN116855642A (en)

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