CN114480728A - LAMP primer group and kit for bedside rapid detection of HPV (human papilloma Virus) and using method thereof - Google Patents
LAMP primer group and kit for bedside rapid detection of HPV (human papilloma Virus) and using method thereof Download PDFInfo
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Abstract
The invention discloses an LAMP primer group for bedside rapid detection of HPV, a kit and a use method thereof, and belongs to the technical field of biological detection. Firstly, an LAMP primer group for detection is provided, so that the detection specificity, sensitivity and the like can be effectively ensured; secondly, the method is applied to the preparation of an HPV detection kit or reagent, and DNA extraction and amplification are combined into a one-step method through the optimization of the optimal proportion of system solution components, so that the LAMP reaction time is shortened, the total reaction time is shortened to be within 40min, and meanwhile, higher sensitivity and excellent specificity are displayed; finally, aiming at the kit, a using method of the kit for rapidly detecting HPV at the bedside is provided, and the using method specifically comprises sample collection, cracking, amplification, discrimination and the like. In addition, in view of the environmental pollution problem, the inventor also designs a closed waste liquid collecting cavity in particular to achieve the aim of eliminating pollution.
Description
Technical Field
The invention relates to a primer group and a kit for HPV detection and a using method thereof, in particular to an LAMP primer group and a kit for rapid detection of HPV at bedside and a using method thereof, belonging to the technical field of biological detection.
Background
Human Papilloma Virus (HPV) is a papilloma vacuole DNA virus belonging to papovaviridae, can be widely spread in human bodies, and can cause neoplasms of skin mucosa, condyloma acuminatum, cervical cancer and the like due to continuous infection, so that the rapid and accurate detection of HPV has an extremely important significance for reducing the incidence rate of cervical cancer. Conventional HPV detection methods include: cell histopathological detection, colposcopic detection, electron microscopy detection, cervical fluoroscopy and the like, and the detection methods have obvious defects: low sensitivity, poor specificity, and the inability to type HPV, leading to clinical routine use. Currently, the clinical conventional HPV detection methods are mainly: real-time fluorescence PCR, hybrid capture-chemiluminescence, PCR capillary electrophoresis fragment analysis and the like, but have the defects of needing professional equipment and professional personnel for operation, being long in time consumption and the like.
The prior art "CN 110607399A, primer combination, kit and method for LAMP amplification to detect HPV and typing" discloses LAMP amplification primer sets comprising HPV16E6, HPV18E6, HPV31E6, HPV33E7, HPV35E6, HPV39E6, HPV45E7, HPV51E6E7, HPV52E6E7, HPV56E6, HPV58E6, HPV59E6, HPV66E6, HPV68E6 and HPV73E6E7, and the above primer combination is used to detect HPV subtypes, but the method has the following disadvantages: (1) DNA extraction is needed, and the time is more than 2 hours; (2) LAMP amplification reaction for 1 hour.
In addition, only HPV16 and HPV18 were detected in "the reaction system and method for detecting HPV16 and 18 based on LAMP and molecular beacon" by CN104805218A, and the following disadvantages existed in this method: (1) the reaction system contains a molecular beacon probe, and the price is higher; (2) the time is long, more than 60 minutes; (3) after LAMP reaction, the fluorescence needs to be observed under the conditions of 4 ℃, 65 ℃ and 95 ℃ by ultraviolet LAMP irradiation, the operation is complicated, and professional equipment is needed: an ultraviolet lamp; the detection time of the CN106148571A reagent kit for detecting human papillomavirus HPV16 and HPV18 is longer, about 1 hour, and an ultraviolet lamp is needed; the CN106939359A primer group and the detection system for detecting the common HPV subtypes by the LAMP method detect HPV6, HPV11, HPV16, HPV42, HPV43 and HPV44, and the related detection method takes longer time, which is more than 1 hour; special equipment is required, for example: a fluorescent quantitative PCR instrument, a vortex mixer, a vibration type constant temperature metal bath, a high-speed low-temperature centrifuge, an electrophoresis instrument, a gel imaging analyzer, a flow lattice instrument, a real-time turbidity tester, an electronic analysis balance, an ultrapure water instrument and the like; the LAMP primer combination and application of CN110093459A for rapidly detecting 13 high-risk HPV takes more than 1 hour, and an S-type curve is drawn by detecting turbidity with a turbidity meter in the reaction process.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an LAMP primer group for rapidly detecting HPV at bedside, a kit and a using method thereof. The inventor develops a large amount of research and development work aiming at a sample LAMP primer group, a lysate and a reaction system, screens the LAMP primer group with strong specificity, optimizes the optimal proportion of the solution components of the system, combines DNA extraction and amplification into a one-step method, shortens the LAMP reaction time, shortens the total reaction time to be within 40min, and simultaneously shows high sensitivity and excellent specificity. In addition, in view of the environmental pollution problem, the inventor also designs a closed waste liquid collecting cavity in particular to achieve the aim of eliminating pollution.
In order to achieve the technical purpose, the following technical scheme is proposed:
the technical scheme provides that: an LAMP primer group for rapidly detecting HPV at bedside comprises the following nucleotide sequence pair combinations:
further, the HPV includes subtypes HPV16, HPV18, HPV33, HPV52, HPV56, HPV58 and HPV 68.
The technical scheme provides that: and (3) applying the LAMP primer group to the preparation of HPV detection reagent.
The technical scheme provides that: and (3) applying the LAMP primer group to the preparation of the HPV detection kit.
The technical scheme provides that: a kit for rapidly detecting HPV beside a bed comprises the LAMP primer group, a lysate and an LAMP amplification system,
the total content of the LAMP primer group is 1-5 mu M;
the lysis solution comprises NaOH, Tris-HCl, SDS, Triton-X100, NP-40 and urea, wherein the NaOH is 5-20mM, the Tris-HCl is 1-5mM, the SDS is 0.05-1%, the Triton-X100 is 0.1-2%, the NP-40 is 1-10%, the urea is 2-10mM, and the total volume of the lysis solution is 50 mu L;
the LAMP amplification system comprises: MgSO (MgSO)4、KCl、（NH4）2SO4Tween-20, betain, dNTPs, Bst3.0 amplidase and EVAgreen, wherein MgSO45-10mM, KCl 25-125mM, (NH)4）2SO40-40mM, Tween-20 0.01-0.5%, and betain0.1-0.25M, 1-5 μ M dNTPs, 0.2-1.0U Bst3.0 amplification enzyme, 0.5-1X EVAgreen and 25 μ L of total volume of the reaction system.
Preferably, the LAMP primer group comprises an inner primer with the concentration of 1.6-2.4 mu M, an outer primer with the concentration of 0.1-0.4 mu M and a loop primer with the concentration of 0.4-0.8 mu M.
Preferably, the lysate comprises 5-10mM NaOH, 1-2mM Tris-HCl, 0.05-0.1% SDS, 0.5-1% Triton-X100, 1-5% NP-40, and 4-6mM urea.
Preferably, the LAMP amplification system comprises 6-7mM MgSO4KCl of 75-125mM, and (NH) of 10-20mM4）2SO40.1-0.4% Tween-20, 0.1-0.25M betain, 1-5. mu.M dNTPs, 0.5-1X EVAgreen and 0.2-1.0U Bst3.0 amplilase.
The technical scheme also provides a using method of the kit for rapidly detecting HPV beside a bed, which comprises the following steps:
1) collecting samples: sample collection using a brush head including but not limited to a vaginal swab or vaginal cytology;
2) cracking: placing the in vitro sample in a sample adding chamber of a microfluidic nucleic acid detection device, and mixing with the prefabricated sample lysate for 10min to obtain lysate;
wherein, the sample lysate comprises NaOH (5-20 mM), Tris-HCl (1-5 mM), Triton-X100 (0.1-2%), NP-40 (1-10%), SDS (0.05-1%) and urea (2-10 mM);
3) amplification: the obtained lysate enters an amplification chamber of a microfluidic nucleic acid detection device, a heating element is turned on, heating is carried out for 30min at 65 ℃, and LAMP amplification is carried out;
wherein the reaction system comprises MgSO4（5-10mM）、KCl （25-125mM）、（NH4）2SO4(0-40 mM), Tween-20 (0.01-0.5%), betain (0.1-0.25M), dNTPs (1-5. mu.M), Bst3.0 amplification enzyme (0.2-1.0U), EVAgreen (0.5-1X) and amplification primers 1-5. mu.M;
4) and (3) discrimination: judging the color of the amplified reaction solution by naked eyes through a transparent observation window on the microfluidic nucleic acid detection device, and determining the reaction solution to be negative if the reaction solution is red; if the reaction solution is orange, the reaction solution is positive;
5) and (3) post-treatment: and (3) introducing the generated waste liquid into a waste liquid collecting chamber of the microfluidic nucleic acid detection device, and then treating the waste liquid with medical waste garbage.
Furthermore, the microfluidic nucleic acid detection device comprises a shell which is arranged in a sealing manner, and a sample adding chamber, an amplification chamber and a waste liquid collecting chamber which are arranged in the shell, wherein the sample adding chamber is arranged above the amplification chamber, the waste liquid collecting chamber is arranged below the amplification chamber, and a passage for unidirectional solution flow can be formed between the sample adding chamber amplification chamber and the waste liquid collecting chamber.
In this technical solution, the terms referred to include:
lamp amplification: loop-mediated isothermal amplification reaction.
In the technical scheme, F3 represents a forward outer primer, B3 represents a reverse outer primer, FIP represents a forward inner primer, BIP represents a reverse inner primer, and LB and LF represent loop primers.
In the technical scheme, the mechanism is as follows:
the sample adding hole in the sample adding chamber contains cell lysate, and after the cell lysate is mixed with a sample, the cell lysate can be cleaved and DNA is released; after entering an amplification chamber, the lysate with DNA is mixed with a prefabricated amplification reaction system to carry out LAMP reaction; after the amplification is finished, the color of the phenol red can be changed by the change of the pH value, and the change of the color is visually distinguished through a transparent observation window: red is negative and orange is positive.
By adopting the technical scheme, the beneficial technical effects brought are as follows:
firstly, in the invention, the DNA does not need to be extracted and purified separately, and the operation of DNA extraction and amplification reaction is a one-step method and can be completed in the same chamber. Based on the kit, the HPV detection operation is simple and convenient, the result is understandable, reading by professional personnel is not needed, and professional training is not needed;
secondly, the LAMP primers are designed to have stronger specificity, the LAMP reaction time is effectively shortened by optimizing the optimal proportion of the solution components of the system and the like, the detection is ensured to be completed within 40min, and meanwhile, higher sensitivity and excellent specificity are displayed;
thirdly, in the invention, the microfluidic nucleic acid detection device is adopted to carry out isothermal amplification without other professional equipment. In addition, the amplification reaction is completed in an independent chamber (amplification chamber), so that aerosol is not easy to form and pollution is not easy to occur. The generated waste liquid finally enters a waste liquid collecting cavity, is totally enclosed and has no environmental pollution;
in the invention, the detection system only needs LAMP primers, amplification enzymes and reaction buffer solution, and other reagents such as probes and the like are not needed, so that the cost is effectively reduced (from hundreds of yuan to tens of yuan);
in the invention, each link is simple and convenient to operate, other special instruments (such as a fluorescent quantitative PCR instrument, a vortex mixer, an ultraviolet lamp and the like) are not needed, the medical treatment process is effectively optimized, the bedside rapid self-test can be basically realized, and great convenience is brought to medical care personnel, family members and the like.
Drawings
FIG. 1 shows the result of HPV16 sensitivity detection in example 7;
FIG. 2 shows the result of HPV18 sensitivity detection in example 7.
Detailed Description
The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.
Example 1
The present embodiment proposes: an LAMP primer group for rapidly detecting HPV at bedside comprises the following nucleotide sequence pair combinations:
among them, HPV concerns include subtypes HPV16, HPV18, HPV33, HPV52, HPV56, HPV58, and HPV 68.
Example 2
Based on embodiment 1, this embodiment proposes: the LAMP primer group is applied to the preparation of the HPV detection reagent.
Example 3
Based on embodiment 1, this embodiment proposes: the LAMP primer group is applied to the preparation of the HPV detection kit.
Example 4
Based on example 1 and example 3, this example proposes: a kit for rapidly detecting HPV beside a bed comprises an LAMP primer group, a lysate and an LAMP amplification system,
the amplification primer is 1-5 mu M;
the lysis solution comprises NaOH, Tris-HCl, SDS, Triton-X100, NP-40 and urea, wherein the NaOH is 5-20mM, the Tris-HCl is 1-5mM, the SDS is 0.05-1%, the Triton-X100 is 0.1-2%, the NP-40 is 1-10%, the urea is 2-10mM, and the total volume of the lysis solution is 50 mu L;
the LAMP amplification system comprises: MgSO (MgSO)4、KCl、（NH4）2SO4Tween-20, betain, dNTPs, Bst3.0 amplidase and EVAgreen, wherein MgSO45-10mM, KCl 25-125mM, (NH)4）2SO40-40mM, 0.01-0.5% Tween-20, 0.1-0.25M beta-ain, 1-5 μ M dNTPs, 0.2-1.0U Bst3.0 amplification enzyme, 0.5-1X EVAgreen and 25 μ L of total volume of the reaction system.
Example 5
Based on embodiment 4, this embodiment preferably proposes:
the LAMP primer group comprises an inner primer with the concentration of 1.6-2.4 MuM, an outer primer with the concentration of 0.1-0.4 MuM and a loop primer with the concentration of 0.4-0.8 MuM;
the lysate comprises 5-10mM NaOH, 1-2mM Tris-HCl, 0.05-0.1% SDS, 0.5-1% Triton-X100, 1-5% NP-40 and 4-6mM urea;
the LAMP amplification system comprises 6-7mM MgSO4KCl of 75-125mM, and (NH) of 10-20mM4）2SO40.1-0.4% Tween-20, 0.1-0.25M betain, 1-5. mu.M dNTPs, 0.5-1X EVAgreen and 0.2-1.0U Bst3.0 amplilase.
Example 6
Based on example 5, this example proposes a method for using a kit for rapid bedside detection of HPV, which specifically includes the following steps:
1) collecting samples: sample collection using a brush head including but not limited to a vaginal swab or vaginal cytology;
2) cracking: placing the in vitro sample in a sample adding chamber of a microfluidic nucleic acid detection device, and mixing with the prefabricated sample lysate for 10min to obtain lysate;
wherein, the sample lysate comprises NaOH (5-20 mM), Tris-HCl (1-5 mM), Triton-X100 (0.1-2%), NP-40 (1-10%), SDS (0.05-1%) and urea (2-10 mM);
3) amplification: the obtained lysate enters an amplification chamber of a microfluidic nucleic acid detection device, a heating element is turned on, heating is carried out for 30min at 65 ℃, and LAMP amplification is carried out;
wherein the reaction system comprises MgSO4（5-10mM）、KCl （25-125mM）、（NH4）2SO4(0-40 mM), Tween-20 (0.01-0.5%), betain (0.1-0.25M), dNTPs (1-5. mu.M), Bst3.0 amplification enzyme (0.2-1.0U), EVAgreen (0.5-1X) and amplification primers 1-5. mu.M;
4) and (3) discrimination: judging the color of the amplified reaction solution by naked eyes through a transparent observation window on the microfluidic nucleic acid detection device, and determining the reaction solution to be negative if the reaction solution is red; if the reaction solution is orange, the reaction solution is positive;
5) and (3) post-treatment: and (3) introducing the generated waste liquid into a waste liquid collecting chamber of the microfluidic nucleic acid detection device, and then treating the waste liquid with medical waste garbage.
The microfluidic nucleic acid detection device comprises a shell, a sample adding chamber, an amplification chamber and a waste liquid collecting chamber, wherein the shell is arranged in a sealing mode, the sample adding chamber, the amplification chamber and the waste liquid collecting chamber are arranged in the shell, the sample adding chamber is arranged above the amplification chamber, the waste liquid collecting chamber is arranged below the amplification chamber, and a path for unidirectional solution flow can be formed between the sample adding chamber amplification chamber and the waste liquid collecting chamber.
Example 7
Based on example 4, this example investigated the sensitivity of the detection of the kit.
Cloning the sequences of HPV16, HPV18, HPV33, HPV52, HPV56, HPV58 and HPV68 into the PUC57 vector respectivelyThe plasmid is used as a template, and digital PCR is used for accurate quantification. Are respectively provided with 108Copy/. mu.L, 107Copy/. mu.L, 106Copy/. mu.L, 105Copy/. mu.L, 104The LAMP amplification was carried out separately using copies/. mu.L as templates, and the results showed that: the detection limit of the detection method is 104Copies/. mu.L.
The results of amplification charts corresponding to HPV16 and HPV18 are shown in FIGS. 1 and 2.
Example 8
Based on example 4, this example uses the specificity of the cross-validation kit detection.
The following HPV plasmid templates were synthesized: HPV16, HPV18, HPV31, HPV33, HPV42, HPV51, HPV52, HPV53, HPV56, HPV58, HPV6, HPV66 and HPV68 were subjected to LAMP amplification as in example 6, and the results are shown in tables 1 to 7: HPV16, HPV18, HPV33, HPV52, HPV56, HPV58 and HPV68 have no cross reaction with other types of HPV, and the specification shows that the specificity is good.
Example 9
Based on the present embodiment 4 and the embodiment 6, 79 collected examples of the cervical part cytology brush heads of the second hospital in western university of sichuan are used in the present embodiment, wherein 55 examples of HPV positives (12 examples of HPV16 positives, 2 examples of HPV18 positives, 10 examples of HPV33 positives, 12 examples of HPV52 positives, 4 examples of HPV56 positives, 13 examples of HPV58 positives and 2 examples of HPV68 positives) and 24 examples of HPV negatives are used to further explain the present invention.
1. 79 cases of cervical part cytology brush heads of a second hospital in western China, Sichuan university are collected;
2. placing the cytology brush head in a sample adding chamber of a portable microfluidic nucleic acid detection device, and mixing the cytology brush head with a prefabricated sample lysate for 10min, wherein the lysate contains the following components: NaOH (5-10 mM), Tris-HCl (1-2 mM), Triton-X100 (0.5-1%), NP-40 (1-5%), SDS (0.05-0.1%), and urea (4-6 mM);
3. the lysate enters an amplification chamber, a heating element is turned on, and the lysate is heated for 30min at 65 ℃ to carry out LAMP amplification;
wherein, the reaction system is as follows: MgSO (MgSO)4（6-7mM）、KCl（75-125mM）、（NH4）2SO4(10-20 mM), Tween-20 (0.1-0.4%), betain (0.1-0.25M), dNTPs (1-5. mu.M), Bst3.0 amplification enzyme (0.2-1.0U), EVAgreen (0.5-1X);
LAMP amplification primers were as follows:
HPV16, forward outer primer: AGACGTTATGACATACATACATTC the flow of the air in the air conditioner,
reverse outer primer: GTCTGCAGAAAACTTTTCCTT the flow of the air in the air conditioner,
forward inner primer:
TTCTAGTGCCTCCTGGGGGAATTCCACTATTTTGGAGGAC，
reverse inner primer: TAACCCAGGCAATTGCTTGTCAAGTGTATTTTTTAAGGGGATCA the flow of the air in the air conditioner,
loop primer: AAAAACATACACCTCCAGCACCTA, respectively;
HPV18, forward outer primer: CACTGAAAGTTCCCATGC the flow of the air in the air conditioner,
reverse outer primer: CAGTATCTACCATATCACCATCT the flow of the air in the air conditioner,
forward inner primer: GCCCAAAATACATAACTGTGTCTGCACGTCTAATGTTTCTGAGGAC the flow of the air in the air conditioner,
reverse inner primer:
GGCTAAAGGCACTGCTTGTAACCAAAACTGTGTTTTTAAGTTCT，
loop primer: GCGTCCTTTATCACAGGGCGATT, respectively;
HPV33, forward outer primer: CTGAGGAAAAACCACGAAC the flow of the air in the air conditioner,
reverse outer primer: ATAAGAACCGCAAACACAG the flow of the air in the air conditioner,
forward inner primer: CCACGCACTGTAGTTCAATGTTATTGCATGATTTGTGCCAA the flow of the air in the air conditioner,
reverse inner primer: TGCAACGATCTGAGGTATATGATTTTCCAAATGGATTTCCCTCTC the flow of the air in the air conditioner,
loop primer: TCTCCAATGCTTGGCACAAA, respectively;
HPV52, forward outer primer: ATAAGGCTGCAGTGTGTG the flow of the air in the air conditioner,
reverse outer primer: CACTTAATGGTTTTTTTACCCTC the flow of the air in the air conditioner,
forward inner primer: CACGCCATATGGATTATTGTCTCTAAGCTACAACGAAGAGAGGT the flow of the air in the air conditioner,
reverse inner primer: ATGTGCCTACGCTTTTTATCTAAGACTTCTAATGTTTTCCCATACAGT, respectively;
HPV56, forward outer primer: GCCTGTGTATTTTTTTAGACGT the flow of the air in the air conditioner,
reverse outer primer: TTTTGGTATTGTCCTTAGTCAC the flow of the air in the air conditioner,
forward inner primer: CTTTGAAACAGGTGTTGGAGGTAGATATTCCCTATTTTTTTGCAGATGG the flow of the air in the air conditioner,
reverse inner primer: CAACGGATTCCTATGTGAAACGCGTAATAGGGATGTCCTACGG the flow of the air in the air conditioner,
forward loop primer: TCACTAGGCCGCCACGT the flow of the air in the air conditioner,
reverse loop primer: TATTTTATCATGCAGGCAGTTCACG, respectively;
HPV58, forward outer primer: ACATTGCATGATTTGTGTCAG the flow of the air in the air conditioner,
reverse outer primer: TGTGTCTCCATATAGCGAAT the flow of the air in the air conditioner,
forward inner primer: GCTGCAAAGTCTTTTTGCATTCAAGCGTTGGAGACATCTGTG the flow of the air in the air conditioner,
reverse inner primer: ATAGAGATGGAAATCCATTTGCAGTCTCACTTATTTTAGATAGCAATCGT, respectively;
HPV68, forward outer primer: TCCTACACAACGACCATAC the flow of the air in the air conditioner,
reverse outer primer: ATGCATTTCAGACACGCT the flow of the air in the air conditioner,
forward inner primer: GCAAAACACACAATTGATGCGAATAACTGCCTGATTTGAGCAC the flow of the air in the air conditioner,
reverse inner primer: AAGGGGAACTGCAAGAAAGAGAGCATACGGTGTACAGTCTC, respectively;
4. through a transparent observation window on the microfluidic nucleic acid detection device, distinguishing by naked eyes: the reaction solution is red, and is negative; the reaction solution was orange, and was positive. The results are shown in tables 8-9, and 51 of the 55 positive samples were judged to be positive; all 24 negative samples were judged negative.
5. And (3) introducing the generated waste liquid into a waste liquid collecting chamber of the microfluidic nucleic acid detection device, and then treating the waste liquid with medical waste garbage.
Example 10
Based on this example 4 and example 6, this example discusses the concentrations of components in the lysate to further illustrate the present invention.
First, discussion of NaOH concentration in lysate
Different NaOH concentration gradients are set, the effect of the lysate is detected by qPCR amplification, the optimal NaOH concentration is judged according to the positive Ct value and the negative Ct value, and the obtained results are shown in the following table 10.
To obtain: based on the above results, 5-10mM was the optimum NaOH concentration.
Second, discussion of Tris-HCl concentration in lysate
Different Tris-HCl concentration gradients are set, the effect of the lysate is detected by qPCR amplification, the optimal Tris-HCl concentration is judged according to the positive Ct value and the negative Ct value, and the obtained results are shown in the following table 11.
To obtain: according to the above results, 1-2mM was the optimum Tris-HCl concentration.
Third, SDS concentration discussion in lysate
Different SDS concentration gradients are set, qPCR amplification is carried out to detect the effect of the lysate, the optimal SDS concentration is judged according to the positive Ct value and the negative Ct value, and the obtained results are shown in the following table 12.
To obtain: from the above results, 0.05-0.1% was the optimum SDS concentration.
Fourth, discussion of Triton-X100 concentration in lysate
Different Triton-X100 concentration gradients are set, the lysis solution effect is detected by qPCR amplification, the optimal Triton-X100 concentration is judged according to the positive Ct value and the negative Ct value, and the obtained results are shown in the following table 13.
To obtain: according to the above results, 0.5-1% is the optimum Triton-X100 concentration.
Fifth, discussion of NP-40 concentration in lysates
Different NP-40 concentration gradients are set, the effect of the lysate is detected by qPCR amplification, the optimal NP-40 concentration is judged according to a positive Ct value and a negative Ct value, and the obtained results are shown in the following table 14.
To obtain: based on the above results, 1-5% is the optimal NP-40 concentration.
Sixthly, discussion of urea concentration in cracking liquid
Different urea concentration gradients are set, the effect of the lysis solution is detected by qPCR amplification, the optimal urea concentration is judged according to the positive Ct value and the negative Ct value, and the obtained results are shown in the following table 15.
To obtain: according to the above results, 4-6mM was the optimum urea concentration.
Example 11
Based on this example 4 and example 6, this example is discussed with respect to the concentration of components in the LAMP amplification system to further illustrate the present invention.
The components in the LAMP amplification system comprise MgSO4、KCl、（NH4）2SO4Tween-20, betain, dNTPs, Bst3.0 amplification enzyme, Evagreen and primers, and screening the reagent concentration with larger influence on amplification due to concentration change; the color indicator of the reaction system is phenol red.
MgSO in LAMP amplification System4Discussion of concentration
Setting different MgSO4The concentration gradient is amplified under a self-matched pH-LAMP reaction system, the optimal magnesium sulfate concentration is judged according to the positive Ct value and the negative Ct value, and the obtained results are shown in the following table 16.
To obtain: total Mg2+The higher the concentration, the more likely non-specific amplification will occur, so that lower Mg concentrations may be selected when the amplification efficiency is not too different2+Concentration; comprehensively considering amplification efficiency and non-specific amplification, selecting optimal Mg2+The concentration is 6-7 mM.
Second, the discussion of KCl concentration in LAMP amplification system
Different KCl concentration gradients are set, amplification is carried out under a self-matched pH-LAMP reaction system, the optimal KCl concentration is judged according to the positive Ct value and the negative Ct value, and the obtained results are shown in the following table 17.
To obtain: the optimal KCl concentration is selected to be 75-125mM by comprehensively considering the amplification efficiency and non-specific amplification.
Third, in LAMP amplification System (NH 4)2SO4Discussion of concentration
Settings are different (NH 4)2SO4Concentration gradient, amplification under self-contained pH-LAMP reaction system, and optimal judgment (NH 4) according to positive Ct value and negative Ct value2SO4The results of concentration are shown in Table 18 below.
To obtain: selecting the best (NH 4) by considering the amplification efficiency and the non-specific amplification2SO4The concentration is 10-20 mM.
Fourth, discussion of Tween-20 concentration in LAMP amplification System
Different Tween-20 concentration gradients are set, amplification is carried out under a self-matched pH-LAMP reaction system, the optimal Tween-20 concentration is judged according to a positive Ct value and a negative Ct value, and the obtained results are shown in the following table 19.
To obtain: the higher the total Tween-20 concentration is, the more easily the non-specific amplification is generated, so that when the amplification efficiency is not greatly different, the lower Tween20 concentration is selected; the optimal concentration of Tween-20 is selected to be 0.1-0.4% by comprehensively considering the amplification efficiency and the non-specific amplification.
Fifth, discussion of Evagreen concentration in LAMP amplification system
Different Evagreen concentration gradients are set, amplification is carried out under a self-matched pH-LAMP reaction system, the optimal Evagreen concentration is judged according to a positive Ct value and a negative Ct value, and the obtained results are shown in the following table 20.
To obtain: based on the above results, the optimum concentration of Eventreen was selected to be 0.5 to 1X.
Sixth, discussion of primer concentration in LAMP amplification system
Different primer concentration gradients are set, amplification is carried out under a self-matched pH-LAMP reaction system, the optimal primer concentration is judged according to a positive Ct value and a negative Ct value, and the obtained results are shown in the following table 21.
To obtain: the amplification efficiency of the inner primer concentration has no obvious change in the gradient of 1.6-2.4 mu M; the influence of the primer concentration inside and outside the gradient range of 0.1-0.4 mu M on the amplification efficiency is relatively low; the amplification efficiency of the loop primer concentration has no significant change in a gradient of 0.4-0.8. mu.M.
Claims (9)
1. An LAMP primer group for rapidly detecting HPV at bedside is characterized by comprising the following nucleotide sequence pair combinations:
。
2. the bedside LAMP primer group for rapid detection of HPV according to claim 1, wherein the HPV comprises subtypes HPV16, HPV18, HPV33, HPV52, HPV56, HPV58 and HPV 68.
3. Use of the LAMP primer group of any one of claims 1-2 in the preparation of HPV detection reagents.
4. The application of the LAMP primer group of any one of claims 1-2 in preparing HPV detection kits.
5. A kit for rapidly detecting HPV at bedside, which is characterized by comprising the LAMP primer group, lysate and LAMP amplification system according to any one of claims 1-2.
6. The kit for rapidly detecting HPV at bedside according to claim 5, wherein the total content of the LAMP primer group is 1-5 μ M;
the lysis solution comprises NaOH, Tris-HCl, SDS, Triton-X100, NP-40 and urea, wherein the NaOH is 5-20mM, the Tris-HCl is 1-5mM, the SDS is 0.05-1%, the Triton-X100 is 0.1-2%, the NP-40 is 1-10%, the urea is 2-10mM, and the total volume of the lysis solution is 50 muL;
the LAMP amplification system comprises: MgSO (MgSO)4、KCl、（NH4）2SO4Tween-20, betain, dNTPs, Bst3.0 amplidase and EVAgreen, wherein MgSO45-10mM, KCl 25-125mM, (NH)4）2SO40-40mM, 0.01-0.5% Tween-20, 0.1-0.25M beta-ain, 1-5 μ M dNTPs, 0.2-1.0U Bst3.0 amplification enzyme, 0.5-1X EVAgreen and 25 μ L of total volume of the reaction system.
7. The kit for bedside rapid detection of HPV according to claim 5, wherein the LAMP primer group comprises an inner primer with a concentration of 1.6-2.4. mu.M, an outer primer with a concentration of 0.1-0.4. mu.M, and a loop primer with a concentration of 0.4-0.8. mu.M;
the lysate comprises 5-10mM NaOH, 1-2mM Tris-HCl, 0.05-0.1% SDS, 0.5-1% Triton-X100, 1-5% NP-40 and 4-6mM urea;
the LAMP amplification system comprises 6-7mM MgSO4KCl of 75-125mM, and (NH) of 10-20mM4）2SO40.1-0.4% Tween-20, 0.1-0.25M betain, 1-5. mu.M dNTPs, 0.5-1X EVAgreen and 0.2-1.0U Bst3.0 amplilase.
8. The use method of the bedside rapid HPV detection kit according to claim 5 is characterized by comprising the following steps:
1) collecting samples: sample collection using a brush head including but not limited to a vaginal swab or vaginal cytology;
2) cracking: placing the in vitro sample in a sample adding chamber of a microfluidic nucleic acid detection device, and mixing with the prefabricated sample lysate for 10min to obtain lysate;
wherein, the sample lysate comprises 5-20mM NaOH, 1-5mM Tris-HCl, 0.1-2% Triton-X100, 1-10% NP-40, 0.05-1% SDS and 2-10mM urea;
3) amplification: the obtained lysate enters an amplification chamber of a microfluidic nucleic acid detection device, a heating element is turned on, heating is carried out for 30min at 65 ℃, and LAMP amplification is carried out;
wherein the reaction system comprises 5-10mM MgSO425-125mM KCl and 0-40mM (NH)4）2SO40.01-0.5% Tween-20, 0.1-0.25M betain, 1-5. mu.M dNTPs, 0.2-1.0U Bst3.0 amplification enzyme, 0.5-1X EVAgreen and 1-5. mu.M amplification primer;
4) and (3) discrimination: judging the color of the amplified reaction solution by naked eyes through a transparent observation window on the microfluidic nucleic acid detection device, and determining the reaction solution to be negative if the reaction solution is red; if the reaction solution is orange, the reaction solution is positive;
5) and (3) post-treatment: and (3) introducing the generated waste liquid into a waste liquid collecting chamber of the microfluidic nucleic acid detection device, and then treating the waste liquid with medical waste garbage.
9. The use method of the bedside rapid HPV detection kit according to claim 8, wherein the microfluidic nucleic acid detection device comprises a housing in a sealed arrangement, and a sample application chamber, an amplification chamber and a waste liquid collection chamber arranged in the housing, wherein the sample application chamber is arranged above the amplification chamber, the waste liquid collection chamber is arranged below the amplification chamber, and a path for unidirectional solution flow is formed between the sample application chamber amplification chamber and the waste liquid collection chamber.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533724A (en) * | 2010-12-30 | 2012-07-04 | 上海复星医学科技发展有限公司 | Cell lysis reagent for extracting and purifying nucleic acids in biological samples |
| CN102952894A (en) * | 2011-08-23 | 2013-03-06 | 中国疾病预防控制中心病毒病预防控制所 | Color based loop-mediated isothermal amplification technology for detecting HPV6, HPV11, HPV16, HPV18, HPV52 and HPV58 |
| CN103184297A (en) * | 2011-12-27 | 2013-07-03 | 上海复星医学科技发展有限公司 | Method and kit for synchronous fluorescence detection of hepatitis and HIV virus nucleic acid |
| CN103184294A (en) * | 2011-12-27 | 2013-07-03 | 上海复星医学科技发展有限公司 | Multi-channel fluorescence PCR detection method and kit for human papillomavirus (HPV) types 16 and 18 |
| CN107557492A (en) * | 2017-09-14 | 2018-01-09 | 温州美众医学检验所 | LAMP detections are combined with primer and its reaction system |
| CN110093459A (en) * | 2019-06-05 | 2019-08-06 | 中国人民解放军总医院第五医学中心 | For quickly detecting the LAMP primer composition and application of 13 kinds of high-risk HPVs |
| CN110607399A (en) * | 2019-09-20 | 2019-12-24 | 西人马（厦门）科技有限公司 | Primer combination, kit and method for LAMP amplification to detect HPV and typing |
| CN113481322A (en) * | 2021-05-18 | 2021-10-08 | 苏州阅微基因技术有限公司 | Composite amplification system and kit for typing detection of 16-valent HPV (human papillomavirus) |
-
2021
- 2021-11-25 CN CN202111408732.0A patent/CN114480728A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533724A (en) * | 2010-12-30 | 2012-07-04 | 上海复星医学科技发展有限公司 | Cell lysis reagent for extracting and purifying nucleic acids in biological samples |
| CN102952894A (en) * | 2011-08-23 | 2013-03-06 | 中国疾病预防控制中心病毒病预防控制所 | Color based loop-mediated isothermal amplification technology for detecting HPV6, HPV11, HPV16, HPV18, HPV52 and HPV58 |
| CN103184297A (en) * | 2011-12-27 | 2013-07-03 | 上海复星医学科技发展有限公司 | Method and kit for synchronous fluorescence detection of hepatitis and HIV virus nucleic acid |
| CN103184294A (en) * | 2011-12-27 | 2013-07-03 | 上海复星医学科技发展有限公司 | Multi-channel fluorescence PCR detection method and kit for human papillomavirus (HPV) types 16 and 18 |
| CN107557492A (en) * | 2017-09-14 | 2018-01-09 | 温州美众医学检验所 | LAMP detections are combined with primer and its reaction system |
| CN110093459A (en) * | 2019-06-05 | 2019-08-06 | 中国人民解放军总医院第五医学中心 | For quickly detecting the LAMP primer composition and application of 13 kinds of high-risk HPVs |
| CN110607399A (en) * | 2019-09-20 | 2019-12-24 | 西人马（厦门）科技有限公司 | Primer combination, kit and method for LAMP amplification to detect HPV and typing |
| CN113481322A (en) * | 2021-05-18 | 2021-10-08 | 苏州阅微基因技术有限公司 | Composite amplification system and kit for typing detection of 16-valent HPV (human papillomavirus) |
Non-Patent Citations (3)
| Title |
|---|
| JIASI WANG等: "Detection of 14 High-Risk Human Papillomaviruses Using Digital LAMP Assays on a Self-Digitization Chip", 《ANAL.CHEM.》, vol. 93, no. 6, pages 3266 - 3272 * |
| 李莉等: "HPV检测技术在***中的研究进展", 《癌症进展》, vol. 16, no. 3, pages 278 - 281 * |
| 马骉: "HPV分型环介导核酸等温扩增检测技术研究", 《中国优秀硕士学位论文全文数据库 医药卫生科级辑》, no. 4, pages 072 - 332 * |
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