CN108929869B

CN108929869B - Preparation method of HPV full-length genome quality control product, amplification primer and detection reagent

Info

Publication number: CN108929869B
Application number: CN201810902257.4A
Authority: CN
Inventors: 卢舟宇; 田洁; 陈永娟; 陈志强; 蔡泽加
Original assignee: Decipher Bioscience Shenzhen Co ltd
Current assignee: Decipher Bioscience Shenzhen Co ltd
Priority date: 2018-08-09
Filing date: 2018-08-09
Publication date: 2022-03-08
Anticipated expiration: 2038-08-09
Also published as: CN108929869A

Abstract

The invention relates to a preparation method of an HPV full-length genome quality control product, an amplification primer and a detection reagent. The preparation method of the HPV full-length genome quality control product comprises the following steps: (1) obtaining a DNA template; (2) carrying out PCR amplification by using an amplification primer; (3) connecting the target DNA fragment with a vector by adopting an HD Cloning connection technology; (4) introducing the plasmid into a competent cell, and then extracting the plasmid; (5) the plasmid was diluted with the human genome. The preparation method of the human papilloma virus full-length genome quality control product can successfully obtain the HPV gene detection quality control product which has no biological infection risk, can simulate clinical samples, can be produced in large quantities, has controllable concentration and is stably stored, and has better applicability.

Description

Preparation method of HPV full-length genome quality control product, amplification primer and detection reagent

Technical Field

The invention relates to a gene detection technology, in particular to a preparation method of an HPV full-length genome quality control product, an amplification primer and a detection reagent.

Background

Human Papillomaviruses (HPV) are a group of small DNA viruses without an envelope, belonging to the family of papillomaviruses. The morphological features of HPV viruses are usually regular icosahedrons with diameters of 52-55 nm. The length of the genome DNA is about 8000bp, and the genome is functionally divided into 3 parts: it includes early transcription region (encoding early proteins such as E1, E2, E4, E5, E6 and E7, which are involved in virus replication, transcription, translation regulation and transformation), late transcription region (encoding major capsid protein L1 and minor capsid protein L2, which are used for virus assembly, wherein L1 accounts for about 80% of capsid protein, which is highly conserved, and has less L2 content and more variation), and long control region (LCR, which contains HPV genomic DNA replication origin and HPV expression required regulatory elements, which regulate virus transcription and replication).

At present, more than 100 HPV genotypes are available, nearly 33% of the genotypes are related to genital tract injury, and the HPV genotypes are clinically classified into low-risk type HPV and high-risk type HPV according to the carcinogenic capacity of the HPV. Low risk HPV infection mainly causes condyloma acuminata, while persistent infection with high risk HPV has been identified as the main cause of cervical lesions. The comprehensive literature reports that high-risk types of HPV comprise HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, 18 types of HPV are shared, and common low-risk types comprise HPV6, 11 and the like.

Since HPV cannot be cultured in conventional cell culture fluids, other classical direct virological diagnostic techniques, such as electron microscopy and immunohistochemistry, are poorly sensitive and specific for routine detection of HPV, and therefore, all HPV detection reagents currently used for diagnosis rely primarily on viral nucleic acid detection. The current common clinical HPV detection methods comprise a PCR-reverse dot hybridization method, a PCR-fluorescent probe method, a hybridization capture method, a flow fluorescent hybridization method, a surface plasmon resonance method, a PCR-mass spectrometry method and the like. Common target sequences are regions L1, E6, E7 and E1, and the target sequence of the partial kit is the whole HPV genome.

The HPV typing and detecting reagents have more types, different principles and characteristics, and different performance indexes such as analysis sensitivity, specificity and the like. The state and government should establish corresponding standard substances, sample trays or quality control products which are traced with international standards to evaluate the performance indexes such as analysis sensitivity, clinical sensitivity, repeatability and the like of the existing and continuously generated new reagents. Laboratory-to-laboratory quality assessments should also be performed to compare the results of laboratory HPV detection or genotyping to improve the level of laboratory detection.

The national biological standard and quality control research institute of the UK establishes HPV16 and 18 international standard substances, countries in the world can obtain the national standard substances by tracing according to the international standard substances, and corresponding HPV genotyping reagent manufacturers can establish enterprise reference substances with unified and comparable quantity units according to the international standard substances or the traced national standard substances, and the enterprise reference substances are used for quantity value tracing and batch matrix detection of each batch of reagents, so that comparability of detection results among different reagent methods is realized. The international standard substance is prepared by adopting a recombinant HPV whole genome plasmid and an HPV negative human genome DNA mixture extracted from a human cervical cell line.

The international standard substances are expensive, less in quantity and not easy to purchase, and only 2 types of HPV16 and 18 cannot cover other common HPV types, and the requirements of reagent standardization, laboratory performance evaluation on a reagent method, indoor quality control and indoor quality evaluation in China cannot be met.

An in-vitro diagnostic reagent national standard substance human papillomavirus complete genome type reference substance (batch number: 360003-201101) is developed by Chinese food and drug assay research institute and is used for quality control and evaluation of a human papillomavirus nucleic acid (genotyping) detection kit (comprising non-L1 regions such as E6, E7 and E1 regions and a complete genome as target sequences) taking the non-L1 region as a target sequence. The reference product comprises 20 types of positive reference products and negative reference products N1-N5. Measuring the HPV whole genome recombinant plasmid concentration of 20 different HPV genotypes by a spectrophotometer, and diluting the HPV whole genome recombinant plasmid of each type to 10 by using 1 ng/mu L human genome TE solution⁶About copies/mL. Comprises 20 types of HPV6, 11, 16, 18, 26, 31, 33, 35, 45, 56, 58, 59, 61, 66, 67, 69, 71, 73, 81 and 82.

The national standard substances mentioned above are expensive, have small yield, contain genotypes which do not cover the common types of HPV39, 52, 68 and the like, and can not meet the requirements of reagent standardization, laboratory performance evaluation of reagent methods, indoor quality control and indoor quality evaluation in China.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: providing a primer for amplifying the full-length genome of HPV;

also provides a reagent for detecting HPV full-length genome by digital PCR;

on the basis, a preparation method of the HPV full-length genome quality control product is also provided; can cover 23 HPV genotypes comprising the HPV39, 52, 68 and the like, and meets the reagent standardization requirement of China.

In order to solve the technical problems, the invention adopts the technical scheme that:

a primer for amplifying the full-length genome of HPV comprises amplification primers for amplifying the whole genomes of HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV82 type, HPV6 type, HPV 11 type, HPV 42 type, HPV 43 type and HPV 81 type, and the nucleotide sequences of the amplification primers are shown as SEQ ID No. 1-46.

According to the above, the full length genome of HPV covering 23 genotypes including HPV39, 52, 68, etc. can be amplified using the primers having the nucleotide sequences shown in SEQ ID Nos. 1-46.

A reagent for detecting the full-length genome of HPV by digital PCR comprises digital PCR upstream primers, downstream primers and probes for detecting HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV82 type, HPV6 type, HPV 11 type, HPV 42 type, HPV 43 type and HPV 81 type, and the nucleotide sequences of the digital PCR upstream primers, the downstream primers and the probes are shown as SEQ ID Nos. 47-48, SEQ ID Nos. 49-50 and SEQ ID No.51 in sequence.

According to the above, the primers and probes with the nucleotide sequences shown in SEQ ID No.47-48, SEQ ID No.49-50 and SEQ ID No.51 are used for digital PCR to detect 23 genotypes of HPV.

A preparation method of an HPV full-length genome quality control product comprises the following steps:

(1) obtaining a DNA template of HPV;

(2) performing PCR amplification using the amplification primers of the primers for amplifying the full-length HPV genome according to claim 1 to obtain a target DNA fragment containing the full-length HPV genome, and purifying the obtained target DNA fragment;

(3) connecting the target DNA fragment with a vector by adopting an HD Cloning connection technology to construct and obtain a plasmid;

(4) introducing the plasmid into a competent cell, then extracting the plasmid and verifying whether the plasmid is matched with sequences of all genotypes of HPV;

(5) and (3) adopting a human genome to dilute the plasmid to obtain the HPV full-length genome quality control product.

The invention has the beneficial effects that:

(1) amplifying HPV whole genome DNA fragments by using the characteristics of high-fidelity DNA polymerase, and establishing a method capable of amplifying 8000bp fragments integrally with high fidelity and high efficiency;

(2) the target fragment is seamlessly and directionally cloned to the target position of the carrier by utilizing the connection characteristic of the HD Cloning enzyme, so that the problem of low connection efficiency is avoided;

(3) the concentration of plasmid DNA is determined by using a digital PCR absolute quantitative technology, and the problem of uncertain concentration of a quality control product is solved;

(4) successfully obtains the HPV gene detection quality control product which has no biological infection risk, can simulate clinical samples, can be produced in large quantities, has controllable concentration and stable preservation, and has better applicability.

Drawings

FIG. 1 is a diagram showing the result of electrophoresis of PCR products after amplification of sample DNA in the method for preparing a quality control product of HPV full-length genome according to the embodiment of the present invention;

FIG. 2 is another electrophoresis result diagram of the PCR product after the amplification of the sample DNA in the method for preparing the HPV full-length genome quality control according to the embodiment of the invention;

FIG. 3 is a schematic structural diagram (containing cloning sites) of a T Vector in the preparation method of the HPV full-length genome quality control product according to the embodiment of the invention;

FIG. 4 is a sample selection fluorescent map in the method for preparing the HPV full-length genome quality control product according to the embodiment of the invention;

FIG. 5 is a formula diagram of a standard curve in the preparation method of the HPV full-length genome quality control product according to the embodiment of the invention;

FIG. 6 is a graph of stability study initial Ct value in the method for preparing a quality control product of HPV full-length genome according to the embodiment of the present invention;

FIG. 7 is a graph showing the Ct value change in stability studies in the method for preparing a HPV full-length genome quality control according to the embodiment of the present invention;

FIG. 8 is a graph showing the result of constant values of 23 types of quality control materials by the digital PCR absolute quantitation method in the preparation method of the HPV full-length genome quality control material in the example of the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

The most key concept of the invention is as follows: the 23 types of human papillomavirus whole genome DNA is cloned into plasmid DNA, thereby realizing the quality control and evaluation of the HPV gene detection kit.

Because the existing HPV standard substances are expensive and have small yield, and the contained genotypes do not cover common types, the requirements of reagent standardization, reagent method performance evaluation in laboratories, indoor quality control and quality evaluation in rooms in China cannot be met. The invention makes statistics of genotypes covered by 63 HPV detection reagents which are certified by the Chinese food and drug administration, selects more than 80 percent of detectable types of the reagents, and comprises 18 high-risk types: HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82; 5 kinds of low-risk types: HPV6, 11, 42, 43, 81.

The main technique that this patent used has:

1. high fidelity long fragment amplification technology: the high-fidelity DNA Polymerase improved PCR enzyme has the special high fidelity and excellent amplification of alpha type DNA Polymerase, and can effectively inhibit nonspecific amplification. By adding an elongation factor and a newly developed specificity-promoting factor, a wide template amount can be amplified rapidly and with high specificity. Efficient amplification can also be performed for high GC content or high AT content sequences that are difficult to amplify with conventional DNA polymerases. In addition, the reaction buffer solution contains the absorption components of the harmful substances of the PCR reaction and the amplification enhancing factors, and the crude extract can also be subjected to high-efficiency PCR amplification. The monoclonal antibody which can inhibit the activity of DNA polymerase and the activity of 3 '-5' exonuclease at normal temperature is added, and the method is suitable for Hot Start PCR.

2. HD Cloning enzyme Cloning technology: the HD Cloning enzyme is a simple and efficient Cloning enzyme, and can rapidly and directionally clone a single or a plurality of DNA fragments into any vectors. The HD Cloning ligation technique is a Cloning technique capable of rapidly Cloning a single or multiple DNA fragments into any vector. The HD Cloning enzyme fuses the DNA fragment and the linearized vector together efficiently and accurately by recognizing the 15bp homologous sequences at the ends of the DNA fragment and the linearized vector. The 15bp homologous sequence is obtained by amplifying the primer designed for the target fragment. Particularly, the method is shown on cloning of long fragments, short nucleotide fragments and multiple fragments, has obvious connection effect and high success rate, does not need restriction enzyme cutting treatment, phosphorylation treatment or connection reaction, and does not add any redundant or unnecessary base sequences to the final recombinant vector.

3. Digital PCR absolute quantification technique: digital PCR generally involves two parts, PCR amplification and fluorescence signal analysis. In the PCR amplification stage, the digital PCR is firstly diluted to a single-molecule level and then is evenly distributed into dozens to tens of thousands of units for reaction. Unlike the method of real-time fluorescence measurement of qPCR for each cycle, digital PCR is to collect the fluorescence signal of each reaction unit after the amplification is finished, the fluorescence signal is recorded as 1, the non-fluorescence signal is recorded as 0, and the reaction unit with the fluorescence signal at least contains one copy. Theoretically, in the case where the concentration of the target DNA in the sample is extremely low, the number of reaction units having a fluorescent signal is equal to the copy number of the target DNA molecule. However, usually, each reaction unit may contain two or more target molecules, and it is necessary to use Poisson distribution function (Poisson distribution) to calculate, and according to the total number of reaction units, the number of units with fluorescence signals, and the dilution factor of the sample, the initial copy number (concentration) of the sample can be obtained.

The key technical problems to be solved by the invention comprise the following:

1. design and amplification of each type specific primer. The design of the primers needs to ensure that non-specific amplification can not occur during amplification of various types of primers, unknown mutation is easy to generate during amplification of common Taq enzyme, the original fidelity is lost, the concentration of the primers needs to be adjusted, and high-fidelity enzyme needs to be used; since the length of the HPV whole genome is about 8000bp, the extension time needs to be prolonged and the cycle number needs to be increased according to the research of the amplification program.

2. And connecting and introducing the PCR product with a vector. Because the length of PCR product is large, if the common T/A clone connection mode is used, the connection efficiency is low and the connection is unstable, the research of ligase, connection system and connection time is needed, the plasmid DNA formed after connection is about 10000bp, the difficulty of introducing competent cells is high, and the research and the solution of the problems of low transformation efficiency, false positive bacteria identification and the like are needed.

3. The concentration of plasmid DNA was determined. Because the concentration of the extracted plasmid stock solution is measured in different ways, the concentration of the target DNA in the quality control product can not be determined, if an ultraviolet spectrophotometer is used for measuring the concentration of the DNA, the luminosity accuracy is +/-0.2 percent, and if a real-time fluorescence quantitative PCR method is used, the problems of fluorescence threshold (threshold) set deviation, standard curve building and the like exist. Therefore, a digital PCR quantitative technology is needed to be utilized to design fluorescent primers and probes, optimize a reaction system, determine the dilution multiple and solve the problem that the concentration cannot be determined.

The invention realizes the preparation of HPV whole genome plasmid by applying PCR-HD Cloning connection principle, and the description is as follows:

selecting cervical exfoliated cells infected with single HPV, determining the type of HPV by a PCR-reverse dot hybridization method, determining the DNA concentration of HPV by fluorescence PCR, and selecting a high-concentration sample with a Ct value below 25 as a DNA template to improve the amplification success rate.

Counting the published HPV gene sequences, finding out the initial position and the termination position of each region, and designing 23 specific pairs of PCR primers aiming at 23 types of gene sequences according to the characteristics of the HPV gene sequences; meanwhile, due to the special requirement of HD Cloning functional enzyme, a 15bp fragment homologous to the vector sequence needs to be added to the 5' end of the primer. The HPV23 types whole genome amplification primer sequences are shown in Table 1:

TABLE 1

(3) Since the length of the HPV whole genome is about 8000bp, the extension time is prolonged to 300s by the amplification program, and the number of cycles is increased to 60; carrying out electrophoresis on the PCR amplification product by an agarose gel electrophoresis apparatus, and determining whether the amplification is successful according to whether a single DNA fragment is obtained and the length of the fragment; the concentration of the PCR product is evaluated by performing gel electrophoresis of DNA of known concentration or gradient concentration simultaneously with the PCR product, and quantifying the PCR product by comparison. Cutting the target fragment and recovering the gel to obtain a purified DNA fragment; the optimized PCR reaction system is shown in Table 2, and the amplification program is shown in Table 3:

TABLE 2

Genomic DNA	2ul
		2×PCR Buffer(Mg²⁺,dNTP plus)	25ul
DNA Polymerase(1.25units/ul)	1ul
		Primer*F(10uM)	1ul
Primer*R(10uM)	1ul
		dH₂O	20ul
Total	50ul

TABLE 3

The annealing temperature and the annealing time have great influence on the PCR amplification efficiency and the specific amplification, and the condition optimization result shows that a nonspecific amplification signal is generated when the annealing temperature is lower; the amplification efficiency is low when the temperature is higher, and the sensitivity is reduced.

(4) Connecting the DNA fragment with the vector by using the characteristics of HD Cloning functional enzyme to form HPV whole genome plasmid DNA, placing the reaction system at 50 ℃ for incubation for 15min, and then placing on ice;

an optimal HD Cloning reaction system was set up, see in particular table 4 below:

TABLE 4

5X HD Enzyme Premix	2μL
		Linearized Vector T	2μL
Purified PCR Fragment	6μL
		Total	10ul

(5) Introducing the plasmid into competent cells: coating on an LB plate containing ampicillin, wherein the concentration of ampicillin is 100 mug/ml, culturing overnight at 37 ℃, picking out monoclone the next day, and determining whether the insert is successful or not through enzyme digestion treatment or PCR screening;

(6) and extracting plasmids, and verifying whether the sequences are matched by sequencing to obtain results shown in table 5, namely a summary table of sequence comparison conditions of the 23 genotypes.

TABLE 5

(7) The plasmid was diluted with human genome (0.5 to 2 ng/. mu.L) and split into quality control disks.

(8) And determining the concentration of the plasmid DNA in each quality control product by using a digital PCR reaction system and an analysis system thereof. HPV23 types of digital PCR primers and probe sequences are shown in Table 6, the 5 'end of the probe P is marked with a reporter fluorophore FAM, and the 3' end is marked with MGB. The optimized digital PCR reaction system is shown in Table 7, and the amplification procedure is shown in Table 8.

TABLE 6

TABLE 7

Each quality control product	2ul
		2X ddPCR mix(Mg²⁺,dNTP plus,Taq,ROX,buffer)	10ul
Primer*F(10uM)	0.2ul
		Primer*R(10uM)	0.2ul
Probe*P(10uM)	0.2ul
		dH₂O	7.4ul
Total	20ul

TABLE 8

For human papillomavirus gene detection, HPV DNA detection is a common diagnosis method at present, HPV typing and detection reagents are more in types, have different principles and characteristics, and have different performance indexes such as analysis sensitivity, specificity and the like. The state and government should also establish corresponding standard substances, sample trays or quality control products which are traced to international standards to evaluate the performance indexes such as analysis sensitivity, clinical sensitivity, repeatability and the like of the existing and continuously generated new reagents. And carrying out the quality evaluation among laboratories, and comparing the results of HPV detection or genotyping in the laboratories so as to improve the detection level of the laboratories. The quality control product has the advantages of simple and easy operation, low production cost and suitability for the detection and evaluation of HPV DNA in China at present.

(1) Study of accuracy

The quality control product of the invention is detected by HPV detection kit, and the obtained results are shown in Table 9, namely the detection result table of 3 HPV detection kits:

TABLE 9

(2) Stability study

The stability is the essential attribute that the in vitro diagnostic reagent must have, is the important index of guaranteeing that the detection reagent is safe and effective in the detection, use course. According to the characteristics of the human papilloma virus quality control product, a reasonable stability research test project is designed to investigate the change condition of main quality indexes of the quality control product with time under different conditions, and a basis is provided for determining the storage condition and the validity period of the quality control product.

a. Transportation stability:

and (3) continuously producing 3 batches of quality control products in a trial manner, carrying out transportation stability and real-time stability study after transportation according to conventional low-temperature transportation conditions, transporting each batch for 3 days, 4 days, 5 days, 6 days, 7 days and 8 days, storing at the temperature of below 15 ℃, and carrying out quality verification at 3 months, 6 months, 9 months, 12 months, 13 months and 14 months. The results show that: after the quality control product is transported at low temperature for 7 days, the quality control product is stored at the temperature of below 15 ℃ below zero for 14 months, and the quality control quality requirement is completely met through quality verification.

Determining that the in-transit transportation time of the quality control product is not more than 7 days according to the principle that the stability of the quality control product is decreased gradually along with the time; the validity period of the quality control product is 12 months, and the quality control performance is stable in the validity period.

The components of the quality control product are replaced by water, frozen at the temperature below-15 ℃ and put into a foam box. And placing an ice bag in the foam box, then filling dry ice, placing a temperature recorder for temperature monitoring, finally sealing and packaging the foam box, and placing the foam box into a carton for box sealing.

Monitoring the transportation of the foam box, and continuously monitoring the temperature change for 4 days in a box of 3kg dry ice; 4kg dry ice box, the temperature change was monitored continuously for 8 days.

b. Transport stability and real-time stability test

According to the result of the simulated transportation test, the quality control product is packed into a foam box according to the method, and an experimental group is arranged, wherein,

group A is as follows: transporting 3kg dry ice for 3 days, taking out, and storing at the temperature below-15 ℃;

group B is as follows: transporting 3kg dry ice for 4 days, taking out, and storing at the temperature below-15 ℃;

group C is: transporting 4kg dry ice for 6 days, taking out, and storing at the temperature below-15 ℃;

group D is: transporting 4kg dry ice for 7 days, taking out, and storing at the temperature below-15 ℃;

group E is: transporting 4kg dry ice for 8 days, taking out, and storing at the temperature below-15 ℃;

the quality control products stored in groups were subjected to quality verification at 3 months, 6 months, 9 months, 12 months, 13 months and 14 months after passing the quality inspection to study the real-time stability, and the obtained results are shown in table 10.

Watch 10

c. Accelerated destruction stability:

the accelerated destructive test mainly inspects the destructive effect of temperature on the quality control performance. And (3) placing the qualified quality control product at the constant temperature of 37 ℃ for 3 days, 5 days, 7 days and 9 days, wherein the results show that: the quality control product has stable performance after being stored for 5 days at 37 ℃, and the performance begins to decline when the temperature is 7 days. To show that the quality control product has limited temperature tolerance, the product should be preserved at a temperature below-15 deg.C. The transportation process requires low temperatures while using dry ice and ice bags.

d. Repeated freeze-thaw stability:

the study was conducted on 3 batches of quality control that had been routinely shipped for 7 days and tested for eligibility, and the results indicated that: the freeze thawing of the quality control product is repeated for 24 times, and the quality control product completely meets the quality control quality inspection requirements through quality inspection. And according to the principle that the stability of the quality control product is decreased gradually along with time, in order to ensure the performance of the quality control product, determining that the repeated freeze thawing of the essence control product should not exceed 20 times. The results of the stability study are shown in Table 11 below.

TABLE 11

	Gradient 1	Gradient 2	Gradient 3	Gradient 4	Gradient 5	Gradient 6
							Transportation stability (Tian)	3	4	5	6	7	8
Storage at-15 deg.C (months)	6	9	10	12	14	15
							Constant temperature preservation at 37 ℃ (Tian)	0	1	3	5	7	9
Repeated freeze thawing (times)	15	18	20	22	24	26

Examples

Referring to FIGS. 1-8, FIGS. 1-2 are diagrams showing the results of electrophoresis of PCR products after sample DNA amplification; FIG. 3 is a schematic diagram of the structure of T Vector (containing cloning sites); FIG. 4 is a sample selection fluorescence spectrum, wherein the curves in FIG. 4 correspond to a standard 1, a sample, a standard 2, a standard 3 and a standard 4 in sequence from left to right; FIG. 5 is a standard curve equation; FIG. 6 is a graph of the initial Ct values for stability studies; FIG. 7 is a graph showing the change in Ct values in stability studies; FIG. 8 is a graph showing the results of the digital PCR absolute quantitation method on the values of 23 types of quality control materials.

The preparation method of the human papilloma virus full-length genome quality control product comprises the following steps:

(1) HPV nucleic acid extraction and typing

The nucleic acid extraction reagent of the applied bioenergy biotechnology (Shenzhen) Limited company has the following model: pathogen DNA (centrifugal column type) is used for extracting cervical exfoliated cells, and the specific method is as follows: fully eluting the cervical brush, taking 0.5mL of cell preservation solution containing the sample, transferring the cell preservation solution into a 1.5mL centrifuge tube, centrifuging the cell preservation solution at 13000rpm for 10 minutes, and sucking and discarding 300 mu L of supernatant (the residual liquid and sediment amount in the tube are about 200 mu L); adding 20 mu LPDE proteinase K into the cell sediment, fully suspending the cell sediment, and centrifuging at low speed for several seconds; then adding 200 mu L of PDE lysate, reversing the upper part and the lower part, uniformly mixing, and centrifuging at low speed for several seconds; (note: PDE proteinase K can not be directly added into PDE lysate) placing the centrifuge tube containing the sample processing solution into a constant temperature of 56 ℃ for 10 min; after completion of lysis, 300. mu.L of absolute ethanol was added, mixed well and centrifuged slightly. Placing a PDE centrifugal column in a PDE collecting tube, and marking and numbering for later use; transfer 1.5mL of the sample treatment solution in the centrifuge tube to the labeled PDE spin column, cover the tube, centrifuge at 12000rpm for 1 min. (note: during the transfer of the sample treatment solution and the addition of the washing solution, the solution should be slowly added in alignment with the center of the PDE spin column, without adhering the solution to the port of the PDE spin column, so as not to affect the quality of DNA purification); discarding the filtrate in the PDE collecting tube, putting the PDE spin column back into the PDE collecting tube, adding 700 μ L of PDE washing liquid, covering the tube cover, and centrifuging at 12000rpm for 1 min; (note: in order to avoid the influence of the liquid attached to the pipe orifice of the PDE spin column on the subsequent washing liquid, the PDE collecting pipe which abandons the filtrate can be reversely buckled and tapped for several times on a paper towel) abandoning the filtrate in the PDE collecting pipe, putting the PDE spin column back into the PDE collecting pipe, adding 700 mu L of absolute ethyl alcohol, covering the pipe cover, and centrifuging at 12000rpm for 1 min; discarding the filtrate in the PDE collecting tube, putting the PDE centrifugal column back into the PDE collecting tube, and carrying out air centrifugation at 14000rpm for 2 min; discarding the PDE collecting pipe, placing the PDE centrifugal column in a clean 1.5mL centrifugal tube, opening the tube cover of the PDE centrifugal column, and airing at room temperature for 5 min; adding 50 μ LPDE eluent into the center of the PDE centrifugal column, covering the tube cover, standing at room temperature for 3min, and centrifuging at 8000rpm for 2 min; discarding PDE spin column, and storing the extracted DNA template at 4 deg.C (within 24 h) or below-18 deg.C for use.

The results obtained by detecting the extracted DNA template by using human papillomavirus genotyping (type 23) detection kit (PCR-reverse dot hybridization method) and human papillomavirus nucleic acid detection kit (PCR-fluorescent probe method) of the sub-energy biotechnology (Shenzhen) Limited are shown in Table 12, and Table 12 is a list of HPV nucleic acid extraction and type determination.

TABLE 12

(2) Obtaining the target fragment

The PCR reaction system is applied to amplify sample DNA, 1% agarose gel is prepared by using 1 XTBE buffer solution, then electrophoresis is carried out on PCR products, the electrophoresis program is 170V 5min, 100V 80min is carried out, and the obtained electrophoresis result is shown in figure 1-2.

(3) Recovering the target fragment

The agarose gel recovery and purification DNA kit is used for recovering the target DNA, and the specific operation is as follows: cutting agarose gel containing target DNA under ultraviolet lamp, about 0.2mg, removing surface liquid with paper towel, adding buffer GM according to 4 gel volumes, mixing, heating at 37 deg.C for 10min, dissolving the gel block at room temperature (15-25 deg.C), and intermittently shaking for mixing to dissolve the gel block. Placing Spin Column on Collection Tube, transferring the solution to the center of Tube, adding dropwise, centrifuging at 12000rpm for 1min, adding the filtrate into Column again, and centrifuging once to improve DNA recovery efficiency. Add 700. mu.L of buffer WB into Column, centrifuge at 12000rpm for 30s at room temperature, repeat once, then place Column on new centrifuge tube, leave it open for 2min at room temperature, add 30. mu.L of TE buffer preheated to 60 ℃ at the center, stand it for 1min at room temperature, and then centrifuge at 12000rpm for 1min at room temperature to elute DNA.

(4) Ligation of the vector to the fragment of interest

And connecting the DNA fragment with the vector by using the characteristic of a connection functional enzyme to form the HPV whole genome plasmid DNA. In general, higher cloning efficiency can be achieved by adding 50-200ng of vector and insert, respectively, regardless of the size of the fragment. The molar ratio of insert to vector was 2: 1, if the PCR product is not connected with enough efficiency, the connection ratio can be optimized to be 3: 1 to 8: 1. the reaction system is incubated for 15min at 50 ℃, and then placed on ice for 5min, so that the connection can be successful.

Taking 100 mu L of DH5 alpha competent cells from a refrigerator at the temperature of-70 ℃, placing the cells in an ice bath for thawing, adding successfully connected plasmid DNA into a competent cell suspension, flicking and uniformly mixing the cells, standing the cells in the ice bath for 30min, placing a centrifugal tube in a water bath at the temperature of 42 ℃ for 60-90sec, quickly transferring the tube into the ice bath to cool the cells for 2-3min, taking the fact that the centrifugal tube cannot be shaken in the process, adding 900 mu L of sterile LB culture medium without antibiotics into the centrifugal tube, uniformly mixing the cells, and carrying out shake culture on the centrifugal tube at the temperature of 37 ℃ for 45min (150rpm) to express related resistance marker genes on the plasmids and recover thalli. Then 100. mu.L of transformed competent cells were plated on an LB plate containing ampicillin at a concentration of 100. mu.g/ml, and the cells were gently spread evenly using a sterile bent glass rod. The plates were left at room temperature until the liquid was absorbed, then incubated overnight at 37 ℃ upside down, and the next day the single clones were picked and expanded.

(5) Plasmids were extracted and verified for sequence matching by digestion, PCR screening or sequencing, with the results shown in Table 4 for comparison of sequence ID.

The plasmid is extracted by using a rapid plasmid miniprep kit (centrifugal column type), 4mL of overnight cultured bacterial liquid is added into a centrifuge tube, centrifuged at 12000rpm for 1min, the supernatant is discarded, 150 mu L of solution P1 is added, the mixture is uniformly shaken, and the cells are suspended and precipitated by measuring, wherein the solution is turbid red. Then 150 μ L of solution P2 was added and mixed by gentle up-down inversion for 6-8 times, but the shaking was not vigorous to avoid contaminating the genomic DNA, at which time the bacterial solution became clear and viscous purple. Adding 350 mu L of solution P5 into a centrifuge tube, immediately and rapidly turning upside down and mixing uniformly for 12-20 times to avoid local precipitation, fully mixing uniformly, wherein flocculent precipitation appears at the moment, the solution is clear yellow, if purple is mixed in yellow, the renaturation is insufficient, and continuously mixing uniformly until the color of the solution is completely changed into clear yellow. The supernatant was then transferred to adsorption column CP3 (adsorption column placed in collection tube) by centrifugation at 12000rpm for 2min, taking care not to aspirate the pellet as much as possible. Centrifuging at 12000rpm for 30sec, pouring off waste liquid in the collection tube, placing adsorption column CP3 into the collection tube, adding 300 μ L rinsing liquid PWT (please check whether absolute ethyl alcohol is added or not) into adsorption column CP3, centrifuging at 12000rpm for 30sec, pouring off waste liquid in the collection tube, placing adsorption column CP3 into the collection tube, placing adsorption column CP3 into the collection tube, centrifuging at 12000rpm for 1min, and removing residual rinsing liquid. The adsorption column CP3 was placed in a clean centrifuge tube, 50-100. mu.L of elution buffer TB was added dropwise to the middle of the adsorption membrane, and the plasmid solution was collected by centrifugation at 12000rpm for 30 sec.

(6) Basic constant value method

Measuring the nucleic acid concentration and purity of the plasmid stock solution by using an absorbance value method, then calculating the concentration of the 23 HPV genotypes according to a copy number calculation formula, and then deducing that the concentration of the 23 HPV genotypes is 10 according to the existing high-risk dilution multiple and copy number calculation formula⁵-10⁷copies/mL. The results are summarized in Table 13. Table 13 shows the results of the measurement of 23 types of quality control materials by the absorbance method.

Watch 13

Using human papillomavirus nucleic acid detection kit (PCR-fluorescent probe method) to make 3 times repeated detection of 5 concentrations of national standard substance human papillomavirus whole gene group type reference material developed by Chinese food and drug detection research institute for external diagnosis reagent (taking average value of 3 times of results when result is analyzed), creating standard curve of gradient dilution result, then substituting detection result of 12 HPV types (respectively: HPV16, 18, 26, 31, 33, 35, 45, 56, 58, 59, 66 and 82) detected by human papillomavirus nucleic acid detection kit (PCR-fluorescent probe method) into standard curve equation of every genotype to obtain concentration range of 10⁵-10⁷copies/mL. The results of the concentration gradient detection of 12 national ginseng are shown in table 14, and the results of the source-identifying of 12 quality control products and national ginseng are shown in table 15.

TABLE 14

Watch 15

The method is characterized in that the method develops the in-vitro diagnostic reagent national standard substance human papilloma virus complete genome type reference pair standard and the absorbance value method and copy number with the Chinese food and drug testing research instituteThe inference method carries out fixed value research on 23 HPV positive quality control products, and the concentration ranges of all the quality control products are as follows: 10⁵-10⁷copies/mL。

(7) Absolute quantitative method

The simple operation flow is as follows by using a digital PCR absolute quantitative method:

1) preparing 24-person digital PCR reaction solution according to the formula of the table 6, and adding the quality control product 1 to the quality control product 23;

2) correctly placing a digital PCR chip, a brush head and a chip cover;

3) and adding the reaction liquid with the sample completely into the sample adding hole at the lower part of the brush head, taking care that no air bubbles exist, and then uniformly coating the reaction liquid on the chip by the brush head. And after finishing smearing, adding 10-15 drops of sealing oil to enable the sealing oil to cover the surface of the chip, covering a chip cover, tightly pressing for at least 15 seconds, taking out the chip, amplifying according to the amplification program in the table 7, taking out the amplified chip after about 2 hours and 30 minutes, and putting the chip into a reader, wherein the reader can automatically read the target DNA concentration of the quality control product. The results of the evaluation of the 23 types of quality control materials by the digital PCR absolute quantification method are summarized in Table 16 and FIG. 8.

TABLE 16

The quantitative research is carried out on 23 HPV positive quality control products by a digital PCR method, and when all the quality control products are diluted by 10 times and added in 2 mu L, the absolute quantitative result is 100 to 400 copies, namely 5X10⁵-2X10⁶The concentration range of copies/mL.

Based on the above, the invention clones the 23 types of human papillomavirus whole genome DNA into plasmid DNA, thereby realizing the quality control and evaluation of the HPV gene detection kit; the method comprises the following steps:

(4) successfully obtains the HPV gene detection quality control product which has no biological infection risk type, can simulate clinical samples, can be produced in large quantities, has controllable concentration and stable preservation, and has better applicability.

In conclusion, the preparation method of the human papillomavirus full-length genome quality control product provided by the invention can successfully obtain the HPV gene detection quality control product which has no biological infection risk, can simulate clinical samples, can be produced in large quantities, has controllable concentration and is stably stored, and has better applicability.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

SEQUENCE LISTING

<110> Yaenergetic Biotechnology (Shenzhen) Limited

<120> preparation method of HPV full-length genome quality control product, amplification primer and detection reagent

<130> 2018

<160> 51

<170> PatentIn version 3.5

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Claims

1. A preparation method of an HPV full-length genome quality control product is characterized by comprising the following steps:

(1) obtaining a DNA template of HPV; the method specifically comprises the following steps: selecting cervical exfoliated cells infected with a single type of HPV, determining the type of HPV by a PCR-reverse dot hybridization method, then determining the DNA concentration of the HPV by fluorescence PCR, and selecting a sample with a Ct value below 25 as a DNA template;

(2) carrying out PCR amplification by using an amplification primer for amplifying the HPV full-length genome to obtain a target DNA fragment containing the HPV full-length genome, and purifying the obtained target DNA fragment; the amplification primers for amplifying the HPV full-length genome comprise amplification primers for amplifying HPV16 type, HPV18 type, HPV26 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV45 type, HPV51 type, HPV52 type, HPV53 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, HPV73 type, HPV82 type, HPV6 type, HPV 11 type, HPV 42 type, HPV 43 type and HPV 81 type whole genomes, and the nucleotide sequences of the amplification primers are shown as SEQ ID No. 1-46; a 15bp fragment which is homologous with a vector sequence is added at the 5' end of the primer;

the specific correspondence is as follows;

the PCR reaction system for PCR using the HPV full-length genomic amplification primers as described above is as follows:

the amplification procedure is as follows:

1 cycle at 94 ℃ for 1 min; 60 cycles at 98 ℃ for 10sec and 58 ℃ for 15sec and 68 ℃ for 5 min; 10min at 68 ℃ for 1 cycle;

the PCR reaction system and the amplification program are that each pair of primers is used for independently amplifying the DNA template of the single type HPV;

2. The method for preparing the HPV full-length genome quality control product according to claim 1, further comprising the step (6): carrying out PCR reaction by adopting the HPV full-length genome quality control product and a digital PCR primer and a probe in a reagent for detecting the HPV full-length genome by digital PCR;

the digital PCR primers and probes of the reagent for detecting the HPV full-length genome by digital PCR comprise digital PCR upstream primers, downstream primers and probes for detecting HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82, 6, 11, 42, 43 and 81, and the nucleotide sequences of the digital PCR upstream primers, the digital PCR downstream primers and the digital PCR probes are sequentially shown as SEQ ID Nos. 47-48, 49-50 and 51.

3. The method for preparing the HPV full-length genome quality control product according to claim 2, wherein the probe is labeled with a reporter Fluorophore (FAM) at the 5 'end and labeled with MGB at the 3' end.

4. The method for preparing the HPV full-length genome quality control product according to claim 2, wherein the reaction system of the PCR reaction in the step (6) is as follows:

5. the method for preparing HPV full-length genome quality control products according to claim 4, wherein the PCR amplification program in the step (6) is: 10min at 95 ℃ for 1 cycle; 30sec at 98 ℃ and 60sec at 58 ℃, 40 cycles.

6. The method for preparing a quality control product of the full-length genome of HPV according to claim 1, wherein the nucleic acid concentration of the obtained quality control product of the full-length genome of HPV is measured.

7. The method for preparing the quality control product of the HPV full-length genome according to claim 1, wherein in the step (3), the obtained plasmid is incubated at 50 ℃ for 15min and then placed on ice.