CN110317901B - Composition, kit and method for high-risk HPV detection and typing - Google Patents
Composition, kit and method for high-risk HPV detection and typing Download PDFInfo
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Abstract
The invention relates to the field of molecular biological detection, in particular to the field of HPV detection. The present invention provides a composition which can specifically detect HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68E 6/E7mRNA, and at the same time, the use of the composition for detecting HPV E6/E7mRNA, a kit comprising the composition and a method for detecting HPV E6/E7mRNA.
Description
Technical Field
The invention belongs to the field of molecular biological detection, and more particularly belongs to the field of HPV detection.
Background
Human papillomaviruses (Human papillomavirus, HPV) are a class of non-enveloped, double-stranded circular DNA viruses of relatively small molecular weight that specifically infect and parasitize epithelial cells of human reproductive organs and other tissue organs. However, not only genital warts or cutaneous warts may appear after HPV infection, but also cervical cancer is more likely to be induced in women, and it has been reported that more than 90% of cervical cancer is caused by HPV infection.
Currently, 4 cervical cancer high-risk HPV detection products are approved by the FDA in the United states, namely Hybrid Capture 2 based on a Hybrid Capture technology, cervista HPV based on an enzyme digestion signal amplification technology, cobas HPV based on a real-time fluorescent PCR technology and Aptima HPV based on a transcription-mediated isothermal amplification technology. The first three detection targets were DNA of HPV, while the last Aptima HPV detection target was HPV E6/E7mRNA.
That is, most of the HPV detection commercial products currently in clinical use are directed against viral DNA. HPV DNA detection is used for cervical cancer screening with higher sensitivity and specificity than cytological examination; however, since most patients can clear by autoimmune function after HPV infection, and HPV infection is a long process from the occurrence of precancerous lesions to canceration, only a small part of HPV infection can cause cervical lesions, so that the detection time point of HPV DNA is too early, and the risk of cervical lesions is difficult to predict.
On the other hand, the expression of oncogenes E6 and E7 is an essential step in causing malignant transformation of cervical cells and progression to cervical cancer, so HPV E6/E7mRNA detection can more accurately predict the likelihood of cell transformation to high grade lesions and cancers.
However, to date, detection products based on HPV E6/E7mRNA are not numerous and are mainly based on Transcription-mediated isothermal amplification (Transmission-mediated amplification, TMA), nucleic acid sequence-dependent amplification (Nucleic acid sequence-based amplification, NASBA) and branched DNA (bDNA).
In particular the number of the elements,
HPV product technology includes procedures such as sample preparation, TMA, hybridization protection detection (Hybridization proteCtionassay, HPA) and dual dynamic detection (Dual kinetic assay, DKA); 14 HR-HPV ( HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) E6/E7mRNA can be detected. Norchip and organism Mei Liai are both NASBA-based detection techniques that detect the 5 highest-risk HPVs ( HPV 16, 18, 31, 33, 45) with the highest cervical cancer relevance. Due to the epidemic of HPV worldwideThe technology for detecting 5 high-risk types can only detect in individual groups, and is inevitably limited by the epidemic characteristics of regional HPV when in use. Quantivirus is produced by Diacarata company in America, and adopts bDNA technology to capture detected gene fragment, signal amplification and chemiluminescence, then the Quantivirus TM luminescence instrument reads photon number, and uses special software to make treatment, then converts the photon number into copy number, and the copy number of detected substance is inversely proportional to light intensity so as to implement quantitative detection of mRNA. Quantiviruses can detect 13 high-risk HPVs ( HPVs 16, 18, 31, 33, 35, 39, 45, 51, 52, 58, 59, 66, 68) and 6 low-risk HPVs ( HPVs 6, 11, 40, 42, 43, 44) E6/E7mRNA simultaneously. However, the technology needs to be provided with a special device Quantivirus luminescence instrument, and certain limitation is brought to the popularization of detection products.
Because the detection methods are based on different technologies, the performances are different, and the detection types are different in coverage, the detection methods are not suitable for the domestic cervical cancer screening requirements, and researches show that the detection methods have a great difference from DNA in aspects of clinical sensitivity and the like, and cannot meet the domestic cervical cancer screening and clinical requirements.
Disclosure of Invention
In view of the above, the invention aims to provide an HPV E6/E7mRNA detection product which is suitable for domestic HPV detection, has comprehensive high-risk coverage and is based on a real-time fluorescent quantitative PCR technology.
As set forth in the background, there are few current detection products based on HPV E6/E7mRNA. Further, real-time fluorescent quantitative PCR detection products based on HPV E6/E7mRNA are relatively fewer. The reason is that firstly, the design difficulty of the primer probe aiming at various types is high, on one hand, the E6/E7 area is short relative to the L1 area, so that the selectable area is less, and the screening difficulty is increased; on the other hand, the total number of primer probes is large for each type, and when the primer probes are combined into a one-tube multiplex PCR detection system, the design of the primers needs to consider the specificity and sensitivity of each type, and direct interaction (such as dimer formation and the like) of the primers and the probes needs to be avoided. Thus, the more types detected, the greater the difficulty in design and verification. As described in the background section, detection of HPV E6/E7mRNA is very important for cervical cancer, and the possibility of a more accurate prediction of cell transition to high grade lesions and cancers is therefore a need for the development of a real-time fluorescent quantitative PCR detection product for HPV E6/E7mRNA.
Through extensive research, the present inventors have provided a composition for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68, the composition comprising the following primers and probes:
an HPV16 type upstream primer shown as SEQ ID NO. 1, an HPV16 type downstream primer shown as SEQ ID NO. 2 and an HPV16 type probe shown as SEQ ID NO. 3;
an HPV18 type upstream primer shown as SEQ ID NO. 4, an HPV18 type downstream primer shown as SEQ ID NO. 5 and an HPV18 type probe shown as SEQ ID NO. 6;
an HPV 31 type upstream primer shown as SEQ ID NO. 7, an HPV 31 type downstream primer shown as SEQ ID NO. 8 and an HPV 31 type probe shown as SEQ ID NO. 9;
an HPV 33 type upstream primer shown as SEQ ID NO. 10, an HPV 33 type downstream primer shown as SEQ ID NO. 11 and an HPV 33 type probe shown as SEQ ID NO. 12;
an HPV 35 type upstream primer shown as SEQ ID NO. 13, an HPV 35 type downstream primer shown as SEQ ID NO. 14 and an HPV 35 type probe shown as SEQ ID NO. 15;
an HPV 39 type upstream primer shown as SEQ ID NO. 16, an HPV 39 type downstream primer shown as SEQ ID NO. 17 and an HPV 39 type probe shown as SEQ ID NO. 18;
an HPV 45 type upstream primer shown as SEQ ID NO. 19, an HPV 45 type downstream primer shown as SEQ ID NO. 20 and an HPV 45 type probe shown as SEQ ID NO. 21;
an HPV 51 type upstream primer shown as SEQ ID NO. 22, an HPV 51 type downstream primer shown as SEQ ID NO. 23 and an HPV 51 type probe shown as SEQ ID NO. 24;
an HPV 52 type upstream primer shown as SEQ ID NO. 25, an HPV 52 type downstream primer shown as SEQ ID NO. 26 and an HPV 52 type probe shown as SEQ ID NO. 27;
HPV53, 56, 66 type upstream primer shown as SEQ ID NO. 28, HPV53, 56, 66 type downstream primer shown as SEQ ID NO. 29 and HPV53, 56, 66 type probe shown as SEQ ID NO. 30;
an HPV 58 type upstream primer shown as SEQ ID NO. 31, an HPV 58 type downstream primer shown as SEQ ID NO. 32 and an HPV 58 type probe shown as SEQ ID NO. 33;
an HPV 59 type upstream primer shown as SEQ ID NO. 34, an HPV 59 type downstream primer shown as SEQ ID NO. 35 and an HPV 59 type probe shown as SEQ ID NO. 36;
an HPV 68 type upstream primer shown as SEQ ID NO. 37, an HPV 68 type downstream primer shown as SEQ ID NO. 38 and an HPV 68 type probe shown as SEQ ID NO. 39.
Using the compositions of the invention, 15 high-risk HPVE6/E7 mRNAs can be detected simultaneously. In fact, as the types of detection increase, the overall difficulty of primer design increases further. The reason is that, firstly, we ensure not only the specificity and sensitivity of each of these detection types, but also that direct interactions of these primers and probes (such as dimer formation, etc.) are avoided; secondly, because of the high homology of the different types of HPV nucleic acid sequences to be detected, on the one hand, highly conserved sequences are selected, and on the other hand, the sequences are required to have a certain level of difference in different strains. It will be appreciated that the more types that are detected, the greater the difficulties faced in design and verification.
Detection of types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68 of HPV can be achieved using the compositions of the invention. Because of the overall detection type, the composition of the invention is capable of covering a wide range of people, in particular, the combination of the invention is especially for HPV detection in Chinese people. For example, cen Yao and the like research on infection status and high-risk type distribution of human papillomavirus of middle female, and the result shows that the total HPV53 type accounts for 5.1% in the north and south of China; kang Junling and the like in the national region of inner Mongolia minority, 1398 samples are researched, and HPV53 type accounts for 8.92%, so that HPV53 type is known to be a high-risk type HPV and is very popular in China (particularly in the minority), and therefore, the composition provided by the invention can further adapt to China and particularly meet the requirements of the national minority due to the fact that 15 HPV types are covered.
In yet another aspect, the use of a set of primers and probes (i.e., SEQ ID NOS: 28-30) in the compositions of the present invention enables simultaneous detection of HPV types 53, 56 and 66, effectively reducing cost by using such a set of primers and probes.
The composition disclosed by the invention uses real-time fluorescence quantitative PCR, is simple in method, accurate in result and easy to popularize, has high sensitivity, has a detection lower limit as low as 400 copies/mL, and is not interfered by other pathogens (chlamydia trachomatis, ureaplasma urealyticum, gonococcus, herpes simplex virus type II, treponema pallidum, candida albicans, trichomonas vaginalis, mycoplasma hominis and HPV common low-risk types 40, 42, 43 and 44) and other HPV genotypes, and has good specificity.
The probes in the compositions of the invention may carry fluorescent reporter groups for real-time fluorescent PCR detection.
In some embodiments, the fluorescent reporter groups on the probes in the compositions of the invention may be selected from FAM, HEX, ROX, VIC, CY, 5-TAMRA, TET, CY and JOE without interference.
In a preferred embodiment, the fluorescent reporter groups of the probes for HPV types 16 and 18 are different, and the fluorescent reporter groups of HPV16 and 18 probes are also different from the rest of the thirteen HPV probes.
Saslow D et al states that: currently, both ACOG and ACS/ASCCP/ASCP suggest HPV16, 18 typing as a standard for patients positive for shunt HPV detection and negative for cytology, i.e., positive HPV16, 18 typing patients suggest colposcopy, or re-screening HPV and cytology within 12 months, whereas existing HPV E6/E7mRNA detection (e.g.,
HPV detection) does not allow specific typing of HPV type 16/18, and thus does not provide guidance for colposcopy. General purpose medicineBy making HPV types 16 and 18 and the rest thirteen HPV probes in the composition have different fluorophores, the invention realizes HPV 16/18 typing and provides guidance for colposcopy.
In a specific embodiment, the fluorescent reporter group of HPV16 probe is CY5, the fluorescent reporter group of HPV18 probe is FAM, and the fluorescent reporter groups of the remaining thirteen HPV probes are ROX.
In a preferred embodiment, the composition of the present invention further comprises: compositions for use as internal standard assays. In particular, a composition for use as an internal standard assay may comprise an internal standard upstream primer, an internal standard downstream primer, and an internal standard probe.
In one embodiment, the internal standard upstream primer has the sequence shown in SEQ ID NO. 40, the internal standard downstream primer has the sequence shown in SEQ ID NO. 41, and the internal standard probe has the sequence shown in SEQ ID NO. 42.
Further, the internal standard probe carries a fluorescent reporter group HEX or VIC.
That is, the composition of the present invention may further include an internal standard primer and an internal standard probe. Which can be used in combination with other primers and probes in the compositions of the invention and do not affect each other. The use of the internal standard primer and the probe can judge whether the detection is effective or not, and further ensure the detection accuracy of the composition.
In a second aspect, the invention also provides the use of a composition as described above in the manufacture of a kit for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68.
In a third aspect, the invention provides a kit comprising a composition as described above.
Further, the kit also comprises reagents required for extracting mRNA and Mg 2+ At least one of dNTPs, reverse transcriptase, DNA polymerase, and PCR buffer.
Still further, the kit further comprises magnetic bead reagents for extracting mRNA.
In a specific embodiment, the specific final concentration of each component isMg 2+ 3 to 5mmol/L, dNTPs to 5mmol/L, 0.05 to 0.2 mu mol/L of each group of upstream and downstream primers, 0.25 to 0.1 mu mol/L of probe, 10 to 70 mu mol/L, DNA of reverse transcriptase and 0.5 to 2mmol/L of polymerase.
In a preferred embodiment, the final concentration of the reverse transcriptase is 30 to 60. Mu. Mol/L.
In order to achieve better amplification, the concentration of the reverse transcriptase in a PCR reaction system is optimized, the optimal reverse transcriptase amount is determined, and the reverse transcription efficiency is improved, so that the detection result is more accurate and sensitive.
In a fourth aspect there is also provided the use of a kit of the invention for HPV detection.
In a fifth aspect, the present invention provides a method for detecting HPV, the method comprising the steps of:
1) Extracting mRNA of a sample to be detected;
2) Performing fluorescent quantitative PCR detection on the mRNA obtained in the step 1) by using the composition or the kit of the invention;
3) The results were obtained and analyzed.
In the present invention, the sample for detection may be wart surface exfoliated cells, female cervical epithelial cells, genital tract secretions, or the like, but is not limited thereto.
In a specific embodiment, the method for extracting mRNA from the sample to be tested in step 1) is a magnetic bead method.
In a specific embodiment, the method of fluorescent quantitative PCR detection in step 2) is one-step reverse transcription fluorescent quantitative PCR.
mRNA is extracted by a magnetic bead method, so that the purity and concentration of the RNA are ensured, and the digestion treatment steps are reduced; the one-step reverse transcription fluorescent quantitative PCR reduces the reverse transcription steps of random primers, so that the method provided by the invention is simple and quick to operate and has wide popularization and application values.
In a specific embodiment, in the step 3), a Ct value is used as a criterion. If the amplification curve of the sample FAM channel is S-shaped, the sample FAM channel has a Ct value which is less than or equal to 39, the sample FAM channel can be judged to be positive to HPV18 type-mRNA, and whether the sample is positive to HPV16 or other 13 high-risk types is judged according to whether amplification curves of CY5 and ROX channels exist; if the sample amplification curve is flat, no Ct value is displayed (Undet), and HPV type 18-mRNA is negative.
As described above, the present invention can employ one-step reverse transcription fluorescent quantitative PCR, and thus makes the operation easier and faster. However, the above advantages are obtained and the requirements for the reaction system are also increased.
Accordingly the present invention provides a fluorescent quantitative PCR reaction system wherein in some embodiments the composition of step 2) has the following concentrations: upstream and downstream primers of 0.05 mu mol/L to 0.2 mu mol/L in each group; probes of 0.25 mu mol/L to 0.1 mu mol/L in each group; preferably, the composition in step 2) has the following concentration: upstream primer and downstream primer of 0.1 mu mol/L in each group; each set of 0.05. Mu. Mol/L probes, where each set of primers and probes represents the primers and probes required to detect one type of HPV.
Further, the following components and concentrations are included in the step 2):
in a specific embodiment, the fluorescent quantitative PCR reaction in step 2) is as follows:
in a sixth aspect, the present invention provides the use of the above detection method for HPV detection.
Drawings
FIG. 1 is a graph showing the results of 3 sets of composition screening for HPV type 16 mRNA;
FIG. 2 is a graph showing the results of HPV type 16 mRNA detection using different amounts of reverse transcriptase;
FIG. 3 is a graph showing the results of HPV type 16 mRNA specific detection;
FIG. 4 is a graph of the sensitivity results of gradient detection of HPV type 16;
FIG. 5 is a graph of the sensitivity results of gradient detection of HPV type 18;
FIG. 6 is a graph of the sensitivity results of gradient detection of HPV type 31;
FIG. 7 is a graph of the sensitivity results of gradient detection of HPV type 33;
FIG. 8 is a graph of the sensitivity results of gradient detection of HPV type 35;
FIG. 9 is a graph of the sensitivity results of gradient detection of HPV type 39;
FIG. 10 is a graph of the sensitivity results of gradient detection of HPV type 45;
FIG. 11 is a graph of the sensitivity results of gradient detection of HPV type 51;
FIG. 12 is a graph of the sensitivity results of gradient detection of HPV type 52;
FIG. 13 is a graph of the sensitivity results of gradient detection of HPV type 53;
FIG. 14 is a graph of the sensitivity results of gradient detection of HPV type 56;
FIG. 15 is a graph of the sensitivity results of gradient detection of HPV type 66;
FIG. 16 is a graph of the sensitivity results of gradient detection of HPV type 58;
FIG. 17 is a graph of the sensitivity results of gradient detection of HPV type 59;
FIG. 18 is a graph of the sensitivity results of gradient detection of HPV type 68;
FIG. 19 is a graph showing the results of two RNA extraction methods for detecting HPV type 16.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Example 1, primers and probes used in the present invention
Homology analysis is performed on the 15 high-risk and beta-action genome sequences searched in Genbank on NCBI by SeqMan and MegAlign software in DNAStar software package, and a plurality of pairs of specific primer pairs are designed. Then, the obtained pairs of primers are subjected to primer5 analysis, primer pairing, neck ring structure, amplification efficiency and mismatch condition are comprehensively considered, 3 sets of primers and corresponding probes are respectively given as examples by search analysis of Blast tools in GenBank database (wherein HPmR366 can detect HPV53, 56 and 66 simultaneously), and the sequences are shown in tables 1-3 in sequence
TABLE 1
TABLE 2
TABLE 3 Table 3
Wherein F and R are forward and reverse primer pairs of amplification primers, GB-F and GB-R are inner reference primer pairs, and P is a detection probe.
Meanwhile, in a reaction system, CY5 and FAM fluorescent markers are adopted for HPV16 type and HPV18 probes, ROX fluorescent markers are adopted for the other 13 types, and an internal standard probe is provided with a fluorescent reporter group HEX or VIC, so that HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68 type infections can be detected simultaneously, and HPV16 and 18 types can be typed.
Example 2 composition screening
The detection kit of the invention independently comprises the following components:
RNA extraction solution I: consists of 0.2 to 1.0 percent (mass/volume) of sodium dodecyl sulfate, 1.0 to 4.0 percent (volume/volume) of triton, 0.2 to 1.0mol/L of guanidine isothiocyanate and 100 to 400 mug/ml of magnetic beads;
RNA extraction solution II: 100 to 300mmol/L of 4-hydroxyethyl piperazine ethane sulfonic acid, pH 6.5+/-0.2 and 100 to 300mmol/L of sodium chloride;
RNA extraction solution III: 0.1 to 1.0 percent (volume/volume) of triton and 100 to 300mmol/L of sodium chloride;
RNA extraction solution IV: mineral oil;
RNA eluate: 0.8 to 1.2mol/L, EDTA 0.1.1 to 1.0mol/L Tris-HCl;
reverse transcription PCR-reaction solution: 10 XHFM buffer reaction buffer 8-16. Mu.l, 0.05 mmol/L-0.2 mmol/L deoxyribonucleoside triphosphate, depc water 22-32. Mu.l, mgcl 2 0.05 to 0.4. Mu.l, 0.05 to 0.2. Mu. Mol/L of a probe for amplifying an upstream primer and a downstream primer of a target polynucleotide, and 0.25 to 0.1. Mu. Mol/L.
Enzyme mixed solution: 1U/. Mu.l to 5U/. Mu.l of thermostable DNA polymerase (Taq enzyme), 0.1U/. Mu.l to 2U/. Mu.l of M-MLV reverse transcriptase.
The 10 Xbuffer comprises 200mmol/L tris hydrochloride solution, 30mmol/L magnesium chloride solution, 500mmol/L potassium chloride solution, 0.2% (v/v) triton solution and 10% (v/v) formamide solution at pH 7.5.
The deoxyribonucleoside triphosphate is dATP, dCTP, dUTP, dGTP or dTTP.
2.1 pseudovirus preparation
Preparation of E6/E7 region pseudoviruses of HPV 15 high-risk type human papillomaviruses 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68, and concentration of 1.00-5.00E+06 copies/ml.
2.2 RNA extraction
Splitting the virus: adding 200 mu l-1 ml of RNA extraction solution 1-mix into each tube, then adding 100 mu l-1 ml of sample to be detected (sample to be detected such as blood plasma, blood serum and the like), covering a tube cover, shaking and mixing for 10 seconds, and performing instantaneous centrifugation (dengue virus negative control and positive control are extracted by referring to the same step);
magnetic beads adsorb nucleic acid: adding 50-400 mu l of RNA extraction solution 2 into each tube, shaking and uniformly mixing for 10 seconds, and standing at room temperature for 10-30 minutes;
removing impurities: after instantaneous centrifugation, placing the centrifuge tube on a magnetic bead separator, and slowly sucking out the solution after 2-5 minutes;
washing: adding 400-1 ml of RNA extraction solution 3 and 100-500 ul of RNA extraction solution 4 into each tube, shaking and uniformly mixing for 3-7 seconds, and placing the centrifuge tube on the separator again after instantaneous centrifugation;
after 2-5 minutes, separating the supernatant into two layers, inserting a suction head into the bottom of the centrifuge tube, slowly sucking out and discarding the liquid from the bottom, standing for 1-3 minutes, and completely sucking out and discarding the residual liquid at the bottom of the tube;
adding 10-100 mul of RNA eluent, eluting magnetic beads on the wall of a centrifugal tube to the bottom of the tube, sucking and beating for 3-4 times, standing for 5-30 minutes at room temperature, placing the centrifugal tube on a separator again for 2-5 minutes, and sucking the eluted RNA into a new 1.5ml sterilizing centrifugal tube.
2.3 PCR reaction system
According to the quantity of sample to be tested, negative control (sterilized normal saline) and positive control (HPV type 16, type 18 and type 45E 6/E7mRNA mixed pseudo virus particles, the concentration of which is 1.00-5.00E+05 copies/ml), taking corresponding quantities of reaction solution, enzyme mixed solution and internal standard according to the proportion (38-44 mu l/human part + 3-5 mu l/human part of enzyme mixed solution), fully and uniformly mixing to obtain PCR-mix, and carrying out instantaneous centrifugation for standby.
Taking the prepared HPV16 type pseudovirus as a sample to be tested, taking 5 μl of the HPV16 type pseudovirus and mixing with a reverse transcription PCR-reaction liquid system (45 μl) of the table 4 (wherein the reaction liquid system respectively comprises the compositions shown in the tables 1, 2 and 3), and performing on-machine detection.
TABLE 4 reverse transcription PCR reaction solution System
2.4 fluorescent PCR reactions (performed on a fluorescent quantitative PCR amplification apparatus)
1) Placing the PCR reaction tube into a sample tank of an amplification instrument, and setting the name of a sample to be detected and the concentration of a quantitative reference according to the corresponding sequence;
2) Selecting a fluorescence detection channel;
3) The fluorescent quantitative PCR reaction conditions are as follows:
2.5 analysis of results
After the reaction is finished, the instrument automatically stores the result, the software of the instrument can be utilized for automatic analysis (the starting value, the ending value and the threshold line value of the base line can be manually adjusted for analysis), and then the Ct value and the fixed value result of the sample are recorded.
Screening of 3 sets of compositions revealed that the Ct value of the HPV type 1 set of compositions was earlier than that of the HPV type 16 set of compositions 2 and 3 sets of compositions, and the results are shown in FIG. 1, so that the set 1 of compositions was screened for detection of HPV mRNA.
EXAMPLE 3 content modulation of different reverse transcriptases
This example discusses the different MMLV reverse transcriptase levels, verifies the effect of reverse transcriptase on amplification, and determines the optimal MMLV reverse transcriptase amount.
Taking HPV type 16 as an example, the E6/E7 region pseudovirus of the known HPV type 16 is diluted by 10 times with Depc water for 3 gradients with the concentration of 1.00-5.00E+06.
Then, 5. Mu.l of each nucleic acid was taken, and 10. Mu. Mol/L, 30. Mu. Mol/L, 50. Mu. Mol/L, or 70. Mu. Mol/L of the reverse transcription PCR-reaction solution (45. Mu.l) of Table 6 containing the composition shown in Table 1 of example 1 was added thereto for detection on the machine.
TABLE 5 reverse transcription PCR reaction solution System
The fluorescence PCR reaction was performed according to the procedure described in example 2.4, and after the reaction was completed, the instrument automatically stored the results, and automatic analysis (or analysis by manually adjusting the starting value, ending value, and threshold line value of the baseline) was performed using the software carried by the instrument, and then the Ct value and the constant result were recorded. The difference of different amounts of reverse transcriptase at high concentration of HPV type 16 is not large, the fluorescence value increases with the increase of the content of reverse transcriptase at low concentration, the result is shown in figure 2, 3S-shaped curves in figure 2 represent detection of 3 HPV types 16 at different concentrations, the difference of enzymes at several concentrations is not large at high concentration of pseudovirus detection, the amplification curves of enzymes at different concentrations are different at low concentration of pseudovirus detection, the curve is smooth at 50 mu mol/L of enzyme amount and Ct value is front, and when the enzyme concentration is increased to 70 mu mol/L, ct value is trailing and fluorescence value is reduced. In the above-described method, the reverse transcriptase amount is detected for each of the other types, and similar results are obtained, so that the amplification of the curve is affected by too much or too little reverse transcriptase, and the reverse transcription efficiency is greatly improved by adjusting the different amounts of reverse transcriptase.
Example 4 specific detection of compositions
To demonstrate the amplification specificity of each specific primer for the target type, all specific primers were reverse transcription amplified with positive cell preservation fluid samples of 15 known high-concentration HPV mRNA, and samples of other pathogens (chlamydia trachomatis, ureaplasma urealyticum, gonococcus, herpes simplex virus type ii, treponema pallidum, candida albicans, trichomonas vaginalis, mycoplasma hominis, HPV common low-risk types 40, 42, 43, 44), other HPV genotypes (national human papillomavirus whole genome component type reference) for a total of 27 cases, wherein each clinical sample was verified for infection type, concentration by third party testing, and the RNA was extracted from 27 cases of samples by the magnetic bead method.
Then, 5. Mu.l of each of the primers of type 16 was mixed with 45. Mu.l of HPmR16-F1, HPmR16-R1 and HPmR16-P1 shown in Table 1 in accordance with the reverse transcription PCR reaction solution system shown in Table 4, and the fluorescent PCR reaction was carried out as described in example 2.4.
After the reaction is finished, the instrument automatically stores the result, the software of the instrument can be utilized for automatic analysis (the starting value, the ending value and the threshold line value of the base line can be manually adjusted for analysis), and then the Ct value and the fixed value result of the sample are recorded. As shown in FIG. 3, the internal standard is amplified normally by adopting HPmR16-F1, HPmR16-R1 and HPmR16-P1 detection, the detection of HPV16 type positive has Ct, the amplification curve is S-shaped, and other sample detection has no amplification curve, which indicates that HPmR16-F1, HPmR16-R1 and HPmR16-P1 can only amplify corresponding HPV16 type samples, and other types have no amplification condition. And respectively carrying out specificity detection on other types according to the description mode to respectively obtain respective Ct values and S-type curves, and the results show that: each primer probe can only amplify corresponding HPV, and other types are not amplified. By detecting the specificity, the accuracy of reagent detection is improved.
Example 5 sensitivity detection
Pseudoviruses of E6/7 region of HPV 15 high-risk human papillomaviruses 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 were used at a concentration of 4.00E+05 copies/ml, subjected to 10-fold gradient dilutions, and subjected to assay using the composition described in Table 1 in example 1, as described in examples 2.2-2.4. The results are shown in FIGS. 4-18. And the results of 20 times of detection were repeated for 4.00E+02 copies/ml and 1.00E+02 copies/ml, as shown in Table 6.
The results show that the gradient of the 10-fold gradient dilution curve of each type is better. The detection was repeated 20 times for each type of 1.00E+02 copies/ml, and it was found that the positive detection rate was inconsistent for each type and could not be detected completely, however, when the detection was repeated 20 times for 4.00E+02 copies/ml, the positive detection rate was 100%, and the specific results are shown in Table 6.
The method has high sensitivity and the detection concentration can be as low as 400 copies/mL.
Table 6 sample was repeated 20 times to examine the results
Example 6 detection Effect of RNA extracted by different RNA extraction methods
In the embodiment, two different methods of phenol chloroform and a magnetic bead method are adopted to extract mRNA of a residual cell preservation solution sample positive to known HPV16 type mRNA, so that a better mRNA extraction method is obtained.
And selecting a high-concentration HPV16 type mRNA positive cell preservation solution sample, diluting 3 gradients and dividing the sample into two parts, and respectively extracting and purifying mRNA by adopting two extraction methods of phenol chloroform and a magnetic bead method.
Phenol chloroform RNA extraction (purchased from Tiangen Co.)
Cells were harvested by centrifugation and the supernatant discarded. Every 5-10×10 6 Animal cells and plant cells were added to 1ml of lysate RZ. Cells were not washed before addition of lysate RZ to avoid degradation of mRNA.
The homogenized sample was left at 15-30℃for 5min to allow complete separation of the nucleic acid protein complexes.
The optional steps are as follows: centrifugation was carried out at 12,000rpm (13,400Xg) for 5min at 4℃and the supernatant was transferred to a new RNase-free centrifuge tube.
Note that: if the sample contains more protein, fat, polysaccharide or muscle, plant nodule, etc., this step may be followed by centrifugation. The pellet obtained by centrifugation includes cell outer membrane, polysaccharide, high molecular weight DNA, and RNA in the supernatant solution.
200 μl chloroform was added, the tube was capped, vigorously shaken for 15sec, and left at room temperature for 3min.
Centrifugation at 12,000rpm (13,400 Xg) at 4℃for 10min, the sample will be divided into three layers: the yellow organic phase, the intermediate layer and the colorless aqueous phase, the RNA being predominantly in the aqueous phase, the volume of the aqueous phase being approximately 50% of the lysate RZ reagent used. The aqueous phase was transferred to a new tube for the next operation.
Slowly add 0.5 volume of absolute ethanol and mix well (precipitation may occur at this time). The resulting solution was transferred to the adsorption column CR3 together with the precipitate, and centrifuged at 12,000rpm (13,400 Xg) at 4℃for 30sec, and if the whole solution and the mixture could not be added to the adsorption column CR3 at one time, the solution was transferred to the adsorption column CR3 in two steps, and centrifuged at 12,000rpm (13,400 Xg) at 4℃for 30sec, the waste liquid in the collection tube was discarded.
To the adsorption column CR3, 500. Mu.l of deproteinized solution RD (please check whether ethanol has been added before use) was added, centrifuged at 12,000rpm (13,400 Xg) at 4℃for 30sec, and the waste solution was discarded, and CR3 was placed in a collection tube.
To the adsorption column CR3, 500. Mu.l of a rinse liquid RW (please check whether ethanol had been added) was added, and the mixture was allowed to stand at room temperature for 2min, and centrifuged at 12,000rpm (13,400 Xg) at 4℃for 30sec, and the waste liquid was discarded.
Repeating the operation step 8
The column was placed in a 2ml collection tube and centrifuged at 12,000rpm (13,400 Xg) at 4℃for 2min to remove residual liquid.
Note that: the purpose of this step is to remove the residual rinse liquid from the column, centrifuge and then place the column CR3 at room temperature for a moment, or air-vent it on an ultra clean bench for a moment, to allow for adequate air-drying. If rinsing liquid remains, the subsequent experimental operations such as RT-PCR and the like can be influenced.
Transferring the CR3 column into a new 1.5ml centrifuge tube, adding 30-100 μl RNase-Free ddH 2 O, 2min at room temperature, 12,000rpm (. About.13,400Xg) at 4℃for 2min.
The volume of the elution buffer should not be less than 30. Mu.l, and too small a volume affects recovery efficiency. And the RNA should be stored at-70℃to prevent degradation.
The extraction of RNA by the magnetic bead method was performed as described in example 2.2 above. Then, RNA extracted by both methods was detected and the results were analyzed according to the procedure of examples 2.3 to 2.5 described above. The mRNA of HPV type 16 with high concentration is extracted by two different methods, and the detection result shows that the Ct value of the high-middle-low concentration magnetic bead method is earlier than that of the phenol chloroform extraction method, and the curves are smooth, as shown in figure 19, the mRNA extracted by the magnetic bead method is positioned at the front of each concentration curve, and the mRNA extracted by the phenol chloroform is positioned at the back of each concentration curve. The other types are respectively subjected to RNA extraction efficiency research according to the above description mode, the Ct value of the magnetic bead method is found to be earlier than that of the phenol chloroform, and the magnetic bead method is compared with the phenol chloroform method, so that the magnetic bead method is more suitable for HPV E6/E7mRNA extraction and PCR detection of a cell preservation fluid sample.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> Santa Hunan Biotechnology Co.Ltd
<120> composition, kit and method for high-risk HPV detection and typing
<160> 118
<170> PatentIn version 3.5
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cactacagca aaccgaggta tatga 25
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ccgagtccga gtaatatcgt agc 23
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caaactctgt atatggagag acactgg 27
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tgtctacgtt tttctgctgg gtt 23
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ctggcaccgc aggcacctta ttaac 25
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tgcagtatgc aaacaatgtt tactgt 26
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gtctttgaca tctatgacac cttatcg 27
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cctctaatgt agtaccatac acagacctgc 30
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gaccctgcac gaattgtgtg 20
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acacactgca gccttatttc atgcac 26
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ctaattgctc gtgacataca aggtcaacc 29
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agctgggttt ctctacgtgt tct 23
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atctgcaaca agacatacat cgaccg 26
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agtagtagaa agctcagcag acgac 25
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cgcatgttta cacttgggtt tc 22
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gcaaaatcat atacctcaga tcgtt 25
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actgcatgat ttgtgcaacg a 21
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agtcatatac ctcactccgc tgtaa 25
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aacaaatttc atggatgctt tcttctacc 29
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cacaacgctg gacaccacct tgc 23
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catcactaca agacgtatct attgcc 26
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ctggaataaa agtctataca tttatggc 28
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caaagcaaca ttggaacgca caga 24
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cctttttaca atacacacac actacctg 28
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cgaacgctgc atgaattatg tgaagc 26
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tgtggcttgt tctgcttgtc c 21
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cctacactgc tatgagcaat taggtgac 28
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gcaggagcgg ccacagcaag ctaga 25
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tccaggacgc agaggagaaa c 21
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tatacctcag atcgctgcaa agtc 24
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tcaggcgttg gagacatctg tgc 23
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cgtatgcagc gtgtctgaaa tg 22
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ggcatctata acagcgtatc agca 24
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ctcatgtaac ggtgtcttgg tttcagc 27
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ctcctgagga gaagtctgcc gttac 25
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acaattccta gtgtgcccat taac 24
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atgtctacgt gtgtgctttg tacgca 26
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agcattccag cagctgtttc tga 23
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caaagaccgt tgtgtccaga a 21
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gcatgtttac acttgggttt c 21
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cgtcctgtcc accttcctcc tatgtt 26
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cctgaaccaa ctgacctata ctgct 25
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cgaactgtgg tgttacaagt gtgac 25
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ctcagatgag gatgaaggct tggacc 26
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ccgctgtgtc cagttgaaaa g 21
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cataacatcg gtggacggtg gac 23
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atgtgttgtc tgaaaccgct gt 22
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caacctgaaa caactgacct acactgc 27
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ccacagcaag ctagacaagc taaac 25
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cacaacattg aatattcctc tgcatgat 28
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gcgacgcttc tactactagt tgca 24
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ctactagcca gacgggacga acaaca 26
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cctgaggaga agtctgccgt tactg 25
Claims (9)
1. A composition for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68, the composition comprising the following primers and probes:
an HPV16 type upstream primer shown as SEQ ID NO. 1, an HPV16 type downstream primer shown as SEQ ID NO. 2 and an HPV16 type probe shown as SEQ ID NO. 3;
an HPV18 type upstream primer shown as SEQ ID NO. 4, an HPV18 type downstream primer shown as SEQ ID NO. 5 and an HPV18 type probe shown as SEQ ID NO. 6;
an HPV 31 type upstream primer shown as SEQ ID NO. 7, an HPV 31 type downstream primer shown as SEQ ID NO. 8 and an HPV 31 type probe shown as SEQ ID NO. 9;
an HPV 33 type upstream primer shown as SEQ ID NO. 10, an HPV 33 type downstream primer shown as SEQ ID NO. 11 and an HPV 33 type probe shown as SEQ ID NO. 12;
an HPV 35 type upstream primer shown as SEQ ID NO. 13, an HPV 35 type downstream primer shown as SEQ ID NO. 14 and an HPV 35 type probe shown as SEQ ID NO. 15;
an HPV 39 type upstream primer shown as SEQ ID NO. 16, an HPV 39 type downstream primer shown as SEQ ID NO. 17 and an HPV 39 type probe shown as SEQ ID NO. 18;
an HPV 45 type upstream primer shown as SEQ ID NO. 19, an HPV 45 type downstream primer shown as SEQ ID NO. 20 and an HPV 45 type probe shown as SEQ ID NO. 21;
an HPV 51 type upstream primer shown as SEQ ID NO. 22, an HPV 51 type downstream primer shown as SEQ ID NO. 23 and an HPV 51 type probe shown as SEQ ID NO. 24;
an HPV 52 type upstream primer shown as SEQ ID NO. 25, an HPV 52 type downstream primer shown as SEQ ID NO. 26 and an HPV 52 type probe shown as SEQ ID NO. 27;
HPV53, 56, 66 type upstream primer shown as SEQ ID NO. 28, HPV53, 56, 66 type downstream primer shown as SEQ ID NO. 29 and HPV53, 56, 66 type probe shown as SEQ ID NO. 30;
an HPV 58 type upstream primer shown as SEQ ID NO. 31, an HPV 58 type downstream primer shown as SEQ ID NO. 32 and an HPV 58 type probe shown as SEQ ID NO. 33;
an HPV 59 type upstream primer shown as SEQ ID NO. 34, an HPV 59 type downstream primer shown as SEQ ID NO. 35 and an HPV 59 type probe shown as SEQ ID NO. 36;
an HPV 68 type upstream primer shown as SEQ ID NO. 37, an HPV 68 type downstream primer shown as SEQ ID NO. 38 and an HPV 68 type probe shown as SEQ ID NO. 39;
wherein each probe carries a fluorescent reporter group, the fluorescent reporter groups of SEQ ID NO. 3 and 6 are different, and the fluorescent reporter groups of SEQ ID NO. 3 and 6 are also different from the fluorescent reporter groups of the rest probes;
wherein the composition further comprises an agent required for extracting mRNA and Mg 2+ dNTPs, reverse transcriptase, DNA polymerase, PCR buffer, the final concentration of each component is: mg of 2+ 3-5 mmol/L, dNTPs-5 mmol/L, 0.05-0.2 mu mol/L of each group of upstream and downstream primers, 0.25-0.1 mu mol/L of probe, 10-70 mu mol/L, DNA of reverse transcriptase and 0.5-2 mmol/L of polymerase.
2. The composition of claim 1, wherein the fluorescent reporter group of HPV type 16 probe is CY5, the fluorescent reporter group of HPV type 18 probe is FAM, and the fluorescent reporter groups of probes of the remaining thirteen HPV types are ROX.
3. The composition of claim 1 or 2, wherein the composition further comprises:
an internal standard upstream primer shown in SEQ ID NO. 40, an internal standard downstream primer shown in SEQ ID NO. 41 and an internal standard probe shown in SEQ ID NO. 42.
4. A composition according to claim 3, wherein the internal standard probe carries a fluorescent reporter group HEX or VIC.
5. Use of a composition according to any one of claims 1 to 4 in the manufacture of a kit for detecting HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68.
6. A kit comprising the composition of any one of claims 1-4.
7. The kit of claim 6, wherein the kit is a one-step reverse transcription fluorescent quantitative PCR.
8. The kit according to claim 7, wherein the final concentration of the reverse transcriptase is 30 to 60. Mu. Mol/L.
9. Use for the preparation of an agent for detecting HPV, said use comprising the steps of:
1) Extracting mRNA of a sample to be detected;
2) Performing fluorescent quantitative PCR on the mRNA obtained in step 1) using the composition of any one of claims 1 to 4 or the kit of any one of claims 6 to 8;
3) The results were obtained and analyzed.
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| CN110592279B (en) * | 2019-09-11 | 2023-04-07 | 圣湘生物科技股份有限公司 | Compositions, kits and methods for detecting HPV |
| CN111088398A (en) * | 2019-12-18 | 2020-05-01 | 苏州阅微基因技术有限公司 | Complex amplification system for nine-valent HPV typing detection and kit thereof |
| CN110938712A (en) * | 2019-12-27 | 2020-03-31 | 苏州药明检测检验有限责任公司 | Primer, probe, kit and method for detecting human papilloma virus based on real-time fluorescent quantitative PCR technology |
| CN111172328A (en) * | 2020-03-06 | 2020-05-19 | 杭州博日科技有限公司 | Complete set of primers, complete set of probes, kit and method for HPV nucleic acid typing detection |
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