CN114480648A

CN114480648A - Kit for quickly detecting methylation of cervical exfoliated cells and detection method thereof

Info

Publication number: CN114480648A
Application number: CN202210107725.5A
Authority: CN
Inventors: 李冰琳; 龙兴宏
Original assignee: Xi'an Qisheng Pharmaceutical Technology Co ltd
Current assignee: Xi'an Qisheng Pharmaceutical Technology Co ltd
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2022-05-13
Anticipated expiration: 2042-01-28
Also published as: CN114480648B

Abstract

The invention discloses a kit for quickly detecting methylation of cervical exfoliated cells and a detection method thereof, wherein the kit comprises a primer probe composition, an enzyme digestion amplification buffer solution, methylation sensitive restriction endonuclease and nuclease-free water, the primer probe composition comprises a ZNF671 primer and a probe, a PAX1 primer and a probe, a FAM19A4 primer and a probe, and an ACTIN primer and a probe.

Description

Kit for quickly detecting methylation of cervical exfoliated cells and detection method thereof

Technical Field

The invention belongs to the technical field of methylation detection, and particularly relates to a kit for quickly detecting methylation of cervical exfoliated cells and a detection method thereof.

Background

Cervical cancer is the most common gynecological malignant tumor, high-risk Human Papilloma Virus (HPV) persistent infection is closely related to the occurrence of cervical cancer, after normal cervical cells are infected with HPV virus, most HPV virus can be eliminated by a human immune system, and only a small number of women can cause cervical cancer premalignant lesion and develop cervical cancer due to persistent infection.

At present, cytological detection methods and HPV-DNA detection are mainly used in the primary screening of cervical lesions; cytological detection methods have been used for most of the century, including pap smear and liquid-based cytology (TCT) developed over a decade, and have a high probability of missed detection due to the fact that the whole detection process is artificially influenced in multiple links from sampling-smear preparation-observation-result interpretation; the HPV-DNA tests only for the presence or absence of HPV virus, which is mostly transient, means that these auto-immune cleared HPV infections are also judged positive, leading to over-treatment and colposcopic referrals.

In addition to the above two methods, methylation detection is also commonly used as a preliminary screening for cervical lesions, and methylation-specific pcr (msp), bisulfite sequencing pcr (bsp), methylation-sensitive high-resolution melting curve analysis (MS-HRM), fluorescence quantification, gene chip methods, and the like are commonly used, and are methylation detection technologies based on sulfite modification pretreatment, which are based on the principle that after DNA reacts with sodium bisulfite, unmethylated cytosine (C) can be converted to uracil (U), while methylated cytosine does not show structural changes, and methylated cytosine is determined by distinguishing structural differences between uracil and methylated cytosine; although the BC strategy provides excellent performance in methylation detection, it still has the disadvantages of tedious pretreatment, time-consuming process, need of multiple reagents and extreme experimental conditions (such as strong acid, strong base and high temperature), and what is worse, the BC strategy has the problems of target degradation, low DNA recovery rate and incomplete conversion, etc., resulting in inaccurate measurement and poor repeatability of methylation detection, especially low methylation abundance.

Therefore, there is an urgent need to explore more sensitive and rapid methylation analysis methods, and Methylation Sensitive Restriction Enzymes (MSRE) are a class of bacterial restriction enzymes that specifically cleave unmethylated CpG dinucleotides within the active site of the enzyme, leaving only methylated DNA as a complete template for PCR or qPCR; the method can be used to determine the CpG methylation frequency of any particular region of interest amplified by PCR, and compared with the sulfite detection method, the detection operation is somewhat simplified, but the detection time is not obviously improved.

Disclosure of Invention

The invention aims to provide a kit for quickly detecting methylation of cervical exfoliated cells and a detection method thereof.

The technical scheme adopted by the invention is that the kit is used for quickly detecting the methylation of the cervical exfoliated cells, and comprises a primer probe composition, an enzyme digestion amplification buffer solution, methylation sensitive restriction endonuclease and nuclease-free water;

the primer probe composition comprises a ZNF671 primer pair, a ZNF671 fluorescent probe, a PAX1 primer pair, a PAX1 fluorescent probe, a FAM19A4 primer pair, a FAM19A4 fluorescent probe, an ACTIN primer pair and an ACTIN fluorescent probe.

Furthermore, the sequence of the ZNF671 primer pair is shown as SEQ ID NO.1-2, and the sequence of the ZNF671 fluorescent probe is shown as SEQ ID NO. 3;

the sequence of the PAX1 primer pair is shown as SEQ ID NO.4-5, and the sequence of the PAX1 fluorescent probe is shown as SEQ ID NO. 6;

the sequence of the FAM19A4 primer pair is shown as SEQ ID NO.7-8, and the sequence of the FAM19A4 fluorescent probe is shown as SEQ ID NO. 9;

the sequence of the ACTIN primer pair is shown in SEQ ID NO.10-11, and the sequence of the ACTIN fluorescent probe is shown in SEQ ID NO. 12.

Further, the fluorescent reporter groups on the ZNF671 fluorescent probe, the PAX1 fluorescent probe, the FAM19A4 fluorescent probe and the ACTIN fluorescent probe are all selected from FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3, NED, Texas Red or JOE;

the ZNF671 fluorescent probe, the PAX1 fluorescent probe and the FAM19A4 fluorescent probe have the same or different fluorescent reporter groups;

the fluorescent reporter group of the ACTIN fluorescent probe is different from that of ZNF671 fluorescent probe, PAX1 fluorescent probe and FAM19A4 fluorescent probe.

Further, the enzyme digestion amplification buffer solution comprises 40mmol/L Tris-HCl, 200mmol/L KCl and 40mmol/L MgCl₂0.4mg/mL recombinant albumin, 0.6% trehalose, 1.5mmol/L HS Taq enzyme, 1mmol/L dNTP.

Further, the methylation sensitive restriction enzyme is selected from one or more of AatII, AccI, AciI, AgeI-HF, BaeI, BglI, BsaAI, HaeII, HhaI, HinfI, HpaII, Hpy166II, HpyCH4IV, NheI-HF, NotI-HF, SacI-HF, SalI-HF, SrfI, TfiFi I, BstUI.

The methylation detection method of the kit for quickly detecting the methylation of the cervical exfoliated cells comprises the following steps:

step S1, measuring the enzyme digestion amplification buffer solution, the primer probe composition and the nuclease-free water respectively, carrying out vortex oscillation for 15S to fully mix the buffer solution, the primer probe composition and the nuclease-free water uniformly, and subpackaging the mixed solution into PCR reaction tubes;

and step S2, adding methylation sensitive restriction enzyme into the mixed solution, blowing uniformly, adding substrate DNA to form a reaction system, performing enzyme digestion and PCR amplification, and performing methylation detection on the amplified product.

Further, the total volume of the reaction system is 25 mu L, and the reaction system comprises 7.5 mu L of enzyme digestion amplification buffer solution, 6 mu L of primer probe composition, 7.5 mu L of nuclease-free water, 2 mu L of methylation sensitive restriction enzyme and 2 mu L of substrate DNA;

the final concentration of each primer pair and the fluorescent probe in the primer probe composition is 0.3-1.5 mu mol/L, the concentration of the methylation sensitive restriction endonuclease is 2.5-12.5U/mu L, and the mass concentration of the substrate DNA is 2.5-250 ng/mu L.

Further, the PCR amplification process is as follows: the reaction is carried out for 20min at 37 ℃, the pre-denaturation is carried out for 5min at 95 ℃, the reaction is carried out for 10s at 95 ℃ and 30s at 60 ℃ for 45 cycles.

The invention has the beneficial effects that: according to the embodiment of the invention, the methylation sensitive restriction enzyme is added into the improved PCR amplification system, so that rapid enzyme digestion and PCR amplification are realized in the same system, the operation process is simple, the transformation process is avoided, the problems of target degradation, low DNA recovery rate, incomplete transformation and the like are reduced, and the sensitivity of methylation detection is greatly improved;

in the embodiment of the invention, excessive methylation sensitive restriction enzyme is used for carrying out enzyme digestion on the DNA of a sample to be detected, meanwhile, the components of an enzyme digestion amplification buffer solution are improved, the activity and the persistence of the methylation sensitive restriction enzyme are enhanced, a hypertonic environment is formed to promote the opening of a secondary structure of nucleic acid, the enzyme digestion and amplification reaction is accelerated, and the detection time is greatly shortened;

in the embodiment of the invention, the PAX1 gene, the FAM19A4 gene and the ZNF671 gene are combined to be used as methylation detection markers, so that the kit has excellent sensitivity and specificity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram of the design of the cleavage sites and primers for PAX1, FAM19A4, ZNF671, ACTIN.

FIG. 2 is a statistical chart of HPV positivity and methylation detection results at various stages.

Fig. 3 is a graph showing effects of embodiment 1 and embodiment 2 of the present invention.

Fig. 4 is an effect diagram of embodiment 3 of the present invention.

Fig. 5 is an effect diagram of embodiment 4 of the present invention.

In FIG. 6, a is a graph showing the effect at a KCl molar concentration of 100mmol/L, b is a graph showing the effect at a KCl molar concentration of 200mmol/L, and c is a graph showing the effect at a KCl molar concentration of 300 mmol/L.

In FIG. 7, a is the result of detection of a positive sample when recombinant albumin and trehalose are added, b is the result of detection of a negative sample when recombinant albumin and trehalose are added, c is the result of detection of a positive sample when recombinant albumin and trehalose are not added, and d is the result of detection of a negative sample when recombinant albumin and trehalose are not added.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The kit for quickly detecting the methylation of the exfoliated cervical cells comprises a primer probe composition, an enzyme digestion diffusion buffer solution and a methylation sensitive restriction enzyme, wherein the components of the components are shown in a table 1:

TABLE 1 Contents of Components of the kit

The primer probe composition is an amplification primer pair and a fluorescent probe of a marker for indicating methylation condition of cervical exfoliated cells, and comprises a PAX1 primer pair, a PAX1 fluorescent probe, a FAM19A4 primer pair, a FAM19A4 fluorescent probe, a ZNF671 primer pair, a ZNF671 fluorescent probe, an ACTIN primer pair and an ACTIN fluorescent probe, wherein the PAX1 gene corresponds to a position 21705875 and 21721721713 on chromosome 20, the FAM19A4 gene corresponds to a position 68932487 and 68932644 on chromosome 3, the ZNF671 gene corresponds to a position 57727335 and 57727452 on chromosome 19, and the ACTIN gene corresponds to a position 553205532159 and 5532150 on chromosome 7.

ZNF671 zinc finger protein is closely related to the processes of expression regulation and control of gene, cell differentiation, malignant transformation of cells and the like, FAM19A4 gene can improve phagocytic capacity of macrophages and plays an important role in infectious diseases, and Pax gene (Paired box genes) is an evolutionarily conserved gene family, is named by coding a pairing domain (Paired domain) consisting of 128 amino acids and is related to the differentiation of cells; the occurrence of cervical cancer is a multi-cause and multi-step process, the PAX1 gene, the FAM19A4 gene and the ZNF671 gene relate to different functions of different signal paths, can comprehensively reflect the risk effects of genetic and non-genetic factors, and the methylation change of the gene combination is earlier than the genetic change in the continuous infection process of HPV, and plays a very important role in the occurrence, the progression and the malignant change process of cancer; these several restriction loci are methylated to different extents in cervical lesions and normal cervical exfoliated cells, i.e., these loci are methylated in cervical lesion exfoliated cells as compared to normal cervical cells.

The ACTIN gene is used as an internal reference gene and does not contain restriction sites of methylation sensitive restriction enzymes, and when a DNA sample is digested by the methylation sensitive restriction enzymes, the sequence of the internal reference gene locus is always kept complete and is used for controlling the DNA concentration, the purity and the quality of the multiple QPCR reaction solution.

The sequences of the PAX1 locus, the FAM19a4 locus, the ZNF671 locus, the ACTIN locus, and the components of the composition are shown in table 2, and the PAX1 locus, the FAM19a4 locus, and the ZNF671 locus contain restriction sites for at least 3 methylation sensitive restriction enzymes; the PAX1 primer pair, the PAX1 fluorescent probe, the FAM19A4 primer pair, the FAM19A4 fluorescent probe, the ZNF671 primer pair and the ZNF671 fluorescent probe do not contain methylase cleavage sites, if the primer pair and the fluorescent probe contain the methylase cleavage sites, the methylase cleavage sites are subjected to methylation modification so as to avoid the methylate sensitive restriction endonuclease from carrying out enzyme cleavage on the primers and the probes, and the fluorescent reporter gene is selected from two or more of FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3, NED, Texas Red and JOE, but is not limited to the above.

TABLE 2 sequences of the substances

In application, two or four fluorescent reporter groups can be selected, namely one fluorescent reporter group is used for the reference gene when two fluorescent reporter groups are selected, three markers share one fluorescent reporter group, the sum of the methylation levels of the groups can be conveniently detected through one fluorescent channel, and the detection efficiency is high; when four markers are selected, namely the reference gene and the three markers, a fluorescence reporter group is used respectively, so that the fluorescence reporter groups are positioned in different channels, mutual interference is avoided, and the detection result is more accurate.

The components of the primer probe composition are mixed in an independent package, and the dosage of each primer and each fluorescent probe is 0.3 mu mol/L-1.5 mu mol/L.

The methylation sensitive restriction endonuclease is one or more of AatII, AccI, AciI, AgeI-HF, BaeI, BglI, BsaAI, HaeII, HhaI, HinfI, HpaII, Hpy166II, HpyCH4IV, NheI-HF, NotI-HF, SacI-HF, SalI-HF, SrfI, TfiFII and BstUI, and as shown in figure 1, the selected marker contains dozens of enzyme cutting sites, the enzyme cutting efficiency can be effectively improved by increasing the variety and the addition amount of the enzyme, and the enzyme cutting time is shortened; the addition amount of each enzyme in the kit is 5-10U, and the total addition amount of the enzyme is less than 25U.

The enzyme digestion amplification buffer solution comprises 20-60mmol/L Tris-HCl, 100-200mmol/L KCl and 20-60mmol/L MgCl₂0.2-0.6mg/mL recombinant albumin, trehalose with the mass volume percentage of 0.4% -0.8%, 1-3mol/L HS Taq enzyme and 1-3mmol/L dNTP; the invention uses recombinant albumin and trehalose as enhancers of enzyme digestion reaction and PCR amplification, is favorable for improving the thermal stability of enzyme, maintains the conformation of methylation sensitive restriction enzyme and amplified HS enzyme, enhances the continuity of enzyme activity, and the trehalose can also form a hypertonic environment to promote the opening of a secondary structure of nucleic acid, is favorable for the implementation of enzyme digestion and amplification reaction, and simultaneously improves the Mg content²⁺The concentration of the methylation sensitive restriction enzyme is increased, but the non-specific amplification of the PCR is increased by doing so, so the invention avoids the generation of the non-specific amplification by a primer design mode.

Example 1

1. Preparing a kit for quickly detecting methylation of cervical exfoliated cells, wherein the kit comprises a primer probe composition, methylation sensitive restriction endonuclease and enzyme digestion amplification buffer solution; the molar concentrations of the ZNF671 upstream primer, the ZNF671 downstream primer and the ZNF671 fluorescent probe in the primer probe composition are all 0.5 mu mol/L, the molar concentrations of the ACTIN upstream primer, the ACTIN downstream primer and the ACTIN fluorescent probe are all 1 mu mol/L, the molar concentrations of the PAX1 upstream primer, the PAX1 downstream primer and the PAX1 fluorescent probe are all 1.2 mu mol/L, and the molar concentrations of the FAM19A4 upstream primer, the FAM19A4 downstream primer and the FAM19A4 fluorescent probe are 0.8 mu mol/L, so that the amplification efficiencies or the fluorescence signal values of all markers are consistent as much as possible, and the unification of result judgment standards is facilitated.

The fluorescent reporter group of the PAX1 gene fluorescent probe is ROX, the fluorescent reporter group of the FAM19A4 gene fluorescent probe is CY5, the fluorescent reporter group of the ZNF671 gene fluorescent probe is FAM, and the fluorescent reporter group of the reference gene ACTIN fluorescent probe is VIC.

The methylation sensitive restriction endonuclease is prepared by diluting three enzymes of AciI, HpaI and HpaII to 10U/. mu.L and mixing the three enzymes in equal volume.

The final concentration of each component in the enzyme digestion amplification buffer solution is as follows: 40mmol/L Tris-HCl, 200mmol/L KCl, 40mmol/L MgCl₂0.4mg/mL recombinant albumin, 0.6% trehalose, 1.5mmol/L HS Taq enzyme, 1mmol/L dNTP.

2. The kit is used for methylation detection of cervical exfoliated cells, and the specific process is as follows:

(one) reagent preparation

(1) Taking out each reagent in advance, carrying out vortex oscillation on the enzyme digestion amplification buffer solution and the primer probe composition for 15s, carrying out low-speed centrifugation for later use, determining the reaction number N, wherein the reaction number N is equal to the number of samples to be detected (N), the number of quality control products (2) and a blank control, and carrying out positive control, negative control and blank control analysis simultaneously in each PCR experiment;

(2) respectively measuring 6 muL of the spare primer probe composition in the step (1), 7.5 muL of enzyme digestion amplification buffer solution and 7.5 muL of nuclease-free water in an EP tube, carrying out vortex oscillation for 15s to fully mix the primer probe composition, the enzyme digestion amplification buffer solution and the nuclease-free water, and subpackaging the reaction solution into PCR reaction tubes according to 21 muL/tube;

(3) mu.L of the methylation sensitive restriction enzyme was added to each PCR reaction tube by a pipette under the liquid surface of the reaction solution, and the mixture was pipetted several times.

(II) sample application detection

Extracting DNA of cervical exfoliated cells as a sample to be detected, adding the DNA of the sample to be detected, a negative/positive control and a blank control into each PCR reaction tube according to the sample adding amount of 2 mu L, tightly covering a tube cover, centrifuging at low speed for 5s to completely throw liquid on the tube wall to the tube bottom, and then immediately carrying out enzyme digestion and PCR amplification reaction.

The positive control is 2% methylated genomic DNA with the concentration of 20 ng/. mu.L and is used for providing reference for low-concentration samples and critical methylation rate, the negative control is 0% methylated genomic DNA with the concentration of 100 ng/. mu.L, the positive control and the negative control provide reference for methylation state judgment of samples to be detected, and meanwhile, the quality control is carried out on the effectiveness of the reaction solution.

The concentration of the DNA of the sample to be detected is 2.5 ng/muL-250 ng/muL, the positive control is formed by diluting the DNA of the human cervical cancer cell strain C33A and the CaSki DNA of the human cervical cell strain to 20 ng/muL and then mixing the two according to the volume ratio of 49: 1; the negative control is prepared by diluting the human cervical cancer cell strain C33A DNA to 100 ng/. mu.L; the blank was nuclease-free water.

(III) enzyme digestion, PCR amplification

And (3) performing amplification by using a PCR instrument, wherein the reaction system is 25 mu L, and the amplification conditions are as follows: 20min at 37 ℃, 5min at 95 ℃, 10s at 95 ℃ and 30s at 60 ℃ for 45 cycles, carrying out enzyme digestion reaction on a reaction system at 37 ℃, carrying out enzyme digestion on cervical exfoliated cell DNA by using methylation sensitive restriction enzyme, cutting off normal gene sequences which are not methylated, not cutting methylated gene sequences, carrying out reaction at 95 ℃ for 5min to inactivate the methylation sensitive restriction enzyme, simultaneously carrying out pre-denaturation on the methylated cervical cell DNA, and carrying out PCR amplification on methylated gene fragments by the system at 95 ℃ for 10s at 60 ℃ for 45 cycles to obtain amplification products.

In the embodiment, the DNA of the sample to be detected is subjected to enzyme digestion by using excessive methylation sensitive restriction enzyme of 10 times, which is beneficial to overcoming the adverse factors caused by different sources, different qualities and different purities of the DNA of the sample to be detected and preventing incomplete enzyme digestion caused by excessive DNA of the sample to be detected; multiple endonucleases are used simultaneously to ensure complete digestion of unmethylated DNA, thereby improving the sensitivity and specificity of methylation detection.

Methylation detection is carried out on the amplification product, the detection result is shown as a curve (r) in figure 3, wherein the reference genes and the fluorescent reporter groups of all the markers are different and are positioned in different channels, the detection results are not interfered with each other, and the detection results of all the target genes can be independently counted and clinically analyzed.

(IV) interpretation of results

(1) Validity determination

Setting the Ct critical value of the methylation detection result to be 36-39, and detecting the methylation state of ZNF671, FAM19A4 and PAX1 by using the kit and the detection method, wherein all channels in the blank control detection result of the kit are No Ct or the Ct value is more than or equal to 40, the detection result of a positive control is positive, the detection result of a negative control is negative, and the specific Ct value is shown in Table 3.

TABLE 3 methylation status detection results of ZNF671, FAM19A4 and PAX1 in quality control substances

(2) Criteria for determination of results

Setting Baseline: baseline is generally set to be 3-15 cycles, and can be specifically adjusted according to actual conditions, wherein the adjustment principle is as follows: selecting a region with a more stable fluorescence signal before exponential amplification, wherein the starting point (Start) avoids the signal fluctuation of the initial stage of fluorescence acquisition, and the End point (End) is reduced by 1-2 cycles compared with the sample Ct with the earliest exponential amplification; threshold setting: the set rule is that the threshold line just exceeds the highest point of the normal negative control, and the result judgment standard is shown in table 4.

TABLE 4 criteria for determination of results

4. Methylation detection of clinical samples Using the kit of this example

(1) Diversion using methylation detection results

The kit assembled in the embodiment is used for respectively carrying out hr-HPV and methylation detection on 284 clinical samples in different stages, the specific detection results are shown in Table 5 and figure 2, the detection results show that the positive rate of hr-HPV in low-grade lesions is high, and the methylation detection can accurately shunt the hr-HPV positive samples, so that the HPV detection specificity is obviously improved.

TABLE 5 clinical samples hr-HPV and methylation detection results

Staging of pathology	hr-HPV positivity	Positive rate of methylation
			Inflammation(s)	77.8％(42/54)	7.1％(4/54)
CIN1	82.4％(28/34)	17.6％(6/34)
			CIN2	84.2％(32/38)	52.6％(20/38)
CIN3	91.5％(86/94)	89.4％(84/94)
			CA	100％(64/64)	100％(64/64)

(2) Methylation detection and TCT result comparison

The AO + inflammation and ASCUS samples are generally considered to be low risk, but partial high-grade lesions can be found from the methylation detection results, which indicates that the methylation detection can primarily screen out potential lesions, improves the accuracy of cytological screening detection, and guides the partial population to perform prevention and further detection and treatment in time, and the specific detection result is compared with data shown in Table 6.

Compared with the prior art, the embodiment combines multiple QPCR amplification and optimized MSRE technology, skillfully combines enzyme digestion and amplification in the same system, reduces tedious operation of sulfite transformation, reduces transformation time (3-4h) and DNA loss in the operation process, realizes high-sensitivity and rapid detection of methylation, and can provide gene angle suggestions for clinical HPV positive and/or cytologically abnormal women by detecting the methylation states of ZNF671, FAM19A and PAX1 in cervical exfoliated cells.

TABLE 6 data of various types of test results

Sample numbering	Cytological diagnosis	Results of methylation detection	Pathological diagnosis
				1	Inflammation(s)	Negative of	CIN1
2	Inflammation(s)	Positive for	CIN3
				3	Inflammation(s)	Negative of	CIN1
4	Inflammation(s)	Negative of	CIN1
				5	Inflammation(s)	Negative of	Is normal
6	Inflammation(s)	Negative of	Is normal
				7	Inflammation(s)	Negative of	CIN1
8	ASCUS	Negative of	CIN2
				9	ASCUS	Negative of	CIN1
10	ASCUS	Negative of	CIN1
				11	ASCUS	Positive for	CIN3
12	ASCUS	Negative of	CIN1
				13	ASCUS	Negative of	CIN1
14	ASCUS	Positive for	CIN3

Example 2

The fluorescent reporter in example 1 was adjusted as follows: the ZNF671 fluorescent probe, the FAM19A4 fluorescent probe and the PAX1 fluorescent probe are marked by the same fluorescent group (such as FAM fluorescent mark), the fluorescent reporter group of the internal reference gene ACTIN fluorescent probe is marked by the other channel (such as VIC fluorescent mark), methylation detection is carried out on an amplification product of a sample to be detected, the detection result is shown as a curve II in figure 3, the methylation sum of the ZNF671 gene, the FAM19A4 gene and the PAX1 gene can be obtained by detecting the FAM fluorescent channel, the detection efficiency is greatly improved, the target gene detection Ct value in the same sample (particularly for a critical value sample) is lower, and when the methylation level of a single marker is lower, if the methylation level sum of the three markers can be detected, a more accurate detection result can also be obtained, so that the detection limit of the methylation level is reduced, and the sensitivity is better.

Example 3

The methylation detection is performed by the method described in example 1 by using various gene combinations as markers to assemble the detection kit, the detection data is shown in table 7 and fig. 4, the percentage data in table 7 and fig. 4 indicate the positive rate proportion at each stage when the methylation detection is performed by using each gene combination, for the population (such as CA) with high risk of canceration, the higher the detected positive rate indicates that the clinical sensitivity of the gene combination to the disease is higher, and for the normal population and the population with low risk of canceration, the lower the detected positive rate indicates that the clinical specificity of the gene combination to the disease is higher, and the table 7 and fig. 4 show that the gene combination provided by the invention has excellent sensitivity and clinical specificity compared with other gene combinations, can accurately detect the pathological changes of cervical cells, and can shunt positive samples.

TABLE 7 Multi-Gene Joint methylation assay data

The ZNF671 in the screened targets has the highest sensitivity for primarily screening cervical cancer and high-order lesions, the combined detection of PAX1, FAM19A4 and ZNF671 can further improve the detection sensitivity of ZNF671 under the condition of ensuring specificity, the complementary action of other targets and ZNF761 is not obvious, and the combined detection with ZNF671 has no great improvement or poor specificity for primarily screening the high-order lesions or the cervical cancer.

Example 4

Preparing an enzyme digestion amplification buffer solution, wherein the concentration of each substance is as follows: 40mmol/L Tris-HCl, 200mmol/L KCl, 0.4Mg/mL recombinant albumin, 0.6% trehalose, 1.5mmol/L HS Taq enzyme, 1mmol/L dNTP, adjusting the concentration of Mg ions in the digestion amplification buffer solution to 10mmol/L, 20mmol/L, 30mmol/L and 40mmol/L respectively, carrying out digestion amplification and methylation detection on the methylation negative sample by using the four digestion amplification buffer solutions, wherein the detection result is shown in figure 5, as shown in figure 5, the value of a target gene signal value delta Rn is gradually reduced along with the increase of the Mg ion concentration, namely the result of detecting false positive is reduced, because when the Mg ion concentration is lower, the activity of the methylation sensitive restriction enzyme is lower, so that the digestion is incomplete, a false positive result is generated, and as the Mg ion concentration is increased, the methylation sensitive restriction endonuclease has improved activity, the enzyme digestion is gradually completed, no false positive result is generated, and the signal value tends to zero.

Example 5

Preparing an enzyme digestion amplification buffer solution, wherein the concentration of each substance is as follows: 40mmol/L Tris-HCl, 40mmol/L MgCl₂0.4mg/mL recombinant albumin, 0.6% trehalose, 1.5mmol/L HS Taq enzyme, 1mmol/L dNTP, adjusting the KCl molar concentrations to 100mmol/L, 200mmol/L, 300mmol/L, respectively, using the three enzyme digestion amplification buffers to perform enzyme digestion amplification and methylation detection on a methylation negative sample, the detection results are shown in FIG. 6, a, b, c in FIG. 6 are detection result graphs when the KCl molar concentrations are 100mmol/L, 200mmol/L, 300mmol/L, respectively, the Ct value in real-time fluorescence PCR means the number of cycles that the fluorescence signal in each reaction tube reaches a set threshold, a in FIG. 6 shows that when the KCl molar concentration is low, the Ct value detected increases, the efficiency showing PCR amplification efficiency is low, and c in FIG. 6 shows that the KCl molar concentration is too high, a false positive detection result appears, which indicates that the enzyme digestion is incomplete, the activity of the methylation sensitive restriction enzyme is inhibited by the high-concentration KCl, and partial normal gene fragments are not completely digested, and the enzyme digestion effect and the PCR amplification efficiency are best when the molar concentration of the KCl is set to be 200mmol/L as shown in b in FIG. 6.

Example 6

Preparing two groups of enzyme digestion amplification buffer solutions, wherein one group is added with 0.4mg/mL recombinant albumin and 0.6% trehalose, the other group is not added, and the concentrations of the other components are 40mmol/L Tris-HCl, 200mmol/L KCl and 40mmol/L MgCl₂1.5mmol/L HS Taq enzyme and 1mmol/L dNTP, and the degree of methylation of the amplification is detected after the enzymatic amplification using the reaction system described in example 1, as shown in FIG. 7, wherein a and b are those to which recombinant albumin and trehalose are addedAs can be seen from FIG. 7, the addition of recombinant albumin and trehalose can reduce the Ct value, improve the PCR amplification efficiency, and improve the detection of positive rate for low-concentration methylated samples.

All the embodiments in the present specification are described in a related manner, and the same and similar parts among the embodiments may be referred to each other, and each embodiment focuses on differences from other embodiments. In particular, for the system embodiment, since it is substantially similar to the method embodiment, the description is simple, and for the relevant points, reference may be made to the partial description of the method embodiment.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

SEQUENCE LISTING

<110> Xian Qinsheng medicine science and technology Limited

<120> kit for quickly detecting methylation of cervical exfoliated cells and detection method thereof

<130> 2022.1.26

<160> 16

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<213> Artificial sequence (Artificial sequence)

<400> 8

aggcaggact cccgatgttt 20

<210> 9

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

tagctgggct cggct 15

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

gaggctgctt gttccagttc a 21

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

tgggtggtgg tcatctttcc 20

<210> 12

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

ctcagccaat gggacct 17

<210> 13

<211> 202

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

aaagggtgac tgagggcccg gaggacgcag cacccacccg cgcggagtcc gttagctccg 60

ccataggacc gtgggcgcgg acagctgccg ggagcggcag gcgtctcgat cggggacgca 120

ggcacttccg tccctgcaga gcatcagacg cgtctcggga cactggggac aacatctcct 180

ccgcgctttc ccaacacctc ca 202

<210> 14

<211> 139

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

gccctccctc tatgcctctc acgcggcggc ggcggcgccc aagctctccc ggactgcgcc 60

gggcccagcc ccggccaccc cggccacccc ggcgccaggc agctggccgg cccgctcgct 120

atgggtaagg ggcgggcga 139

<210> 15

<211> 158

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

tgcccgcagc cgcgcgcgca gtcggtgcgc cccgtccagg gcggcgcgca gctagcagtg 60

cggagccgag cccagctagc gcgctgacca ggaggggccg ggcccgccgc ggaacgcgct 120

gattggccgg gccgcccggc tgctcctgtc cctgcctg 158

<210> 16

<211> 92

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

gaggctgctt gttccagttc acggtacaag gctgtgctca gccaatggga cctgctcctc 60

ccttgaaggt tgcagaggcc acagcctggt gg 92

Claims

1. The kit for quickly detecting the methylation of the exfoliated cervical cells is characterized by comprising a primer probe composition, an enzyme digestion amplification buffer solution, methylation sensitive restriction endonuclease and nuclease-free water;

2. The kit for rapidly detecting methylation of exfoliated cervical cells according to claim 1, wherein the sequences of the ZNF671 primer pair are shown as SEQ ID No.1-2, and the sequence of the ZNF671 fluorescent probe is shown as SEQ ID No. 3;

the sequence of the ACTIN primer pair is shown as SEQ ID NO.10-11, and the sequence of the ACTIN fluorescent probe is shown as SEQ ID NO. 12.

3. The kit for rapid detection of methylation of cervical exfoliated cells of claim 1, wherein the fluorescent reporter groups on the ZNF671 fluorescent probe, the PAX1 fluorescent probe, the FAM19A4 fluorescent probe and the ACTIN fluorescent probe are all selected from FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3, NED, Texas Red and JOE;

4. The kit for rapid detection of methylation of cervical exfoliated cells according to claim 1, wherein the enzymatic amplification buffer comprises 40mmol/L Tris-HCl, 200mmol/L KCl, 40mmol/L MgCl₂0.4mg/mL recombinant albumin, 0.6% trehalose, 1.5mmol/L HS Taq enzyme, 1mmol/L dNTP.

5. The kit for rapid detection of methylation of exfoliated cervical cells according to claim 1, wherein the methylation sensitive restriction enzyme is selected from one or more of AatII, AccI, AciI, AgeI-HF, BaeI, BglI, BsaAI, HaeII, HhaI, HinfI, HpaII, Hpy166II, HpyCH4IV, NheI-HF, NotI-HF, SacI-HF, SalI-HF, SrfI, Tfifi, BstUI.

6. The methylation detection method by using the kit for the rapid detection of the methylation of the cervical exfoliated cells according to any one of claims 1 to 5, wherein the method comprises the following steps:

7. The methylation detection method according to claim 6, wherein the total volume of the reaction system is 25 μ L, and the reaction system comprises 7.5 μ L of enzyme digestion amplification buffer solution, 6 μ L of primer probe composition, 7.5 μ L of nuclease-free water, 2 μ L of methylation-sensitive restriction enzyme and 2 μ L of substrate DNA;

8. The methylation detection method of claim 7, wherein the PCR amplification process is as follows: the reaction is carried out for 20min at 37 ℃, the pre-denaturation is carried out for 5min at 95 ℃, the reaction is carried out for 10s at 95 ℃ and 30s at 60 ℃ for 45 cycles.