CN116949222A - Detection primer, detection probe, kit and method for accurate stage of invasive cervical squamous carcinoma - Google Patents

Detection primer, detection probe, kit and method for accurate stage of invasive cervical squamous carcinoma Download PDF

Info

Publication number
CN116949222A
CN116949222A CN202311160451.7A CN202311160451A CN116949222A CN 116949222 A CN116949222 A CN 116949222A CN 202311160451 A CN202311160451 A CN 202311160451A CN 116949222 A CN116949222 A CN 116949222A
Authority
CN
China
Prior art keywords
seq
detection
squamous carcinoma
cervical squamous
invasive cervical
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202311160451.7A
Other languages
Chinese (zh)
Inventor
陈涛
孙如美
刘栋
吴涛
程雅婷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Jinyu Translational Medical Research Institute Co ltd
Original Assignee
Guangzhou Jinyu Translational Medical Research Institute Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Jinyu Translational Medical Research Institute Co ltd filed Critical Guangzhou Jinyu Translational Medical Research Institute Co ltd
Priority to CN202311160451.7A priority Critical patent/CN116949222A/en
Publication of CN116949222A publication Critical patent/CN116949222A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Virology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a detection primer, a detection probe, a kit and a method for accurate stage of invasive cervical squamous carcinoma, wherein the detection primer comprises the following components: SEQ ID No.6 and SEQ ID No.7 for HPV16, SEQ ID No.9 and SEQ ID No.10 for HPV18, SEQ ID No.12 and SEQ ID No.13 for HPV33, SEQ ID No.15 and SEQ ID No.16 for HPV52, SEQ ID No.18 and SEQ ID No.19 for HPV 58. The invention can clearly determine the correlation between the DNA load of the ctHPV and the occurrence and development of cervical cancer, thereby realizing the accurate differentiation of invasive cervical squamous carcinoma precancerous lesions, early cervical cancer and late cervical cancer.

Description

Detection primer, detection probe, kit and method for accurate stage of invasive cervical squamous carcinoma
Technical Field
The invention belongs to the technical field of medical detection, and particularly relates to a detection primer, a detection probe, a kit and a method for accurate stage detection of invasive cervical squamous carcinoma based on the DNA load of peripheral blood ctHPV.
Background
Cervical cancer is the fourth most common cancer in women worldwide, with about 70% of squamous carcinomas, about 20% of adenocarcinomas, and about 10% of adenosquamous carcinomas. The cervical cancer onset process is sequentially divided into: hyperplasia, atypical hyperplasia, carcinoma in situ, and invasive carcinoma, wherein cervical atypical hyperplasia and carcinoma in situ are collectively referred to as CIN. Furthermore, according to the clinical staging criteria for cervical cancer of the International gynaecological and obstetrics Union (FIGO), cervical invasive carcinoma is generally staged 1 to 4 corresponding to Roman numerals I, II, III and IV, with stages IA1-IB2 being early cervical cancer and stages IB3-IVA being late cervical cancer.
99.7% of cervical cancers are associated with persistent infection by human papilloma virus (Human Papillomavirus, HPV), a circular double-stranded DNA virus, more than 200 subtypes exist. The HPV genome is divided into 3 regions: non-coding region, early transcribed region (including E1, E2, E4, E5, E6, E7 genes), late transcribed region (including L1, L2 genes).
At present, cytology/HPV detection, colposcopy and cervical biopsy are main methods for clinically diagnosing cervical cancer and precancerous lesions, wherein colposcopy or direct-view cervical biopsy is a final diagnosis gold standard, but colposcopy or direct-view cervical biopsy currently lacks uniform international image diagnosis standards, the examination process is influenced by subjective judgment and clinical experience of clinicians, and cervical biopsy belongs to invasive puncture examination, and wound infection risk exists, so that a certain wound is caused to a patient, and the patient is easy to contradict psychology.
With the development of new technology, cervical cancer staging methods based on cervical exfoliated cell gene detection and cervical cancer staging methods based on digital PCR detection of peripheral blood ctHPV DNA are successively presented. The cervical cancer staging method based on cervical exfoliated cell gene detection illustrates the correlation between P53 and P16 and cervical cancer occurrence and development from the perspective of exfoliated cells, and provides a new basis for cervical cancer staging diagnosis, however, the method can only distinguish precancerous lesions and cervical cancer, and cannot further stage cervical cancer accurately. According to the cervical cancer stage method for detecting the peripheral blood ctHPV DNA based on digital PCR, circulating tumor DNA (ctDNA) is extracted from peripheral blood, specific primers and probes are designed based on an E7 region of HPV viral sequences, and the correlation between the ctHPV DNA load and the occurrence and development of cervical cancer is detected by using a digital PCR technology, but the method can only effectively detect the ctHPV DNA in cervical cancer later patients, and can not accurately distinguish cervical cancer early stages from cervical cancer later stages.
Therefore, it is very significant to search for a method that can achieve accurate staging of precancerous lesions, early cervical cancer and late cervical cancer.
Disclosure of Invention
Based on the above, the invention aims to provide a detection primer, a detection probe, a kit and a method for accurate stage division of invasive cervical squamous carcinoma.
The technical scheme for realizing the aim of the invention comprises the following steps.
In a first aspect of the invention, there is provided a detection primer for accurate staging of invasive cervical squamous carcinoma, comprising: SEQ ID No.6 and SEQ ID No.7 for HPV16, SEQ ID No.9 and SEQ ID No.10 for HPV18, SEQ ID No.12 and SEQ ID No.13 for HPV33, SEQ ID No.15 and SEQ ID No.16 for HPV52, SEQ ID No.18 and SEQ ID No.19 for HPV 58.
In a second aspect of the present invention, there is provided a detection probe for accurate staging of invasive cervical squamous carcinoma, comprising: SEQ ID No.8 for HPV16, SEQ ID No.11 for HPV18, SEQ ID No.14 for HPV33, SEQ ID No.17 for HPV52, SEQ ID No.20 for HPV 58.
In a third aspect of the invention, a detection kit for accurate stage division of invasive cervical squamous carcinoma is provided, and the kit comprises the detection primer and the detection probe.
According to a fourth aspect of the invention, a detection method for accurate stage detection of invasive cervical squamous carcinoma is provided, ctDNA of a plasma sample to be detected is extracted, and ddPCR is adopted for detection by using the detection primer and the detection probe.
According to five high-risk types of HPV16/18/33/52/58, a low-conservation area E6 is selected to design a specific detection primer and a detection probe, and a digital PCR technology is adopted to detect the DNA load of ctHPV in peripheral blood, so that the correlation between the DNA load of ctHPV and the occurrence and development of cervical cancer can be clarified, and the accurate differentiation of invasive precancerous lesions of cervical squamous carcinoma, early cervical cancer and late cervical cancer is realized.
Drawings
FIG. 1 shows the sequence alignment results of five high risk genotypes of HPV16/18/33/52/58 of the present invention.
FIG. 2 shows the signal results of FAM channel in the test example of the present invention.
FIG. 3 shows the signal results of HEX channel in the test example of the present invention.
FIG. 4 shows the results of FAM channel droplet count calculation in the test example of the present invention.
FIG. 5 shows the results of the test of clinical samples in comparative examples of the present invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Green and Sambrook et al, molecular cloning, an experimental guideline (Molecular Cloning: A Laboratory Manual, 2013), or by the manufacturer's recommendations. The various chemicals commonly used in the examples are commercially available.
According to the invention, firstly, according to the epidemiological characteristics of HPV infection in China, five high-risk typing differential sequence areas of HPV16/18/33/52/58 (the sequences are respectively shown as SEQ ID NO. 1-SEQ ID NO.5, and the sequence comparison result is shown as shown in figure 1), a specific detection primer and a detection probe are designed aiming at a low-conservation area E6, a digital PCR technology is adopted to detect the DNA load of ctHPV in peripheral blood (a sample is obtained in a non-invasive mode, the trauma caused by a gynecological examination method sampling mode is avoided, compared with colposcope or direct-view cervical tissue biopsy pathological examination, the detection of the DNA load of the ctHPV in the peripheral blood has the characteristics of noninvasive or minimally invasive, and compared with the detection primer and the detection probe which are designed aiming at the area E7, the specific detection primer and the detection probe are adopted to detect, so that the correlation between the DNA load of ctHPV and the development of cervical cancer can be clear, and the accurate differentiation of the pre-cervical squamous carcinoma lesions, early cervical carcinoma and late cervical carcinoma can be realized. The invention designs the specific detection primer and the detection probe only aiming at E6 of the early transcription region which is considered by the traditional method, so that the invasive cervical squamous carcinoma can be detected, and the precancerous lesions, the early cervical carcinoma and the late cervical carcinoma can be distinguished.
>HPV16(SEQ ID NO.1)
atgtttcaggacccacaggagcgacccagaaagttaccacagttatgcacagagctgcaaacaactatacatgatataatat
tagaatgtgtgtactgcaagcaacagttactgcgacgtgaggtatatgactttgcttttcgggatttatgcatagtatatagaga
tgggaatccatatgctgtatgtgataaatgtttaaagttttattctaaaattagtgagtatagacattattgttatagtttgtatggaa
caacattagaacagcaatacaacaaaccgttgtgtgatttgttaattaggtgtattaactgtcaaaagccactgtgtcctgaag
aaaagcaaagacatctggacaaaaagcaaagattccataatataaggggtcggtggaccggtcgatgtatgtcttgttgca
gatcatcaagaacacgtagagaaacccagctgtaa
>HPV18(SEQ ID NO.2)
atggcgcgctttgaggatccaacacggcgaccctacaagctacctgatctgtgcacggaactgaacacttcactgcaaga
catagaaataacctgtgtatattgcaagacagtattggaacttacagaggtatttgaatttgcatttaaagatttatttgtggtgta
tagagacagtataccccatgctgcatgccataaatgtatagatttttattctagaattagagaattaagacattattcagactctg
tgtatggagacacattggaaaaactaactaacactgggttatacaatttattaataaggtgcctgcggtgccagaaaccgttg
aatccagcagaaaaacttagacaccttaatgaaaaacgacgatttcacaacatagctgggcactatagaggccagtgccat
tcgtgctgcaaccgagcacgacaggaacgactccaacgacgcagagaaacacaagtataa
>HPV33(SEQ ID NO.3)
atgtttcaagacactgaggaaaaaccacgaacattgcatgatttgtgccaagcattggagacaactatacacaacattgaac
tacagtgcgtggaatgcaaaaaacctttgcaacgatctgaggtatatgattttgcatttgcagatttaacagttgtatatagaga
gggaaatccatttggaatatgtaaactgtgtttgcggttcttatctaaaattagtgaatatagacattataattattctgtatatgga
aatacattagaacaaacagttaaaaaacctttaaatgaaatattaattaggtgtattatatgtcaaagacctttgtgtcctcaaga
aaaaaaacgacatgtggatttaaacaaacgatttcataatatttcgggtcgttgggcagggcgctgtgcggcgtgttggagg
tcccgacgtagagaaactgcactgtga
>HPV52(SEQ ID NO.4)
atgtttgaggatccagcaacacgaccccggaccctgcacgaattgtgtgaggtgctggaagaatcggtgcatgaaataag
gctgcagtgtgtgcagtgcaaaaaagagctacaacgaagagaggtatacaagtttctatttacagatttacgaatagtatata
gagacaataatccatatggcgtgtgtattatgtgcctacgctttttatctaagataagtgaatataggcattatcaatattcactgt
atgggaaaacattagaagagagggtaaaaaaaccattaagtgaaataactattagatgtataatttgtcaaacgccattatgt
cctgaagaaaaagaaagacatgttaatgcaaacaagcgatttcataatattatgggtcgttggacagggcgctgttcagagt
gttggagaccccgacctgtgacccaagtgtaa
>HPV58(SEQ ID NO.5)
Atgttccaggacgcagaggagaaaccacggacattgcatgatttgtgtcaggcgttggagacatctgtgcatgaaatcga
attgaaatgcgttgaatgcaaaaagactttgcagcgatctgaggtatatgactttgtatttgcagatttaagaatagtgtataga
gatggaaatccatttgcagtatgtaaagtgtgcttacgattgctatctaaaataagtgagtatagacattataattattcgctatat
ggagacacattagaacaaacactaaaaaagtgtttaaatgaaatattaattagatgtattatttgtcaaagaccattgtgtccac
aagaaaaaaaaaggcatgtggatttaaacaaaaggtttcataatatttcgggtcgttggacagggcgctgtgcagtgtgttg
gagaccccgacgtagacaaacacaagtgtaa
In some embodiments of the invention, a detection primer for accurate stage of invasive cervical squamous carcinoma is disclosed, comprising: SEQ ID No.6 and SEQ ID No.7 for HPV16, SEQ ID No.9 and SEQ ID No.10 for HPV18, SEQ ID No.12 and SEQ ID No.13 for HPV33, SEQ ID No.15 and SEQ ID No.16 for HPV52, SEQ ID No.18 and SEQ ID No.19 for HPV 58.
In some of these embodiments, the detection primer further comprises SEQ ID No.21 and SEQ ID No.22 detected against the reference gene FGFR 1.
In some embodiments of the invention, a detection probe for precise staging of invasive cervical squamous carcinoma is disclosed, comprising: SEQ ID No.8 for HPV16, SEQ ID No.11 for HPV18, SEQ ID No.14 for HPV33, SEQ ID No.17 for HPV52, SEQ ID No.20 for HPV 58.
In some of these embodiments, the 5' end of the detection probe is modified with a fluorescent-labeled FAM.
In some of these embodiments, the detection probe further comprises SEQ ID No.23 for detection of the reference gene FGFR 1.
In some of these embodiments, the 5' end of the detection probe is modified with a fluorescent label HEX.
In other embodiments of the present invention, a detection kit for precise staging of invasive cervical squamous carcinoma is disclosed, comprising the detection primer and the detection probe described above.
In some of these embodiments, the detection primer operates at a concentration of 400nM to 1400nM, preferably 900nM, and the detection probe operates at a concentration of 150nM to 350nM, preferably 250nM.
In other embodiments of the present invention, a method for detecting precise stage of invasive cervical squamous carcinoma is disclosed, comprising the steps of: ctDNA of the plasma sample to be tested was extracted, and detection was performed by ddPCR using the above-described detection primer and detection probe.
In some of these embodiments, the reaction system of ddPCR comprises: 5.4. Mu.L of ctDNA template, 10. Mu.L of ddPCR Supermix, 0.9. Mu.L of 20. Mu.M detection primer each, 0.5. Mu.L of 20. Mu.M detection probe each, and no nuclease water added to 20. Mu.L.
In some of these embodiments, the ddPCR reaction procedure is: 95 ℃ for 5min;94 ℃ for 30s and 55 ℃ for 1min, and the cycle is 40 times; 98 ℃ for 10min; the temperature rise and fall speed is less than or equal to 2.5 ℃/s.
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1 detection primers and probes for accurate staging of invasive cervical squamous carcinoma
The HPV virus has more than 200 subtypes, five high-risk types of HPV16/18/33/52/58 are screened according to epidemiological characteristics of HPV infection in China, sequences with differences among the five high-risk types are screened through sequence comparison, and specific primers and probes for detecting HPV16/18/33/52/58 and specific primers and probes for an internal reference gene FGFR1 are designed by using Primer5.0 primer design software aiming at an E6 region, wherein the sequences are shown in tables 1 and 2 respectively. Wherein, the probes are double fluorescent probes FAM and HEX, the 5 'end is a fluorescent group, and the 3' end is a quenching group; the FAM probe is used for detecting HPV16/18/33/52/58, and the HEX probe is used for detecting a reference gene FGFR 1.
TABLE 1
TABLE 2
Example 2 detection method for accurate stage of invasive cervical squamous carcinoma
The method comprises the following steps:
1. collecting a sample
Collecting 10mL of a sample (blood) to be detected in a free DNA preservation tube; centrifuge at 3000g for 10min at 4 ℃; centrifuging 16000g of supernatant at 4deg.C for 10-15 min, removing precipitate, collecting supernatant, and preserving at-80deg.C.
2. Extraction of circulating tumor DNA (ctDNA)
ctDNA was extracted from plasma samples using QIAamp circulating nucleic acid extraction kit according to the procedure of its instructions, ctDNA concentration was determined using Qubit 2.0 and ctDNA was kept as a sample at-20 ℃ for later use.
3. ddPCR detection
ddPCR detection was performed using a Berle QX200 model ddPCR instrument.
(1) Preparing a reaction solution: mu.L of ctDNA template, 10 mu.L of ddPCR Supermix, 0.9 mu.L of 20 mu.M upstream and downstream primer, and 0.5 mu.L of 10 mu M probe were added to prepare 20 mu.L of reaction solution; mixing, and centrifuging briefly.
(2) Droplet generation: placing DG8 cartridge into a DG8 cartridge holder, adding all reaction liquid into a DG8 cartridge middle sample adding hole, adding 70 mu LDroplet Generation Oil into 8 holes at the bottom of the DG8 cartridge, filling a rubber pad (gasset), placing the DG8 cartridge holder into a droplet generator to generate droplets, slowly transferring the droplets into a 96-well plate, and sealing the droplets by using a preheated PX1 heat sealer, wherein the operation procedures are as follows: 180 deg.c for 5s.
(3) And (3) PCR reaction: after sealing the membrane of the 96-well plate, carrying out PCR reaction within 30min or placing the membrane in a refrigerator at 4 ℃ for 4 hours, wherein the reaction procedure is as follows: 95 ℃ for 5min;94 ℃ for 30s and 55 ℃ for 1min, and the cycle is 40 times; 98 ℃ for 10min; preserving at 4 ℃; the temperature rise and fall speed is less than or equal to 2.5 ℃/s;
(4) Droplet detection: the 96-well plate with the PCR reaction completed is placed in a droplet reader, quantaSoft (Bio-Rad) software is opened, and droplet analysis is performed after setting the sample information.
4. QuantaSoft software (Bio-Rad) quantitative analysis
And analyzing the running result by using QuantaSoft, and checking parameters such as a channel, a scattered point cluster map, concentration data, the number of events and the like.
5. Statistical analysis
And (5) integrating the detection result and comparing the pathological result, and performing statistical analysis.
Example 3 practical application of the method for detecting invasive cervical squamous carcinoma of the invention
In this example, 20 samples (and negative quality control nuclease-free water) were collected from 5 cases of patients with HPV16 infection and early stage cervical cancer (stages IA1-IB 2), patients with HPV16 infection and late stage cervical cancer (stages IB 3-IV), and samples of the population without HPV infection, and 10mL of blood was taken from the free DNA storage tube; centrifuge at 3000g for 10min at 4 ℃; centrifuging 16000g of supernatant at 4deg.C for 10-15 min, removing precipitate, collecting supernatant, and preserving at-80deg.C.
The result judging method comprises the following steps: the FAM channel is a probe for detecting HPV16 type, the blue is a positive droplet, the black is a negative droplet, wherein samples of patients which are not infected with HPV, infected with HPV16 and of which the pathological type is a precancerous lesion (CIN 1-3) stage are not signaled in the FAM channel, the number of positive droplets is 0, the samples of patients which are infected with HPV16 and of which the pathological type is invasive cervical squamous carcinoma (comprising IA1-IB2 stage and IB3-IV stage) are signaled in the FAM channel (figure 2); the HEX channel is a probe for detecting the reference gene FGFR1, green is positive droplet, black is negative droplet, and the reference gene should be positive except for negative quality control (fig. 3).
By adopting the detection method of the embodiment 2 of the invention, the detection of invasive cervical squamous carcinoma is carried out on each detection sample. Negative control samples and pre-cancerous lesions samples, no signal was detected in the FAM channel. Both early invasive cervical cancer (stages IA1-IB 2) and late invasive cervical cancer (stages IB 3-IV) samples detected a signal in the FAM channel. The number of FAM channel droplets was calculated by software and the results are shown in fig. 4. In early invasive cervical cancer (IA 1-IB2 stage) samples, the copy number of HPV16 in each milliliter of blood plasma is 1-24 copies, and in late stage cervical squamous carcinoma (IB 3-IV stage) samples, the copy number of HPV16 in each milliliter of blood plasma is 57-601 copies, so that early stage invasive cervical squamous carcinoma and late stage invasive cervical squamous carcinoma can be well distinguished.
Comparative example A method for detecting invasive cervical squamous carcinoma
This comparative example designed specific primers and probes based on the E7 region of HPV16 and HPV18 viral sequences, the sequences being shown in Table 3.
TABLE 3 Table 3
Using the detection primers and probes of Table 3, ctHPV DNA analysis of clinical samples using the method of the article "The Diagnostic Value ofCirculating Cell-Free HPV DNA in Plasma from Cervical Cancer Patients (Cells 2022,11,2170.Https:// doi. Org/10.3390/Cells 11142170)" found that the detection rate of ctHPV DNA was only 10.0% in early cervical squamous carcinoma patients (stages IA1-IB 2), 63.3% in late cervical squamous carcinoma patients (stages IB 3-IV), and that the copy number of ctHPV DNA could not be detected or detected in samples (within the box) of some clinically confirmed advanced cervical carcinoma patients was comparable to that of cervical carcinoma early stages (FIG. 5), and therefore, the early and late cervical carcinoma stages could not be distinguished based on the copy number of ctHPV DNA, and the purpose of accurate pre-cancerous lesions, early cervical carcinoma stages, and late cervical carcinoma stages could not be achieved.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
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.

Claims (10)

1. A detection primer for accurate stage of invasive cervical squamous carcinoma, comprising: SEQ ID No.6 and SEQ ID No.7 for HPV16, SEQ ID No.9 and SEQ ID No.10 for HPV18, SEQ ID No.12 and SEQ ID No.13 for HPV33, SEQ ID No.15 and SEQ ID No.16 for HPV52, SEQ ID No.18 and SEQ ID No.19 for HPV 58.
2. The detection primer for precise staging of invasive cervical squamous carcinoma according to claim 1, characterized by further comprising SEQ ID NO.21 and SEQ ID NO.22 for detection of internal reference gene FGFR 1.
3. A detection probe for precise stage of invasive cervical squamous carcinoma, comprising: SEQ ID No.8 for HPV16, SEQ ID No.11 for HPV18, SEQ ID No.14 for HPV33, SEQ ID No.17 for HPV52, SEQ ID No.20 for HPV 58.
4. The detection probe for precise staging of invasive cervical squamous carcinoma according to claim 3, characterized in that the 5' end of the detection probe is modified with fluorescent label FAM.
5. The probe for detecting precise stage of invasive cervical squamous carcinoma according to claim 3, further comprising SEQ ID NO.23 for detecting the reference gene FGFR 1.
6. The detection probe for precise staging of invasive cervical squamous carcinoma according to claim 5, characterized in that the 5' end of the detection probe is modified with fluorescent label HEX.
7. A kit for detecting accurate stage of invasive cervical squamous carcinoma, characterized in that the kit comprises the detection primer according to claim 1 or 2 and the detection probe according to any one of claims 3 to 6.
8. The kit for accurate stage detection of invasive cervical squamous carcinoma according to claim 6, wherein the working concentration of the detection primer is 400nM to 1400nM and the working concentration of the detection probe is 150nM to 350nM.
9. The method for detecting the accurate stage of invasive cervical squamous carcinoma is characterized by comprising the following steps of: ctDNA of a plasma sample to be tested is extracted and detected by ddPCR using the detection primer according to claim 1 or 2 and the detection probe according to claim 3 or 4.
10. The method for detecting precise stages of invasive cervical squamous carcinoma according to claim 8, wherein the reaction system of ddPCR includes: ddPCR Supermix 10. Mu.L, 20. Mu.M detection primers each 0.9. Mu.L, 10. Mu.M detection probes each 0.5. Mu. L, ctDNA template 5.4. Mu.L, and nuclease-free water to 20. Mu.L; and/or the ddPCR reaction procedure is: 95 ℃ for 5min;94 ℃ for 30s and 55 ℃ for 1min, and the cycle is 40 times; 98 ℃ for 10min; the temperature rise and fall speed is less than or equal to 2.5 ℃/s.
CN202311160451.7A 2023-09-08 2023-09-08 Detection primer, detection probe, kit and method for accurate stage of invasive cervical squamous carcinoma Pending CN116949222A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311160451.7A CN116949222A (en) 2023-09-08 2023-09-08 Detection primer, detection probe, kit and method for accurate stage of invasive cervical squamous carcinoma

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311160451.7A CN116949222A (en) 2023-09-08 2023-09-08 Detection primer, detection probe, kit and method for accurate stage of invasive cervical squamous carcinoma

Publications (1)

Publication Number Publication Date
CN116949222A true CN116949222A (en) 2023-10-27

Family

ID=88444671

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311160451.7A Pending CN116949222A (en) 2023-09-08 2023-09-08 Detection primer, detection probe, kit and method for accurate stage of invasive cervical squamous carcinoma

Country Status (1)

Country Link
CN (1) CN116949222A (en)

Similar Documents

Publication Publication Date Title
CN101487063B (en) Human papilloma virus infection gene amplification fluorescent detection kit
CN104818342B (en) Detection kit, detection architecture and method for 19 kinds of high-risk human mammilla papillomavirus (HPV)
CN107841576A (en) 14 kinds of Combining high-risk human papillomavirus E6/E7 areas mRNA detection kits
CN110628953B (en) Kit for human papilloma virus typing detection
CN104560962B (en) Fluorescent PCR kit and its primer special group for detecting HPV
CN108179226B (en) Nucleic acid composition for detecting human papilloma virus, application thereof and kit
US9157129B2 (en) Methods for detecting human papillomavirus-associated cancers
EP2150629B1 (en) Identification and quantification of oncogenic hpv nucleic acids by means of real-time pcr assays
CN108728578A (en) Detect the method and kit of a variety of high-risk human mammilla papillomavirus and its hypotype simultaneously in single tube reaction
CN112575123A (en) Primer combination, probe combination and human papilloma virus nucleic acid detection kit
CN108085419B (en) probe and primer composition
CN116949222A (en) Detection primer, detection probe, kit and method for accurate stage of invasive cervical squamous carcinoma
CN114214415A (en) Primer probe combination for methylation detection of cervical cancer related genes and application thereof
CN113584225A (en) Primer and probe combination for detecting HPV (human papillomavirus), reagent for typing detection of HPV and application of HPV primer and probe combination
CN113817833A (en) Kit for detecting cervical cell gene methylation based on fluorescent quantitative PCR technology and application
CN107828920B (en) Kit for realizing typing of multiple high-risk human papilloma viruses
CN117925916A (en) Application of E6 gene of HPV DNA in preparation of invasive cervical cancer diagnosis product
Abdul-Samad et al. The molecular detection of HPV infection in samples of Iraqi women with abnormal cervical smears
CN113584229B (en) Primer probe combination, kit and application for isothermal nucleic acid amplification detection of human papilloma virus type 6 and/or type 11
CN108841956B (en) Use of long-chain non-coding RNAs
Hachim et al. Molecular Detection of High-Risk Human Papillomavirus Genotypes from Cervical Lesions in Baghdad
CN117757991B (en) POCT detection method for DNA and mRNA of HPV16/18 virus gene in cervical exfoliated cells
CN116103406B (en) Primer probe combination for detecting cervical high-level squamous intraepithelial lesions and application
CN116064805A (en) Cervical cancer related gene methylation detection probe combination, kit and detection method
CN111876526A (en) Microfluidic chip for detecting HPV (human papillomavirus) virus and typing

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination