CN111876526A - Microfluidic chip for detecting HPV (human papillomavirus) virus and typing - Google Patents
Microfluidic chip for detecting HPV (human papillomavirus) virus and typing Download PDFInfo
- Publication number
- CN111876526A CN111876526A CN202010786653.2A CN202010786653A CN111876526A CN 111876526 A CN111876526 A CN 111876526A CN 202010786653 A CN202010786653 A CN 202010786653A CN 111876526 A CN111876526 A CN 111876526A
- Authority
- CN
- China
- Prior art keywords
- dna
- hpv
- globin
- artificial sequence
- microfluidic chip
- 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
Links
Images
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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0605—Valves, specific forms thereof check valves
-
- 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)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Clinical Laboratory Science (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Dispersion Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a microfluidic chip for detecting HPV virus and typing, which is characterized in that a high-specificity MGB probe method is utilized, specific primers and probes of various types are designed in the HPV gene E6E 7 area, the primers and probes and an enzyme premix which can be freeze-dried into powder are embedded in the microfluidic chip, and the obtained microfluidic chip is used for detecting HPV virus.
Description
Technical Field
The invention belongs to the technical field of medical detection, and particularly relates to a micro-fluidic chip for detecting HPV (human papillomavirus) viruses and typing.
Background
Human Papillomavirus (HPV), belonging to the family of papillomaviruses, is a small molecule, non-enveloped, circular double-stranded DNA virus containing approximately 7900 pairs of bases (bp) and consisting of 3 gene regions in total, including an Early Region (E Region), a Late Region (L Region), and a noncoding Region (UCR) or Upstream Regulatory Region (URR). The E region is 7 genes of E6, E7, E1, E2, E3, E4 and E5 in sequence, and is involved in the replication, transcription and encoding of viral DNA, and maintenance of high copy number of viruses in cells, wherein E6 and E7 are main oncogenes of HPV, and are related to the transformation function of viral cells and carcinogenicity. HPV infects humans by direct or indirect contact with contaminated articles or by sexual transmission. The virus not only has host specificity, but also has tissue specificity, and can only infect skin and mucosal epithelial cells of human, causing various papilloma or wart of human skin and reproductive tract epithelial hyperplastic injury.
Results of studies on a global scale show that 70-80% of women will have at least one HPV infection during their lifetime, and the presence of high-risk HPV DNA is detected in 99.7% of cervical cancer patients, with 80% of HPV16, 18, 45 and 31 infections. Low risk HPV types are generally associated with condyloma acuminata or low grade squamous intraepithelial lesions, rarely causing invasive carcinoma. Therefore, HPV detection is very important for detection and typing of HPV virus. With regard to the division of high-risk types and low-risk types, many international organizations give reference suggestions, and 13 genotypes such as HPV16, 18,31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and the like are classified as high-risk types, and 5 genotypes such as 26, 53, 66, 73, 82 and the like are classified as medium-risk types according to research results of WHO International cancer research Institute (IARC) and other international organizations.
The diagnosis of cervical cancer in the traditional art has mainly followed "three-step" diagnostic procedures, i.e., cervical cytology, colposcopy, and histopathological examination. The traditional early screening of female cervical cancer mainly depends on the three-step diagnosis procedure, when three detection methods are used, pathological cells are easy to separate in the cervical cell sampling process, the cell morphology is easy to change or the pathological cells are easily covered by other inflammatory cells and blood cells in the flaking process, and meanwhile, the result judgment is too dependent on individual subjective judgment, so that the disease missed diagnosis is easy to occur. Although the detection result of the DNA hybridization technology is high, the detection cost and the detection process are complex, and the detection process is easy to generate errors and generate false negative results; although the Q-PCR fluorescent dye detection technology is low in cost, the Q-PCR fluorescent dye detection technology has a remarkable defect that the specificity is very easily influenced by nonspecific gene amplification and primer dimer formation, so that the qPCR method combining double-stranded DNA with the fluorescent dye cannot be effectively applied to clinical detection. However, the MGB probe method qPCR can effectively solve the problem of non-specificity of the qPCR combining the double-stranded DNA with the fluorescent dye, has the advantages of extremely strong specificity, high sensitivity, good repeatability, accurate quantification, high speed and the like, and can become an important tool for molecular diagnosis in future clinical detection and microfluidic chip detection.
Meanwhile, the invention is applied to HPV virus detection and typing of the microfluidic chip, and the microfluidic chip has the advantages of simple operation, low price, small sample consumption, high analysis speed and no dependence on expensive instruments, thereby being very suitable for rapid detection and analysis of diseases in economically undeveloped areas. The chip can be expanded to the rapid detection of any other pathogen, and has very important significance for the basic prevention and control of some acute infectious pathogens.
Disclosure of Invention
The invention aims to provide a micro-fluidic chip for detecting HPV viruses and typing, and particularly relates to a method for detecting and typing 20 types of high-risk and low-risk HPV viruses based on the micro-fluidic chip. The MGB probe method with the characteristics of strong stability, high specificity and the like is adopted, the type specific primers which are designed in E6 and E7 areas with high conservation and difficult loss are combined with a Q-PCR primer, a probe and an enzyme premix which are independently developed and can be lyophilized into powder to be embedded in a microfluidic chip, and finally the HPV virus can be detected through the microfluidic chip.
In order to achieve the purpose, the invention adopts the following technical scheme:
a micro-fluidic chip for detecting HPV virus and typing comprises specific primers and probes for detecting HPV virus and typing, wherein the sequences of the primers and the probes are as follows:
1.HPV16E-F：AGGAGCGACCCAGAAAGTTACC
2.HPV16E-R：ACAGCATATGGATTCCCATCTC
3.HPV16EFam-F:CAACAGTTACTGCGACG；
4.HPV18E-F: CAACACGGCGACCCTACAAG
5.HPV18E-R: TACATTTATGGCATGCAGCATG
6.HPV18EFam-F: CACTTCACTGCAAGACA
7.HPV31E-F: GAAAGACCTCGGAAATTGCATG
8.HPV31E-R: TGGTGTGTCGTCCCTATATACT
9.HPV31EFam-F: CGGCATTGGAAATACCC
10.HPV33E-F: ATTGCATGATTTGTGCCAAGC
11.HPV33E-R: ATTCCAAATGGATTTCCCTCTC
12.HPV33EFam-R: ACATTGAACTACAGTGCG
13.HPV35E-F: CAGCGGAGTGAGGTATATGAC
14.HPV35E-R: CTAACGTTTCTCCATACACAC
15.HPV35EFam-F: CATATGGCTGGCCTTC
16.HPV39E-F: ATGGCGCGATTTCACAATCCTG
17.HPV39E-R: TCGTCTGCAATAGACACAGGCT
18.HPV39EFam-F: ACAACGCTGGACACCA
19.HPV45E-F: TACGAGCAATTAAGCGAGTCAG
20.HPV45E-R: CTGTAAGCTCAATTCTGCCGTC
21.HPV45EFam-F: AGTCATGCACAACTAC
22.HPV51E-F: gtacacgacaacgtaacgaaacc
23.HPV51E-R: cgtctttctggtagctggtc
24.HPV51EFam-F: TGTAGTATTGCATTTAACACCAC
25.HPV52E-F: ATGGACAAGCAGAACAAGCCAC
26.HPV52E-R: AACTTGTAATGTGCCCAACAGC
27.HPV52EFam-F: ACGGACCTTCGTACTC
28.HPV56E-F: AGGTGCTACAGATGTCAAAGT
29.HPV56E-R: CTGTAGATTCTCTAGGTTCTC
30.HPV56EFam-F：CTAATAGCACATGGTTG
31.HPV58E-F: ATGTGACAGCTCAGACGAGG
32.HPV58E-R: CGGTTGTTGTACTGTTGATACAC
33.HPV58EFam-F：ATGGACAAGCACAACCG
34.HPV59E-F: GAAGTTGACCTTGTGTGCTACG
35.HPV59E-R: ACACAATGTTGTGACGCTGTGG
36.HPV59EFam-F：CTCCATCTGGTTCATC
37.HPV68E-F: ACAACCCTGAGGAACGGCCAT
38.HPV68E-R: GGCAAATTCATATACCTCTGTC
39.HPV68EFam-R：TCCAATGTCCTGCACA
40.HPV26E-F: GAGATAGGAGTCCGTATGCTGC
41.HPV26E-R: GGCATTTGACATCTATGACACC
42.HPV26EFam-F：TGCAACATTAGAAGCC
43.HPV53E-F: AGTGTATAGAGACGGGTATCCG
44.HPV53E-R: GGATGTTGACATCTGTAGCACC
45.HPV53EFam-R：CCCGTACACTGAACAA
46.HPV66E-F: ATTCAGCAATACACAGGAACGT
47.HPV66E-R: TTTAACTCAATACATGCAAACCT
48.HPV66EFam-F：CTGAGCGAGGTATTAC
49.HPV73E-F: GACAGACAAGCTGAACGAGAG
50.HPV73E-R: CACTCTTAAATCAGCTTTGTTGC
51.HPV73EFam-F：ACTGACACTTCGTGCA
52.HPV82E-F: GCCTGAAGAAAAGCAAAAGG
53.HPV82E-R: GGTGTTAACTCCAACACTATGTC
54.HPV82EFam-R:TTGCACACTGTCCCGTCC
55.HPV6E-F: GGATATGCAACAACTGTTGAAG
56.HPV6E-R: CCCTTCCACGTACAATTTAGC
57.HPV6EFam-F:TACTAACCAAGGCACGGT
58.HPV11E-F: CAGTGCGTGTTTTGCAGGAATG
59.HPV11E-R: CCCTTGCAGTTCTAAGCAACAG
60.HPV11EFam-R:AAGTTGTCTCGCCACACA
61.Globin-F: GGCTCATGGCAAGAAAGTGC
62.Globin-R: CAAGCGTCCCATAGACTCACC
63.Globin-Hex-BHQ1-F: TGGCCTGGCTCACCTGGACAACCTC。
the microfluidic chip comprises Q-PCR reaction liquid embedded in the microfluidic chip, wherein the reaction liquid is as follows: the HPV-F0.3 mMol/ml, the HPV-R0.3 mMol/ml, the HPV probe 0.25 mu Mol/ml, the Globin-F0.25 mu Mol/ml, the Globin-R0.25 mu Mol/ml, the Globin-Hex-BHQ 1-F0.2 mu Mol/ml, the DNA polymerase 2000U/ml, the 10 XTris-HCL reaction buffer solution 1X, and the DNA Sample 10 not less than 104Copy number/reaction, ddH2O make up to 3 uL.
A HPV virus detection and typing method based on a microfluidic chip comprises the following steps:
A. selection and determination of detection type; B. primer design of the HPV E6 and E7 regions; C. selection and design of MGB probes; D. independently developing a Q-PCR primer, a probe and an enzyme premix which can be freeze-dried into powder and embedded in a microfluidic chip; E. exploration and determination of the detection volume; F. and (4) designing and optimizing detection conditions.
The method specifically comprises the following steps:
(1) selection and determination of detection type: the detection types of the method cover 12 carcinogen types (HPV 16, 18,31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) and can also distinguish other 8 important types ( HPV 6, 11,26, 53, 66, 68, 73 and 82). The flux is large, and the detection is comprehensive;
(2) sequence analysis and primer design of HPV virus E6 and E7 regions: the method adopts the E6 and E7 region targeted detection, and avoids the problems of false negative, misdiagnosis and missed diagnosis caused by the frequent loss of the L1 region in the high-risk HPV integration process. Through multiple comparison among all detection types, the type specific primers are designed, so that the problem of cross contamination is effectively avoided;
(3) selection and design of MGB probes: the invention detects HPV virus by MGB probe method, which greatly improves the detection specificity. Through multiple comparison among all detection types, a type specific probe is designed, so that the problem of cross contamination is effectively avoided;
(4) the independent research and development of the Q-PCR primer and the probe which can be freeze-dried into powder and embedded in the microfluidic chip and the enzyme premix: the Q-PCR reaction solution used in the invention can be lyophilized, so that the problem of embedding the primer probe and the reaction buffer solution together is solved, and the subsequent detection is convenient and efficient;
(5) exploration and determination of the detection volume: 3ul of detection system is adopted, so that the reagent cost is greatly reduced;
(6) design and optimization of detection conditions: in the invention, the primers, the probes, the reaction solution and the like are embedded in the chip in advance, so that the detection and the typing only need to add the pre-mixed genome of the cervical exfoliated cells and the DNA polymerase in one step. A one-way valve is arranged between each reaction chamber in the chip, so that PCR reactions are mutually independent, and cross contamination is avoided.
The invention detects HPV virus by MGB probe method, solves the nonspecific problem of fluorescent dye qPCR method, and reduces the HPV detection cost by micro-fluidic chip.
The primers and probes designed by the invention analyze the sequences of various types of HPV by bioinformatics, and design specific primers of various types in the region of HPV virus gene E6E 7 by using biological software (snapGene, DNAman and Vector NTI), wherein the designed primers and probes have high specificity. Since the general PCR primer is used for amplifying the HPV L1 region, but the L1 region is usually lost in the process of integrating high-risk HPV, false negative, misdiagnosis and missed diagnosis are very easy to occur in the detection.
The invention also applies a Q-PCR primer and a probe which are independently developed and can be lyophilized into powder to be embedded in the microfluidic chip, and an enzyme premix. The invention solves the problem of embedding primers and probes when the microfluidic chip is used for typing HPV viruses.
Usually, the detection volume required by the Q-PCR molecular detection of HPV is 25ul, but the detection system can be 3ul on the microfluidic chip, thereby greatly reducing the detection cost. Meanwhile, in the traditional HPV molecular detection and typing, a plurality of PCR reactions need to be carried out on the same sample independently, so that the labor, time and material consumption and required equipment are more, and the microfluidic chip can finish the sample detection at one time, so that the problems are successfully solved, and the limitation of the traditional detection is changed.
In the invention, the primers, the probes, the reaction solution and the like are embedded in the chip in advance, so that the detection and the typing only need to add the pre-mixed genome of the cervical exfoliated cells and the DNA polymerase in one step. A one-way valve is arranged between each reaction chamber in the chip, so that PCR reactions are mutually independent, and cross contamination is avoided.
According to the invention, the cervical exfoliated cell genome fluid enters the Q-PCR reaction chamber of the microfluidic chip by virtue of the piston which is stretched backwards, and is not required to be pre-treated into vacuum or form negative pressure, and the situation that the cervical exfoliated cell genome fluid cannot enter the Q-PCR chamber due to vacuum or negative pressure leakage or insufficient negative pressure can be avoided by stretching the piston.
The invention has the advantages that:
1. the primers and probes used for HPV detection and typing have high specificity and sensitivity and good repeatability.
2. The invention detects HPV virus by the micro-fluidic chip, and has simple operation, lower cost and short period.
3. The invention relates to 20 types of HPV types with high risk and low risk, large flux and comprehensive detection.
4. The Q-PCR reaction solution used by the invention can be lyophilized, so that the problem of embedding the primer probe and the reaction buffer solution together is solved, and the subsequent detection is convenient and efficient.
Drawings
FIG. 1 is a diagram of the result of fluorescent quantitative PCR specific to HPV16 and Globin internal reference primer and probe.
FIG. 2 is a diagram of the result of fluorescent quantitative PCR specific to HPV18 and Globin internal reference primer and probe.
FIG. 3 is a diagram of the result of fluorescent quantitative PCR specific to HPV31 and Globin internal reference primer and probe.
FIG. 4 is a diagram of the result of fluorescent quantitative PCR specific to HPV33 and Globin internal reference primer and probe.
FIG. 5 is a diagram of the result of fluorescent quantitative PCR specific to HPV35 and Globin internal reference primer and probe.
FIG. 6 is a diagram of the result of fluorescent quantitative PCR specific to HPV39 and Globin internal reference primer and probe.
FIG. 7 is a diagram of the result of fluorescent quantitative PCR specific to HPV45 and Globin internal reference primer and probe.
FIG. 8 is a diagram of the result of fluorescent quantitative PCR specific to HPV51 and Globin internal reference primer and probe.
FIG. 9 is a graph showing the result of fluorescent quantitative PCR specific to HP52 and Globin internal reference primer and probe.
FIG. 10 is a diagram of the result of fluorescent quantitative PCR specific to HPV56 and Globin internal reference primer and probe.
FIG. 11 is a diagram of the result of fluorescent quantitative PCR specific to HPV58 and Globin internal reference primer and probe.
FIG. 12 is a diagram of the result of fluorescent quantitative PCR specific to HPV59 and Globin internal reference primer and probe.
FIG. 13 is a diagram of the result of fluorescent quantitative PCR specific to HPV6 and Globin internal reference primer and probe.
FIG. 14 is a diagram of the result of fluorescent quantitative PCR specific to HPV11 and Globin internal reference primer and probe.
FIG. 15 is a diagram of the result of fluorescent quantitative PCR specific to HPV26 and Globin internal reference primer and probe.
FIG. 16 is a diagram of the result of fluorescent quantitative PCR specific to HPV53 and Globin internal reference primer and probe.
FIG. 17 is a diagram of the result of fluorescent quantitative PCR specific to HPV66 and Globin internal reference primer and probe.
FIG. 18 is a diagram of the result of fluorescent quantitative PCR specific to HPV68 and Globin internal reference primer and probe.
FIG. 19 is a diagram of the result of fluorescent quantitative PCR specific to HPV73 and Globin internal reference primer and probe.
FIG. 20 is a diagram of the result of fluorescent quantitative PCR specific to HPV82 and Globin internal reference primer and probe.
FIG. 21 validation of HPV16 primer and probe detection sensitivity.
FIG. 22 verification of HPV18 primer and probe detection sensitivity.
FIG. 23 is a cross-validation chart of HPV16 primer and probe specificity.
FIG. 24 is a cross-validation chart of HPV18 primer and probe specificity.
Fig. 25 top view of a microfluidic chip. 1. A sample inlet hole; 2. a sample inlet one-way valve; 3. a microfluidic conduit; 4. a microfluidic conduit check valve; a Q-PCR reaction chamber; 6. an injection tube; 7. volume scale; 8. an injection piston; 9. a stretching arm; 10. a stretch arm direction icon; 11. a sample inlet hole cover; p. positive control reaction zone, S. experimental group reaction zone.
Fig. 26 is a side view of a microfluidic chip. 1. A sample inlet hole; 2. a sample inlet one-way valve; 3. a microfluidic conduit; 4. a microfluidic conduit check valve; a Q-PCR reaction chamber; 6. an injection tube; 7. volume scale; 8. an injection piston; 9. a stretching arm; 10. a stretch arm direction icon; 11. a sample inlet hole cover; p. positive control reaction zone, S. experimental group reaction zone.
Detailed Description
Example 1
1. The sequences of the primers and the probes are shown in a sequence table.
System and component concentrations:
3.10 XTris-HCL reaction buffer component concentration:
Q-PCR reaction conditions: 1.94 ℃ for 3 min; 2.94 ℃ for 5 s; 3.60 ℃ for 30 s; plate Read; go to 2, 45 Cycle.
And (3) detection results: HPV detection results and positive determination:
(1) the test result of the positive control of the quality control standard I is positive, and Ct is less than or equal to 35.00. The result of the negative control is negative, and Ct has no value or is not less than 39.00. ③ the amplification curve should have an obvious exponential phase; the baseline and exponential phases should have sharp inflection points. And fourthly, the 3 conditions are simultaneously met, otherwise, the test is invalid.
(2) As a result, it was judged that (i) the Ct value was zero or that the specimen having a Ct value of not less than 39.00 was negative. ② the specimen with Ct less than 35.00 and amplification curve without obvious exponential phase or baseline and exponential phase without obvious clear inflection point is negative. And the Ct is less than or equal to 35.00, the amplification curve has an obvious exponential phase, and the specimen with obvious and clear inflection points at the baseline and the exponential phase is positive. And fourthly, the specimen with the Ct more than 35.00 and less than 39.00 is tested repeatedly, the result of the repeated test is positive with the Ct less than 39.00, otherwise, the result is negative.
20 human papillomavirus DNA plasmids (types are respectively HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, HPV82, HPV6 and HPV 11) in an HPV genotyping quality control panel established by Chinese medicine biological product identification according to 104The amount of Copies/reaction, internal reference is the human tubaeidae gene Globin, the system is 10ul, wherein Fam is the HPV virus fluorescent amplification signal, and Hex is the internal reference fluorescent amplification signal. All HPV types in the detection result can detect standard amplification signals, and Ct values are all smaller than 35.
Verification of HPV typing detection sensitivity:
respectively diluting 20 human papillomavirus DNA plasmids (types are respectively HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, HPV82, HPV6 and HPV 11) in an HPV genotyping quality control panel established by Chinese medicine biological product identification to prepare a plurality of samples, wherein the concentrations are respectively 105Copies/µL、104Copies/µL、103Copies/µL、102Copies/µL、101Copies/mu L is a DNA template, and the detection is carried out according to a detection system of 10ul (including an internal reference), and the result is 105Copies/µL、104HPV signals and internal reference signals can be detected in Copies/mu L. Therefore, the Q-PCR differential typing detection sensitivity was 104Copies/µL。
3. Verification of HPV typing detection specificity: (Cross validation)
20 human papillomavirus DNA plasmids (types are respectively HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, HPV82, HPV6 and HPV 11) in an HPV genotyping quality control panel established by Chinese medicine biological product identification according to 104Copies/reaction amount, system 10ul for cross validation of 20 HPV primers and probes, respectively. In the result, all HPV typing primers and probe cross-validation result signals are single and are signals of corresponding HPV types.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fuzhou university
West horse West Mak West Mak medical science and technology Co Ltd
<120> a microfluidic chip for detecting HPV virus and typing
<130>63
<160>63
<170>PatentIn version 3.3
<210>1
<211>22
<212>DNA
<213> Artificial sequence
<400>1
aggagcgacc cagaaagtta cc 22
<210>2
<211>22
<212>DNA
<213> Artificial sequence
<400>2
acagcatatg gattcccatc tc 22
<210>3
<211>17
<212>DNA
<213> Artificial sequence
<400>3
caacagttac tgcgacg 17
<210>4
<211>20
<212>DNA
<213> Artificial sequence
<400>4
<210>5
<211>22
<212>DNA
<213> Artificial sequence
<400>5
tacatttatg gcatgcagca tg 22
<210>6
<211>17
<212>DNA
<213> Artificial sequence
<400>6
cacttcactg caagaca 17
<210>7
<211>22
<212>DNA
<213> Artificial sequence
<400>7
gaaagacctc ggaaattgca tg 22
<210>8
<211>22
<212>DNA
<213> Artificial sequence
<400>8
tggtgtgtcg tccctatata ct 22
<210>9
<211>17
<212>DNA
<213> Artificial sequence
<400>9
cggcattgga aataccc 17
<210>10
<211>21
<212>DNA
<213> Artificial sequence
<400>10
attgcatgat ttgtgccaag c 21
<210>11
<211>22
<212>DNA
<213> Artificial sequence
<400>11
attccaaatg gatttccctc tc 22
<210>12
<211>18
<212>DNA
<213> Artificial sequence
<400>12
acattgaact acagtgcg 18
<210>13
<211>21
<212>DNA
<213> Artificial sequence
<400>13
cagcggagtg aggtatatga c 21
<210>14
<211>21
<212>DNA
<213> Artificial sequence
<400>14
ctaacgtttc tccatacaca c 21
<210>15
<211>16
<212>DNA
<213> Artificial sequence
<400>15
catatggctg gccttc 16
<210>16
<211>22
<212>DNA
<213> Artificial sequence
<400>16
atggcgcgat ttcacaatcc tg 22
<210>17
<211>22
<212>DNA
<213> Artificial sequence
<400>17
tcgtctgcaa tagacacagg ct 22
<210>18
<211>16
<212>DNA
<213> Artificial sequence
<400>18
<210>19
<211>22
<212>DNA
<213> Artificial sequence
<400>19
tacgagcaat taagcgagtc ag 22
<210>20
<211>22
<212>DNA
<213> Artificial sequence
<400>20
ctgtaagctc aattctgccg tc 22
<210>21
<211>16
<212>DNA
<213> Artificial sequence
<400>21
<210>22
<211>23
<212>DNA
<213> Artificial sequence
<400>22
gtacacgaca acgtaacgaa acc 23
<210>23
<211>20
<212>DNA
<213> Artificial sequence
<400>23
cgtctttctg gtagctggtc 20
<210>24
<211>23
<212>DNA
<213> Artificial sequence
<400>24
tgtagtattg catttaacac cac 23
<210>25
<211>22
<212>DNA
<213> Artificial sequence
<400>25
atggacaagc agaacaagcc ac 22
<210>26
<211>22
<212>DNA
<213> Artificial sequence
<400>26
aacttgtaat gtgcccaaca gc 22
<210>27
<211>16
<212>DNA
<213> Artificial sequence
<400>27
acggaccttc gtactc 16
<210>28
<211>21
<212>DNA
<213> Artificial sequence
<400>28
aggtgctaca gatgtcaaag t 21
<210>29
<211>21
<212>DNA
<213> Artificial sequence
<400>29
ctgtagattc tctaggttct c 21
<210>30
<211>17
<212>DNA
<213> Artificial sequence
<400>30
ctaatagcac atggttg 17
<210>31
<211>20
<212>DNA
<213> Artificial sequence
<400>31
<210>32
<211>23
<212>DNA
<213> Artificial sequence
<400>32
cggttgttgt actgttgata cac 23
<210>33
<211>17
<212>DNA
<213> Artificial sequence
<400>33
atggacaagc acaaccg 17
<210>34
<211>22
<212>DNA
<213> Artificial sequence
<400>34
gaagttgacc ttgtgtgcta cg 22
<210>35
<211>22
<212>DNA
<213> Artificial sequence
<400>35
acacaatgtt gtgacgctgt gg 22
<210>36
<211>16
<212>DNA
<213> Artificial sequence
<400>36
ctccatctgg ttcatc 16
<210>37
<211>21
<212>DNA
<213> Artificial sequence
<400>37
acaaccctga ggaacggcca t 21
<210>38
<211>22
<212>DNA
<213> Artificial sequence
<400>38
ggcaaattca tatacctctg tc 22
<210>39
<211>16
<212>DNA
<213> Artificial sequence
<400>39
<210>40
<211>22
<212>DNA
<213> Artificial sequence
<400>40
gagataggag tccgtatgct gc 22
<210>41
<211>22
<212>DNA
<213> Artificial sequence
<400>41
ggcatttgac atctatgaca cc 22
<210>42
<211>16
<212>DNA
<213> Artificial sequence
<400>42
<210>43
<211>22
<212>DNA
<213> Artificial sequence
<400>43
agtgtataga gacgggtatc cg 22
<210>44
<211>22
<212>DNA
<213> Artificial sequence
<400>44
ggatgttgac atctgtagca cc 22
<210>45
<211>16
<212>DNA
<213> Artificial sequence
<400>45
<210>46
<211>22
<212>DNA
<213> Artificial sequence
<400>46
attcagcaat acacaggaac gt 22
<210>47
<211>23
<212>DNA
<213> Artificial sequence
<400>47
tttaactcaa tacatgcaaa cct 23
<210>48
<211>16
<212>DNA
<213> Artificial sequence
<400>48
<210>49
<211>21
<212>DNA
<213> Artificial sequence
<400>49
gacagacaag ctgaacgaga g 21
<210>50
<211>23
<212>DNA
<213> Artificial sequence
<400>50
cactcttaaa tcagctttgt tgc 23
<210>51
<211>16
<212>DNA
<213> Artificial sequence
<400>51
<210>52
<211>20
<212>DNA
<213> Artificial sequence
<400>52
<210>53
<211>23
<212>DNA
<213> Artificial sequence
<400>53
ggtgttaact ccaacactat gtc 23
<210>54
<211>18
<212>DNA
<213> Artificial sequence
<400>54
ttgcacactg tcccgtcc 18
<210>55
<211>22
<212>DNA
<213> Artificial sequence
<400>55
ggatatgcaa caactgttga ag 22
<210>56
<211>21
<212>DNA
<213> Artificial sequence
<400>56
cccttccacg tacaatttag c 21
<210>57
<211>18
<212>DNA
<213> Artificial sequence
<400>57
tactaaccaa ggcacggt 18
<210>58
<211>22
<212>DNA
<213> Artificial sequence
<400>58
cagtgcgtgt tttgcaggaa tg 22
<210>59
<211>22
<212>DNA
<213> Artificial sequence
<400>59
cccttgcagt tctaagcaac ag 22
<210>60
<211>18
<212>DNA
<213> Artificial sequence
<400>60
aagttgtctc gccacaca 18
<210>61
<211>20
<212>DNA
<213> Artificial sequence
<400>61
<210>62
<211>21
<212>DNA
<213> Artificial sequence
<400>62
caagcgtccc atagactcac c 21
<210>63
<211>25
<212>DNA
<213> Artificial sequence
<400>63
tggcctggct cacctggaca acctc 25
Claims (3)
1. A HPV virus detection and typing primer and probe based on a microfluidic chip are characterized in that: the primer and probe sequences are specifically as follows:
1）.HPV16E-F：AGGAGCGACCCAGAAAGTTACC，
2）.HPV16E-R：ACAGCATATGGATTCCCATCTC，
3）.HPV16EFam-F:CAACAGTTACTGCGACG；
4）.HPV18E-F: CAACACGGCGACCCTACAAG，
5）.HPV18E-R: TACATTTATGGCATGCAGCATG，
6）.HPV18EFam-F: CACTTCACTGCAAGACA；
7）.HPV31E-F: GAAAGACCTCGGAAATTGCATG，
8）.HPV31E-R: TGGTGTGTCGTCCCTATATACT，
9）.HPV31EFam-F: CGGCATTGGAAATACCC；
10）.HPV33E-F: ATTGCATGATTTGTGCCAAGC，
11）.HPV33E-R: ATTCCAAATGGATTTCCCTCTC，
12）.HPV33EFam-R: ACATTGAACTACAGTGCG；
13）.HPV35E-F: CAGCGGAGTGAGGTATATGAC，
14）.HPV35E-R: CTAACGTTTCTCCATACACAC，
15）.HPV35EFam-F: CATATGGCTGGCCTTC；
16）.HPV39E-F: ATGGCGCGATTTCACAATCCTG，
17）.HPV39E-R: TCGTCTGCAATAGACACAGGCT，
18）.HPV39EFam-F: ACAACGCTGGACACCA；
19）.HPV45E-F: TACGAGCAATTAAGCGAGTCAG，
20）.HPV45E-R: CTGTAAGCTCAATTCTGCCGTC，
21）.HPV45EFam-F: AGTCATGCACAACTAC；
22）.HPV51E-F: gtacacgacaacgtaacgaaacc，
23）.HPV51E-R: cgtctttctggtagctggtc，
24）.HPV51EFam-F: TGTAGTATTGCATTTAACACCAC；
25）.HPV52E-F: ATGGACAAGCAGAACAAGCCAC，
26）.HPV52E-R: AACTTGTAATGTGCCCAACAGC，
27）.HPV52EFam-F: ACGGACCTTCGTACTC；
28）.HPV56E-F: AGGTGCTACAGATGTCAAAGT，
29）.HPV56E-R: CTGTAGATTCTCTAGGTTCTC，
30）.HPV56EFam-F：CTAATAGCACATGGTTG；
31）.HPV58E-F: ATGTGACAGCTCAGACGAGG，
32）.HPV58E-R: CGGTTGTTGTACTGTTGATACAC，
33）.HPV58EFam-F：ATGGACAAGCACAACCG；
34）.HPV59E-F: GAAGTTGACCTTGTGTGCTACG，
35）.HPV59E-R: ACACAATGTTGTGACGCTGTGG，
36）.HPV59EFam-F：CTCCATCTGGTTCATC；
37）.HPV68E-F: ACAACCCTGAGGAACGGCCAT，
38）.HPV68E-R: GGCAAATTCATATACCTCTGTC，
39）.HPV68EFam-R：TCCAATGTCCTGCACA；
40）.HPV26E-F: GAGATAGGAGTCCGTATGCTGC，
41）.HPV26E-R: GGCATTTGACATCTATGACACC，
42）.HPV26EFam-F：TGCAACATTAGAAGCC；
43）.HPV53E-F: AGTGTATAGAGACGGGTATCCG，
44）.HPV53E-R: GGATGTTGACATCTGTAGCACC，
45）.HPV53EFam-R：CCCGTACACTGAACAA；
46）.HPV66E-F: ATTCAGCAATACACAGGAACGT，
47）.HPV66E-R: TTTAACTCAATACATGCAAACCT，
48）.HPV66EFam-F：CTGAGCGAGGTATTAC；
49）.HPV73E-F: GACAGACAAGCTGAACGAGAG，
50）.HPV73E-R: CACTCTTAAATCAGCTTTGTTGC，
51）.HPV73EFam-F：ACTGACACTTCGTGCA；
52）.HPV82E-F: GCCTGAAGAAAAGCAAAAGG，
53）.HPV82E-R: GGTGTTAACTCCAACACTATGTC，
54）.HPV82EFam-R:TTGCACACTGTCCCGTCC；
55）.HPV6E-F: GGATATGCAACAACTGTTGAAG，
56）.HPV6E-R: CCCTTCCACGTACAATTTAGC，
57）.HPV6EFam-F:TACTAACCAAGGCACGGT；
58）.HPV11E-F: CAGTGCGTGTTTTGCAGGAATG，
59）.HPV11E-R: CCCTTGCAGTTCTAAGCAACAG，
60）.HPV11EFam-R:AAGTTGTCTCGCCACACA；
61）.Globin-F: GGCTCATGGCAAGAAAGTGC，
62）.Globin-R: CAAGCGTCCCATAGACTCACC，
63）.Globin-Hex-BHQ1-F: TGGCCTGGCTCACCTGGACAACCTC。
2. a microfluidic chip for detecting HPV virus and typing comprising the primers and probes of claim 1.
3. The microfluidic chip of claim 1, wherein: the device comprises Q-PCR reaction liquid embedded in a microfluidic chip, wherein the reaction liquid is as follows: the primers of claim 1, wherein the primers comprise HPV-F0.3 mMol/ml, HPV-R0.3 mMol/ml, probes 0.25 mMol/ml, Globin-F0.25 mMol/ml, Globin-R0.25 mMol/ml, Globin-Hex-BHQ 1-F0.2 mMol/ml, DNA polymerase 2000U/ml, 10 XTris-HCL reaction buffer 1X, and DNA Sample is more than or equal to 104Copy number/reaction, ddH2O make up to 3 uL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010786653.2A CN111876526A (en) | 2020-08-07 | 2020-08-07 | Microfluidic chip for detecting HPV (human papillomavirus) virus and typing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010786653.2A CN111876526A (en) | 2020-08-07 | 2020-08-07 | Microfluidic chip for detecting HPV (human papillomavirus) virus and typing |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111876526A true CN111876526A (en) | 2020-11-03 |
Family
ID=73211861
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010786653.2A Pending CN111876526A (en) | 2020-08-07 | 2020-08-07 | Microfluidic chip for detecting HPV (human papillomavirus) virus and typing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111876526A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107641663A (en) * | 2017-07-28 | 2018-01-30 | 中国药科大学 | For detecting the primer and probe and kit of high-risk human mammilla papillomavirus oncogene E6/E7DNA partings |
| CN109207639A (en) * | 2018-10-22 | 2019-01-15 | 南通国际旅行卫生保健门诊部 | Respiratory pathogen rapid fluorescence PCR detection kit and its primer combination of probe |
| US20190232289A1 (en) * | 2016-09-23 | 2019-08-01 | ArcherDX, Inc. | Fluidic system and related methods |
| CN110607399A (en) * | 2019-09-20 | 2019-12-24 | 西人马（厦门）科技有限公司 | Primer combination, kit and method for LAMP amplification to detect HPV and typing |
-
2020
- 2020-08-07 CN CN202010786653.2A patent/CN111876526A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190232289A1 (en) * | 2016-09-23 | 2019-08-01 | ArcherDX, Inc. | Fluidic system and related methods |
| CN107641663A (en) * | 2017-07-28 | 2018-01-30 | 中国药科大学 | For detecting the primer and probe and kit of high-risk human mammilla papillomavirus oncogene E6/E7DNA partings |
| CN109207639A (en) * | 2018-10-22 | 2019-01-15 | 南通国际旅行卫生保健门诊部 | Respiratory pathogen rapid fluorescence PCR detection kit and its primer combination of probe |
| CN110607399A (en) * | 2019-09-20 | 2019-12-24 | 西人马（厦门）科技有限公司 | Primer combination, kit and method for LAMP amplification to detect HPV and typing |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Poljak et al. | Commercially available molecular tests for human papillomaviruses (HPV): 2015 update | |
| CN104818342B (en) | Detection kit, detection architecture and method for 19 kinds of high-risk human mammilla papillomavirus (HPV) | |
| CN105603121B (en) | For detecting method, oligonucleotides and the kit of high-risk HPV | |
| CN105087827B (en) | Detection primer, probe and the kit of 16 kinds of type HPV viruses | |
| EP3187596B1 (en) | Method for detecting and typing high-risk human papillomaviruses | |
| Shen-Gunther et al. | HPV molecular assays: defining analytical and clinical performance characteristics for cervical cytology specimens | |
| CN110592279B (en) | Compositions, kits and methods for detecting HPV | |
| CN101796198A (en) | Composition comprising an oligonucleotide mixture for improved detection of human papillomavirus genotypes | |
| CN112575123B (en) | Primer combination, probe combination and human papillomavirus nucleic acid detection kit | |
| Ding et al. | Distribution of human papillomavirus 58 and 52 E6/E7 variants in cervical neoplasia in Chinese women | |
| Kim et al. | Development and evaluation of a highly sensitive human papillomavirus genotyping DNA chip | |
| CN108179226B (en) | Nucleic acid composition for detecting human papilloma virus, application thereof and kit | |
| CN109609696B (en) | Nucleic acid reagent, kit, system and method for detecting human papilloma virus | |
| CN105803110A (en) | Kit capable of carrying out typing and detection on many kinds of human papilloma viruses simultaneously and applications of kit | |
| CN111172328A (en) | Complete set of primers, complete set of probes, kit and method for HPV nucleic acid typing detection | |
| CN101886137B (en) | Method for qualitatively detecting high-risk HPV by using fluorescence quantitative PCR and kit thereof | |
| CN110607399A (en) | Primer combination, kit and method for LAMP amplification to detect HPV and typing | |
| CN104560962A (en) | Fluorescent PCR kit for detecting human papilloma virus and special primer group for kit | |
| EP2150629B1 (en) | Identification and quantification of oncogenic hpv nucleic acids by means of real-time pcr assays | |
| CN108085419B (en) | probe and primer composition | |
| CN111876526A (en) | Microfluidic chip for detecting HPV (human papillomavirus) virus and typing | |
| CN111593140B (en) | High-risk human papilloma virus detection and typing kit | |
| Garland et al. | Methods for HPV detection: Polymerase chain reaction assays | |
| Tinelli et al. | HPV viral activity by mRNA-HPV molecular analysis to screen the transforming infections in precancer cervical lesions | |
| Tadokoro et al. | Comparative quantitative analysis of 14 types of human papillomavirus by real-time polymerase chain reaction monitoring Invader reaction (Q-Invader assay) |
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 |