CN111876526A - A microfluidic chip for detection and typing of HPV virus - Google Patents

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

A microfluidic chip for detection and typing of HPV virus

technical field

The invention belongs to the technical field of medical detection, and in particular relates to a microfluidic chip for detecting and typing HPV viruses.

Background technique

Human papillomavirus (Human Paillomavirus, HPV), belonging to the papillomavirus family, is a small molecule, non-encapsulated, circular double-stranded DNA virus, containing about 7900 base pairs (bp), a total of 3 Gene region composition, including early region (Early Region, E region), late region (Late Region, L region) region and non-coding region (UncodingRegion, UCR) or upstream regulatory region (URR). The E region consists of 7 genes in order, E6, E7, E1, E2, E3, E4 and E5, which are involved in viral DNA replication, transcription, encoding viral proteins, and maintaining a high copy number of intracellular viruses. Among them, E6 and E7 It is the main oncogene of HPV, which is related to the transformation function of virus cells and carcinogenicity. HPV infects humans through direct or indirect contact with contaminated items or through sexual transmission. The virus is not only host-specific, but also tissue-specific, and can only infect human skin and mucosal epithelial cells, causing a variety of papilloma or warts of human skin and proliferative damage to the reproductive tract epithelium.

Global research results show that 70% to 80% of women will have at least one HPV infection in their lifetime, and the presence of high-risk HPV DNA has been detected in 99.7% of cervical cancer patients, including HPV16, 18, and HPV types. Type 45 and 31 infections accounted for 80%. Low-risk HPV types are generally associated with condyloma acuminatum or low-grade squamous intraepithelial lesions and rarely cause invasive carcinoma. Therefore, HPV detection is extremely important for the detection and typing of HPV virus. Regarding the classification of high-risk and low-risk types, many international institutions have given reference recommendations. According to the research results of the WHO International Agency for Research on Cancer (IARC) and other international organizations, HPV16, 18, 31, 33, 35, 39 13 genotypes including 45, 51, 52, 56, 58, 59, and 68 were classified as high-risk types, and 5 genotypes including 26, 53, 66, 73, and 82 were classified as medium-risk types.

The traditional diagnosis of cervical cancer mainly follows the "three-step" diagnostic procedure, namely cervical cytology, colposcopy and histopathological examination. The traditional early screening of cervical cancer in women mainly relies on this "three-step" diagnostic procedure. When three detection methods are used, the diseased cells are easily detached during the cervical cell sampling process, and the cell morphology is also easily changed during the preparation process. Changes or are covered by other inflammatory cells and blood cells, and the result judgment is too dependent on personal subjective judgment, so it is easy to miss the diagnosis of the disease. Of course, there are also some modern molecular detection technologies, such as DNA hybridization detection technology, Q-PCR fluorescent dye detection technology, etc. Although DNA hybridization technology has high detection results, the detection cost and detection process are complicated, and it is very easy to make errors in the detection process. , resulting in false negative results; Q-PCR fluorescent dye detection technology, although low in cost, has a significant disadvantage that its specificity is very susceptible to non-specific gene amplification and primer dimer formation, which makes double-stranded DNA qPCR methods combined with fluorescent dyes have not been able to be effectively applied in clinical detection. However, MGB probe qPCR can effectively solve the non-specific problem of double-stranded DNA-binding fluorescent dye qPCR. MGB probe qPCR has the advantages of strong specificity, high sensitivity, good repeatability, accurate quantification, and fast speed. It can be used in the future. It has become an important tool for molecular diagnosis in clinical detection and microfluidic chip detection.

At the same time, the invention is applied to the HPV virus detection and typing of the microfluidic chip. The microfluidic chip has the advantages of simple operation, low price, low sample consumption, fast analysis speed, and does not depend on expensive instruments, and is very suitable for the treatment of underdeveloped areas. Rapid detection and analysis of disease. The chip can be extended to the rapid detection of any other pathogens, which is of great significance for the grass-roots prevention and control of some acute infectious pathogens.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a microfluidic chip for detecting and typing HPV viruses, and specifically relates to a method for detecting and typing HPV viruses containing 20 types of high-risk and low-risk types based on a microfluidic chip. The MGB probe method with strong stability and high specificity is adopted, and the type-specific primers designed by the E6 and E7 regions that are highly conservative and less prone to loss, combined with a self-developed lyophilized powder that can be embedded in The Q-PCR primers and probes and the enzyme premix in the microfluidic chip finally realize the detection of HPV virus through the microfluidic chip, with simple operation, low cost and short cycle.

To achieve the above object, the present invention adopts the following technical solutions:

A microfluidic chip for detecting HPV virus and typing, comprising specific primers and probes for detecting HPV virus and typing, the primers and probe sequences 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: CGGCATGGAAATACCC

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 includes a Q-PCR reaction solution embedded in the microfluidic chip, and the reaction solution is: the above-mentioned HPV-F 0.3 mMol/ml, HPV-R 0.3 mMol/ml, HPV probe 0.25µMol/ml, Globin-F 0.25µMol/ml, Globin-R 0.25µMol/ml, Globin-Hex-BHQ1-F 0.2µMol/ml, DNA polymerase 2000U/ml, 10X Tris-HCL reaction buffer 1X, DNA Sample ≥10 ⁴ copies/reaction, ddH ₂ O made up to 3uL.

A method for HPV virus detection and typing based on a microfluidic chip, comprising the following methods:

A. Selection and determination of detection type; B. Design of primers for HPV virus E6 and E7 regions; C. Selection and design of MGB probes; Independent research and development of PCR primers, probes and enzyme master mixes; E. Exploration and determination of detection volume; F. Design and optimization of detection conditions.

Specifically, the following methods are included:

(1) Selection and determination of detection types: The detection types of this method cover 12 types of carcinogens (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) ), and can also distinguish other 8 important types (HPV6, 11, 26, 53, 66, 68, 73, 82). Large flux, comprehensive detection;

(2) Sequence analysis and primer design of E6 and E7 regions of HPV virus: This method adopts targeted detection of E6 and E7 regions to avoid the frequent loss of L1 region during the integration of high-risk HPV, resulting in false negatives, misdiagnosis and missed diagnosis. Type-specific primers are designed through multiple alignments between all detection types, which effectively avoids the problem of cross-contamination;

(3) Selection and design of MGB probe: The present invention detects HPV virus by the MGB probe method, which greatly improves the detection specificity. Type-specific probes are designed through multiple alignments between all detection types, effectively avoiding the problem of cross-contamination;

(4) Independent research and development of Q-PCR primers and probes and enzyme premixes that can be lyophilized into powder and embedded in microfluidic chips: The Q-PCR reaction solution used in the present invention can be lyophilized, which solves the problem of primer probes and primers. The problem that the reaction buffer is embedded together makes the subsequent detection convenient and efficient;

(5) Exploration and determination of detection volume: 3ul detection system is adopted, which greatly reduces the cost of reagents;

(6) Design and optimization of detection conditions: In the present invention, primers, probes and reaction solutions are pre-embedded in the chip, so that detection and typing only need to add pre-mixed cervical exfoliated cell genome and DNA polymerase in one step. Can. A one-way valve is arranged between each reaction chamber in the chip, so that the PCR reactions are independent of each other and cross-contamination is avoided.

The invention detects the HPV virus by the MGB probe method, solves the non-specific problem of the fluorescent dye qPCR method, and at the same time relies on the microfluidic chip to reduce the cost of HPV detection.

The primers and probes designed in the present invention analyze various types of HPV sequences through bioinformatics, and use biological software (snapGene, DNAman, Vector NTI) to design specific primers for each type in the E6E7 region of the HPV virus gene, and the designed primers and Probe specificity is high. Because general-purpose PCR primers are used to amplify the HPV L1 region, but the L1 region is often lost during the integration of high-risk HPV, the detection is very prone to false negatives, misdiagnosis and missed diagnosis.

The invention also uses a self-developed Q-PCR primer, probe and enzyme premix that can be freeze-dried into powder and embedded in a microfluidic chip. There is no lyophilized PCR reaction solution on the market for the time being, and the invention solves the problem of embedding primers and probes when the microfluidic chip is used for HPV virus typing.

Usually the detection volume required for HPV Q-PCR molecular detection is 25ul, but a 3ul detection system can be achieved on the microfluidic chip, which greatly reduces the detection cost. At the same time, in the traditional HPV molecular detection and typing, the same sample needs to do multiple PCR reactions separately, which is labor-intensive, time-consuming, consumables, and required equipment, while the microfluidic chip can complete the sample detection at one time. It solves such problems and changes the limitations of traditional detection.

In the present invention, primers, probes, reaction solution, etc. are pre-embedded in the chip, so that detection and typing only need to add pre-mixed cervical exfoliated cell genome and DNA polymerase in one step. A one-way valve is arranged between each reaction chamber in the chip, so that the PCR reactions are independent of each other and cross-contamination is avoided.

In the present invention, the genomic fluid of cervical exfoliated cells enters the Q-PCR reaction chamber of the microfluidic chip by means of a piston that is stretched backwards, and does not need to be pre-processed into vacuum or negative pressure, and the use of piston stretching can avoid vacuum or negative pressure leakage pressure , or insufficient negative pressure to prevent cervical exfoliated cell genomic fluid from entering the Q-PCR chamber.

The advantages of the present invention are:

1. The primers and probes used in the HPV detection and typing of the present invention have high specificity and sensitivity and good repeatability.

2. The present invention relies on a microfluidic chip to detect HPV virus, with simple operation, low cost and short cycle.

3. The HPV typing of the present invention involves a total of 20 types of high-risk and low-risk types, with large throughput and comprehensive detection.

4. The Q-PCR reaction solution used in the present invention can be freeze-dried, which solves the problem that the primer probe and the reaction buffer are embedded together, and makes subsequent detection convenient and efficient.

Description of drawings

Figure 1 The results of HPV16 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 2 The results of HPV18 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 3. The results of HPV31 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 4 The results of HPV33 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 5. The results of HPV35 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Fig. 6 The results of HPV39 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 7 The results of HPV45 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 8. The results of HPV51 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 9 Results of HP52 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Fig. 10 The results of HPV56 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 11 Result of HPV58 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 12 Result of HPV59 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 13 Result of HPV6 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 14 Result of HPV11 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 15 Result of HPV26 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 16 Result of HPV53 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 17 The results of HPV66 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 18 Result of HPV68 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 19 HPV73 and Globin internal reference primers and probe-specific fluorescence quantitative PCR results.

Figure 20 Result of HPV82 and Globin internal reference primers and probe-specific fluorescence quantitative PCR.

Figure 21 Validation of the detection sensitivity of HPV16 primers and probes.

Figure 22 Validation of the detection sensitivity of HPV18 primers and probes.

Figure 23 HPV16 primer and probe-specific cross-validation results.

Figure 24 HPV18 primer and probe-specific cross-validation results.

Figure 25 is a top view of the microfluidic chip. 1. Injection hole; 2. Injection hole one-way valve; 3. Microfluidic pipeline; 4. Microfluidic pipeline one-way valve; 5. Q-PCR reaction chamber; 6. Syringe; 7. Volume scale; 8. injection piston; 9. stretching arm; 10. stretching arm direction icon; 11. injection hole cover; P. positive control reaction area; S. experimental group reaction area.

Figure 26. Side view of the microfluidic chip. 1. Injection hole; 2. Injection hole one-way valve; 3. Microfluidic pipeline; 4. Microfluidic pipeline one-way valve; 5. Q-PCR reaction chamber; 6. Syringe; 7. Volume scale; 8. injection piston; 9. stretching arm; 10. stretching arm direction icon; 11. injection hole cover; P. positive control reaction area; S. experimental group reaction area.

Detailed ways

Example 1

1. The primer and probe sequences are shown in the sequence table.

System and component concentrations:

3. 10X Tris-HCl reaction buffer component concentration:

4. Q-PCR reaction conditions: 1. 94°C, 3min; 2. 94°C, 5s; 3. 60°C, 30s; 4. Plate Read; 5. go to 2, 45 Cycle.

Test results: 1. HPV test results and positive judgment:

(1) Quality control standard ① The determination result of the positive control is positive, and Ct≤35.00. ② The determination result of the negative control is negative, Ct has no value or Ct≥39.00. ③ The amplification curve should have a distinct exponential phase; the baseline and exponential phase should have distinct inflection points. ④ The above three conditions should be met at the same time, otherwise the test will be invalid.

(2) Judgment of results ① Specimens with no Ct value or Ct≥39.00 are negative. ② Specimens with Ct<35.00 but no obvious exponential phase of the amplification curve or no clear inflection point between the baseline and exponential phase are negative. ③ Ct≤35.00, and the amplification curve has an obvious exponential phase, and the specimens with a clear inflection point in the baseline and exponential phase are positive. ④ The specimens with 35.00<Ct<39.00 were tested again, and the retest results were positive if Ct<39.00, otherwise they were all negative.

The 20 human papillomavirus DNA plasmids (types: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56) in the HPV genotyping quality control disk established by the China Institute of Pharmaceutical and Biological Products were used. , HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, HPV82, HPV6, HPV11), according to the amount of 10 ⁴ Copies/reaction, the internal reference is the human housekeeping gene Globin, the system is 10ul, and Fam is the HPV virus fluorescence amplification signal, Hex is the internal reference fluorescence amplification signal. All HPV types in the test results can detect standard amplification signals and the Ct values are less than 35.

2. HPV typing detection sensitivity verification:

The 20 human papillomavirus DNA plasmids (types: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56) in the HPV genotyping quality control disk established by the China Institute of Pharmaceutical and Biological Products , HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, HPV82, HPV6, HPV11) were diluted respectively to prepare several samples with concentrations of 10 ⁵ Copies/µL, 10 ⁴ Copies/µL, 10 ³ Copies/µL , 10 ² Copies/µL, 10 ¹ Copies/µL, which are DNA templates, were detected according to the 10ul detection system (including internal reference), and HPV could be detected in both 10 ⁵ Copies/µL and 10 ⁴ Copies/µL. signal and internal reference signal. Therefore, the detection sensitivity of Q-PCR specific typing was 10 ⁴ Copies/µL.

3. Validation of HPV typing test specificity: (cross-validation)

The 20 human papillomavirus DNA plasmids (types: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56) in the HPV genotyping quality control disk established by the China Institute of Pharmaceutical and Biological Products , HPV58, HPV59, HPV68, HPV26, HPV53, HPV66, HPV73, HPV82, HPV6, HPV11), according to the amount of 10 ⁴ Copies/reaction, the system was 10ul to carry out cross-validation of 20 HPV primers and probes respectively. In the results, all HPV typing primers and probe cross-validation results showed that the signal was single and the signal of the corresponding HPV type.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

<110> Fuzhou University

Western Human Horse Dazhou (Pingxiang) Medical Technology Co., Ltd.

<120> A microfluidic chip for detection and typing of HPV virus

<130> 63

<160> 63

<170> PatentIn version 3.3

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ggatatgcaa caactgttga ag 22

<210> 56

<211> 21

<212> DNA

<213> Artificial sequences

<400> 56

cccttccacg tacaatttag c 21

<210> 57

<211> 18

<212> DNA

<213> Artificial sequences

<400> 57

tactaaccaa ggcacggt 18

<210> 58

<211> 22

<212> DNA

<213> Artificial sequences

<400> 58

cagtgcgtgt tttgcaggaa tg 22

<210> 59

<211> 22

<212> DNA

<213> Artificial sequences

<400> 59

cccttgcagt tctaagcaac ag 22

<210> 60

<211> 18

<212> DNA

<213> Artificial sequences

<400> 60

aagttgtctc gccacaca 18

<210> 61

<211> 20

<212> DNA

<213> Artificial sequences

<400> 61

ggctcatggc aagaaagtgc 20

<210> 62

<211> 21

<212> DNA

<213> Artificial sequences

<400> 62

caagcgtccc atagactcac c 21

<210> 63

<211> 25

<212> DNA

<213> Artificial sequences

<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.

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