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CN-121992149-A - Multiple RPA-CRISPR detection system for human papilloma virus typing

CN121992149ACN 121992149 ACN121992149 ACN 121992149ACN-121992149-A

Abstract

The invention provides a multiple RPA-CRISPR detection system aiming at human papilloma virus typing, which comprises an RPA isothermal amplification module, a CRISPR multiple detection module and a signal output module, wherein the RPA isothermal amplification module comprises RPA amplification primers targeting eight HPV subtypes of 16, 18, 11, 31, 33, 45, 52 and 58 and 2 freeze-dried spheres, so that one-tube multiple amplification is realized on a sample, the CRISPR multiple detection module comprises a multiple microcavity device, a detection system pre-buried in the multiple microcavity device and a constant-temperature heating pad, and the signal output module is a lateral flow chromatographic test strip. The invention also provides application of the multiple RPA-CRISPR detection system and a detection method. According to the invention, the microfluidic chip is used for integrating a plurality of reaction steps and reagents, so that POC detection of the eight high-risk HPVs is realized.

Inventors

  • CHEN RUI
  • PAN XING
  • Ding shijia
  • LI JUAN

Assignees

  • 重庆医科大学

Dates

Publication Date
20260508
Application Date
20260126

Claims (10)

  1. 1. A multiple RPA-CRISPR detection system for human papilloma virus typing is characterized by comprising an RPA isothermal amplification module, a CRISPR multiple detection module and a signal output module, wherein the RPA isothermal amplification module comprises RPA amplification primers targeting eight HPV subtypes 16, 18, 11, 31, 33, 45, 52 and 58 and 2 freeze-dried spheres to realize one-tube multiple amplification of a sample, the CRISPR multiple detection module comprises a multiple microcavity device, a detection system embedded in the multiple microcavity device and a constant-temperature heating pad, and the signal output module is a lateral flow chromatography test strip.
  2. 2. The multiplex RPA-CRISPR detection system as recited in claim 1 wherein said RPA amplification primers comprise forward and reverse primers and said RPA amplification primer sequence is set forth in SEQ ID NO. 1-16.
  3. 3. The multiplex RPA-CRISPR detection system for human papillomavirus typing according to claim 1, wherein the detection system of the CRISPR multiplex detection module comprises cas12a, crRNA and NEBbuffer r 2.1.1, and the crRNA sequence is shown as SEQ ID NO. 17-24.
  4. 4. The multiplex RPA-CRISPR detection system as recited in claim 3 wherein said detection system further comprises a reporter probe, said reporter probe being a fluorescent probe FQ, said fluorescent probe having a sequence of 5'-FAM-TTATT-3' -BHQ1, a 5 '-end-labeled FAM group, and a 3' -end-labeled quenching group.
  5. 5. The multiplex RPA-CRISPR detection system for human papillomavirus typing according to claim 3, wherein the detection system further comprises a report probe and ddH 2 O, wherein the report probe is a test strip probe FB, the test strip probe sequence is 5'-FAM-CGCGCGCG-3' -Biotin, a FAM group is marked at the 5 'end, and a Biotin group is marked at the 3' end.
  6. 6. The multiple RPA-CRISPR detection system for human papilloma virus typing, which is characterized in that the lateral flow chromatography test strip comprises a sample pad, a conjugate pad, an NC membrane and an absorption pad, wherein the conjugate pad is coated with latex microspheres coupled with anti-FAM antibodies, the NC membrane is sequentially provided with a detection T line and a quality control C line, and the T line is used for fixing streptavidin and the C line is used for fixing secondary antibodies.
  7. 7. Use of a multiplex RPA-CRISPR detection system according to any of claims 1-6 for the detection of human papillomavirus typing for non-diagnostic purposes.
  8. 8. A method according to any one of claims 1-6 for detection of human papillomavirus typing non-disease diagnostic purposes based on a multiplex RPA-CRISPR detection system for human papillomavirus typing, characterized by comprising the steps of: s1, extracting DNA in a sample to be detected; s2, performing multiple RPA reaction, namely adding an RPA amplification primer, magnesium ions and 2 freeze-dried balls into a reaction device by taking DNA in an extracted sample to be detected as a template, and reacting for 10-20 min at 39 ℃; s3, CRISPR reaction, namely after RPA reaction is finished, injecting a mixture of an RPA reaction product and enzyme-free water into the multi-microcavity device, and reacting for 10-20 min at 48 ℃ on a heating pad; s4, after the CRISPR reaction is finished, adding enzyme-free water again, reversing the device to enable the mixed solution to flow to a test paper tank for the lateral flow chromatography detection, and observing the result after 5 minutes.
  9. 9. The method of claim 8, wherein the amplification reaction system comprises the following reagents and amounts of 2.5. Mu.L of 10. Mu.M forward primer, 2.5. Mu.L of 10. Mu.M reverse primer and 45. Mu.L of the sample DNA template to be tested.
  10. 10. The method of claim 8, wherein the CRISPR reaction system in step S3 comprises 1. Mu.M cas12a 1. Mu.L, 2. Mu.M crRNA 1. Mu.L, 10. Mu.M NEBbuffer r 2.1.1. Mu.L, and reporter probe 2. Mu. L, RPA product 2. Mu. L, ddH 2 O12. Mu.L.

Description

Multiple RPA-CRISPR detection system for human papilloma virus typing Technical Field The invention belongs to the technical field of multiplex nucleic acid detection, and particularly relates to a multiplex RPA-CRISPR detection system for human papillomavirus typing. Background Cervical cancer is a global health problem and is a major cause of cancer-related death in women worldwide. About 60.4 ten thousand women were diagnosed with cervical cancer in 2020, and about 34.2 ten thousand women died from cervical cancer. Although the incidence of cervical cancer is reduced in high income countries with cervical cancer screening and vaccine popularization, the middle and low income countries still bear a huge cervical cancer burden. More than 85% of new cases and deaths are concentrated in areas where resources are low. It is estimated that more than 95% of cervical cancers are caused by HPV. HPV nucleic acid molecule detection methods include Southern blotting, northern blotting, reverse dot blotting, in situ blotting, hybridization Capture (HC) II, polymerase Chain Reaction (PCR) and microarray technology. All of the above methods have the disadvantages of being time consuming, cumbersome, relying on expensive instrumentation and specialized operators, and are not suitable for use in low resource areas. Early diagnosis of cervical HPV infection requires low cost, high operability, high sensitivity and specific detection methods. Thus, establishing an HPV point-of-care test (POCT) that is independent of laboratory equipment and professionals, even allowing for self-testing at home, can significantly increase the popularity of HPV screening. The method can meet the requirements of basic medical market and household HPV screening, and has great market potential. In recent years, CRISPR/Cas systems have an incomparable advantage in the field of nucleic acid diagnostics, with higher specificity, sensitivity and without expensive instrumentation. Typically, the target nucleic acid is amplified using techniques such as PCR, LAMP, or RPA, followed by the use of CRISPR RNA (crRNA) to recognize the amplicon, the crRNA activating CRISPR CAS enzyme, cis-cleaving the target DNA and trans-cleaving the reporter. The trans-cleavage activity can generate continuous cleavage for the reporter for 0.07-7 times per second, so that the signal output is obviously enhanced, and meanwhile, the visualization of the detection result can be realized by combining with the lateral flow chromatography, an additional reading device is not needed, and the POCT scene is more adapted. At present, the mature HPV POCT commercial products such as HPV detection test paper and the like adopt a cytological staining related principle, the sensitivity and the specificity are far lower than those of a molecular detection means, the antibody antigen related detection means have a detection window period and are easy to cause false positive or false negative false detection, and other HPV nucleic acid POC detection means which are under research and are only based on isothermal amplification technology such as LAMP, RCA, RAA and the like have the technical bottlenecks of low detection flux, incapability of ensuring the specificity, lack of accurate typing means and the like, so that the sensitive multiple typing detection is difficult to realize. The research and development of the new generation of HPV POC detection technology based on the molecular principle are urgent. Disclosure of Invention In order to solve the problems in the prior art, the invention provides a multiple RPA-CRISPR detection system for human papillomavirus typing, which adopts the technology of combining RPA with CRISPR/Cas12a, and covers 90% of cervical cancer cases aiming at eight main HPV subtypes of HPV16, 18, 11, 31, 33, 45, 52 and 58 types, thereby effectively solving the technical problem of low amplification flux caused by primer interaction during RPA multiple amplification. The invention realizes an eight-fold RPA amplification system in a reaction tube, realizes the typing detection of eight high-risk HPVs through eight crRNAs, and realizes that the detection depth can reach 10 copy/mu L in 40min reaction time. According to the invention, the microfluidic chip is used for integrating a plurality of reaction steps and reagents, so that POC detection of the eight high-risk HPVs is realized, POC development of high-risk genotyping detection of the HPVs is promoted to a great extent, the popularization rate of the HPV detection is promoted, and the standardization, the precision and the popularization of risk layering management of HPV patients are assisted. The invention solves the technical problems by adopting the following technical scheme: The invention provides a multiple RPA-CRISPR detection system aiming at human papilloma virus typing, which comprises an RPA isothermal amplification module, a CRISPR multiple detection module and a signal output module, wherein the R