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Surface enhanced Raman spectrum detection method for gene based on CRISPR/Cas12a protein

A surface-enhanced Raman and spectral detection technology, which is applied in the field of spectral detection of virus genes, can solve the problems of weakening and SERS signal enhancement, and achieve the effects of high success rate, high sensitivity detection, and simple operation

Pending Publication Date: 2021-12-03
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of SERS technology in nucleic acid detection has been reported, most of which are based on the change in the aggregation shape of metal nanoparticles caused by nucleic acid hybridization, which leads to the enhancement or weakening of the SERS signal.

Method used

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  • Surface enhanced Raman spectrum detection method for gene based on CRISPR/Cas12a protein
  • Surface enhanced Raman spectrum detection method for gene based on CRISPR/Cas12a protein
  • Surface enhanced Raman spectrum detection method for gene based on CRISPR/Cas12a protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 CRISPR / Cas12a combined with SERS technology for detection of HPV16 nucleic acid

[0053] In this embodiment, the metal nanoparticle probe is a gold nanoparticle probe.

[0054] In this embodiment, the Raman signaling molecule is MBN.

[0055] In this example, the DNA 1 The sequence is SH-AAAAAAAAAAACACACTCACACACAC.

[0056] In this example, the DNA 2 The sequence is SH-AAAAAAAAAACCTCACCACCAACAC.

[0057] In this embodiment, the sequence of the substrate ssDNA is GTGTGAGTGTGTGTGGTGTTGGTGGTGAGG.

[0058] 在该实施例中,所述靶标核酸为HPV 16 DNA具体序列为CTATGGATTACAAACAAACACAATTGTGTTTAATTGGTTGCAAACCACCTATAGGGGAACACTGGGGCAAAGGATCCCCATGTACCAATGTTGCAGTAAATCCAGGTGATTGTCCACCATTAGAGTTAATAAACACAGTTATTCAGGATGGTGATATGGTTGATACTGGCTTTGGTGCTATGGACTTTACTACATTACAGGCTAACAAAAGTGAAGTTCCACTGGATATTTGTACATCTA。

[0059] In this embodiment, the crRNA is the corresponding crRNA of HPV 16 16 , the specific sequence is UAAUUUCUACUAAGUGUAGAUCUACAUUACAGGCUAACAAA.

[0060] (1) Preparation of gold nanopa...

Embodiment 2

[0083] Example 2 CRISPR / Cas12a combined with SERS technology for detection of HPV18 nucleic acid

[0084] In this embodiment, the metal nanoparticle probe is a gold nanoparticle probe.

[0085] In this embodiment, the Raman signaling molecule is MBN.

[0086] In this example, the DNA 1 The sequence is SH-AAAAAAAAAAACACACTCACACACAC.

[0087] In this example, the DNA 2 The sequence is SH-AAAAAAAAAACCTCACCACCAACAC.

[0088] In this embodiment, the sequence of the substrate ssDNA is GTGTGAGTGTGTGTGGTGTTGGTGGTGAGG.

[0089] 在该实施例中,所述靶标核酸为HPV 18 DNA片段,具体序列为GGGAACACTGGGCTAAAGGCACTGCTTGTAAATCGCGTCCTTTATCACAGGGCGATTGCCCCCCTTTAGAACTTAAAAACACAGTTTTGGAAGATGGTGATATGGTAGATACTGGATATGGTGCCATGGACTTTAGTACATTGCAAGATACTAAATGTGAGGTACCATTGGATATTTGTCAGTCTATTTGTAAATATCCTGATTATTTACAAATGTCTGCAGATCCTTATGGGGATTCCA。

[0090] In this embodiment, the crRNA is HPV 18 crRNA 18 , the specific sequence is UAAUUUCUACUAAGUGUAGAUGUACAUUGCAAGAUACUAAA.

[0091] Steps (1) and (2) to prepare gold nanoparticles ...

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Abstract

The invention discloses a surface enhanced Raman spectrum detection method for a gene based on CRISPR / Cas12a protein. Target virus nucleic acid, Cas12a and crRNA are utilized to form a ternary complex, and a substrate (i.e., ssDNA) in a cleavage system is opened. The ssDNA is a bridging complementary sequence of DNA1 and DNA2 on a probe 1 and a probe 2. Whether the probe 1 and the probe 2 are bridged or not can cause change of SERS signals, so that whether a sample to be detected contains target nucleic acid or not can be known. Accurate and rapid detection of viral nucleic acid can be realized by using the specific crRNA. The CRISPR@Cas12a is combined with the SERS technology, so that the biggest advantage is that a tedious modification preparation process is avoided, and the SERS probe can stably exist for a long time. According to the method, the key step of signal amplification occurs on a solution phase of ssDNA instead of an interface of a nano assembly, so that the method is more stable and higher in trans-cutting efficiency, the sensitivity and repeatability of the detection method can be guaranteed, and the method has important significance in early monitoring, diagnosis, prevention and control of diseases.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a spectral detection method for viral genes based on CRISPR / Cas12a protein and surface-enhanced Raman scattering (SERS). Background technique [0002] Rapid diagnosis of virus types can effectively reduce the large-scale spread and infection of viral infectious diseases, and provide an important scientific basis for its prevention and control policies in time. Traditional viral nucleic acid detection methods generally have problems such as harsh detection conditions, expensive or time-consuming detection, and sensitivity to be improved. Therefore, it is urgent to develop rapid, highly sensitive, and specific detection technologies for viral nucleic acids. [0003] In recent years, surface-enhanced Raman scattering (Surface-enhanced Raman Scattering, SERS) biosensing technology has been widely used in biological detection and analysis due to its ultra-high sensi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6816C12Q1/70C12R1/93
CPCC12Q1/6816C12Q1/708C12Q2521/301C12Q2563/155C12Q2565/515C12Q2565/632
Inventor 徐抒平苏艾玲梁重阳徐蔚青
Owner JILIN UNIV
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