CN-122012675-A - Rapid detection method for echinococcus lycoris based on RPA-CRISPR/Cas

Abstract

The invention discloses a rapid detection method for echinococcus lycocanal taenia based on RPA-CRISPR/Cas, and belongs to the technical field of molecular diagnosis. The method comprises the steps of extracting DNA of a sample to be detected, carrying out RPA isothermal amplification by using a specific primer, reacting a reaction system comprising buffer solution, an upstream primer, a downstream primer, RPA enzyme, magnesium acetate and template DNA for 20 minutes at 39 ℃, mixing an amplified product with LbCas a protein, sgRNA and a fluorescent reporter probe, carrying out CRISPR/Cas reaction for 30 minutes at 37 ℃, and judging a result through a real-time fluorescent signal. The invention realizes constant-temperature rapid amplification by utilizing RPA, combines high specificity identification and cutting activity of CRISPR/Cas12a, can realize high-sensitivity and high-specificity detection of echinococcus spinosus under field conditions, has sensitivity up to single-digit copy number, is simple and convenient to operate, does not need complex instruments, and is suitable for basic medical treatment and field quarantine.

Inventors

• CAI QIGANG
• WANG LIPING
• WU XIAODONG

Assignees

• 青海省畜牧兽医科学院

Dates

Publication Date

20260512

Application Date

20260206

Claims (1)

1. 1. The rapid detection method for echinococcus lycopersicum based on RPA-CRISPR/Cas is characterized by comprising the following steps: S1, extracting DNA of a sample to be detected; S2, RPA amplification, namely configuring an RPA reaction system, and amplifying by an RPA method to obtain an amplified product; s3.CRISPR/Cas system reaction detection, namely adding a fluorescent reporter probe, cas12a and sgRNA into the amplification product to perform CRISPR reaction detection and read detection signals; Wherein the Cas12a protein is LbCas a, the RPA primer combination F-5'-CATGCAGTGAGTTAGATGGTAAGCGTTGGTT-3', R-5'-AACCACAATAACAATTCTAGCCACACTACCT-3', and the sgRNA has the sequence UAAUUUCUACUAAGUGUAGAUAAAUACUCGUAAUUGAGUGUUGA. Also comprises a fluorescent report probe with the sequence of 5'-TTATT-3'. The kit also comprises an RPA amplification reagent, which comprises RPA enzyme and magnesium acetate; The reaction system for RPA amplification comprises an RPA buffer solution, ddH 2 O, an upstream primer, a downstream primer, RPA enzyme lyophilized powder, template DNA and magnesium acetate, wherein the reaction condition for RPA amplification is that the reaction is carried out at 39 ℃ and 20min, the reaction system for CRISPR/Cas system reaction comprises 10X LbCas12a Reaction Buffer, lbCas12a, a template, a fluorescent reporter probe and sgRNA, and the reaction condition for CRISPR/Cas system reaction is that the reaction is carried out at 37 ℃ and 30 min; The RPA amplification reaction system comprises 29.5 mu L of RPA buffer solution, 2.5 mu L of 280 mM magnesium acetate, 2.4 mu L of 10 mu M upstream primer, 2.4 mu L of 10 mu M downstream primer, 3 mu L of DNA template and RPA enzyme freeze-dried powder, wherein the ratio of the components of the CRISPR/Cas system reaction system is prepared according to the following proportion that the RPA reaction product is 2 mu L, 10X LbCas12a Reaction Buffer mu L,1 mu M LbCas12 a1 mu L,10 mu M fluorescent report probe is 1 mu L and 1 mu M sgRNA is 1 mu L, ddH 2 O is supplemented to 20 mu L, and the reaction condition is that a FAM channel fluorescent signal is collected every 30 s by a real-time fluorescent quantitative PCR instrument at a qPCR instrument setting program of 37 ℃ and 30 min.

Description

Rapid detection method for echinococcus lycoris based on RPA-CRISPR/Cas Technical Field The invention relates to the technical field of molecular diagnosis, in particular to a method for rapidly detecting echinococcus spinosa based on combination of Recombinase Polymerase Amplification (RPA) and clustered regular interval short palindromic repeated sequences/CRISPR related proteins (CRISPR/Cas), which is suitable for clinical diagnosis, livestock quarantine and epidemiological investigation of echinococcus spinosa. Background Echinococcus disease (Echinococcosis shiquicus) is a parasitic disease caused by larvae of Echinococcus (Echinococcus shiquicus), is a special parasitic disease in China, is first found and identified in Qinghai-Tibet plateau, and is limited to the Qinghai-Tibet plateau in China at present. The main intermediate host is a high-altitude mouse rabbit (Plateau pika, ochotona curzoniae) at present, and is mainly parasitic on organs such as liver, lung and the like, and the final host is dogs such as foxes and the like. At present, the detection method of echinococcus disease has obvious shortboards that the detection of the ova mainly includes microscopic examination after floating sedimentation is carried out on excrement of a final host, but the discharge of adult pregnancies of the final host is irregular, the ova are not uniformly dispersed in the excrement of the host and are easy to leak detection, the method is very similar to other echinococcus ova in morphology and difficult to distinguish under an optical microscope, the conventional PCR detection depends on expensive equipment and complex temperature cycle operation, the detection period is long, the influence of inhibitors in samples is large, and the requirements of on-site rapid detection and epidemiological investigation in remote pastures cannot be met. Therefore, there is a need to develop a method suitable for on-site, rapid and accurate detection to overcome the shortcomings of the existing detection techniques. Disclosure of Invention Aiming at the defects of the echinococcus spinosus detection method in the prior art, the invention provides a rapid detection method based on RPA-CRISPR/Cas, realizes high-specificity, high-sensitivity and constant-temperature rapid detection of the echinococcus spinosus, and meets the requirements of clinical diagnosis and on-site quarantine. The invention aims to provide a rapid detection method for echinococcus lycoris based on RPA-CRISPR/Cas, which comprises the following steps: S1, extracting DNA of a sample to be detected; S2, RPA amplification, namely configuring an RPA reaction system, and amplifying by an RPA method to obtain an amplified product; S3.CRISPR/Cas system reaction detection, namely taking the amplification product, adding a fluorescent reporter probe, cas12a and sgRNA, carrying out CRISPR reaction detection, and reading a detection signal. Wherein the Cas12a protein is LbCas a, the RPA primer combination F-5'-CATGCAGTGAGTTAGATGGTAAGCGTTGGTT-3', R-5'-AACCACAATAACAATTCTAGCCACACTACCT-3', and the sgRNA has the sequence UAAUUUCUACUAAGUGUAGAUAAAUACUCGUAAUUGAGUGUUGA. Also comprises a fluorescent report probe with the sequence of 5'-TTATT-3'. The kit also comprises an RPA amplification reagent, which comprises RPA enzyme and magnesium acetate; The reaction system for RPA amplification comprises an RPA buffer solution, ddH 2 O, an upstream primer, a downstream primer, RPA enzyme lyophilized powder, template DNA and magnesium acetate, wherein the reaction condition for RPA amplification is that the reaction is carried out at 39 ℃ and 20min, the reaction system for CRISPR/Cas system reaction comprises 10X LbCas12a Reaction Buffer, lbCas12a, a template, a fluorescent reporter probe and sgRNA, and the reaction condition for CRISPR/Cas system reaction is that the reaction is carried out at 37 ℃ and 30 min. The RPA amplification reaction system comprises 29.5 mu L of RPA buffer solution, 2.5 mu L of 280 mM magnesium acetate, 2.4 mu L of 10 mu M upstream primer, 2.4 mu L of 10 mu M downstream primer, 3 mu L of DNA template and RPA enzyme freeze-dried powder. The CRISPR/Cas system reaction system comprises the following components in proportion, wherein the proportion of each component of the reaction system is prepared according to the proportion of 2 mu L of RPA reaction products, 10X LbCas, a Reaction Buffer mu L, 1 mu M LbCas a, 1 mu L of 10 mu M fluorescent reporter probes and 1 mu L of 1 mu M sgRNA, ddH 2 O are supplemented to 20 mu L, and the reaction condition is that a real-time fluorescent quantitative PCR instrument is used for collecting FAM channel fluorescent signals every 30 s at the temperature of 37 ℃ in a qPCR instrument setting program of 30 min. The CRISPR/Cas (Clustered regularly interspaced short palindromic repeats, CRISPR, cas protein) system (especially Cas12a protein) has high specific sequence recognition capability and 'trans-cleavage' activity, can non-sp