CN118547109A - A crRNA, LAMP-CRISPR/Cas12a detection kit for detecting influenza virus typing, method and application - Google Patents

Abstract

The invention discloses a crRNA and LAMP-CRISPR/Cas12a detection kit for detecting influenza virus typing, a method and application thereof, and relates to the technical field of biology. The LAMP primer and crRNA sequence for parting identification are screened and optimized by combining the LAMP technology and the CRSPR technology, so that the accuracy and the sensitivity of influenza parting detection are further improved, the isolation and the integration operation of the LAMP amplification and the CRSPR detection process are realized by means of a centrifugal microfluidic chip or an EP tube, a common influenza virus parting method based on LAMP-CRISPR/cas12a is established, and an accurate, quick, sensitive and simple solution is provided for influenza virus parting identification.

Description

A crRNA, LAMP-CRISPR/Cas12a test kit for detecting influenza virus typing, method and application

Technical Field

The present invention relates to the field of biotechnology, and in particular to a crRNA, LAMP-CRISPR/Cas12a detection kit, method and application for detecting influenza virus typing.

Background Art

Influenza virus is highly contagious, the general population is susceptible, and the morbidity rate is high. It has caused many outbreaks throughout the world in history and is an important public health issue of global concern. There are multiple subtypes of influenza virus, and each subtype requires different clinical intervention measures. It is worth noting that H1N1, H3N2, and influenza B virus (IBV) have been widely circulated in humans, and the zoonotic transmission of other subtypes (such as H5N1 and H7N9) occasionally poses a major threat to human health. Therefore, there is an urgent need for an accurate and rapid solution to distinguish these influenza virus subtypes to control the spread of the virus and prevent further deterioration of the disease.

Since influenza viruses are prone to mutation, conventional viral nucleic acid detection methods based on polymerase chain reaction (PCR) or isothermal amplification principles are prone to introduce base mismatches during the amplification process, resulting in the inability to accurately identify mutant genes, causing "false negative" test results. In recent years, with the discovery of the trans-cleavage activity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-related protein Cas12a, some teams have developed CRISPR-based molecular diagnostic technology (CRISPR-based diagnostic, CRISPR-Dx) using CRISPR/Cas12 and CRISPR/Cas13 enzyme systems. Due to its strong specificity, high reaction efficiency and fast speed, it is considered to be the next generation of molecular diagnostic technology with the most application prospects.

Isothermal nucleic acid amplification technology has the advantages of no need for a thermal cycler, simple operation, short amplification time, and low requirements for reaction conditions. The isothermal amplification technology is combined with the CRISPR/Cas12a detection system to complete the amplification of the target gene fragment to be tested in a short time through isothermal amplification, and then use the highly specific crRNA in the CRISPR/Cas12a system to recognize and bind to the target sequence, giving full play to the advantages of both, making it a rare nucleic acid detection technology that is accurate, fast, sensitive, and simple.

The Chinese patent application document with publication number CN105274252A discloses a real-time detection kit and method for influenza virus, which includes a detection composition with a pH value of 7.0-7.2, and also includes N-acetylneuraminic acid-firefly luciferin conjugate or its salt and firefly luciferase. The real-time detection method includes: combining the influenza virus with the detection composition under an environment with a pH value of 7.0-7.2, and then detecting the firefly luciferin signal. However, the patented method cannot detect multiple influenza viruses at the same time, and the sensitivity is poor.

Summary of the invention

The technical problem to be solved by the present invention is how to provide a LAMP-CRISPR/Cas12a detection kit and method for influenza virus typing.

The present invention solves the above technical problems through the following technical means:

The first aspect of the present invention provides a crRNA capable of specifically detecting influenza virus typing, wherein the influenza virus typing includes one or more of influenza A virus H1N1, H3N2, H5N1, H7N9 and influenza B virus (IBV); the crRNA sequence for detecting H1N1 virus is shown in SEQ ID NO: 1; the crRNA sequence for detecting H3N2 virus is shown in SEQ ID NO: 7; the crRNA sequence for detecting H5N1 virus is shown in SEQ ID NO: 13; the crRNA sequence for detecting H7N9 virus is shown in SEQ ID NO: 20; the crRNA sequence for detecting IBV virus is shown in SEQ ID NO: 27.

Beneficial effects: The crRNA used for typing detection of influenza A virus H1N1, H3N2, H5N1, H7N9 and influenza B virus (IBV) in the present invention is obtained through multiple screening and optimization, and can specifically detect the conserved sequence in the corresponding typing in the amplified product, which improves the accuracy of the detection result compared with the traditional fluorescent PCR detection method.

The second aspect of the present invention proposes LAMP amplification primers for the above-mentioned crRNA sequences of each type. The sequences of the LAMP amplification primers for amplifying H1N1 virus are shown in SEQ ID NOs: 2-6; the sequences of the LAMP amplification primers for amplifying H3N2 are shown in SEQ ID NOs: 8-13; the sequences of the LAMP amplification primers for amplifying H5N1 virus are shown in SEQ ID NOs: 14-19; the sequences of the LAMP amplification primers for amplifying H7N9 virus are shown in SEQ ID NOs: 21-26; the sequences of the LAMP amplification primers for amplifying IBV virus are shown in SEQ ID NOs: 28-33.

Wherein SEQ ID NO:2-6 means SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and the same applies to the others.

Beneficial effects: The LAMP amplification primer sequences used for the typing detection of influenza A virus H1N1, H3N2, H5N1, H7N9 and influenza B virus (IBV) in the present invention have been screened and optimized many times to determine the best primer combination, which has the advantages of high detection sensitivity and short detection time. At the same time, it can be combined with crRNA sequence and CRISPR/Cas12a system for accurate and rapid detection of influenza typing.

The third aspect of the present invention proposes a kit for detecting influenza virus typing based on LAMP-CRISPR/Cas12a, the kit comprising an amplification system containing the above-mentioned LAMP amplification primers and a CRISPR/Cas12a detection system containing the above-mentioned crRNA for detecting influenza virus typing.

Preferably, the amplification system further contains DNA polymerase, reverse transcriptase and LAMP Premix Buffer.

Preferably, the CRISPR/Cas12a detection system also contains CRISPR/Cas12a protein and a ssDNA reporter fluorescent probe.

Preferably, the kit utilizes a centrifugal microfluidic chip or an EP tube for detection.

Beneficial effects: The present invention combines loop-mediated isothermal amplification (LAMP) technology with the CRISPR/Cas12a system and applies it to common influenza virus typing detection. The optimized LAMP isothermal amplification system and CRISPR/Cas12a detection system show high sensitivity and high specificity in improving the reliability of the results.

The fourth aspect of the present invention proposes the use of the above-mentioned kit in detecting influenza virus typing for purposes other than disease diagnosis.

A fifth aspect of the present invention provides a method for detecting influenza virus typing using the above kit for non-disease diagnosis purposes, comprising the following steps:

(1) extracting nucleic acid from the sample to be tested;

(2) amplifying the nucleic acid in the sample to be tested using LAMP amplification primers;

(3) Using the CRISPR/Cas12a detection system to identify the amplified product and cut the ssDNA reporter fluorescent probe, the fluorescence signal is detected using a fluorescence detector to determine whether the sample contains the corresponding influenza virus type.

Preferably, when the kit adopts centrifugal microfluidic chip detection, the amplification system is pre-embedded in the chip amplification cavity in the form of freeze-dried powder, and the CRISPR/Cas12a detection system is pre-embedded in the independent detection cavity of the chip. The nucleic acid extracted in step (1) is added to the liquid reservoir, and the nucleic acid extracted in step (1) is shunted to the chip amplification cavity for LAMP amplification reaction by centrifugation. After the amplification is completed, centrifugation is performed, and the amplification product enters the independent detection cavity of the chip. Finally, the fluorescence signal of the detection cavity is detected. When the independent detection cavity emits fluorescence, it indicates that the sample to be tested contains the influenza virus of the subtype. Otherwise, it indicates that the sample to be tested does not contain the influenza virus of the subtype.

Preferably, when the kit adopts EP tube detection, the CRISPR/Cas12a detection system is dripped into the inner wall of the tube cap, the amplification primers and the sample to be tested are added to the bottom of the tube, and the tube is sealed with paraffin oil. After the amplification reaction is completed, the CRISPR/Cas12a detection reagent is mixed with the LAMP amplification product by centrifugation for reaction, and finally the fluorescence signal of the reaction system is detected. When the reaction system emits fluorescence, it indicates that the sample to be tested contains the influenza virus of the subtype, otherwise, it indicates that the sample to be tested does not contain the influenza virus of the subtype.

Preferably, in step (2), an isothermal amplification system is used to amplify the product at 62° C. for 40 minutes.

Preferably, in step (3), a CRISPR/Cas12a detection system is used and the detection result is obtained by reacting at 42° C. for 5 minutes.

Beneficial effects: The present invention aims at typing common influenza viruses, establishes a rapid typing method based on LAMP-CRISPR/Cas12a, adopts a centrifugal microfluidic chip or EP tube, separates the two processes of LAMP amplification and CRISPR/Cas12a detection in physical space, avoids mutual interference of the two reactions, and after the amplification is completed, mixes the amplification product with the CRISPR/Cas12a detection system by centrifugation, avoids aerosol contamination caused by exposure of the amplification product.

The advantages of the present invention are:

The present invention combines the CRISPR/Cas system with loop-mediated isothermal amplification (LAMP) technology and applies it to common influenza virus typing detection, showing high sensitivity and high specificity in improving the reliability of diagnostic results.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram of crRNA screening for five influenza typings in Example 1 of the present invention;

FIG2 is a diagram of five influenza typing LAMP primer screenings in Example 2 of the present invention;

FIG3 is a schematic diagram of a single-tube LAMP-CRISPR/Cas12a method according to Example 3 of the present invention;

FIG4 is a comparison chart of the sensitivity of the LAMP-CRISPR/Cas12a kit in detecting five common influenza virus typings in Example 4 of the present invention;

FIG5 is a schematic diagram of the process of the centrifugal microfluidic chip LAMP-CRISPR/Cas12a method in Example 5 of the present invention;

Figure 6 is a graph showing the results of clinical sample detection using the centrifugal microfluidic chip LAMP-CRISPR/Cas12a method in Example 6 of the present invention.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

Example 1: Screening of LAMP primers and crRNA

Based on the National Center for Biotechnology Information (NCBI), the sequences of 5 influenza virus subtypes were obtained, and the DNAMAN software was used for sequence alignment to ensure sequence conservation. 8 corresponding crRNAs were designed for each subtype, and the amplified products were used to evaluate the binding efficiency and reaction time of each crRNA, and two candidate crRNAs with high CRISPR/Cas12a cutting efficiency were selected for the subsequent LAMP primer screening process. The results are shown in Figure 1. The curve is the reaction curve of each crRNA, and the relative position of each crRNA is above it, and the boxed ones are the best crRNAs corresponding to each typing.

Based on the location of the candidate crRNA in the genome, LAMP primers containing crRNA sequences were designed. The amplification efficiency and sensitivity of the three sets of LAMP primers for each genotype were tested to select the best LAMP primers. The results are shown in Figure 2, where 1, 2, 3, and 4 represent the corresponding target sequence concentrations of 1000 copies/μL, 100 copies/μL, 10 copies/μL, and 0, respectively. The optimal LAMP primers and crRNA sequences corresponding to the five genotypes were finally determined as shown in Table 1.

Example 2: Single-tube method for detecting five influenza virus typing

The CRISPR/Cas12a detection system includes 1.25 μL (4 μM) crRNA, 1 μL (15 μM) ssDNA reporter fluorescent probe, 1.25 μL (2 μM) Cas12a, 1.25 μL (40 U/μL) RNA inhibitor and 1 μL (1 M) MgSO ₄ .

The LAMP amplification system included 1 μL (10 μM) outer primer, 1 μL (40 μM) inner primer, 2 μL (10 μM) loop primer, 2 μL (8 U/μL) Bst 2.0HS DNA polymerase, 0.375 μL (200 U/μL) reverse transcriptase and 5 μL (5×) LAMP Premix Buffer-T8AN.

The single-tube method was used to perform LAMP-CRISPR/Cas12a detection on five influenza virus typings. This method temporarily isolates the amplification reaction and the detection reaction by means of physical space separation. The specific principle is shown in Figure 3. The entire reaction is carried out in a closed EP tube. The CRISPR/Cas12a detection system is added to the tube cap, and the LAMP amplification reagent and the sample to be tested are added to the bottom of the EP tube and sealed with paraffin oil. After LAMP amplification at 62°C for 40 minutes, the CRISPR/Cas12a detection reagent is mixed with the LAMP amplification product by centrifugation, and the EP tube is placed on the LightCycler 96 system and reacted at 42°C for 5 minutes.

Example 3: LAMP-CRISPR/Cas12a detects five influenza virus plasmids with high sensitivity

The sensitivity of the established LAMP-CRSPR/Cas12a system detection was verified by the single-tube method. Synthetic plasmid DNA was used as a template, and the concentration was adjusted and gradiently diluted with EASY Dilution, so that the concentration of each group of templates was 10 ³ copies/μL, 10 ² copies/μL, and 10 ¹ copies/μL, respectively. Subsequently, plasmid DNA of each series of concentrations was used as a template, and the corresponding LAMP-CRSPR/Cas12a detection experiment was performed in a LightCycler 96 fluorescence quantitative PCR instrument using the best cRNA and the corresponding LAMP primers, and its sensitivity was verified by detecting the fluorescence intensity for 5 minutes. Figure 4 is the sensitivity fluorescence value of LAMP-CRSPR/Cas12a detection of H1N1, H3N2, H5N1, H7N9 and IBV, respectively. In this embodiment, the sensitivity of five influenza virus typing detections using the LAMP-CRSPR/Cas12a detection system can reach 10 copies/μL, which is better than similar methods.

Example 4: Detection of five influenza virus typing using a centrifugal microfluidic chip

Centrifugal microfluidic chips were used to perform LAMP-CRISPR/Cas12a multiplex detection on five influenza virus typings. The specific experimental operation is shown in Figure 5. Use a pipette to inject 70μL of the collected and extracted sample into the centrifugal microfluidic chip. After the sample is added, the chip is sealed with a cover sheet to perform the detection experiment. The sealed chip is placed on a centrifuge and centrifuged at 3000rmp for 5s. The sample is distributed into the detection chamber by centrifugation. The RT/LAMP reaction is performed at 62℃ for 40min. The chip is placed on the centrifuge again and centrifuged at 9500rmp for 15s. The amplified product is placed in the detection chamber by centrifugation. The CRISPR reaction is performed at 42℃ for 5min. After the reaction, the end point fluorescence picture is collected.

Example 5: Centrifugal microfluidic chip for detecting influenza virus clinical samples

In order to verify the clinical practicality of the established LAMP-CRSPR/Cas12a system for centrifugal microfluidic chip detection, we tested 38 clinical samples (including 22 positive samples and 16 negative samples). As shown in Figure 6a, the clinical samples were first tested using the gold standard qRT-PCR, and the CT value was obtained. Subsequently, the clinical samples were tested in the microfluidic chip based on LAMP-CRSPR/Cas12a, and the heat map showed the influenza typing identification results of the clinical samples, as shown in Figure 6b. The results showed that among the positive samples, there were 1 H1N1, 11 H3N2, 5 H5N1, 2 H7N9 and 2 IBV. It is worth noting that there was also a sample containing both H5N1 and IBV viruses. The positive or negative results determined by this detection method are consistent with the qRT-PCR results. This shows that our detection method can effectively realize the influenza virus typing detection of clinical samples.

Table 1 shows the crRNA and LAMP primer sequences of the five influenza virus typing genes screened

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A crRNA capable of specifically detecting influenza virus typing, characterized in that the influenza virus typing includes one or more of influenza A virus H1N1, H3N2, H5N1, H7N9 and influenza B virus (IBV); the crRNA sequence for detecting H1N1 virus is shown in SEQ ID NO: 1; the crRNA sequence for detecting H3N2 virus is shown in SEQ ID NO: 7; the crRNA sequence for detecting H5N1 virus is shown in SEQ ID NO: 13; the crRNA sequence for detecting H7N9 virus is shown in SEQ ID NO: 20; the crRNA sequence for detecting IBV virus is shown in SEQ ID NO: 27. 2. The LAMP amplification primer capable of specifically detecting crRNA for influenza virus typing according to claim 1, characterized in that the sequence of the LAMP amplification primer for amplifying H1N1 virus is shown in SEQ ID NO: 2-6; the sequence of the LAMP amplification primer for amplifying H3N2 is shown in SEQ ID NO: 8-13; the sequence of the LAMP amplification primer for amplifying H5N1 virus is shown in SEQ ID NO: 14-19; the sequence of the LAMP amplification primer for amplifying H7N9 virus is shown in SEQ ID NO: 21-26; the sequence of the LAMP amplification primer for amplifying IBV virus is shown in SEQ ID NO: 28-33. 3. A LAMP-CRISPR/Cas12a sequence composition for influenza virus typing detection, characterized in that it comprises the crRNA for detecting influenza virus typing according to claim 1 and the LAMP amplification primer according to claim 2. 4. A kit for detecting influenza virus typing based on LAMP-CRISPR/Cas12a, characterized in that: the kit comprises an amplification system containing the LAMP amplification primers described in claim 2 and a CRISPR/Cas12a detection system containing crRNA for detecting influenza virus typing according to claim 1. 5. The kit for detecting influenza virus typing according to claim 4, characterized in that: the amplification system also contains DNA polymerase, reverse transcriptase and LAMP Premix Buffer; the CRISPR/Cas12a detection system also contains CRISPR/Cas12a protein and ssDNA reporter fluorescent probe. 6. The kit for detecting influenza virus typing according to claim 5, characterized in that: the amplification system includes 1 μL (10 μM) outer primers, 1 μL (40 μM) inner primers, 2 μL (10 μM) loop primers, 2 μL (8 U/μL) Bst 2.0HS DNA polymerase, 0.375 μL (200 U/μL) reverse transcriptase and 5 μL (5×) LAMP Premix Buffer-T8AN; the CRISPR/Cas12a detection system includes 1.25 μL (4 μM) crRNA, 1 μL (15 μM) ssDNA reporter fluorescent probe, 1.25 μL (2 μM) Cas12a, 1.25 μL (40 U/μL) RNA inhibitor and 1 μL (1 M) MgSO ₄ . 7. The kit for detecting influenza virus typing according to claim 4, characterized in that the kit utilizes a centrifugal microfluidic chip or an EP tube for detection. 8. Use of the kit for detecting influenza virus typing according to any one of claims 4 to 7 in detecting influenza virus typing for purposes other than disease diagnosis. 9. A method for detecting influenza virus typing using the kit according to any one of claims 4 to 7 for purposes other than disease diagnosis, characterized in that it comprises the following steps: (1) extracting nucleic acid from the sample to be tested; (2) amplifying the nucleic acid in the sample to be tested using LAMP amplification primers; (3) Using the CRISPR/Cas12a detection system to cut the amplified product, using a fluorescence detector to detect the fluorescent signal, and determine whether the sample to be tested contains influenza virus typing. 10. The influenza virus typing detection method according to claim 9, characterized in that: When the kit adopts a centrifugal microfluidic chip for detection, the amplification system is pre-embedded in the chip amplification cavity in the form of a lyophilized powder, the CRISPR/Cas12a detection system is pre-embedded in the independent detection cavity of the chip, the nucleic acid extracted in step (1) is added to the liquid reservoir, and the nucleic acid extracted in step (1) is shunted to the chip amplification cavity for LAMP amplification reaction by centrifugation. After the amplification is completed, centrifugation is performed, and the amplification product enters the independent detection cavity of the chip. Finally, the fluorescence signal of the detection cavity is detected. When the independent detection cavity emits fluorescence, it indicates that the sample to be tested contains the influenza virus of the subtype, otherwise, it indicates that the sample to be tested does not contain the influenza virus of the subtype; When the kit adopts EP tube detection, the CRISPR/Cas12a detection system is dripped into the inner wall of the tube cap, the amplification primer and the sample to be tested are added to the bottom of the tube, and the tube is sealed with paraffin oil. After the amplification reaction is completed, the CRISPR/Cas12a detection reagent and the LAMP amplification product are mixed by centrifugation to react, and finally the fluorescence signal of the reaction system is detected. When the reaction system emits fluorescence, it indicates that the sample to be tested contains the influenza virus of the subtype, otherwise, it indicates that the sample to be tested does not contain the influenza virus of the subtype.