Kit for detecting haemophilus influenzae based on PCR / RAA-CRISPR-Cas13a

Abstract

The invention relates to the technical field of biology, and particularly discloses a kit for detecting haemophilus influenzae based on PCR (Polymerase Chain Reaction) / RAA-CRISPR-Cas13a. The invention discloses a kit for detecting a haemophilus influenzae gene on the basis of PCR (Polymerase Chain Reaction) / RAA-CRISPR-Cas13a. The kit comprises a primer pair for specifically amplifying haemophilus influenzae genes and crRNA, the sequence of the crRNA is composed of an anchoring sequence used for being combined with Cas13a protein and a guide sequence of a target sequence of the haemophilus influenzae genes, and the guide sequence is shown as the 39-66th site of SEQ ID No.28. The invention constructs a method for detecting haemophilus influenzae genes by combining a PCR or RAA technology with a CRISPR-Cas13a technology, the method is simple, convenient and rapid, has higher sensitivity and specificity, can reach a single copy level in sensitivity, and can be used for clinically and rapidly identifying whether the haemophilus influenzae is infected or not.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a kit for detecting Haemophilus influenzae based on PCR / RAA-CRISPR-Cas13a. Background Art

[0002] Haemophilus influenzae (HI) is a Gram-negative pathogen that is mainly distributed in the human nose, pharynx and nasopharynx. It can often cause diseases such as pneumonia, otitis media, acute sinusitis, and even central nervous system infection.

[0003] Currently, the traditional bacterial culture process has high environmental requirements, is time-consuming, and has a low success rate in isolating single colonies, making it difficult to meet the needs of rapid testing. Real-time fluorescence PCR (RT-PCR), a commonly used molecular detection technology in clinical practice, has high sensitivity and specificity. However, when detecting samples with low concentrations of the target gene, missed detection may still occur, and detection relies on the preparation of a standard curve. The fully automatic rapid microbial mass spectrometry detection system (MALDI-TOF) can achieve rapid detection of bacterial proteomics, but it needs to be completed on the basis of bacterial culture. Therefore, it is of great significance to explore new, efficient and accurate HI molecular detection methods.

[0004] In recent years, gene editing technology has developed rapidly, and gene editing systems of different subtypes of CRISPR-Cas have expanded their applications in clinical testing, basic research, and biomedical fields. CRISPR-Cas13 belongs to type VI of the two types of CRISPR systems. Unlike Cas9, which specifically cuts DNA, this system can specifically bind to and cut RNA under the guidance of gRNA, and can also edit it. The Cas13a effector protein is an RNA-guided CRISPR effector protein with the property of "incidental cleavage." It can not only specifically target and cut single-stranded RNA, but also remain active after cutting the target sequence and continue to cut other non-target RNAs. Utilizing this incidental cleavage activity of Cas13a, it is applied to molecular diagnostic tests. By adding a single-stranded RNA probe containing a reporter group to the detection system, when Cas13a recognizes the presence of the target sequence, it will enter an enzymatically "activated" state, and then cut the single-stranded RNA probe to release the fluorescent reporter group, emitting a fluorescent signal, and achieving the purpose of detecting the target sequence. This technology is expected to improve the effectiveness of HI detection. Summary of the Invention

[0005] One of the objectives of the present invention is to provide a method for accurately, sensitively, simply and rapidly detecting Haemophilus influenzae genes.

[0006] The present invention provides a kit for detecting Haemophilus influenzae, which includes a primer pair and crRNA for specifically amplifying Haemophilus influenzae genes, wherein the sequence of the crRNA consists of an anchor sequence for binding to the Cas13a protein and a guide sequence targeting the Haemophilus influenzae gene target sequence, and the guide sequence is shown in positions 39-66 of SEQ ID No. 28.

[0007] The nucleotide sequence of the Haemophilus influenzae gene target site is shown in SEQ ID No. 18.

[0008] In the kit of the present invention, the nucleotide sequence of the crRNA is shown as SEQ ID No.28.

[0009] Among them, positions 1-38 of SEQ ID No. 28 are anchor sequences for binding to Cas13a protein; positions 39-66 of SEQ ID No. 28 are guide sequences targeting the Haemophilus influenzae gene target sequence (SEQ ID No. 18).

[0010] In the kit of the present invention, the primer pair is any one of the following: A1) a PCR primer pair, the PCR primer pair consisting of primer HI-PCR-F3 and primer HI-PCR-R1, wherein the primer HI-PCR-F3 is a single-stranded DNA molecule represented by SEQ ID No. 5; and the primer HI-PCR-R1 is a single-stranded DNA molecule represented by SEQ ID No. 6; A2) an RAA primer pair, the RAA primer pair consisting of primer HI-RAA-F2 and primer HI-RAA-R3, wherein the primer HI-RAA-F2 is a single-stranded DNA molecule represented by SEQ ID No. 10; and the primer HI-RAA-R3 is a single-stranded DNA molecule represented by SEQ ID No. 14.

[0011] In the kit of the present invention, the kit further comprises Cas13a protein, preferably LwCas13a protein.

[0012] In the above-mentioned kit, the Cas13a protein can exist independently or in the form of a complex with the crRNA of the present invention.

[0013] In the kit of the present invention, the kit further includes a reporter RNA labeled with a fluorescent group, or further includes one or more of NTP (such as NTP Mix), T7 RNA polymerase, RNase inhibitor, buffer (such as HEPES Buffer Solution), MgCl2 and RNase-free water.

[0014] The reporter RNA may be an RNA molecule having a signal reporting function, and when the RNA molecule is degraded, it can report a positive signal and be detected.

[0015] In one embodiment of the present invention, the reporter RNA is RNaseAlert™ QC System v2 (Invitrogen™ product, catalog number 4479769).

[0016] Furthermore, the kit may also include a readable carrier recording the method for detecting Haemophilus influenzae genes described herein. The readable carrier may be instructions for practicing the method of the present invention (e.g., printed instructions) or a computer-readable medium (e.g., a floppy disk, CD, etc.) on which information is recorded.

[0017] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0018] The present invention discloses a nucleic acid detection technology based on CRIPSR-Cas13a, the most important mechanism of which is that the Cas13a protein can identify RNA fragments with a targeting sequence with the help of a guide RNA, and then the activated RNase activity that is not restricted by the sequence, by adding a signal reporter molecule caused by RNA chain degradation in the reaction system, ultimately achieving signal recognition of RNA fragments with a targeting sequence. The present invention successfully achieved rapid, efficient, highly specific, and sensitive Haemophilus influenzae gene detection using PCR technology or RAA technology in conjunction with CRISPR-Cas13a technology.

[0019] The present invention also provides crRNA molecules as described above.

[0020] The present invention also provides a primer pair for detecting Haemophilus influenzae, as described above.

[0021] The present invention also provides a composition for detecting Haemophilus influenzae, which is composed of the above-mentioned primer pair (PCR primer pair or RAA primer pair) and a crRNA molecule.

[0022] Furthermore, the composition may be composed of the above-mentioned PCR primer pair and the above-mentioned crRNA molecule.

[0023] Furthermore, the composition may be composed of the above-mentioned RAA primer pair and the above-mentioned crRNA molecule.

[0024] The present invention also provides a method for detecting Haemophilus influenzae for purposes other than disease diagnosis or treatment, the method comprising the following steps: B1) Extract DNA from the sample to be tested; B2) using the DNA as a template, performing PCR amplification using primers HI-PCR-F3 and HI-PCR-R1 to obtain a PCR amplification product; or using the DNA as a template, performing RAA amplification using primers HI-RAA-F2 and HI-RAA-R3 to obtain a RAA amplification product; The primers HI-PCR-F3 and HI-PCR-R1, the primers HI-RAA-F2 and HI-RAA-R3 are as described above; B3) Detection using the CRISPR-Cas13a detection system; The CRISPR-Cas13a detection system includes the above-mentioned crRNA molecule, or further includes Cas13a protein and / or T7 RNA polymerase.

[0025] In the method of the present invention, the CRISPR-Cas13a detection system includes: PCR amplification product or RAA amplification product, LwCas13a protein, NTP, T7 RNA polymerase, RNase inhibitor, crRNA molecule, buffer, MgCl2, reporter RNA and RNase-free water.

[0026] Furthermore, in step B2), the reaction conditions for the PCR amplification may be: pre-denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s, and extension at 72°C for 10 min.

[0027] Furthermore, in step B2), the reaction system (25 µL) for the PCR amplification can be: 1 µL of the sample DNA to be tested, 2 µL of the primer HI-PCR-F3 10 µM, 2 µL of the primer HI-PCR-R1 10 µM, 14.5 µL of 2×Taq mix, and 5.5 µL of ddH2O.

[0028] The 2×Taq mix was a product of Biomed, with the product number MT211-02.

[0029] Furthermore, in step B2), the reaction conditions for performing the RAA amplification may be: reacting at 39° C. for 20-40 minutes (eg, 30 minutes).

[0030] Furthermore, in step B2), the reaction system (47.5 µL) for performing the RAA amplification can be: 5 µL of the sample DNA to be tested, 2 µL of the primer HI-RAA-F2 10 µM, 2 µL of the primer HI-RAA-R3 10 µM, and 38.5 µL of A Buffer.

[0031] Buffer A is a component of the RAA Nucleic Acid Amplification Kit (Hangzhou Zhongce Biotechnology Co., Ltd., catalog number S001ZC).

[0032] Furthermore, the method for detecting using the CRISPR-Cas13a detection system in step B3) is as follows: ① Prepare a CRISPR-Cas13a detection system containing the following components: RAA amplification product or PCR amplification product obtained in step B2), Cas13a protein, crRNA, reporter RNA, NTP, T7 RNA polymerase, RNase inhibitor, RNase-free water, MgCl2, and CRISPR-Cas13a detection reaction buffer; ② Perform the reaction; ③ Detect a positive signal.

[0033] Furthermore, in one embodiment of the present invention, the CRISPR-Cas13a detection system (25 µL) can be: template 5 µL, LwCas13a protein (1X) 1 µL, NTP Mix (10 mM) 2 µL, T7 RNA polymerase (50,000 U / ml) 0.5 µL, RNase inhibitor (Murine RNase inhibitor) (40 U / ml) 1 µL, HI-crRNA-2 (100 ng / µL) 1.5 µL, HEPES Buffer Solution (1 M) 0.5 µL, MgCl2 (1 M) 0.25 µL, RNAse Alertv2 (125 nM) 2.5 µL, and RNase-free water (RNase free water) is added to 25 µL.

[0034] Furthermore, in step ②, the reaction conditions may be: 37° C., and in step ③, the positive signal detection may be: detecting the positive signal once every 2 minutes, for a total of 30 detections.

[0035] Furthermore, the method further includes determining whether the sample to be tested contains the Haemophilus influenzae gene based on the presence or absence of a positive signal, and / or determining the concentration of the Haemophilus influenzae gene in the sample to be tested based on the strength of the positive signal.

[0036] The method of determining whether the sample to be tested contains the Haemophilus influenzae gene based on the presence or absence of a positive signal is as follows: if there is a positive signal, it is determined that the sample to be tested contains or is a candidate for containing the Haemophilus influenzae gene; if there is no positive signal, it is determined that the sample to be tested does not contain or is a candidate for not containing the Haemophilus influenzae gene.

[0037] The concentration of the Haemophilus influenzae gene in the sample to be tested is determined according to the strength of the positive signal: the stronger the positive signal, the higher the content of the Haemophilus influenzae gene in the sample to be tested; the weaker the positive signal, the lower the content of the Haemophilus influenzae gene in the sample to be tested.

[0038] In the present invention, the positive signal may be a fluorescent signal. Within the same detection time, a positive result (positive signal) is determined when the fluorescence intensity value of the experimental group is more than 3 times higher than the fluorescence intensity value of the negative control (ddH2O).

[0039] Furthermore, the sample to be tested may be purified bacterial colonies, serum, plasma or environmental samples.

[0040] The present invention also provides any of the following applications of the above-mentioned kit or crRNA molecule or primer pair or composition: C1) Use in detecting Haemophilus influenzae genes for non-disease diagnosis or treatment purposes; C2) Use in the preparation of a product for detecting Haemophilus influenzae genes.

[0041] The purposes of the above-mentioned applications and methods of the present invention may be disease diagnosis, disease prognosis and / or disease treatment, or they may be non-disease diagnosis, non-disease prognosis and non-disease treatment.

[0042] The detection method of the present invention for non-disease diagnosis or treatment purposes can be used to detect Haemophilus influenzae genes in the environment, or to be used in the efficacy evaluation of products for treating Haemophilus influenzae.

[0043] The beneficial effects of the present invention are at least: The present invention provides a kit, a composition and applications thereof for detecting Haemophilus influenzae genes based on PCR / RAA-CRISPR-Cas13a (PCR-CRISPR-Cas13a and RAA-CRISPR-Cas13a).

[0044] The method provided by the present invention is simple, rapid, and has high sensitivity and specificity. The sensitivity can reach the single copy level, effectively eliminating the occurrence of false positive events of non-target amplified fragments in the previous nucleic acid amplification reaction. It can be used for clinical rapid identification of Haemophilus influenzae strains, or for detecting whether Haemophilus influenzae infection is present in clinical samples, thereby providing patients with reasonable antibiotic treatment to shorten the treatment cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a graph showing the agarose electrophoresis detection results of the PCR amplification product in step six of Example 1.

[0046] Figure 2 This is a graph showing the agarose electrophoresis detection results of the RAA amplification product in step seven of Example 1.

[0047] Figure 3 For Example 2, 10 3 The results of PCR-CRISPR screening of five crRNA sequences using a plasmid with a concentration of 100 μg / mL.

[0048] Figure 4 This is the sensitivity detection result of the PCR-CRISPR-Cas13a method in Example 4.

[0049] Figure 5 This is the sensitivity detection result of the RAA-CRISPR-Cas13a method in Example 4.

[0050] Figure 6 This is the repeatability experimental result of the PCR-CRISPR-Cas13a method in Example 4.

[0051] Figure 7 This is the reproducibility experimental result of the RAA-CRISPR-Cas13a method in Example 4.

[0052] Figure 8 These are the clinical sample experimental results of the PCR-CRISPR-Cas13a method in Example 4.

[0053] Figure 9 These are the clinical sample experimental results of the RAA-CRISPR-Cas13a method in Example 4.

[0054] Figure 10 This is the specific experimental result of the PCR-CRISPR-Cas13a method in Example 4.

[0055] Figure 11 This is the specific experimental result of the RAA-CRISPR-Cas13a method in Example 4. DETAILED DESCRIPTION

[0056] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0057] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available or prepared according to conventional methods in the art.

[0058] Example 1 Detection of Haemophilus influenzae genes based on PCR / RAA-CRISPR-cas13a The basic principle of the method for detecting Haemophilus influenzae genes based on PCR / RAA-CRISPR-Cas13a provided by the present invention is: first, the Haemophilus influenzae gene is transcribed into single-stranded RNA, then the crRNA of CRISPR specifically binds to the target fragment, and finally, the reporter RNA with a fluorescent signal is cleaved by Cas13a enzyme activity to detect the Haemophilus influenzae gene through the fluorescent signal.

[0059] 1. Preparation of Plasmid Standards The Haemophilus influenzae plasmid pUC57-HI was synthesized by Biomed Biotechnology at a concentration of 200 ng / µL. The plasmid is 3359 bp in length.

[0060] Copy number calculation formula: 6.02×10 23 ×200 (ng / µL)×10 -9 / 3359×660=5.4×10 10 copies / µL; Dilution: Take 4 µL of plasmid and add it to 18 µL of water to obtain a concentration of 1 × 10 10 copies / µL plasmid standard. Then serially dilute to 10 9 copies / µL, 10 8 copies / µL, 10 7 copies / µL, 10 6 copies / µL, 10 5 copies / µL, 10 4 copies / µL, 10 3 copies / µL, 10 2 copies / µL, 10 1 copies / µL, 10 0 copies / µL.

[0061] Plasmid pUC57-HI is a recombinant vector obtained by replacing the fragment (small fragment) between the EcoRV restriction endonuclease recognition sites of the pUC57 vector with the DNA fragment shown in SEQ ID No. 2 in the sequence listing, while keeping the other nucleotide sequences of the pUC57 vector unchanged.

[0062] The nucleotide sequence of the recombinant vector pUC57-HI is shown in SEQ ID No.1.

[0063] 2. Design of PCR and RAA Primers The P6 outer mold protein (ompP6) gene of Haemophilus influenzae was selected as the detection target, and its gene sequence was subjected to conservation analysis. Fifty-two Haemophilus influenzae ompP6 gene sequence information was downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov / ). The sequences were subjected to bioinformatics analysis using MEGA7 software, and the conserved sequence was obtained as shown in SEQ ID No. 2.

[0064] Primers were designed targeting the conserved sequences described above. A T7 transcription sequence was included at the 5' end of the forward primer, allowing the double-stranded DNA (dsDNA) amplified by PCR and RAA to be recognized and transcribed by T7 RNA polymerase. The PCR and RAA primer sequences are shown in Tables 1 and 2, respectively, and were synthesized by Sangon Biotech (Shanghai) Co., Ltd. For primer sequence HI-PCR-F3, degenerate bases R (A / G) were introduced, while for primer sequences HI-RAA-F3, HI-RAA-R2, and HI-RAA-R3, degenerate bases Y (C / T) were introduced to enhance target sequence recognition.

[0065] Table 1 PCR amplification primers for Haemophilus influenzae genes Note: The underlined parts in the table are T7 sequences.

[0066] Table 2 Primers for amplification of Haemophilus influenzae gene RAA Note: The underlined parts in the table are T7 sequences.

[0067] 3. Related reagents The genome extraction kit in the present invention is a product of Tiangen Company, the DNA extraction kit (item number DP315), 2×Taq mix (item number MT211-02), and RAA nucleic acid amplification kit (item number S001ZC) are products of Hangzhou Zhongce Biotechnology Co., Ltd., and ddH2O is a product of Bomade Company.

[0068] IV. Obtaining PCR amplification products Using the plasmid standard obtained in step 1 as a template, PCR amplification was performed using the primers designed in Table 1 to obtain a PCR amplification product. The PCR amplification system is shown in Table 3.

[0069] Table 3 PCR amplification system Among them, HI-PCR-F is the forward primer for PCR amplification of Haemophilus influenzae genes, and HI-PCR-R is the reverse primer for PCR amplification of Haemophilus influenzae genes.

[0070] PCR reaction conditions: 95°C pre-denaturation for 5 min, 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, 35 cycles, and 72°C extension for 10 min. PCR amplification products were obtained.

[0071] 5. Obtaining RAA Amplification Products Using the plasmid standard obtained in step 1 as a template, RAA amplification was performed using the primers designed in Table 2 to obtain RAA amplification products. The RAA amplification system is shown in Table 4.

[0072] Table 4 RAA amplification system Among them, HI-RAA-F is the forward primer for amplifying the RAA gene of Haemophilus influenzae, and HI-RAA-R is the reverse primer for amplifying the RAA gene of Haemophilus influenzae.

[0073] The specific amplification steps are as follows: Add 47.5 µL of the mixed solution to the reaction unit provided with the RAA amplification kit to fully dissolve the lyophilized powder. Add 2.5 µL of B Buffer to the cap of each reaction tube. Close the cap, invert 5-6 times to mix thoroughly, and quickly centrifuge for 10 seconds. Incubate the reaction tubes at 39°C for 30 minutes to obtain the RAA amplification product.

[0074] 6. Screening of PCR Primers With 10 3 The plasmid standard obtained in step 1 was used as a template and PCR amplification was performed using the method described in step 4. The primers were the combinations of primers shown in Table 1, namely F1R1 (primer HI-PCR-F1 and primer HI-PCR-R1), F1R2 (primer HI-PCR-F1 and primer HI-PCR-R2), F1R3 (primer HI-PCR-F1 and primer HI-PCR-R3), F2R1 (primer HI-PCR-F2 and primer PCR amplification products were obtained using primers F1 (primer HI-PCR-R1), F2R2 (primer HI-PCR-F2 and primer HI-PCR-R2), F2R3 (primer HI-PCR-F3 and primer HI-PCR-R3), F3R1 (primer HI-PCR-F3 and primer HI-PCR-R1), F3R2 (primer HI-PCR-F3 and primer HI-PCR-R2), and F3R3 (primer HI-PCR-F3 and primer HI-PCR-R3). A negative control was used for the amplification product using water as the template.

[0075] After PCR, take 5 μL of PCR amplification product, add 1 μL of 6× Loading Buffer, mix well, and then perform agarose gel electrophoresis.

[0076] Test results such as Figure 1 As shown, the results show that the combination of primers HI-PCR-F3 and HI-PCR-R1 (i.e., SEQ ID No. 5 and SEQ ID No. 6) has a high amplification efficiency. Therefore, the combination of primers HI-PCR-F3 and HI-PCR-R1 is used as the optimal PCR amplification primer pair for amplifying the HI gene.

[0077] VII. Screening of RAA Primers With 10 4 The plasmid standard obtained in step 1 was used as a template and RAA amplification was performed using the method described in step 5. The primers were the primer combinations shown in Table 2, namely F1R1 (primer HI-RAA-F1 and primer HI-RAA-R1), F1R2 (primer HI-RAA-F1 and primer HI-RAA-R2), F1R3 (primer HI-RAA-F1 and primer HI-RAA-R3), F2R1 (primer HI-RAA-F2 and primer The RAA amplification products were obtained by PCR amplification using primers HI-RAA-R1, F2R2 (primer HI-RAA-F2 and primer HI-RAA-R2), F2R3 (primer HI-RAA-F2 and primer HI-RAA-R3), F3R1 (primer HI-RAA-F3 and primer HI-RAA-R1), F3R2 (primer HI-RAA-F3 and primer HI-RAA-R2), and F3R3 (primer HI-RAA-F3 and primer HI-RAA-R3). The amplification products using water as the template were used as a negative control and subjected to agarose gel electrophoresis. The test results are shown in Figure 2. Figure 2 As shown, the results show that the combination of primers HI-RAA-F2 and HI-RAA-R3 (i.e., SEQ ID No. 10 and SEQ ID No. 14) has a high amplification efficiency. Therefore, the combination of HI-RAA-F2 and HI-RAA-R3 (F2R3 combination) is used as the optimal RAA amplification primer for amplifying the HI gene.

[0078] Example 2 Design and screening of crRNA for the present invention 1. Design of crRNA Based on the PCR primers selected in Example 1 (primers HI-PCR-F3 and HI-PCR-R1 in Table 1), sequence alignment analysis results, and crRNA design principles (reference PMID: 27246147), five crRNAs were designed from the Haemophilus influenzae gene sequence (SEQ ID No. 2): HI-crRNA-1, HI-crRNA-2, HI-crRNA-3, HI-crRNA-4, and HI-crRNA-5. The crRNA sequence consists of a repeat sequence at the 5' end and the reverse complement of the target sequence at the 3' end. The repeat sequence is 38 nt: 5'-GGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAAC-3' (SEQ ID No. 15), which can bind to the LwCas13a protein. The crRNAs were obtained by T7 in vitro amplification and transcription. The specific sequences are shown in Table 5 and were synthesized by Shanghai Sangon Co., Ltd.

[0079] Table 5 Primer sequences required for crRNA preparation 2. Preparation of crRNA 1. PCR amplification The synthesized sequence was diluted to 10 µM with ddH2O and the PCR reaction system was prepared. The PCR reaction system was prepared as shown in Table 6.

[0080] Table 6 PCR amplification system PCR reaction conditions: thermal denaturation at 95°C for 5 min; 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s; automatic extension at 72°C for 10 min; and storage of PCR products at 4°C.

[0081] 2. Purification of PCR Products The PCR product obtained in step 1 was purified using Tris-equilibrated phenol as follows: 500 µL of Tris-equilibrated phenol (Haoxiang Bio) was added to an equal volume of chloroform, vortexed, and briefly centrifuged. The supernatant was discarded. 130 µL of the phenol-chloroform mixture was added to the PCR product, mixed, and centrifuged at 12,000 rpm for 1 min. The supernatant was transferred to a new 1.5 mL centrifuge tube, and anhydrous ethanol was added to a 3:7 (volume ratio) supernatant:ethanol ratio. The mixture was vortexed and centrifuged at 12,000 rpm for 10 min. The supernatant was discarded. 200 µL of 75% ethanol was added, and the mixture was centrifuged at 12,000 rpm for 10 min. This step was repeated three times. The resulting pellet was air-dried at room temperature (approximately 10 min), and 40 µL of RNase-free water was added. The concentration was determined using an ND5000 micro-spectrophotometer and stored at -20°C.

[0082] 3. Transcription Take 1 µg of the purified PCR product obtained in step 2 and transcribe crRNA using the T7 transcription kit (NEB). The crRNA transcription system is shown in Table 7.

[0083] Table 7 crRNA transcription system After mixing the above crRNA transcription system, transcribe at 37°C overnight and use DNase I to remove excess DNA: add 20 µL of RNase-free water and 2 µL of DNase I to the transcription product obtained in the previous step, mix well, and incubate at 37°C for 15 min to obtain crRNA.

[0084] 4. Purification of crRNA Purify the crRNA obtained by transcription in step 3 according to the Agencourt RNA Clean XP instructions (Beckman Coulter). Specific steps are as follows: Vortex the magnetic beads to mix thoroughly. Add 1.8 times the volume of magnetic beads to the transcript. Pipet 10 times or vortex 30 seconds to mix the beads and transcription system. Incubate at room temperature for 5 minutes. Place the reaction system on a magnetic rack and let it stand for 5-10 minutes to separate the beads. Gently aspirate the liquid to avoid removing the beads. Add 200 µL of 70% ethanol (prepared in RNase-free water) to the beads. Incubate at room temperature for 30 seconds. Aspirate the ethanol. Repeat this process to wash the beads three times. Allow the system to air dry at room temperature to remove the ethanol (approximately 10 minutes). Add 50 µL of RNase-free water, vortex for 30 seconds or pipette 10 times, aspirate the supernatant, and place it in an RNase-free 1.5 mL centrifuge tube. Measure the concentration of the purified crRNA using an ND5000 spectrophotometer and store in aliquots at -80°C for later use.

[0085] The sequences of the five crRNAs obtained are as follows: crRNA1 (HI-crRNA-1): 5'-GGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACgccuaauuuaccagcaucaacaccuuua-3' (SEQ ID No. 27); crRNA2 (HI-crRNA-2): 5'-GGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACccuaauuuaccagcaucaacaccuuuac-3'' (SEQ ID No. 28); crRNA3 (HI-crRNA-3): 5'-GGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACcuaauuuaccagcaucaacaccuuuacc-3' (SEQ ID No. 29); crRNA4 (HI-crRNA-4): 5'-GGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACuaauuuaccagcaucaacaccuuuacca-3' (SEQ ID No. 30); crRNA5 (HI-crRNA-5): 5'-GGGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACaauuuaccagcaucaacaccuuuaccag-3' (SEQ ID No. 31).

[0086] 3. Screening of crRNA 1. With 10 3 The plasmid standard obtained in step 1 of Example 1 at a concentration of 100 copies / µL was used as a template, and HI-PCR-F3 and HI-PCR-R1 (SEQ ID No. 5 and SEQ ID No. 6) in Table 1 were used as primers. PCR amplification was performed according to the method in step 4 of Example 1 to obtain a PCR amplification product.

[0087] 2. After PCR amplification, 5 μL of the amplified product was taken and used to detect the Haemophilus influenzae gene using different crRNAs according to the method in Example 3. At the same time, the amplified product using water as a template was set as a negative control.

[0088] The test results show that when using 10 3The fluorescence value of HI-crRNA-2 (as shown in SEQ ID No. 28, positions 1-38 are anchor sequences for binding to Cas13a protein, and positions 39-66 are guide sequences for binding to PCR / RAA amplification products) was higher than that of the other four crRNAs ( Figure 3 ). Therefore, HI-crRNA-2 is used as the preferred crRNA for Haemophilus influenzae gene detection.

[0089] Example 3 Establishment of a CRISPR-Cas13a-based method for detecting Haemophilus influenzae genes In Example 3, two methods for detecting Haemophilus influenzae genes based on CRISPR-Cas13a were established. One method was a PCR-CRISPR-Cas13a-based method for detecting Haemophilus influenzae genes (referred to as the PCR-CRISPR-Cas13a method), and the other was a RAA-CRISPR-Cas13a-based method for detecting Haemophilus influenzae genes (referred to as the RAA-CRISPR-Cas13a method). Specifically: The PCR-CRISPR-Cas13a method includes the following steps: (1) Extract DNA from the sample to be tested; (2) Using the DNA as a template, PCR amplification was performed using primers HI-PCR-F3 (SEQ ID No. 5) and primer HI-PCR-R1 (SEQ ID No. 6) to obtain a PCR amplification product; (3) Detection using the CRISPR-Cas13a detection system.

[0090] The PCR amplification system and PCR reaction conditions in step (2) above are the same as those in step 4 of Example 1.

[0091] The RAA-CRISPR-Cas13a method includes the following steps: (1) Extract DNA from the sample to be tested; (2) Using the DNA as a template, RAA amplification was performed using primers HI-RAA-F2 (SEQ ID No. 10) and HI-RAA-R3 (SEQ ID No. 14) to obtain a RAA amplification product; (3) Detection using the CRISPR-Cas13a detection system.

[0092] The RAA amplification system and RAA amplification method in step (2) above are the same as those in step 5 of Example 1.

[0093] The CRISPR-Cas13a detection systems of the PCR-CRISPR-Cas13a method and the RAA-CRISPR-Cas13a method are the same except for the templates, while the other components and their amounts are the same.

[0094] In the CRISPR-Cas13a detection system of the PCR-CRISPR-Cas13a method, the template is a PCR amplification product obtained by PCR amplification using primers HI-PCR-F3 (SEQ ID No. 5) and primer HI-PCR-R1 (SEQ ID No. 6).

[0095] The template in the CRISPR-Cas13a detection system of the RAA-CRISPR-Cas13a method is the RAA amplification product obtained by RAA amplification using primers HI-RAA-F2 (SEQ ID No. 10) and primer HI-RAA-R3 (SEQ ID No. 14).

[0096] The preparation and detection method of the CRISPR-Cas13a detection system are as follows: 1. Preparation of CRISPR-Cas13a detection system Using the HI amplification product (PCR amplification product) obtained in step 4 of Example 1 as a template, a CRISPR-Cas13a detection system was prepared according to Table 8, wherein the crRNA was HI-crRNA-2 (SEQ ID No. 28).

[0097] Using the HI amplification product (RAA amplification product) obtained in step 5 of Example 1 as a template, a CRISPR-Cas13a detection system was prepared according to Table 8, wherein the crRNA was HI-crRNA-2 (SEQ ID No. 28).

[0098] The negative control was prepared by replacing the amplification products in Table 8 with ddH2O and keeping other reagent components unchanged.

[0099] Table 8 CRISPR-Cas13a detection system Related reagents: LwCas13a protein is a product of Hangzhou Zhongce Biotechnology Co., Ltd. The reporter RNA is the RNaseAlert™ QC System v2, a product of Invitrogen™, catalog number 4479769. It contains reporter RNA, which produces a fluorescent signal when degraded. NTP Mix is ​​a product of BBI, catalog number B600056-0500. Murine RNase inhibitor and T7 RNA polymerase are products of New England Biolabs, catalog numbers M0314S and M0251S, respectively. HEPES Buffer Solution is a product of Gibco, catalog number 15630-106.

[0100] 2. Fluorescence intensity detection Place the PCR tube containing the reaction system prepared above into a fluorescence quantitative PCR instrument and detect changes in the fluorescence signal in the FAM channel. Set the instrument to 37°C and read the fluorescence intensity every 2 minutes for 30 readings.

[0101] Result determination: within the same detection time, if the fluorescence intensity value of the experimental group is more than 3 times higher than that of the negative control (ddH2O), it is determined to be a positive result (positive signal).

[0102] Furthermore, whether the sample to be tested contains the Haemophilus influenzae gene is determined based on the presence or absence of a positive signal, and / or the concentration of the Haemophilus influenzae gene in the sample to be tested is determined based on the strength of the positive signal: (1) If there is a positive signal, it is determined that the sample to be tested contains or is a candidate for containing the Haemophilus influenzae gene; if there is no positive signal, it is determined that the sample to be tested does not contain or is a candidate for not containing the Haemophilus influenzae gene; (2) The stronger the positive signal, the higher the content of Haemophilus influenzae genes in the sample to be tested; the weaker the positive signal, the lower the content of Haemophilus influenzae genes in the sample to be tested.

[0103] Example 4 Sensitivity, specificity and repeatability experiments of Haemophilus influenzae gene detection method based on CRISPR-Cas13a system 1. Sensitivity test Using the gradient diluted plasmid standard in Example 1 as a template, the two methods in Example 3 were used to detect plasmids containing different concentrations of Haemophilus influenzae to test the sensitivity of the method of the present invention. The specific steps are as follows: 1. According to the methods in steps 4 and 5 of Example 1, PCR and RAA amplification were performed on Haemophilus influenzae plasmids of different concentrations to obtain PCR amplification products and RAA amplification products.

[0104] 2. After PCR and RAA amplification, 5 μL of the amplified product was taken to detect the Haemophilus influenzae plasmid according to the method in Example 3. The specific steps are as follows: 4 The plasmid was gradiently diluted to 1 copy / μl, and water was used as a negative control. The method of the present invention (refer to Example 3) was used as a template for detection. After detection using the PCR-CRISPR-Cas13a method (refer to Example 3), it was shown that at 1 copy / μl, the fluorescence value was significantly different from that of the negative control after 30 minutes of detection, indicating that the sensitivity of the PCR-CRISPR-Cas13a method of the present invention is good and can be as low as 1 copy / μl ( Figure 4 ). Detection using the RAA-CRISPR-Cas13a method (refer to Example 3) showed that at 1 copy / μl, the fluorescence value was significantly different from that of the negative control after 30 minutes of detection, indicating that the RAA-CRISPR-Cas13a method of the present invention has good sensitivity and can be as low as 1 copy / μl ( Figure 5 ).

[0105] 2. Repeatability Experiment The method of the present invention was used to perform PCR-CRISPR-Cas13a and RAA-CRISPR-Cas13a detection on each sample using a gradient dilution of the plasmid standard pUC57-HI (i.e., the standard prepared in step 1 of Example 1). The samples were repeated 6 times (referring to Example 1), and the results showed no significant difference. Figure 6 、 Figure 7 shown.

[0106] 3. Clinical Sample Testing The two methods of the present invention (PCR-CRISPR-Cas13a method and RAA-CRISPR-Cas13a method) were used to extract DNA from 4 sputum samples with HI-positive bacterial culture, and then the PCR-CRISPR-Cas13a method and RAA-CRISPR-Cas13a method were used respectively, and positive signals were detected in all four samples. All four samples were from Beijing Chuiyangliu Hospital. The clinical sample test results of the PCR-CRISPR-Cas13a method are as follows Figure 8 As shown, the clinical sample detection results of the RAA-CRISPR-Cas13a method are as follows Figure 9 shown.

[0107] 4. Specificity Experiment The HI standard strain (ATCC 49247) (B80837 Mingzhou Biotechnology) and five non-HI strains, including Streptococcus pneumoniae (ATCC 49619), Klebsiella pneumoniae (ATCC 700603), Staphylococcus aureus (ATCC 29213), Acinetobacter baumannii (ATCC 700603), and Escherichia coli (ATCC 25922), were obtained from Beijing Chuiyangliu Hospital.

[0108] DNA extracted from the six strains was used as a template to test the specificity of the method described in Example 3 (PCR-CRISPR-Cas13a and RAA-CRISPR-Cas13a). The specific steps are as follows: (1) Using DNA from one HI standard strain and DNA from five non-HI strains as detection templates, PCR amplification and RAA amplification were performed according to the methods in steps 4 and 5 of Example 1, respectively, to obtain PCR amplification products and RAA amplification products.

[0109] (2) Take 5 μL of the above amplified product and detect it according to the method in Example 3. At the same time, set the amplified product with water as the template as a negative control.

[0110] The test results showed that the fluorescence signal of the experimental group of Haemophilus influenzae strains gradually increased after the reaction started, while the fluorescence intensity of the negative control group (ddH2O) and the experimental group of non-Haemophilus influenzae strains did not increase over time. After 30 minutes of detection, the fluorescence intensity of the experimental group of Haemophilus influenzae strains was significantly higher than that of the negative control group and the non-Haemophilus influenzae strain group. The specific detection results of the PCR-CRISPR-Cas13a method are shown in Figure 2. Figure 10 As shown; the specific detection results of the RAA-CRISPR-Cas13a method are shown Figure 11 This shows that the method for detecting Haemophilus influenzae genes based on the CRISPR-Cas13a system of the present invention has high specificity and no cross reaction occurs during the detection process.

[0111] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A kit for detecting Haemophilus influenzae, characterized in that: The kit includes a primer pair and crRNA for specifically amplifying the Haemophilus influenzae gene, wherein the crRNA sequence consists of an anchor sequence for binding to the Cas13a protein and a guide sequence targeting the Haemophilus influenzae gene target sequence, and the guide sequence is shown in positions 39-66 of SEQ ID No.

28.

2. The kit according to claim 1, wherein The nucleotide sequence of the crRNA is shown in SEQ ID No.

28.

3. The kit according to claim 1 or 2, characterized in that The primer pair is any one of the following: A1) a PCR primer pair, the PCR primer pair consisting of primer HI-PCR-F3 and primer HI-PCR-R1, wherein the primer HI-PCR-F3 is a single-stranded DNA molecule represented by SEQ ID No. 5; and the primer HI-PCR-R1 is a single-stranded DNA molecule represented by SEQ ID No. 6; A2) an RAA primer pair, the RAA primer pair consisting of primer HI-RAA-F2 and primer HI-RAA-R3, wherein the primer HI-RAA-F2 is a single-stranded DNA molecule represented by SEQ ID No. 10; and the primer HI-RAA-R3 is a single-stranded DNA molecule represented by SEQ ID No.

14.

4. The kit according to any one of claims 1 to 3, characterized in that The kit further comprises a Cas13a protein, preferably a LwCas13a protein; And / or, the kit further comprises a reporter RNA labeled with a fluorescent group, or further comprises one or more of NTP, T7 RNA polymerase, RNase inhibitor, buffer, MgCl2 and RNase-free water.

5. crRNA molecule, characterized in that As claimed in claim 1 or 2.

6. A primer pair for detecting Haemophilus influenzae, characterized in that: As described in claim 3.

7. A composition for detecting Haemophilus influenzae, characterized in that: The composition consists of the primer pair according to claim 6 and the crRNA molecule according to claim 5.

8. A method for detecting Haemophilus influenzae for purposes other than disease diagnosis or treatment, characterized in that: The method comprises the following steps: B1) Extract DNA from the sample to be tested; B2) using the DNA as a template, performing PCR amplification using primers HI-PCR-F3 and HI-PCR-R1 to obtain a PCR amplification product; or using the DNA as a template, performing RAA amplification using primers HI-RAA-F2 and HI-RAA-R3 to obtain a RAA amplification product; The primers HI-PCR-F3 and HI-PCR-R1, the primers HI-RAA-F2 and HI-RAA-R3 are as described in claim 3; B3) Detection using the CRISPR-Cas13a detection system; The CRISPR-Cas13a detection system includes the crRNA molecule according to claim 5, or further includes Cas13a protein and / or T7 RNA polymerase.

9. The method according to claim 8, characterized in that The CRISPR-Cas13a detection system includes: PCR amplification product or RAA amplification product, LwCas13a protein, NTP, T7 RNA polymerase, RNase inhibitor, crRNA molecule, buffer, MgCl2, reporter RNA and RNase-free water.

10. Any of the following uses of the kit according to any one of claims 1 to 4, the crRNA molecule according to claim 5, the primer pair according to claim 6, or the composition according to claim 7: C1) Use in detecting Haemophilus influenzae genes for non-disease diagnosis or treatment purposes; C2) Use in the preparation of a product for detecting Haemophilus influenzae genes.

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