Method for detecting group B streptococcus based on CRISPR / Cas12a

Abstract

The invention discloses a method for rapidly and accurately detecting group B streptococcus (GBS) based on CRISPR / Cas12a, when nucleic acid of the GBS forms a ternary complex with Cas12a and crRNA, a RuvC structural domain of the Cas12a in the complex shows DNase activity, a labeled single-stranded DNA probe can be cut, and the Cas12a can be used for rapidly and accurately detecting the group B streptococcus. The detection is further performed by adopting methods such as fluorescence detection, lateral flow chromatography detection or lateral flow chromatography detection matched with an intelligent mobile terminal and the like. The method disclosed by the invention can efficiently, accurately and quickly detect GBS, has the advantages of simplicity in operation, adaptability to various detection scenes and the like, and has important significance on early monitoring and diagnosis of GBS and antibiotic treatment decision.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapid detection of pathogenic microorganisms and their nucleic acids. Specifically, it relates to a method for rapid and accurate identification and detection of Group B Streptococcus (GBS) using a CRISPR / Cas12a system. Background Art

[0002] Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is a Gram-positive β-hemolytic streptococcus that usually parasitizes the vagina and gastrointestinal tract of women. As a conditional pathogen, GBS does not cause disease in the general healthy population. However, when pregnant women are infected with GBS, there is a risk of vertical transmission of the bacteria to newborns through the delivery process. It is estimated that the probability of newborns being infected with GBS is approximately between 1% and 2%, which may lead to severe local and systemic infections, thereby causing serious diseases such as neonatal pneumonia, sepsis, and meningitis. In view of this, GBS testing for pregnant women and subsequent preventive interventions are essential to reduce the risk of neonatal infection and its complications. Western developed countries such as the United States and European countries generally recommend the implementation of prenatal GBS screening and the use of antibiotics during delivery. However, there are differences in the choice of screening timing: the Centers for Disease Control (CDC) in the United States recommends GBS culture screening at 35-37 weeks of gestation, while the EU consensus prefers the use of rapid GBS testing technology during labor.

[0003] In the "Expert Consensus on Prevention of Perinatal Group B Streptococcus (China)" issued in my country in 2021, it is recommended to screen pregnant women with GBS at 35 to 37 weeks of gestation. The laboratory detection method recommended in this consensus is: first, use the broth medium of the selective enrichment medium for enrichment culture, and then inoculate the culture on the blood agar medium for further culture. Then, GBS is accurately identified and detected by advanced technical means such as latex agglutination test, chromogenic culture medium method, DNA probe technology or nucleic acid amplification testing (NAAT). At present, the GBS detection methods used in clinical practice have certain limitations. The traditional bacterial culture method, as a classic method and means for GBS detection, is limited by the detection principle and has poor detection specificity. In addition, this method requires 18 to 24 hours of culture time to observe the results, and it is even more difficult to detect low concentrations of bacteria. At the same time, microbiological culture methods are often accompanied by high false positive and false negative rates in clinical testing. The latex agglutination method and colloidal gold immunochromatography based on antigen detection face the challenge of insufficient sensitivity and specificity. Although nucleic acid detection technologies such as qPCR have gradually become the main means of detecting GBS infection in pregnant women due to their high sensitivity, excellent specificity, good repeatability, rapidity and safety, they rely on expensive qPCR amplifiers and require professional technicians to operate, which limits their popularization and application in primary medical units and families. Therefore, the development of a simple, fast and highly sensitive detection method that can be screened in central laboratories and is suitable for point-of-care testing (POCT) is the future development direction of GBS nucleic acid detection.

[0004] Clustered regularly interspaced short palindromic repeats (CRISPR) is a natural immune defense mechanism found in archaea. CRISPR-Cas12a is an important type of CRISPR system, which only requires crRNA to accurately target its substrate. It not only has cis activity that specifically cuts double-stranded DNA, but also obtains trans activity after identifying the target gene and non-specifically degrades single-stranded DNA. The triggering of this trans activity provides Cas12a with a powerful nucleic acid detection potential. The targeting specificity of crRNA and the triggering of cis and trans activities are the key to the application of Cas12a in nucleic acid detection. crRNA consists of a hairpin structure and a segment of nucleotides complementary to double-stranded nucleic acids. Generally, the longer the complementary sequence, the better the crRNA cutting activity, and the shorter the complementary sequence, the better the crRNA recognition specificity, but short crRNA may lead to loss of activity. In addition, the 4-5 bases after the hairpin structure have a greater effect on RNA activity. If the nucleic acid mutates at this position, the cutting activity will not be activated. Therefore, a reasonable and conservative RNA sequence is designed to ensure the detection effect.

[0005] Recently, domestic and foreign scholars have mainly integrated fluorescence detection systems with CRISPR / Cas12a technology to develop new GBS detection methods. These methods have shown good performance when applied to clinical sample detection. However, this detection system usually relies on high-cost fluorescence detection equipment that requires professional operating skills, which to a certain extent limits its wide application. In addition, Yu et al. successfully achieved naked-eye visualization detection of GBS by combining G4 chain technology with CRISPR / Cas12a. Although this method provides a solution for GBS detection without complex instruments, direct visual interpretation results may be affected by environmental conditions and subjective judgment, thereby affecting the accuracy and repeatability of the test results. Considering the importance of perinatal GBS screening and prevention and control monitoring, as well as the actual limitations of pregnant women's physical condition and doctor-patient ratio, GBS detection not only needs to have high accuracy and sensitivity, but also should be able to be performed in primary medical institutions and even home environments outside professional laboratories. However, existing GBS detection methods based on CRISPR / Cas12a still face usage restrictions in these environments, especially in the absence of professional optical equipment. Summary of the invention

[0006] The present invention aims to solve the limitations and shortcomings of existing GBS screening, monitoring and prevention and control technologies, thereby providing a detection method with high sensitivity and high specificity. The method is simple to operate, fast to detect and can accurately detect GBS. Furthermore, the method also supports digital analysis of the test results, and provides a systematic solution for result output and data management based on mobile smart terminals. The detection method established by the present invention is not only suitable for professional testing institutions, but also suitable for prenatal testing of pregnant women in various environments such as primary medical institutions and even families. It does not require expensive instruments and professional laboratory conditions, and is of great significance for the detection and prevention of perinatal GBS infection in pregnant women.

[0007] The purpose of the present invention is to provide a crRNA for GBS detection with ideal detection effect and high sensitivity and accuracy.

[0008] Another object of the present invention is to provide the use of the crRNA in detecting Group B Streptococcus.

[0009] Another object of the present invention is to provide the use of the crRNA in the preparation of a Group B Streptococcus detection reagent.

[0010] Another object of the present invention is to provide a detection system for detecting group B streptococci based on CRISPR / Cas12a, comprising the crRNA, ERA amplification primer pair and a labeled single-stranded DNA probe.

[0011] Preferably, the ERA amplification primer pair ERA-F and ERA-R, the nucleic acid sequences are shown in SEQ ID No 2, 3.

[0012] The detection system of the present invention is a fluorescence detection system, and the single-stranded DNA probe is labeled with a fluorescent group and a quenching group.

[0013] In a specific example, the labeled single-stranded DNA probe is 5'-FAM-TTATT-BHQ1-3'.

[0014] The detection system of the present invention may be a lateral flow chromatography detection system, and the single-stranded DNA probe is labeled with a fluorescent group and a biotin group.

[0015] In a specific example, the labeled single-stranded DNA probe is 5'-FAM-TTATT-Bio-3'.

[0016] The lateral flow chromatography detection system also includes a colloidal gold lateral flow chromatography carrier for detecting the integrity of the labeled single-stranded DNA probe. The lateral flow chromatography detection system can also be integrated with a smart mobile terminal, including a miniaturized portable device for reading and analyzing colloidal gold lateral flow chromatography cards, a smart mobile terminal application applet for transmitting and managing data analysis results, and the like.

[0017] Another object of the present invention is to provide a CRISPR / Cas12a system for detecting GBS.

[0018] Another object of the present invention is to provide a fluorescence detection method for rapid and accurate detection of GBS based on CRISPR / Cas12a.

[0019] Another object of the present invention is to provide a lateral flow chromatography test paper detection method for rapid and accurate detection of GBS based on CRISPR / Cas12a.

[0020] Another object of the present invention is to provide a lateral flow chromatography detection method based on CRISPR / Cas12a for rapid and accurate detection of GBS integrated with an intelligent mobile terminal.

[0021] Another object of the present invention is to provide a fluorescence detection kit for rapid and accurate detection of GBS based on CRISPR / Cas12a.

[0022] Another object of the present invention is to provide a lateral flow chromatography test strip and test card for rapid and accurate detection of GBS based on CRISPR / Cas12a.

[0023] Another object of the present invention is to provide a systematic solution for digital analysis, result output and data management of lateral flow chromatography test strips based on CRISPR / Cas12a for rapid and accurate detection of GBS by smart mobile terminals.

[0024] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0025] The present invention has found that when GBS nucleic acid forms a ternary complex with Cas12a and crRNA, the RuvC domain of Cas12a in the complex exercises DNase activity to cut the single-stranded DNA probe labeled with a fluorescent reagent and a quenching group. By detecting the fluorescence recovery, it can be known whether the sample to be tested contains GBS nucleic acid. Similarly, when the single-stranded DNA probe labeled with a fluorescent reagent and biotin is cut by the activated Cas12a, the test result can be obtained by lateral flow chromatography test paper. On this basis, the portable lateral flow chromatography test paper reader included in the present technology can accurately capture the optical signals of the quality control line (C line) and the detection line (T line) on a specific model of test paper card, and compare it with the performance parameters of different batches of test paper cards pre-established in the cloud server, and then use the gray value ratio (T / C) of the detection line to the quality control line to calculate and analyze the GBS test results. After the calculation is completed, the test results will be transmitted back through a specially designed application applet and saved on the user's smart mobile device (such as Figure 1 As shown). Based on this, the present invention has established a method for rapid and accurate detection of group B streptococci based on CRISPR / Cas12a, which includes three systems: fluorescence detection, lateral flow chromatography detection, and intelligent mobile terminal digital detection. High-quality crRNA with high accuracy, high detection efficiency, and high sensitivity is designed to ensure the GBS detection effect.

[0026] Therefore, the present invention provides a crRNA for detecting Group B Streptococcus, the nucleotide sequence of which is shown in SEQ ID No: 1.

[0027] The use of the crRNA in detecting Group B Streptococcus and in preparing GBS detection reagents are also within the scope of protection of the present invention.

[0028] Based on this, a CRISPR / Cas12a system for detecting group B streptococci is also provided, comprising the above-mentioned crRNA, an ERA amplification primer pair and a labeled single-stranded DNA probe.

[0029] Preferably, the ERA amplification primer pair ERA-F and ERA-R have nucleic acid sequences as shown in SEQ ID Nos: 3 and 4.

[0030] The detection system of the present invention is a fluorescence detection system, and the single-stranded DNA probe is labeled with a fluorescent group and a quenching group.

[0031] In a specific example, the labeled single-stranded DNA probe is 5'-FAM-TTATT-BHQ1-3'.

[0032] The detection system of the present invention may be a lateral flow chromatography detection system, and the single-stranded DNA probe is labeled with a fluorescent group and a biotin group.

[0033] In a specific example, the labeled single-stranded DNA probe is 5'-FAM-TTATT-Bio-3'.

[0034] The lateral flow chromatography detection system also includes a colloidal gold lateral flow chromatography carrier for detecting the integrity of the labeled single-stranded DNA probe. The lateral flow chromatography detection system can also be integrated with a smart mobile terminal, including a miniaturized portable device for reading and analyzing colloidal gold lateral flow chromatography cards, a smart mobile terminal application applet for transmitting and managing data analysis results, and the like.

[0035] In addition, the CRISPR / Cas12a system also includes reagents required for the ERA amplification system and the CRISPR / Cas12a detection system.

[0036] Specifically, as an optional solution, the ERA amplification system is as follows:

[0037]

[0038]

[0039] The CRISPR / Cas12a detection system is as follows:

[0040]

[0041] In addition, based on the above technical achievements, the present invention also covers GBS detection kits and related products, which include the CRISPR / Cas12a system and three corresponding supporting detection schemes. These schemes use the CRISPR / Cas12a system to achieve rapid and accurate detection of GBS. At the same time, the present invention also relates to GBS detection lateral flow test strips and test cards constructed based on the CRISPR / Cas12a detection principle, as well as a set of corresponding digital system solutions for data collection, analysis, and return and management of test results based on mobile smart devices and their working principles. All these components and technical solutions belong to the scope of protection of the present invention.

[0042] The present invention has the following beneficial effects:

[0043] The present invention provides for the first time a technology based on CRISPR / Cas12a that can quickly and accurately detect GBS nucleic acid. This technology includes fluorescence detection, lateral flow test paper detection, and result analysis combined with smart mobile terminals, which can all be used as a method for detecting GBS infection in pregnant women during the perinatal period. Its biggest advantage is that it is suitable for use in multiple scenarios. The fluorescence detection system is suitable for general screening of GBS infection in medical institutions, and the test paper detection system is suitable for rapid prenatal diagnosis in primary medical institutions. The smart mobile terminal digital detection system provides an excellent detection solution for primary medical institutions and personal home detection, and at the same time lays the foundation for the establishment of primary medical data files and the tracking of personal medical records.

[0044] Another advantage of the detection method established by the present invention is that the fluorescence and test paper detection system based on CRISPR / Cas12a is highly sensitive and can detect 1copy / μL of bacterial genome or 3000cfu / ml of bacterial samples. Compared with the "gold standard" fluorescent quantitative PCR for nucleic acid detection, the detection instrument is simple, low-cost, and does not require professional laboratory conditions.

[0045] Another advantage of the detection method established by the present invention is that the lateral flow chromatography test paper detection system based on CRISPR / Cas12a can realize visual detection and interpretation. Without the assistance of instruments, the color changes of the T line and the C line can be directly observed by the naked eye to obtain the detection results.

[0046] Another advantage of the detection method established by the present invention is that the lateral flow chromatography test paper detection system combined with the micro optical signal detector can realize data analysis and storage of the intelligent mobile terminal, eliminating human errors and environmental interference.

[0047] Another advantage of the detection method established by the present invention is that detection sites with good effects can be screened, so that the specificity and sensitivity of the detection method established by the present invention are further improved.

[0048] Another advantage of the detection method established by the present invention is that crRNA with good effect can be screened, so that the sensitivity of the detection method established by the present invention is further improved.

[0049] Another advantage of the detection method established by the present invention is that the detection time is short, and both detection systems based on CRISPR / Cas12a only need 40-50 minutes to obtain the detection results. Therefore, the detection method established by the present invention is more conducive to the rapid clinical prenatal detection of GBS. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of the GBS detection system of Crispr / Cas12a described in the present invention.

[0051] Figure 2 Schematic diagram of the digital detection system for smart mobile terminals.

[0052] Figure 3 To screen the detection effects of the two designed crRNAs, the fluorescence ratio of the positive plasmid / control plasmid was used as an indicator.

[0053] Figure 4 Sensitivity analysis results of the Crispr / Cas12a-based GBS fluorescence detection system.

[0054] Figure 5 These are the sensitivity analysis results of the GBS intelligent terminal digital detection system based on Crispr / Cas12a.

[0055] Figure 6 This is the specificity analysis result of the GBS fluorescence detection system based on Crispr / Cas12a.

[0056] Figure 7 These are the specific analysis results of the GBS intelligent terminal digital detection system based on Crispr / Cas12a.

[0057] Figure 8 Simulate the sensitivity analysis results of clinical sample detection for the Crispr / Cas12a-based GBS fluorescence detection system.

[0058] Fig. 9 The sensitivity analysis results of clinical sample detection were simulated for the Crispr / Cas12a-based GBS intelligent terminal digital detection system.

[0059] Fig.10 Comparison of the results of clinical samples detected by Crispr / Cas12a-based GBS intelligent terminal digital detection system and culture method DETAILED DESCRIPTION

[0060] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0061] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0062] Statistical analysis of the following examples of the present invention: All experiments were repeated at least 3 times independently, and the results were expressed as mean values ​​and standard errors, using one-way analysis of variance and T test analysis. All statistical analyses used P < 0.05 as the test standard for significant statistical differences, and the analysis software was GraphPad Prism 9.5.

[0063] Example 1 Construction of pathogen genome positive plasmid

[0064] In the process of establishing the detection method, the determination of the detection target is crucial. First, the CFB gene, which is highly conserved within the GBS species and highly specific between species, was determined as the detection target through NCBI blast. The conserved gene CFB was synthesized and constructed on the PUC57 vector to obtain the recombinant plasmid PUC57-CFB, which simulates the pathogen genome. At the same time, the PUC57 plasmid was used as a negative control. The above plasmids were transformed into E. coli BL21 (DH5α) to construct genetically engineered bacteria for preservation.

[0065] Example 2 Design of crRNA

[0066] The core of the CRISPR / Cas12a detection method lies in crRNA, so the quality of crRNA is directly related to the sensitivity and accuracy of the detection method. CrRNA targeting the detection sequence PAM was designed for the conserved region of the CFB gene, and the 23nt of the genome after the PAM sequence was complementary. The nucleotide sequence of crRNA1 is shown in SEQ ID No.2, and the nucleotide sequence of crRNA2 is shown in SEQ ID No.1.

[0067] (1) Prepare in vitro transcription template (Template DNA), and obtain the transcription template using primer complementary PCR. The reaction system is shown in Table 1

[0068] The complementary primer sequences are T7-F, T7-R1 and T7-R2 sequences such as SEQ ID No. 5-7.

[0069] Table 1

[0070]

[0071] Add the above components to a 200 μL centrifuge tube, mix well and centrifuge. Place in a PCR instrument for PCR. The reaction conditions are shown in Table 2. The primers were synthesized by Qingke Biotechnology.

[0072] Table 2

[0073]

[0074] (2) In vitro transcription

[0075] Using HiScribe TM Transcription was performed using the T7 Quick High Yield RNA Synthesis Kit. The reaction system is shown in Table 3.

[0076] Table 3

[0077]

[0078] Add the above components to a 200μL centrifuge tube, flick to mix and centrifuge. Place in a 37℃ constant temperature water bath and react for 12 hours; then add 30μl of nuclease-free water and 2μl of DNaseI to each tube and incubate at 37℃ for 15 minutes to obtain crRNA.

[0079] (3) Purification of crRNA

[0080] RNA Clean&Concentrator TM -5 (Zymo Research) kit was used to purify crRNA, and the concentration was measured on NanoDrop. The purified crRNA was frozen in a -80°C refrigerator.

[0081] Example 3 Establishment of CRISPR / Cas12a method for detecting GBS

[0082] 1. Amplification of target fragment

[0083] (1) Extraction of positive genetic engineering bacterial plasmids

[0084] Take 4 ml of overnight cultured BL21(DH5α)-PUC57-CFB and BL21(DH5α)PUC57 LB culture medium, use Plasmid Mini Kit ID6943, and process according to the operating procedures to extract plasmids to simulate pathogens.

[0085] (2) ERA amplification

[0086] ERA-F / ERA-R designed for ERA amplification. ERA amplification system is shown in Table 4

[0087] Table 4

[0088]

[0089] Add the above 48 μL of the mixed solution to the reaction tube containing the lyophilized enzyme powder, blow the lyophilized enzyme powder until it is completely dissolved, then add 2 μL of magnesium acetate solution to the reaction tube, mix well, and place the reaction tube in a 38°C constant temperature incubator for 20 minutes to complete ERA amplification. The ERA amplification product was subjected to 1.5% agarose gel nucleic acid electrophoresis to observe the band position and brightness depth. The basic nucleic acid amplification kit (ERA method) was purchased from Suzhou Xianda Gene Technology Co., Ltd.

[0090] 2. CRISPR / Cas12a detection of GBS fluorescence detection system

[0091] The fluorescence detection system is shown in Table 5

[0092] Table 5

[0093]

[0094] The components other than the probe and ERA amplification product were added to the 96-well black board in sequence, centrifuged in the dark, and placed in a 37°C constant temperature biochemical incubator. After incubation for 20 minutes, probe F5Q and ERA amplification product were added. The fluorescence value was read using a SpectraMaxiD3 microplate reader. The sequence of the synthesized probe F5Q is 5'-FAM-TTATT-BHQ1-3', and its detection conditions are λex: 485nm; λem: 535nm.

[0095] 3. CRISPR / Cas12a Detection of GBS Lateral Flow Chromatography Detection System

[0096] The lateral flow chromatography detection system is based on the fluorescence detection system. The reaction systems of the two are the same. Only the F5Q probe needs to be replaced with the F5B probe. The difference is that after adding the probe and the ERA amplification product to the lateral flow chromatography test paper, incubate at 37°C for 20 minutes, take 8μL of the reaction system and add 90μL PB (78mmol / LNaH2PO4, 122mmol / L Na2HPO4). Add 80μL of the detection solution to the sample pad of the test paper. Wait for five minutes and observe the color changes of the T line and C line of the test paper.

[0097] Example 4 Screening of efficient detection sites and crRNA

[0098] The positive standard was used as a template to perform ERA amplification using the ERA method and conditions in Example 3. The two RNAs in Example 2 were used for fluorescence detection. The results are shown in FIG. Figure 3 As shown, after 30 minutes of reaction, the crRNA2 detection group had a higher signal-to-noise ratio, indicating that the detection effect of crRNA2 was better.

[0099] Example 5 Preparation of Lateral Flow Chromatography Test Paper

[0100] The colloidal gold suspension was prepared by citric acid reduction method. Prepare colloidal gold labeled FITC antibody (hytest), adjust the pH value of the colloidal gold suspension with K2CO3, add mouse anti-FITC antibody, stir at room temperature for 45 minutes, and then add bovine serum albumin (BSA, 1%, w / v) to block the active sites on the colloidal gold that are not bound to the antibody. Collect the colloidal gold labeled antibody by centrifugation at 5000g for 30 minutes, resuspend it in gold diluent (78mmol / L NaH2PO4, 122mmol / L Na2HPO4, 0.1% Tween20 v / v, 10% sucrose w / v), evenly spread it on the gold pad, and dry it in a drying room for 2.5 hours. Spray streptavidin and goat anti-mouse antibody solution evenly on the NC membrane to form a control line and a test line, and dry it in a drying room for 4 hours. The sample pad is treated together with the dried gold pad, NC membrane and absorption pad and assembled on the back card. The assembled full-size test strips were cut into 3 mm lateral flow chromatography test strips.

[0101] Example 6 Digital Detection System for Smart Mobile Terminal

[0102] The lateral flow chromatography test strip prepared in Example 5 is loaded into a positioning card shell that is compatible with the micro optical signal detector, and a QR code is generated according to the production batch. When using it, the user only needs to scan the QR code, and the background of the mini program can match this test strip with the production batch, and call the current batch standard curve for standby. Use the mini program on the smart mobile terminal to scan the QR code on the detector to establish a Bluetooth connection. When the detection solution in Example 3 is added dropwise to the sample loading hole of the positioning card shell, it is immediately inserted into the micro detector. After the detector automatically counts down, it reads the grayscale value. The detector transmits data to the mobile device via Bluetooth and is recognized by the mini program. The mini program compares the detection data with the background standard curve, obtains the detection results and displays them on the mini program page. The detection process is as follows Figure 2 .

[0103] Example 7 Sensitivity analysis of Crispr / Cas12a detection of GBS

[0104] The genome was extracted from group B streptococci inoculated on blood agar plates and the concentration was determined and serially diluted to 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 The diluted genome was tested using the fluorescence detection system and test paper detection system in Example 3. The results are as follows Figure 4As shown in Figure 5, 1 copy / μL of the genome in the fluorescence detection system can still increase the fluorescence value and is significantly different from the negative control. The test paper detection system can also successfully detect 1 copy / μL of the genome.

[0105] Example 8 Specificity analysis of Crispr / Cas12a detection of GBS

[0106] In addition to the effective signal amplification efficiency of Cas12a, the high selectivity of the Cas12a system is another important advantage. Determine the specificity of the detection system. Detect the genomes of different bacteria, including bacteria of the same genus (such as Streptococcus pneumoniae, Streptococcus viridans), microorganisms commonly found in vaginal swabs (such as Enterococcus faecalis, Enterococcus faecium, Neisseria gonorrhoeae), and bacteria commonly found in nosocomial infections (such as Pseudomonas aeruginosa and Acinetobacter baumannii). The results are as follows Figure 6 , 7 showed that only the genome of GBS was positive, indicating that the detection method has a high specificity.

[0107] Example 9: Sensitivity analysis of simulated clinical sample detection

[0108] GBS liquid medium with known colony counts was serially diluted and added to the swab eluate to a final concentration of 3 × 10 1 cfu / ml, 3×10 2 cfu / ml, 3×10 3 cfu / ml, 3×10 4 cfu / ml, 3×10 5 cfu / ml, 3×10 6 cfu / ml to simulate clinical samples with different numbers of GBS infection. The genomes in the above samples were extracted and tested using the detection systems in Examples 3 and 6. The results are shown in Figure 8 , 9. Both detection systems can detect samples infected with 3000 cfu / ml.

[0109] Example 10 Clinical sample detection

[0110] 11 clinical samples were rapidly tested using crRNA2 and the intelligent terminal digital detection system in Example 6. The test results were compared with the gold standard culture method for GBS detection. Fig.10 The test results of the intelligent mobile terminal digital detection system were consistent with those of the culture method.

Claims

1. A crRNA for detecting Group B Streptococcus, characterized in that The nucleotide sequence is shown in SEQ ID No:

1.

2. The use of crRNA according to claim 1 in detecting Group B Streptococcus.

3. Application of crRNA described in claim 1 in the preparation of a Group B Streptococcus detection reagent.

4. A detection system for detecting group B streptococci based on CRISPR / Cas12a, characterized in that It comprises the crRNA according to claim 1, an ERA amplification primer pair and a labeled single-stranded DNA probe.

5. The detection system according to claim 4, characterized in that The ERA amplification primer pair ERA-F and ERA-R, the nucleic acid sequences are shown in SEQ ID No3, 4.

6. The detection system according to claim 4, characterized in that The detection system is a fluorescence detection system, and the single-stranded DNA probe is labeled with a fluorescent group and a quenching group.

7. The detection system according to claim 6, characterized in that The labeled single-stranded DNA probe is 5'-FAM-TTATT-BHQ1-3'.

8. The detection system according to claim 4, characterized in that The detection system is a lateral flow chromatography detection system, and the single-stranded DNA probe is labeled with a fluorescent group and a biotin group.

9. The detection system according to claim 8, characterized in that The labeled single-stranded DNA probe is 5'-FAM-TTATT-Bio-3'.

10. The detection system according to claim 8, characterized in that: Also included is a colloidal gold lateral flow chromatography carrier for detecting the integrity of the labeled single-stranded DNA probe.

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